US20210369758A1 - DOUBLE-STRANDED OLIGO RNA STRUCTURE COMPRISING miRNA - Google Patents
DOUBLE-STRANDED OLIGO RNA STRUCTURE COMPRISING miRNA Download PDFInfo
- Publication number
- US20210369758A1 US20210369758A1 US17/402,081 US202117402081A US2021369758A1 US 20210369758 A1 US20210369758 A1 US 20210369758A1 US 202117402081 A US202117402081 A US 202117402081A US 2021369758 A1 US2021369758 A1 US 2021369758A1
- Authority
- US
- United States
- Prior art keywords
- mir
- mirna
- seq
- bond
- cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000002679 microRNA Substances 0.000 title claims abstract description 112
- 108091070501 miRNA Proteins 0.000 title claims abstract description 111
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 71
- 201000011510 cancer Diseases 0.000 claims abstract description 53
- 238000000034 method Methods 0.000 claims abstract description 29
- 108091063295 miR-8078 stem-loop Proteins 0.000 claims abstract description 28
- 230000006907 apoptotic process Effects 0.000 claims abstract description 18
- 230000001939 inductive effect Effects 0.000 claims abstract description 10
- 239000000463 material Substances 0.000 claims description 67
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 59
- 239000000178 monomer Substances 0.000 claims description 34
- 208000020816 lung neoplasm Diseases 0.000 claims description 23
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 22
- 201000005202 lung cancer Diseases 0.000 claims description 22
- 108091034117 Oligonucleotide Proteins 0.000 claims description 20
- 230000002209 hydrophobic effect Effects 0.000 claims description 20
- 150000001875 compounds Chemical class 0.000 claims description 17
- 239000002202 Polyethylene glycol Substances 0.000 claims description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims description 10
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims description 7
- 230000001404 mediated effect Effects 0.000 claims description 7
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 6
- 125000005456 glyceride group Chemical group 0.000 claims description 6
- 150000003431 steroids Chemical class 0.000 claims description 4
- 229910052717 sulfur Inorganic materials 0.000 claims description 4
- 235000012000 cholesterol Nutrition 0.000 claims description 3
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 3
- 239000000194 fatty acid Substances 0.000 claims description 3
- 229930195729 fatty acid Natural products 0.000 claims description 3
- 150000004665 fatty acids Chemical class 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 229930195734 saturated hydrocarbon Natural products 0.000 claims description 3
- 229930195735 unsaturated hydrocarbon Natural products 0.000 claims description 3
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims description 2
- QYIXCDOBOSTCEI-QCYZZNICSA-N (5alpha)-cholestan-3beta-ol Chemical compound C([C@@H]1CC2)[C@@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@H](C)CCCC(C)C)[C@@]2(C)CC1 QYIXCDOBOSTCEI-QCYZZNICSA-N 0.000 claims description 2
- YEYCQJVCAMFWCO-UHFFFAOYSA-N 3beta-cholesteryl formate Natural products C1C=C2CC(OC=O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 YEYCQJVCAMFWCO-UHFFFAOYSA-N 0.000 claims description 2
- 239000004380 Cholic acid Substances 0.000 claims description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 claims description 2
- YEYCQJVCAMFWCO-PXBBAZSNSA-N [(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-yl] formate Chemical compound C1C=C2C[C@@H](OC=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 YEYCQJVCAMFWCO-PXBBAZSNSA-N 0.000 claims description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 2
- QYIXCDOBOSTCEI-UHFFFAOYSA-N alpha-cholestanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 QYIXCDOBOSTCEI-UHFFFAOYSA-N 0.000 claims description 2
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims description 2
- 235000019416 cholic acid Nutrition 0.000 claims description 2
- 229960002471 cholic acid Drugs 0.000 claims description 2
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims description 2
- RBNPOMFGQQGHHO-UHFFFAOYSA-N glyceric acid Chemical compound OCC(O)C(O)=O RBNPOMFGQQGHHO-UHFFFAOYSA-N 0.000 claims description 2
- 150000003904 phospholipids Chemical class 0.000 claims description 2
- 229920001451 polypropylene glycol Polymers 0.000 claims description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 claims description 2
- 108091086868 miR-4477a stem-loop Proteins 0.000 abstract description 27
- 108091055411 miR-3670-1 stem-loop Proteins 0.000 abstract description 26
- 108091039169 miR-3670-2 stem-loop Proteins 0.000 abstract description 26
- 108091024959 miR-3670-3 stem-loop Proteins 0.000 abstract description 26
- 108091045722 miR-3670-4 stem-loop Proteins 0.000 abstract description 26
- 239000000203 mixture Substances 0.000 abstract description 22
- 230000001093 anti-cancer Effects 0.000 abstract description 12
- 230000002401 inhibitory effect Effects 0.000 abstract description 12
- 239000003937 drug carrier Substances 0.000 abstract description 6
- 239000008194 pharmaceutical composition Substances 0.000 abstract description 5
- 230000009702 cancer cell proliferation Effects 0.000 abstract 1
- 108700011259 MicroRNAs Proteins 0.000 description 89
- 210000004027 cell Anatomy 0.000 description 82
- 108020004999 messenger RNA Proteins 0.000 description 51
- 108090000623 proteins and genes Proteins 0.000 description 49
- 108020004459 Small interfering RNA Proteins 0.000 description 33
- 230000014509 gene expression Effects 0.000 description 33
- 230000000295 complement effect Effects 0.000 description 23
- 108020005345 3' Untranslated Regions Proteins 0.000 description 15
- 230000000875 corresponding effect Effects 0.000 description 15
- 230000000694 effects Effects 0.000 description 15
- 238000012216 screening Methods 0.000 description 15
- 239000000126 substance Substances 0.000 description 15
- 239000002246 antineoplastic agent Substances 0.000 description 13
- 108091029119 miR-34a stem-loop Proteins 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- 238000001727 in vivo Methods 0.000 description 12
- 101000702707 Homo sapiens Smad nuclear-interacting protein 1 Proteins 0.000 description 11
- 102100030914 Smad nuclear-interacting protein 1 Human genes 0.000 description 11
- 230000006870 function Effects 0.000 description 11
- 230000012010 growth Effects 0.000 description 11
- 230000000692 anti-sense effect Effects 0.000 description 10
- 230000027455 binding Effects 0.000 description 10
- 239000002105 nanoparticle Substances 0.000 description 10
- 210000004881 tumor cell Anatomy 0.000 description 10
- 108091081021 Sense strand Proteins 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 230000007246 mechanism Effects 0.000 description 9
- 230000011664 signaling Effects 0.000 description 9
- 206010027476 Metastases Diseases 0.000 description 8
- 230000030833 cell death Effects 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 230000009401 metastasis Effects 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 230000004083 survival effect Effects 0.000 description 8
- 230000004614 tumor growth Effects 0.000 description 8
- 108010051975 Glycogen Synthase Kinase 3 beta Proteins 0.000 description 7
- 102100038104 Glycogen synthase kinase-3 beta Human genes 0.000 description 7
- 239000004480 active ingredient Substances 0.000 description 7
- 230000010261 cell growth Effects 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 102100024581 Alpha-taxilin Human genes 0.000 description 6
- 102000008130 Cyclic AMP-Dependent Protein Kinases Human genes 0.000 description 6
- 108010049894 Cyclic AMP-Dependent Protein Kinases Proteins 0.000 description 6
- 108060001084 Luciferase Proteins 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 229940041181 antineoplastic drug Drugs 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 230000009368 gene silencing by RNA Effects 0.000 description 6
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 230000014616 translation Effects 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 108090000672 Annexin A5 Proteins 0.000 description 5
- 102000004121 Annexin A5 Human genes 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 5
- 102100023768 Constitutive coactivator of PPAR-gamma-like protein 1 Human genes 0.000 description 5
- 102100032917 E3 SUMO-protein ligase CBX4 Human genes 0.000 description 5
- 108050004787 GREB1 Proteins 0.000 description 5
- 102000016251 GREB1 Human genes 0.000 description 5
- 102100039788 GTPase NRas Human genes 0.000 description 5
- 101000779390 Homo sapiens A-kinase anchor protein 11 Proteins 0.000 description 5
- 101001048826 Homo sapiens Constitutive coactivator of PPAR-gamma-like protein 1 Proteins 0.000 description 5
- 101000797579 Homo sapiens E3 SUMO-protein ligase CBX4 Proteins 0.000 description 5
- 101000872865 Homo sapiens E3 ubiquitin-protein ligase HECTD3 Proteins 0.000 description 5
- 101000744505 Homo sapiens GTPase NRas Proteins 0.000 description 5
- 101001008953 Homo sapiens Kinesin-like protein KIF11 Proteins 0.000 description 5
- 101001089248 Homo sapiens Receptor-interacting serine/threonine-protein kinase 4 Proteins 0.000 description 5
- 102100027629 Kinesin-like protein KIF11 Human genes 0.000 description 5
- 239000005089 Luciferase Substances 0.000 description 5
- 108091007773 MIR100 Proteins 0.000 description 5
- 102100033734 Receptor-interacting serine/threonine-protein kinase 4 Human genes 0.000 description 5
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 5
- 102000013814 Wnt Human genes 0.000 description 5
- 108050003627 Wnt Proteins 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 230000034994 death Effects 0.000 description 5
- 230000001419 dependent effect Effects 0.000 description 5
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Substances OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 108091091360 miR-125b stem-loop Proteins 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 102100033811 A-kinase anchor protein 11 Human genes 0.000 description 4
- 108010050543 Calcium-Sensing Receptors Proteins 0.000 description 4
- 102100031116 Disintegrin and metalloproteinase domain-containing protein 19 Human genes 0.000 description 4
- 102100035650 Extracellular calcium-sensing receptor Human genes 0.000 description 4
- 102100039820 Frizzled-4 Human genes 0.000 description 4
- 101000760787 Homo sapiens Alpha-taxilin Proteins 0.000 description 4
- 101000777464 Homo sapiens Disintegrin and metalloproteinase domain-containing protein 19 Proteins 0.000 description 4
- 101000885581 Homo sapiens Frizzled-4 Proteins 0.000 description 4
- 101000766332 Homo sapiens Tribbles homolog 1 Proteins 0.000 description 4
- 108091056531 Homo sapiens miR-3670-1 stem-loop Proteins 0.000 description 4
- 108091090313 Homo sapiens miR-3670-2 stem-loop Proteins 0.000 description 4
- 108091045539 Homo sapiens miR-3670-3 stem-loop Proteins 0.000 description 4
- 108091045540 Homo sapiens miR-3670-4 stem-loop Proteins 0.000 description 4
- 108091055302 Homo sapiens miR-4477a stem-loop Proteins 0.000 description 4
- 108091080241 Homo sapiens miR-8078 stem-loop Proteins 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 102100026387 Tribbles homolog 1 Human genes 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000032823 cell division Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000002651 drug therapy Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 230000009711 regulatory function Effects 0.000 description 4
- 102000004091 Caspase-8 Human genes 0.000 description 3
- 108090000538 Caspase-8 Proteins 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 3
- 102100034678 E3 ubiquitin-protein ligase HECTD3 Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108090000331 Firefly luciferases Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102100022057 Hepatocyte nuclear factor 1-alpha Human genes 0.000 description 3
- 101001045751 Homo sapiens Hepatocyte nuclear factor 1-alpha Proteins 0.000 description 3
- 108091093037 Peptide nucleic acid Proteins 0.000 description 3
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 3
- 108091030071 RNAI Proteins 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 3
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000005907 cancer growth Effects 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000030279 gene silencing Effects 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 3
- IIRDTKBZINWQAW-UHFFFAOYSA-N hexaethylene glycol Chemical compound OCCOCCOCCOCCOCCOCCO IIRDTKBZINWQAW-UHFFFAOYSA-N 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 108091074487 miR-34 stem-loop Proteins 0.000 description 3
- 108091092493 miR-34-1 stem-loop Proteins 0.000 description 3
- 108091059780 miR-34-2 stem-loop Proteins 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- JUJBNYBVVQSIOU-UHFFFAOYSA-M sodium;4-[2-(4-iodophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=N1 JUJBNYBVVQSIOU-UHFFFAOYSA-M 0.000 description 3
- 239000000829 suppository Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 2
- 108020003589 5' Untranslated Regions Proteins 0.000 description 2
- 101710159080 Aconitate hydratase A Proteins 0.000 description 2
- 101710159078 Aconitate hydratase B Proteins 0.000 description 2
- 101710168463 Alpha-taxilin Proteins 0.000 description 2
- 108091032955 Bacterial small RNA Proteins 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N CCCCC Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 102100028624 Cytoskeleton-associated protein 5 Human genes 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 101000766864 Homo sapiens Cytoskeleton-associated protein 5 Proteins 0.000 description 2
- 108091044979 Homo sapiens miR-1244-1 stem-loop Proteins 0.000 description 2
- 108091034013 Homo sapiens miR-1244-2 stem-loop Proteins 0.000 description 2
- 108091034014 Homo sapiens miR-1244-3 stem-loop Proteins 0.000 description 2
- 108091045543 Homo sapiens miR-1244-4 stem-loop Proteins 0.000 description 2
- 108091044695 Homo sapiens miR-1248 stem-loop Proteins 0.000 description 2
- 108091061957 Homo sapiens miR-1298 stem-loop Proteins 0.000 description 2
- 108091055169 Homo sapiens miR-2392 stem-loop Proteins 0.000 description 2
- 108091056662 Homo sapiens miR-23c stem-loop Proteins 0.000 description 2
- 108091072916 Homo sapiens miR-3119-1 stem-loop Proteins 0.000 description 2
- 108091072913 Homo sapiens miR-3119-2 stem-loop Proteins 0.000 description 2
- 108091072954 Homo sapiens miR-3164 stem-loop Proteins 0.000 description 2
- 108091072682 Homo sapiens miR-3188 stem-loop Proteins 0.000 description 2
- 108091033969 Homo sapiens miR-3609 stem-loop Proteins 0.000 description 2
- 108091056762 Homo sapiens miR-3612 stem-loop Proteins 0.000 description 2
- 108091056641 Homo sapiens miR-3662 stem-loop Proteins 0.000 description 2
- 108091035136 Homo sapiens miR-378c stem-loop Proteins 0.000 description 2
- 108091054894 Homo sapiens miR-3943 stem-loop Proteins 0.000 description 2
- 108091055649 Homo sapiens miR-4424 stem-loop Proteins 0.000 description 2
- 108091055315 Homo sapiens miR-4477b stem-loop Proteins 0.000 description 2
- 108091064348 Homo sapiens miR-4765 stem-loop Proteins 0.000 description 2
- 108091064326 Homo sapiens miR-4773-1 stem-loop Proteins 0.000 description 2
- 108091064327 Homo sapiens miR-4773-2 stem-loop Proteins 0.000 description 2
- 108091063648 Homo sapiens miR-4999 stem-loop Proteins 0.000 description 2
- 108091054782 Homo sapiens miR-548aa-1 stem-loop Proteins 0.000 description 2
- 108091054818 Homo sapiens miR-548aa-2 stem-loop Proteins 0.000 description 2
- 108091072958 Homo sapiens miR-548u stem-loop Proteins 0.000 description 2
- 108091088900 Homo sapiens miR-5697 stem-loop Proteins 0.000 description 2
- 108091089075 Homo sapiens miR-5705 stem-loop Proteins 0.000 description 2
- 108091089071 Homo sapiens miR-5707 stem-loop Proteins 0.000 description 2
- 108091063730 Homo sapiens miR-571 stem-loop Proteins 0.000 description 2
- 108091061624 Homo sapiens miR-641 stem-loop Proteins 0.000 description 2
- 108091080430 Homo sapiens miR-8053 stem-loop Proteins 0.000 description 2
- 108091054455 MAP kinase family Proteins 0.000 description 2
- 102000043136 MAP kinase family Human genes 0.000 description 2
- 108700026676 Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Proteins 0.000 description 2
- 102000057613 Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Human genes 0.000 description 2
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 2
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 2
- 101710105008 RNA-binding protein Proteins 0.000 description 2
- 108010052090 Renilla Luciferases Proteins 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 102000004389 Ribonucleoproteins Human genes 0.000 description 2
- 108010081734 Ribonucleoproteins Proteins 0.000 description 2
- 102100028029 SCL-interrupting locus protein Human genes 0.000 description 2
- 208000000453 Skin Neoplasms Diseases 0.000 description 2
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 2
- 108091023045 Untranslated Region Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000008436 biogenesis Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 230000022131 cell cycle Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 2
- 229960004316 cisplatin Drugs 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 230000002222 downregulating effect Effects 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 229920001477 hydrophilic polymer Polymers 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000006882 induction of apoptosis Effects 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 229920000831 ionic polymer Polymers 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 239000000693 micelle Substances 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000000394 mitotic effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000003607 modifier Substances 0.000 description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000009437 off-target effect Effects 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 108091007428 primary miRNA Proteins 0.000 description 2
- 230000007115 recruitment Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091092562 ribozyme Proteins 0.000 description 2
- 201000000849 skin cancer Diseases 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 229940043263 traditional drug Drugs 0.000 description 2
- -1 troches Substances 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010011122 A Kinase Anchor Proteins Proteins 0.000 description 1
- 102000014022 A Kinase Anchor Proteins Human genes 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 229920000945 Amylopectin Polymers 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- 101150017888 Bcl2 gene Proteins 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- UIDGHUYTAIPOLJ-UHFFFAOYSA-N CCC(C)(C)C1=NCCO1 Chemical compound CCC(C)(C)C1=NCCO1 UIDGHUYTAIPOLJ-UHFFFAOYSA-N 0.000 description 1
- SMLDDNFMIZNKNQ-UHFFFAOYSA-N CCC(C)(C)N1CCCC1=O Chemical compound CCC(C)(C)N1CCCC1=O SMLDDNFMIZNKNQ-UHFFFAOYSA-N 0.000 description 1
- 101150109761 CKAP5 gene Proteins 0.000 description 1
- 101710167800 Capsid assembly scaffolding protein Proteins 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 108010058546 Cyclin D1 Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 1
- 101001008948 Dictyostelium discoideum Kinesin-related protein 13 Proteins 0.000 description 1
- 102000017944 Dishevelled Human genes 0.000 description 1
- 108050007016 Dishevelled Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108091035710 E-box Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000792859 Enema Species 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 102100024165 G1/S-specific cyclin-D1 Human genes 0.000 description 1
- 230000010337 G2 phase Effects 0.000 description 1
- 208000022072 Gallbladder Neoplasms Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010020382 Hepatocyte Nuclear Factor 1-alpha Proteins 0.000 description 1
- 102100039996 Histone deacetylase 1 Human genes 0.000 description 1
- 101100220774 Homo sapiens CKAP5 gene Proteins 0.000 description 1
- 101001035024 Homo sapiens Histone deacetylase 1 Proteins 0.000 description 1
- 101000984753 Homo sapiens Serine/threonine-protein kinase B-raf Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 102100022875 Hypoxia-inducible factor 1-alpha Human genes 0.000 description 1
- 108050009527 Hypoxia-inducible factor-1 alpha Proteins 0.000 description 1
- 108091029795 Intergenic region Proteins 0.000 description 1
- 102000007482 Interleukin-13 Receptor alpha2 Subunit Human genes 0.000 description 1
- 108010085418 Interleukin-13 Receptor alpha2 Subunit Proteins 0.000 description 1
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 102000011781 Karyopherins Human genes 0.000 description 1
- 108010062228 Karyopherins Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 102100022699 Lymphoid enhancer-binding factor 1 Human genes 0.000 description 1
- 108090001093 Lymphoid enhancer-binding factor 1 Proteins 0.000 description 1
- 101150113681 MALT1 gene Proteins 0.000 description 1
- 102000019149 MAP kinase activity proteins Human genes 0.000 description 1
- 108040008097 MAP kinase activity proteins Proteins 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091030146 MiRBase Proteins 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 102000038030 PI3Ks Human genes 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 108010068086 Polyubiquitin Proteins 0.000 description 1
- 102100037935 Polyubiquitin-C Human genes 0.000 description 1
- 101710130420 Probable capsid assembly scaffolding protein Proteins 0.000 description 1
- 101710092482 Protein kinase 4 Proteins 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 101710204410 Scaffold protein Proteins 0.000 description 1
- 102100027103 Serine/threonine-protein kinase B-raf Human genes 0.000 description 1
- 102100023085 Serine/threonine-protein kinase mTOR Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 101150044133 Stil gene Proteins 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 108010083176 Twist-Related Protein 2 Proteins 0.000 description 1
- 102100031720 Twist-related protein 2 Human genes 0.000 description 1
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 1
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 101001091265 Xenopus laevis Kinesin-like protein KIF11-A Proteins 0.000 description 1
- 101001091264 Xenopus laevis Kinesin-like protein KIF11-B Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 239000003098 androgen Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 230000003140 astrocytic effect Effects 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000009036 biliary tract cancer Diseases 0.000 description 1
- 208000020790 biliary tract neoplasm Diseases 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 239000007963 capsule composition Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000023359 cell cycle switching, meiotic to mitotic cell cycle Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000010109 chemoembolization Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 1
- 229940038472 dicalcium phosphate Drugs 0.000 description 1
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000007905 drug manufacturing Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000007920 enema Substances 0.000 description 1
- 229940095399 enema Drugs 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- MVPICKVDHDWCJQ-UHFFFAOYSA-N ethyl 3-pyrrolidin-1-ylpropanoate Chemical compound CCOC(=O)CCN1CCCC1 MVPICKVDHDWCJQ-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000010685 fatty oil Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000003682 fluorination reaction Methods 0.000 description 1
- 230000003485 founder effect Effects 0.000 description 1
- 201000010175 gallbladder cancer Diseases 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000007902 hard capsule Substances 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 208000026037 malignant tumor of neck Diseases 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000006198 methoxylation reaction Methods 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 108091033783 miR-153 stem-loop Proteins 0.000 description 1
- 108091049021 miR-3677 stem-loop Proteins 0.000 description 1
- 108091058694 miR-4765 stem-loop Proteins 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 230000036456 mitotic arrest Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000035407 negative regulation of cell proliferation Effects 0.000 description 1
- 230000009826 neoplastic cell growth Effects 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000765 poly(2-oxazolines) Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 208000015347 renal cell adenocarcinoma Diseases 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940045902 sodium stearyl fumarate Drugs 0.000 description 1
- 239000007901 soft capsule Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 102000009076 src-Family Kinases Human genes 0.000 description 1
- 108010087686 src-Family Kinases Proteins 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000002511 suppository base Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 201000002743 tongue squamous cell carcinoma Diseases 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 108091006108 transcriptional coactivators Proteins 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/25—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids with polyoxyalkylated alcohols, e.g. esters of polyethylene glycol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/50—Methods for regulating/modulating their activity
Definitions
- This application includes an electronically submitted sequence listing in .txt format.
- the .txt file contains a sequence listing entitled “448DIV_SeqListing_ST25.txt” created on Aug. 13, 2021 and is 689,034 bytes in size.
- the sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
- the present invention relates to a double-stranded oligo RNA structure containing double-stranded miRNA and a composition for preventing or treating cancer containing the same. More specifically, the present invention relates to an anti-cancer pharmaceutical composition containing a double-stranded oligo RNA structure comprising miR-3670, miR-4477a and miR-8078, effectively inhibiting proliferation of cancer cells or inducing apoptosis of cancer cells, and a pharmaceutically acceptable carrier.
- Surgical removal of tumors has been used as an effective and traditional treatment method for diseases caused by failure of normal control of genes, which are typically referred to as cancer.
- anticancer drug therapy is used due to impossibility of surgical operation.
- Anticancer drugs used for drug therapy are generally monomolecular substances synthesized by an organic or inorganic method.
- Anticancer drugs have been developed and used in order to inhibit activities of proteins by effectively binding to the proteins that disrupt signal transduction pathways through over-expression of phosphorylation factor proteins contained in signal transduction pathways.
- Such traditional drug therapy involves many side effects, which include that the substance used as a drug is an artificially synthesized exogenous substance and that actions of anticancer substances target already over-expressed proteins.
- siRNAs small interfering RNAs
- RISCs RNA Induced Silencing complexes
- RISCs function as RNA enzyme scissors, that is, RISCs cleave messenger RNAs (hereafter referred to as “mRNAs”) to inhibit the production of proteins from mRNAs.
- the siRNAs contained in RISCs bind to mRNAs having sequences complementary to the siRNAs sequences to form double-stranded RNAs.
- the RISCs act as RNA enzyme scissors to cleave the target mRNAs, thus preventing mRNAs from acting as templates to repeatedly produce proteins.
- siRNA-based anti-cancer agents are considered to be more advanced than the above-mentioned anticancer agents in that the siRNA-based anti-cancer agents block mRNAs before protein production and use RNAs and intracellular RISC systems.
- off-target effects there are side effects called “off-target effects” that cannot be solved by siRNA-based technologies (Jackson, A. L. et al. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. Rna 12, 1179-87, 2006, Jackson, A. L. et al. Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. Rna 12, 1197-205, 2006, Jackson, A. L. et al.
- RNAi Nat Biotechnol 21, 635-7, 2003, Nielsen, C. B. et al. Determinants of targeting by endogenous and exogenous microRNAs and siRNAs. Rna 13, 1894-910, 2007, Peek, A. S. & Behlke, M. A. Design of active small interfering RNAs. Curr Opin Mol Ther 9, 110-8, 2007).
- siRNA-based anti-cancer agents degrade mRNAs that bind complementarily to siRNA sequences.
- siRNA-based anti-cancer agents bind to even mRNAs which are not complementary to the entirety of siRNA sequences and are complementary to the part thereof, thus causing degradation, which is called a “off-target effect”, meaning causing degradation of non-target mRNAs.
- miRNAs microRNAs
- Oncotarget 5 872-81, 2014, van Rooij, E., Purcell, A. L. & Levin, A. A. Developing MicroRNA Therapeutics. Circulation Research 110, 496-507, 2012, Burnett, J. C. & Rossi, J. J. RNA-based therapeutics: current progress and future prospects.
- miRNAs are RNAs composed of 16 to 27 nucleotides, which are classified as protein non-coding RNAs as compared to messenger RNAs (mRNAs) that are translated into proteins (Carthew, R. W. & Sontheimer, E. J. Origins and Mechanisms of miRNAs and siRNAs. Cell 136, 642-55, 2009, MacFarlane, L.-A. & Murphy, P. R. MicroRNA: Biogenesis, Function and Role in Cancer. Current Genomics 11, 537-561, 2010, Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-33, 2009).
- miRNAs are recorded in the genomes of higher plant and animal cells and are known to play a key role in regulating cell metabolism and functions, including cell production, growth, differentiation and death. To date, about 2,000 types of miRNAs have been found in the human genome, the function of many of these miRNAs are still unknown.
- miRNAs are transcribed from the genomes into RNAs by an RNA polymerase called “Pol II”, the initial length of which is too variable to be specified (Carthew, R. W. & Sontheimer, E. J. Origins and Mechanisms of miRNAs and siRNAs. Cell 136, 642-55, 2009, Brodersen, P. & Voinnet, O. Revisiting the principles of microRNA target recognition and mode of action. Nat Rev Mol Cell Biol 10, 141-148, 2009). This is due to the diversity of the location of the miRNA in the genome.
- miRNA is produced in many ways, for example, miRNA is located in an intron, which is a part not involved in the protein production of mRNAs, and is transcribed at the same time as in production of mRNAs, or miRNA is located in the intergenic region on the genome and is transcribed independently (Malone, C. D. & Hannon, G. J. Small RNAs as guardians of the genome. Cell 136, 656-68, 2009). miRNA produced at an early stage is called “primary microRNA”, and the primary miR is edited into the precursor miRNA (precursor miRNA, pre-miR) by an RNA-cleaving enzyme (RNase) called “Drosha” in the nucleus (Bartel, D. P.
- RNase RNA-cleaving enzyme
- Pre-miR has an RNA hairpin structure consisting of approximately 70 to 80 nucleotides.
- Pre-miR inside the cell nucleus is transported from the nucleus to the cytoplasm by exportin proteins, and is secondarily processed by another RNA-cleaving enzyme (RNase) called “Dicer” in the cytoplasm to produce double-stranded mature microRNAs (hereinafter referred to as “miRs” without any other modifiers) composed of 16 to 27 nucleotides.
- RNase RNA-cleaving enzyme
- miRs double-stranded mature microRNAs
- RNA of one strand of the double-stranded miRs is selectively determined, has activity by binding to the ribonucleoprotein complex, RISC, and binds to the target mRNA using the miR sequence.
- mRNAs can be divided into three parts based on involvement in protein production. That is, mRNAs can be divided into a coding region having protein translation information, and the 5′ and 3′ parts of the coding region having no protein translation information, respectively, 5′-UTR (untranslated region) and 3′-UTR.
- siRNAs which cause degradation of target mRNAs using complementary sequences, act regardless of the 5′-UTR, 3′-UTR and coding parts of the mRNA, whereas miRs bind primarily to the 3′-UTR ((Carthew, R. W. & Sontheimer, E. J. Origins and Mechanisms of miRNAs and siRNAs. Cell 136, 642-55, 2009., Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-33, 2009).
- siRNAs distinguished from siRNAs, in addition to their binding sites to the mRNAs, are that the siRNAs bind primarily to mRNAs containing sequences complementary to the entire siRNA sequence, while, regarding the miRNAs, seed region sequences with a limited size, located at nucleotides at positions of 2 to 8 from the 5′ end, are mainly used to recognize target mRNAs, so the sequence of the entire miRNA does not have a perfectly complementary sequence with the target gene, and although it contains even a part of the non-complementary sequence, it does not affect miRNA activity (Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-33, 2009).
- the sequence size of the seed region is 6 to 8 nucleotides, there are various mRNA types having the sequence complementary thereto in the 3′ UTR. For this reason, it is possible to simultaneously control several types of mRNAs with one type of miRNA.
- the properties of these miRNAs impart, to miRNAs, functions as efficient regulators that are involved in controlling various cell physiological properties that entail cell division, growth, differentiation and death.
- the functions of miRNAs as regulators are advantageous in achieving effective anticancer effects because siRNAs target the suppression of single gene expression, while miRNAs simultaneously inhibit the expression of many cancer-inducing genes.
- a large number of mRNAs contain portions of the 3′ UTR to which one or more types of miRNAs are likely to bind, and one bioinformatic calculation shows that protein production of approximately 30% of the total mRNA is regulated by miRNAs.
- miRNAs act as major regulators in signaling pathways of mRNAs can be identified in that they play a key role in major diseases including cancer (MacFarlane, L.-A. & Murphy, P. R. MicroRNA: Biogenesis, Function and Role in Cancer. Current Genomics 11, 537-561. 2010, Malone, C. D. & Hannon, G. J. Small RNAs as guardians of the genome. Cell 136, 656-68. 2009, Nicoloso, M. S., Spizzo, R., Shimizu, M., Rossi, S. & Calin, G.A. MicroRNAs—the micro steering wheel of tumour metastases. Nat Rev Cancer 9, 293-302. 2009, Landi, D., Gemignani, F.
- miRNAs as therapeutic agents based on the aforementioned close correlation of miRNAs with cancer.
- clinical testing is underway to identify the ability of miRNAs called “miR-34a” to inhibit proliferation of cancer cells and induce apoptosis (Wiggins, J. F. et al. Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34. Cancer Res 70, 5923-30. 2010, Bader, A. G. et al. miR-34 Regulated Genes and Pathways as Targets for Therapeutic Intervention. Google Patents, 2009, Hermeking, H. The miR-34 family in cancer and apoptosis. Cell Death Differ 17, 193-9. 2010, Chang, T. C. et al. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 26, 745-52. 2007).
- RNA oligo structures containing miRNA sequences can be used. It is known that a high efficiency can be induced in vivo by linking a chemical substance or the like to the terminal site of RNA oligos so as to provide enhanced pharmacokinetic characteristics (Nature 11; 432(7014):173-8, 2004). The stability of the RNA oligos depends on the nature of chemical substance bound to the ends of sense (passenger) or antisense (guide) strands of RNA oligos.
- an RNA oligo which is conjugated with a polymer compound such as polyethylene glycol (PEG), interacts with an anionic phosphate group of oligo in the presence of a cationic substance to form a complex, which can become a carrier with improved stability (J Control Release 129 (2): 107-16, 2008).
- a polymer compound such as polyethylene glycol (PEG)
- PEG polyethylene glycol
- micelles composed of polymer complexes are extremely small in size, very uniform in distribution and are spontaneously formed, as compared with other systems used as drug delivery carriers, such as microspheres or nanoparticles, thus having advantages of easy quality control of preparations and reproducibility.
- RNA oligos are simply bonded to a hydrophilic substance (polyethylene glycol (PEG)) as a biocompatible polymer via a simple covalent bond or a linker-mediated covalent bond
- PEG polyethylene glycol
- chemical modification of oligo and conjugation of polyethylene glycol (PEG) still do not solve the drawbacks of low in vivo stability and non-favorable delivery to target organs.
- double-stranded oligo RNA structures in which hydrophilic and hydrophobic substances are bonded to double-stranded oligo RNAs have been developed. These structures form self-assembled nanoparticles called “SANiRNATM” (self-assembled micelle inhibitory RNAs) via hydrophobic interaction of hydrophobic substances (See Korean Patent No. 10-1224828), and this SAMiRNATM technology has advantages of obtaining much smaller and more homogenous nanoparticles than conventional delivery technologies.
- SANiRNATM self-assembled micelle inhibitory RNAs
- the present inventors found miR-3670, miR-4477a and miR-3477a with excellent anticancer efficacy, and identified that these miRNAs and double-stranded oligo RNA structures containing the same effectively inhibited the expression of a number of genes known as cancer-inducing genes to achieve anticancer effects, thus completing the present invention.
- the present invention provides a double-stranded oligo RNA structure comprising the following Formula (1):
- A represents a hydrophilic material
- B represents a hydrophobic material
- X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond
- R represents at least one miRNA selected from the group consisting of miR-3670, miR-4477a, and miR-8078.
- the present invention also provides a composition for preventing or treating cancer comprising the oligonucleotide structure.
- FIG. 1 is a graph showing test results regarding inhibitory activities against protein expression by miR-34a, miR-100 and miR-125b, which are representatively selected from the entire screening library, and each mRNA 3′ UTR whose expression is known to be inhibited by miR-34a, miR-100 and miR-125b is inserted into the 3′ UTR of the luciferase expression vector, in order to identify the activity of the miRNAs;
- FIG. 2 is a graph showing results, when treating NCI-H460 lung cancer cell lines with a screening library composed of 1,700 kinds of miRNAs, quantifying cell growth using Resazurin reagent and then converting the resulting value into a relative growth value;
- FIG. 3 is a graph showing results when selecting about 50 kinds of miRNAs showing excellent efficacy in NCI-H460 cell lines and measuring relative inhibitory activity against cancer cell growth with WST-1 reagent using the miRNAs;
- FIG. 4 shows results, when injecting miR-34a, miR-3670, miR-8078 and miR-4477a by transfection into cells in order to measure apoptotic effects in lung cancer cell lines, staining Annexin V labeled with FITC dye and then analyzing the degree of cell death with a flow cytometer;
- FIG. 5 shows measurement results regarding effects of miRNAs on clustering ability of lung cancer cell lines, after injecting each miRNA by transfection into lung cancer cell lines and culturing in soft agar for 2 weeks;
- FIG. 6 shows cell death performance represented by Z-score when selecting target candidate groups using the miRNA target prediction software, TargetScan, and reducing the intracellular content using siRNAs targeting the target candidate groups;
- FIG. 7 shows results of qPCR analysis indicating the degree of inhibition of expression levels of genes identified in FIG. 6 by miRNA
- FIG. 8 shows the degree of inhibition against the expression of luciferase proteins by miRNAs by cloning the genes identified in FIG. 6 on the 3′ UTR of luciferase;
- FIGS. 9A and 9B are images showing comparison in induction of apoptosis mechanisms, when treating lung cancer cell lines with an oligo RNA structure containing a miRNA sequence, and then staining with Annexin V.
- miRNAs having better efficacies than miR-34a known to have anticancer effects are found and anti-cancer effects thereof are identified.
- 1,700 types of miRNA screening libraries are synthesized (Example 1), NCI-H460 (lung cancer cell line) cells are treated with the miRNA screening libraries, and the ability to suppress growth of cancer cells is measured, as a result, miR-3670, miR-4477a and miR-8078 having the following base sequences (Table 3) have found to have better efficacies than miR-34a, so that it is identifed that miR-3670, miR-4477a and miR-8078 have excellent anticancer efficacies ( FIGS. 4 to 8 ).
- the present invention is directed to a double-stranded oligo RNA structure that comprises at least one miRNA selected from the group consisting of miR-3670, miR-4477a and miR-8078, and the structure represented by the following Formula (1):
- A represents a hydrophilic material
- B represents a hydrophobic material
- X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond
- R represents at least one miRNA selected from the group consisting of miR-3670, miR-4477a, and miR-8078.
- miR-3670 may be a double stranded RNA comprising a base sequence of SEQ ID NO. 35; and a base sequence of SEQ ID NO. 36 or SEQ ID NO. 67.
- miR-4477a may be a double stranded RNA comprising a base sequence of SEQ ID NO. 43; and a base sequence of SEQ ID NO. 44 or SEQ ID NO. 68.
- miR-8078 may be a double stranded RNA comprising a base sequence of SEQ ID NO. 65; and a base sequence of SEQ ID NO. 66 or SEQ ID NO. 69.
- the template strand of miR-3670 may be represented by SEQ ID NO. 35.
- miRNAs are ultimately active in vivo as single-strands, but should be fed into cells in the form of double strands with a base sequence having a similar base size for binding to RISC.
- the antisense binding to the active sequence has a complementary sequence to the active sequence.
- the complementary sequence has a perfect complementary sequence or an in vivo endogenous sequence. All sequences having double strands or bases located at 3′ of one strand may not have a base bond with an opposite (the other) sequence, which is referred to as a 3′ overhang. That is, the perfect complementary sequence of miR-3670 is represented by SEQ ID NO. 36.
- endogenous complementary sequence of miR-3670 may be represented by SEQ ID NO. 67.
- the seed region corresponding from the 2 nd base to the 8 th -9 th bases of miRNA active sequences are major active factors, and long double strands containing the same may be produced and used upon the production of double stranded RNAs.
- active sequences of miR-4477a and miR-8078 and complementary sequences that form double strands with the active sequences are represented as follows. As described above, these double strands may include a 3′ overhang and may be double strands with a long sequence including the seed region.
- miR-4477a (MIMAT0019004 SEQ ID NO. 43) 5′-CUAUUAAGGACAUUUGUGAUUC-3′ Perfect complementary sequence of miR-4477a (SEQ ID NO. 44) 5′-AUCACAAAUGUCCUUAAUAGUU-3′ Endogenous complementary sequence of miR-4477a (SEQ ID NO. 68) 5′-AUCACAAAUGUCCUUAAUGGCA-3′ miR-8078 (MIMAT0031005, SEQ ID NO. 65) 5′-GGUCUAGGCCCGGUGAGAGACUC Perfect complementary sequence of miR-8078 (SEQ ID NO. 66) 5′-GUCUCUCACCGGGCCUAGACCUU Endogenous complementary sequence of miR-8078 (SEQ ID NO. 69) 5′-CUCCACCGGGCUGACCGGCCUG-3′
- the miRNAs discovered through the library screening according to the present invention were found to provide anticancer efficacies by controlling genes commonly known to play a key role in the induction, production and growth of cancer.
- the feature of miRNAs is that one type of miRNA can simultaneously control expression of a plurality of mRNAs. This feature can be identified by the present invention as well and is useful for the development of oligo-based anti-cancer drugs.
- the miR-3670 of the present invention simultaneously inhibits expressions of CBX4, NRAS, CASR, TXLNA, SNIP1, HNF1A, FZD4, TRIB1, ADMA19 and CKAP5, miR-8078 inhibits expressions of GREB1, HECTD3 and RIPK4, and miR-4477a simultaneously inhibits expressions of STIL, KIF11, AKAP11, and FAM120A ( FIG. 6 ).
- the target genes inhibited by miRNAs of the present invention are known to have the following functions.
- CBX4 (polycomb chromobox 4) is involved in angiogenesis of tumors and facilitation of metastasis thereof, and NRAS is known to play a key role in tumor growth and cell division (Orouji, E. et al. MAP Kinase pathway gene copy alterations in NRAS/BRAF wild-type advanced melanoma. Int J Cancer (2015); Zheng, C. et al. MicroRNA-195 functions as a tumor suppressor by inhibiting CBX4 in hepatocellular carcinoma. Oncol Rep 33, 1115-22 (2015); Jiao, H. K. et al.
- CASR is found to be over-expressed in tumors and is required for metastasis of tumors, and TXLNA is known to be involved in the growth and metastasis of tumors.
- Clinical results have been reported that the survival rate of patients with high expression rates of TXLNA is low (Mashidori, T., Shirataki, H., Kamai, T., Nakamura, F. & Yoshida, K. Increased alpha-taxilin protein expression is associated with the metastatic and invasive potential of renal cell cancer. Biomed Res 32, 103-10 (2011); Tennakoon, S., Aggarwal, A. & Kallay, E. The calcium-sensing receptor and the hallmarks of cancer. Biochim Biophys Acta (2015); Ohtomo, N. et al. Expression of alpha-taxilin in hepatocellular carcinoma correlates with growth activity and malignant potential of the tumor. Int J Oncol 37, 1417-23 (2010)).
- SNIP1 known as a transcriptional coactivator, promotes the expression of cyclin D1, which is essential for cell growth and division. It is known that the anticancer prognosis of patients with a high expression level of SNIP1 is bad.
- SNIP1 is known to function to promote tumor growth in combination with c-Myc, which acts as a major regulator of cell proliferation (Li, Q. et al. SNIP1: a new activator of HSE signaling pathway. Mol Cell Biochem 362, 1-6 (2012); Fujii, M. et al. SNIP1 is a candidate modifier of the transcriptional activity of c-Myc on E box-dependent target genes. Mol Cell 24, 771-83 (2006); Roche, K.
- HNF1A and FZD4 are constituent factors of the Wnt signaling system that is deeply involved in the growth and survival of tumors.
- the Wnt signaling system has been intensively researched in tumor biology and its importance is widely known.
- TRIB1 is known to play a role in growth and metastasis of tumor cells and inhibition of cell apoptosis, and is known as a factor regulating the MAPK signaling system which is one of the main signaling pathways of tumor growth (Pecina-Slaus, N. et al.
- Wnt signaling transcription factors TCF-1 and LEF-1 are upregulated in malignant astrocytic brain tumors. Histol Histopathol 29, 1557-64 (2014); Ueno, K. et al.
- Tumor suppressor microRNA-493 decreases cell motility and migration ability in human bladder cancer cells by downregulating RhoC and FZD4.
- TRIB1 Int J Cancer 135, 541-50 (2014); Soubeyrand, S., Naing, T., Martinuk, A. & McPherson, R. ERK1/2 regulates hepatocyte Trib1 in response to mitochondrial dysfunction. Biochim Biophys Acta 1833, 3405-14 (2013)).
- HECTD3 is known as an E3 ubiquitin ligase that inhibits tumor death by inducing degradation of caspase-8 by attaching polyubiquitin to caspase-8 which facilitates cell apoptosis and increases drug resistance to cisplatin anticancer drugs by stabilizing MALT1 proteins.
- RIPK4 has been reported as receptor-interacting protein kinase 4 to induce accumulation of ⁇ -catenin, which is a cell growth signaling factor, and to activate the Wnt signaling system. It has been found that artificial elimination of RIPK4 can inhibit tumor growth in tumor animal models (Li, Y. et al.
- the HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by promoting K63-linked polyubiquitination of caspase-8. Cell Death Dis 4, e935 (2013); Li, Y. et al. The HECTD3 E3 ubiquitin ligase suppresses cisplatin-induced apoptosis via stabilizing MALT1. Neoplasia 15, 39-48 (2013); Huang, X. et al. Phosphorylation of Dishevelled by protein kinase RIPK4 regulates Wnt signaling. Science 339, 1441-5 (2013)).
- the STIL gene as an essential element in the transition from the G2 phase to the M phase during the cell cycle is observed to be highly expressed in various types of cancer and is known to be necessary for tumor proliferation and survival.
- KIF11 is also reported to be one of factors necessary for the growth and metastasis of tumor cells and is known to inhibit growth of tumors by inhibiting the activity of KIF11 (Erez, A. et al. Sil overexpression in lung cancer characterizes tumors with increased mitotic activity. Oncogene 23, 5371-7 (2004); Erez, A. et al.
- the SIL gene is essential for mitotic entry and survival of cancer cells. Cancer Res 67, 4022-7 (2007); Tang, Y., Orth, J.D., Xie, T.
- AKAP220 protein organizes signaling elements that impact cell migration. J Biol Chem 286, 39269-81 (2011); Whiting, J. L. et al. Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3beta to A-kinase Anchoring Protein 220. J Biol Chem 290, 19445-57 (2015); Tanji, C. et al. A-kinase anchoring protein AKAP220 binds to glycogen synthase kinase-3beta (GSK-3beta) and mediates protein kinase A-dependent inhibition of GSK-3beta.
- GSK-3beta glycogen synthase kinase-3beta
- miR-3670 simultaneously inhibits expression of CBX4, NRAS, CASR, TXLNA, SNIP1, HNF1A, FZD4, TRIB1, ADMA19 and CKAP5
- miR-8078 inhibits expression of GREB1, HECTD3 and RIPK4
- miR-4477a simultaneously inhibits expression of STIL, KIF11, AKAP11 and FAM120A.
- the mRNAs of the genes include a perfect match wherein the corresponding miRNA regions are complementary 100% to base sequences, as well as mismatch wherein the corresponding miRNA regions are inconsistent with some base sequences.
- miRNAs For these miRNAs, the match of the seed regions is the most important, and miRNAs preferably have at least 70%, more preferably at least 80%, even more preferably at least 90%, still even more preferably 95% or more, and most preferably 100%, of an identity with a part of mRNA sequences of the corresponding genes.
- miRNAs may be duplexes, include single molecule polynucleotide, and be antisense oligonucleotides or microRNAs (miRNAs), but are not limited thereto.
- the conjugates wherein hydrophilic and hydrophobic materials are bound to RNA oligos
- the conjugates wherein hydrophilic and hydrophobic materials are bound to both ends of RNA oligos, can efficiently deliver RNA oligos in vivo and improve stability.
- Self-assembled nanoparticles are formed through hydrophobic interactions of hydrophobic materials. These nanoparticles have extremely excellent in vivo delivery efficiency and in vivo stability, and the improvement in structures provides very uniform particle sizes and easy quality control (QC), thus having an advantage of simple drug manufacturing process.
- the hydrophilic material in the double-stranded oligo RNA structures comprising miRNAs according to the present invention is represented by (A) n , (A m -J) n or (J-A m ) n , wherein A represents a hydrophilic material monomer, n represents 1 to 200, m represents 1 to 15, and J represents a linker that links m hydrophilic material monomers to one another, or links m hydrophilic material monomers to oligonucleotides.
- the double-stranded oligo RNA structure according to the present invention has the structure represented by the following Formula (1′):
- A, B, X and Y are as defined in Formula (1), S represents a sense strand of specific miRNA regarding the corresponding gene and AS represents an antisense strand of specific miRNA regarding the corresponding gene.
- the double-stranded oligo RNA structure comprising miRNAs according to the present invention may be a double-stranded oligo RNA structure comprising the structure represented by the following Formula (2):
- A, B, X, Y and R are as defined in Formula (1).
- the double-stranded oligo RNA structure has a structure represented by the following Formula (2′):
- the hydrophilic material may be a cationic or non-ionic polymer material having a molecular weight of 200 to 10,000, preferably a non-ionic polymer substance having a molecular weight of 1,000 to 2,000.
- a non-ionic hydrophilic polymer compound for example, polyethylene glycol, polyvinylpyrrolidone or polyoxazoline is preferably used, but the present invention is not limited thereto.
- the double-stranded oligo RNA structure according to the present invention has a structure represented by the following Formula (3) or Formula (4):
- A represents a hydrophilic material monomer
- n 1 to 200
- J represents a linker that links m hydrophilic material monomers to one another, or links m hydrophilic material monomer to oligonucleotide
- X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond
- R represents specific miRNA according to the present invention.
- the double-stranded oligo RNA structure comprising miRNA according to the present invention may have a structure represented by Formula (3′):
- A, B, J, m, n, X and Y are as defined in Formula (3), S represents a sense strand of specific miRNA regarding the corresponding gene, and AS represents an antisense strand of specific miRNA regarding the corresponding gene.
- the double-stranded oligo RNA structure comprising miRNAs according to the present invention has a structure represented by the following Formula (4′):
- A, B, J, m, n, X and Y are as defined in Formula (4), S represents a sense strand of specific miRNA regarding the corresponding gene, and AS represents an antisense strand of specific miRNA regarding the corresponding gene.
- any monomer of non-ionic hydrophilic polymers may be used as the hydrophilic material monomer (A) in Formula (3) and Formula (4) without particular limitation so long as it satisfies the objects of the present invention.
- Preferred is a monomer selected from Compounds (1) to (3) shown in Table 1, and more preferred is a monomer of Compound (1).
- G in Compound (1) is preferably selected from CH 2 , O, S and NH.
- the monomer represented by compound (1) has advantages of having a variety of functional groups which would be introduced, exhibiting excellent bio-compatibility, for example, providing better in vivo affinity and inducing less immune reactions, and improving in vivo stability and delivery efficiency of oligonucleotide contained in the structure according to Formula (3) and Formula (4), thus being very suitable for the manufacture of the structure according to the present invention.
- the hydrophilic material in Formula (3) and Formula (4) preferably has a total molecular weight of 1,000 to 2,000. Accordingly, for example, wherein hexaethylene glycol in Compound (1), that is, G is 0, and m is 6, is used in Formula (3) and Formula (4), the molecular weight of the hexaethylene glycol spacer is 344 and thus the number of repeats (n) is preferably 3 to 5. According to the present invention, in Formula (3) and Formula (4), a repeat unit represented by (A m -J) or (J-A m ), that is, a hydrophilic material block may be used as an appropriate number represented by “n”.
- the linker which mediates the bond between the hydrophilic material monomers, used for respective hydrophilic material blocks, may be identical or different.
- the linker (J) is preferably selected from the group consisting of PO 3 ⁇ , SO 3 and CO 2 , but is not limited thereto. Depending on the monomer of the used hydrophilic material or the like, any linker may be used so long as it satisfies the object of the present invention and is obvious to those skilled in the art.
- the entirety or part of the hydrophilic material monomer may be modified to have a functional group required for bonding to other substances such as a target specific ligand.
- one to three phosphate groups may be bonded to the 5′ end of the antisense strand of a double-stranded oligo RNA structure comprising specific miRNA for the gene.
- the double-stranded oligo RNA structure comprising miRNA has a structure represented by the following Formula (3′′) or Formula (4′′):
- the hydrophobic material (B) functions to form nanoparticles having an oligonucleotide structure represented by Formula (1) through hydrophobic interaction.
- the hydrophobic material preferably has a molecular weight of 250 to 1,000, and may be a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, C 12 to C 50 unsaturated or saturated hydrocarbon, diacyl-phosphatidylcholine, fatty acid, phospholipid, lipopolyamine or the like, but is not limited thereto.
- Any hydrophobic material may be used without limitation so long as it satisfies the objects of the present invention, which is obvious to those skilled in the art to which the present invention pertains.
- the steroid derivative may be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate and cholestanylamine, and the glyceride derivative may be selected from mono-, di- and tri-glyceride and the like, wherein the fatty acid of glyceride is preferably a C 12 to C 50 unsaturated or saturated fatty acid.
- saturated or unsaturated hydrocarbon or cholesterol is preferred for easy binding to oligonucleotide during synthesis of the oligonucleotide structures according to the present invention.
- the hydrophobic material is bonded to the distal end of the hydrophilic mateiral and may be bonded to the sense strand or antisense strand of miRNAs.
- the hydrophilic material, the hydrophilic material block or the hydrophobic material is bonded to oligonucleotide through a simple covalent bond or linker-mediated covalent bond (X or Y).
- the covalent bond may be a non-degradable or degradable bond.
- the non-degradable bond include an amide bond or a phosphoryl bond
- the degradable bond includes a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, an enzyme-degradable bond or the like, but is not limited thereto.
- the miRNA oligo structures according to the present invention were produced and in vitro treated with lung cancer cell lines, and the cell lines were stained with Annexin V and were analyzed by flow cytometry. As can be seen from FIGS. 9A and 9B , when nanoparticles were used to improve in vivo stability using the RNA structure, apoptosis of cell lines can be concentration-dependently induced.
- the present invention relates to a composition for preventing or treating cancer comprising the oligonucleotide structure.
- the present invention also relates to a method for preventing or treating cancer comprising a step of administering the oligonucleotide structure.
- the cancer is at least one selected from the group consisting of primary cancer such as lung cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, gall bladder cancer, biliary tract cancer, breast cancer, leukemia, esophageal cancer, non-Hodgkin's lymphoma, thyroid cancer, cervical cancer and skin cancer, and metastatic cancer caused by metastasis from primary cancer to other organs, and tumor-associated cell diseases caused by promotion of abnormal excessive cell division, but the present invention is not limited thereto.
- primary cancer such as lung cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, gall bladder cancer, biliary tract cancer, breast cancer, leukemia, esophageal cancer, non-Hodgkin's lymphoma, thyroid cancer, cervical cancer and skin cancer
- metastatic cancer caused by metastasis from primary cancer to other organs
- tumor-associated cell diseases caused by promotion of abnormal excessive cell division
- the miRNA sequence that can be used as an active ingredient of the composition for treating cancer provided by the present invention is a sequence derived from the human genome, however, a miRNA sequence obtained from the genome derived from another animal also can be used without limiting the genome of the miRNA
- the miRNA can be used in the form of various miRNA derivatives (miRNA mimics) that generate the bioequivalence efficacy of miRNAs, and can be modified miRNAs containing miRNA sequences containing the same seed region. At this time, the length of sequence 1 or sequence 2 can be reduced, and a short derivative consisting of 15 nucleotides can also be used.
- miRNA mimics miRNA mimics
- the miRNA derivatives for miRNAs may partially include a phosphorothiolate structure in which O in the RNA phosphate backbone structure is replaced with another element such as sulfur, and can be used in the forms wherein DNA, PNA (peptide nucleic acid) and LNA (locked nucleic acid) molecules are entirely or partially replaced with RNA and can be used in the forms wherein the 2′ hydroxyl group of RNA sugar is replaced with various functional structures, and examples of such modifications include, but are not limited to, methylation, methoxylation, fluorination and the like.
- the miRNA is not limited to mature miRNA and the double stranded RNA of the miRNA derivative derived therefrom, and can be used in the form of a miRNA precursor and, for the miRNA precursor, aforementioned partial or entire replacement of the RNA phosphate backbone structure and RNA nucleic acid with DNA, PNA, LNA and the like, and modification of the 2′ hydroxyl group of the RNA sugar molecule are possible.
- the miRNA can be used in the form of precursor miRNA or primary miRNA (pri-miRNA) and can be synthesized by a chemical method or delivered in the form of a plasmid to cells which express the same.
- methods for delivering miRNAs to cells cultured on culture dishes include, but are not limited to, mixing with cationic lipids, using electrical stimulation, and using viruses.
- composition for treating cancer comprising the miRNA as an active ingredient may be a pharmaceutical composition further containing a pharmaceutically acceptable carrier and may be formulated together with a carrier.
- pharmaceutically acceptable carrier refers to a carrier or diluent that does not impair biological activities or properties of an administered compound without stimulating an organism.
- Acceptable pharmaceutical carriers for compositions which are formulated into liquid solutions, are sterilized and biocompatible and examples thereof include saline, sterile water, Ringer's solution, buffered saline, albumin injection solutions, dextrose solutions, maltodextrin solutions, glycerol, ethanol and mixtures thereof. If necessary, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added.
- injectable solutions such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.
- composition for preventing or treating cancer comprising the miRNA and the pharmaceutically acceptable carrier can be applied to any formulation containing the same as an active ingredient and can be prepared for oral or parenteral formulation.
- the pharmaceutical formulation may include formulations suitable for oral, rectal, nasal, topical (including under the cheek and tongue), subcutaneous, vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or inhalation or insufflation.
- formulations for oral administration containing the composition of the present invention as an active ingredient include tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs.
- a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrating agent such as corn starch or sweet potato starch, a lubricant such as calcium stearate, sodium stearyl fumarate or polyethyleneglycol wax can be incorporated, and capsule formulations may further contain a liquid carrier such as a fatty oil, in addition to the above-mentioned ingredients.
- a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin
- an excipient such as dicalcium phosphate
- a disintegrating agent such as corn starch or sweet potato starch
- a lubricant such as calcium stearate, sodium stearyl fumarate or polyethyleneglycol wax
- capsule formulations may further contain a liquid
- compositions for parenteral administration containing the composition of the present invention as an active ingredient include injection forms such as subcutaneous injection, intravenous injection or intramuscular injection, suppository or spray forms such as aerosols inhalable through a breathing apparatus.
- injection forms such as subcutaneous injection, intravenous injection or intramuscular injection, suppository or spray forms such as aerosols inhalable through a breathing apparatus.
- the compositions of the present invention can be mixed in water with stabilizers or buffers to prepare solutions or suspensions and the solutions or suspensions can be formulated on the basis of an ampule or vial unit for administration.
- compositions for rectal administration such as suppositories containing a conventional suppository base such as cocoa butter or other glycerides or enema preparations can be formulated.
- an additive such as a propellant may be mixed such that a water-dispersed concentrate or wet powder is dispersed.
- miRBase As 21-version human miRNA sequences provided from the miRNA database, miRBase (www.mirbase.org), double-stranded sequences of miRNA were synthesized by a solid synthesis method used for common synthesis of oligo from 1,700 miRNA screening libraries, based on the stem-loop structure. Each strand of the synthesized miRNA was purified by reverse phase separation using a C18 resin. Whether or not the intended sequence was synthesized for all synthesized miRNA strands was detected and identified with a MALDI-TOF mass spectrometer. In order to prepare double-stranded miRNAs, the synthesized miRNA strands and the corresponding complementary strands were heated in the presence of a salt at 95° C.
- Sequences 1 of respective tested double-stranded miRNAs are represented by SEQ ID NO. 70 to SEQ ID NO. 1797 in this order and Sequences 2 thereof are represented by SEQ ID NO. 1798 to 3525 in this order.
- miR-34a, miR-100 and miR-125b were selectively selected from about 1,700 types of miRNA screening libraries. miRNAs have been selected based on the large number of studies previously performed on the types of target mRNAs that control functions and expression and the binding sites that bind to each mRNA 3′ UTR.
- the 3′ UTR sites of Bcl2, mTOR and Lin28b mRNAs, which are known to be regulated by miR-34a, miR-100 and miR-125b, respectively, are replaced with the 3′ UTR sites of the firefly luciferase vector to produce vectors corresponding to respective miRNAs.
- the HEK-293T cell lines were co-transfected with each vector and miR control group, or miR-34a, miR-100 and miR-125b corresponding to each vector, using an intracellular delivery reagent of oligo, Lipofectamine 2000 (Invitrogen) (three replicate samples) and cultured at 37° C. and 5% (v/v) carbon dioxide for 24 hours.
- the activity of luciferase was measured using a luminometer (Thermo Scientific) to identify the activity of the synthesized miRNA ( FIG. 1 ).
- RNAiMAX reagent Invitrogen
- the cells were further cultured for 24 hours under the same conditions as the cell culture conditions described above, and the fluorescence value generated by adding Resazurin reagent (Promega) was measured using a fluorescence meter (Fluoremeter, Tecan).
- x i is the measured value of each well
- ⁇ is the mean value of the whole well of the plate
- ⁇ is the standard deviation.
- the standard deviation multiple, z i , of each well was the mean value obtained from the three plate replicates, and was used to select 50 primary candidate miRNAs having a z value less than ⁇ 2 ( FIG. 2 , Table 2).
- hsa-miR-3119 strand 1 UGGCUUUUAACUUUGAUGGC 23 SEQ ID NO. hsa-miR-3119 strand 2 CAUCAAAGUUAAAAGCCAUU 24 SEQ ID NO. hsa-miR-3164 strand 1 UGUGACUUUAAGGGAAAUGGCG 25 SEQ ID NO. hsa-miR-3164 strand 2 CCAUUUCCCUUAAAGUCACAUU 26 SEQ ID NO. hsa-miR-3188 strand 1 AGAGGCUUUGUGCGGAUACGGGG 27 SEQ ID NO. hsa-miR-3188 strand 2 CCGUAUCCGCACAAAGCCUCUUU 28 SEQ ID NO.
- hsa-miR-4477a strand 2 AUCACAAAUGUCCUUAAUAGUU 44
- SEQ ID NO. hsa-miR-4477b strand 2 CAAUCACAAAUGUCCUUAAUUU 46
- the method used for screening is to measure the degree of relative inhibition of cell proliferation by measuring the number of cells in a quantitative sense.
- the mechanisms that inhibit cell proliferation include a method of reducing the cell cycle rate and a method of inducing apoptosis.
- the degree of apoptosis was analyzed by flow cytometry (fluorescence activated cell sorter (FACS)). For this purpose, cells were seeded on a 6-well plate, and the miRNA was injected into the cells using RNAiMAX reagent. Then, cells were cultured under the conditions described above for 48 hours.
- FACS fluorescence activated cell sorter
- miR-4477a-IC IC AUCACAAAUGUCCUUAAUGGCA 68 SEQ ID NO. hsa-miR-8078 strand 1 GGUCUAGGCCCGGUGAGAGACUC 65 SEQ ID NO. hsa-miR-8078 strand 2 GUCUCUCACCGGGCCUAGACCUU 66 SEQ ID NO. miR-8078-IC IC CUCCACCGGGCUGACCGGCCUG 69
- NCI-H460 lung cancer cell lines were treated with control miRNA, miR-34a, miR-8078, miR-3670, miR-4477a and miR-4765, cultured for 24 hours, mixed with soft agar, and cultured on a 6 well-plate for 2 weeks. The cells were stained with a crystal violet dye and the numbers of clusters were counted ( FIG. 5 ). Results showed that the cells treated with miR-8078 and miR-3670 formed almost no clusters, and that the cells treated with miR-4477a showed about 30% of cluster formation ability as compared with the control.
- Target mRNAs whose protein expression is controlled by miRNA, have a sequence partially complementary to the sequence of the miRNAs.
- the sequence of the seed region for miRNAs is particularly important, because it binds to mRNA having a sequence complementary to the seed region sequence to inhibit gene expression.
- the seed region sequence is relatively short, i.e., 8 to 9 bases, the mRNA targeted by the miRNA is estimated using software.
- the target genes predicted through software were treated with siRNA to reduce intracellular content and were selected by determining whether or not cell growth was inhibited.
- TargetScan was used as a target prediction software generally used in the art in order to predict the target mRNA of miRNA, and the total 600 types of genes were selected as estimated targets of miR-3670, miR-4477a and miR-8078.
- Three siRNAs were synthesized for each gene selected, and the same experiment was conducted as in Example 3 using the siRNAs. Cells were seeded on 96-well plates, treated with each siRNA and cultured for 48 hours, and then cell proliferation was measured using a resazurin reagent. The Z-score of each gene was calculated from the mean of the measurement values of a total of about 1,800 (600 genes ⁇ 3 kinds of siRNAs) in the same manner as in Example 3 and shown in FIG. 6 .
- miRNAs The action mode of miRNAs deteriorates production of proteins from mRNAs and, at the same time, causes the degradation of most of the target mRNAs. Accordingly, miRNAs are injected into cells and the contents of mRNAs, which are the targets of miRNAs, are analyzed using qPCR, and the decrease in the content is measured, which can be used as a criterion for determining the target mRNA of miRNAs.
- RNAs were extracted from respective cells to quantitatively measure RNA contents ( FIG. 7 ). Results showed that the predicted target mRNA contents of miR-3670, miR-4477a and miR-8078 were remarkably lowered.
- luciferase assay is commonly used as a method for directly measuring the relationships between miRNAs and target mRNAs.
- the TargetScan software provides the 3′ UTR sequence containing the miRNA-binding sequence.
- the 3′ UTR sequence was inserted by gene cloning into the 3′ UTR of firefly luciferase as described in Example 2, and the vector produced in this manner was transfected into human embryonic kidney (HEK) cells simultaneously with the corresponding miRNA to measure the expression level of luciferase in the vector.
- HEK human embryonic kidney
- renilla luciferase was also transfected simultaneously to calibrate the transfection efficiency.
- miRNA, firefly luciferase and Renilla luciferase were simultaneously injected and cultured for 48 hours and then measured with a luminometer ( FIG. 8 ). Results showed that each target mRNA is directly controlled by the corresponding miRNA.
- the double-stranded oligo RNA structure produced according to the present invention has a structure represented by the following Formula (5):
- S represents a sense strand of miRNA
- AS represents an anti-sense strand of miRNA
- PO 4 represents a phosphate group
- ethylene glycol is a hydrophilic material monomer
- hexaethylene glycols are bonded to through a phosphate group (PO 3 ⁇ ) as a linker (J)
- C 24 represents a tetradocosane containing a disulfide bond as a hydrophobic material
- 5′ and 3′ represent the end directions of double-stranded oligo RNA.
- a phosphodiester bond constituting an RNA backbone structure using ⁇ -cyanoethyl phosphoamidite and DMT-hexaethyl glycol-CPG as a support is linked, so an oligo RNA-hydrophilic material structure containing the sense strand wherein hexaethylene glycol is bonded to the 3′ end is synthesized, and then tetradodecanoic acid containing a disulfide bond is bonded at the 5′ end, to form a desired RNA-polymer structure of the sense strand.
- an antisense strand of the sequence complementary to the sense strand was prepared through the aforementioned reaction.
- the RNA oligo structure was prepared by the method in accordance with Example 10 in order to ensure in vivo stability of the miRNA selected through Examples described above.
- the lung cancer cell lines were seeded and cultured on a 6-well plate.
- the nanoparticles with different concentrations for respective wells were added to a culture medium.
- the cells were stained with Annexin V labeled with an FIT-C fluorescent dye, and the degree of apoptosis was analyzed by flow cytometry.
- miRNAs as the RNA structure kill cells dependent upon the concentration of treated miRNAs.
- the double-stranded oligo RNA structure and the composition for treating cancer containing the same according to the present invention include at least one miRNA selected from the group consisting of miR-3670, miR-4477a and miR-8078, thereby being widely used as an anti-cancer therapeutic agent because of improved anti-cancer effects, as compared to the pharmaceutical composition for treating cancer containing miR-34a and other miRNAs as an active ingredient.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Emergency Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to a double-stranded oligo RNA structure comprising double-stranded miRNA, and a composition for preventing or treating cancer, containing the same. More specifically, the present invention relates to an anti-cancer pharmaceutical composition, containing: a double-stranded oligo RNA structure comprising miR-3670, miR-4477a and miR-8078, and characterized by a method for effectively inhibiting cancer cell proliferation or inducing cancer cell apoptosis; and a pharmaceutically acceptable carrier.
Description
- This is a divisional under 35 USC § 120 of U.S. patent application Ser. No. 16/302,670 filed Nov. 18, 2018, which is a U.S. national phase under 35 USC § 371 of International Patent Application No. PCT/KR17/09271 filed Aug. 24, 2017, which in turn claims priority under 35 U.S.C. § 119 of Korean Patent Application No. 10-2016-0107937 filed Aug. 24, 2016. The disclosures of all such patent applications are hereby incorporated herein by reference in their respective entireties, for all purposes.
- This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “448DIV_SeqListing_ST25.txt” created on Aug. 13, 2021 and is 689,034 bytes in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
- The present invention relates to a double-stranded oligo RNA structure containing double-stranded miRNA and a composition for preventing or treating cancer containing the same. More specifically, the present invention relates to an anti-cancer pharmaceutical composition containing a double-stranded oligo RNA structure comprising miR-3670, miR-4477a and miR-8078, effectively inhibiting proliferation of cancer cells or inducing apoptosis of cancer cells, and a pharmaceutically acceptable carrier.
- Surgical removal of tumors has been used as an effective and traditional treatment method for diseases caused by failure of normal control of genes, which are typically referred to as cancer. However, when primary cancer metastasizes to other organs, anticancer drug therapy is used due to impossibility of surgical operation. Anticancer drugs used for drug therapy are generally monomolecular substances synthesized by an organic or inorganic method. Anticancer drugs have been developed and used in order to inhibit activities of proteins by effectively binding to the proteins that disrupt signal transduction pathways through over-expression of phosphorylation factor proteins contained in signal transduction pathways. Such traditional drug therapy involves many side effects, which include that the substance used as a drug is an artificially synthesized exogenous substance and that actions of anticancer substances target already over-expressed proteins.
- The development of drug therapies to replace the traditional drug treatment methods has progressed in many ways, one of which is the use of small interfering RNAs (hereinafter referred to as “siRNAs”). (Iorns, E., Lord, C. J., Turner, N. & Ashworth, A. Utilizing RNA interference to enhance cancer drug discovery. Nat Rev Drug Discov 6, 556-68. 2007). siRNAs are single-stranded RNAs including 16 to 27 nucleotides, which act as constituent components of ribonucleoproteins known as RISCs (RNA Induced Silencing complexes) (Tomari, Y. & Zamore, P. D. Perspective: machines for RNAi. Genes Dev 19, 517-29, 2005, Chu, C. Y. & Rana, T. M. Potent RNAi by short RNA triggers. Rna 14, 1714-9, 2008, Mittal, V. Improving the efficiency of RNA interference in mammals. Nat Rev Genet 5, 355-65, 2004, Reynolds, A. et al. Rational siRNA design for RNA interference. Nat Biotechnol 22, 326-30. 2004). RISCs function as RNA enzyme scissors, that is, RISCs cleave messenger RNAs (hereafter referred to as “mRNAs”) to inhibit the production of proteins from mRNAs. The siRNAs contained in RISCs bind to mRNAs having sequences complementary to the siRNAs sequences to form double-stranded RNAs. The RISCs act as RNA enzyme scissors to cleave the target mRNAs, thus preventing mRNAs from acting as templates to repeatedly produce proteins.
- Such siRNA-based anti-cancer agents are considered to be more advanced than the above-mentioned anticancer agents in that the siRNA-based anti-cancer agents block mRNAs before protein production and use RNAs and intracellular RISC systems. However, there are side effects called “off-target effects” that cannot be solved by siRNA-based technologies (Jackson, A. L. et al. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. Rna 12, 1179-87, 2006, Jackson, A. L. et al. Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. Rna 12, 1197-205, 2006, Jackson, A. L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21, 635-7, 2003, Nielsen, C. B. et al. Determinants of targeting by endogenous and exogenous microRNAs and siRNAs. Rna 13, 1894-910, 2007, Peek, A. S. & Behlke, M. A. Design of active small interfering RNAs. Curr Opin Mol Ther 9, 110-8, 2007). As described above, siRNA-based anti-cancer agents degrade mRNAs that bind complementarily to siRNA sequences. However, the siRNA-based anti-cancer agents bind to even mRNAs which are not complementary to the entirety of siRNA sequences and are complementary to the part thereof, thus causing degradation, which is called a “off-target effect”, meaning causing degradation of non-target mRNAs.
- In order to overcome technical difficulties of the aforementioned siRNA-based anticancer drugs, research is underway to use microRNAs (hereinafter referred to as miRNAs) as therapeutic agents (Agostini, M. & Knight, R. A. miR-34: from bench to bedside. Oncotarget 5, 872-81, 2014, van Rooij, E., Purcell, A. L. & Levin, A. A. Developing MicroRNA Therapeutics. Circulation Research 110, 496-507, 2012, Burnett, J. C. & Rossi, J. J. RNA-based therapeutics: current progress and future prospects. Chem Biol 19, 60-71, 2012, Dangwal, S. & Thum, T. microRNA therapeutics in cardiovascular disease models. Annu Rev Pharmacol Toxicol 54, 185-203, 2014). miRNAs are RNAs composed of 16 to 27 nucleotides, which are classified as protein non-coding RNAs as compared to messenger RNAs (mRNAs) that are translated into proteins (Carthew, R. W. & Sontheimer, E. J. Origins and Mechanisms of miRNAs and siRNAs. Cell 136, 642-55, 2009, MacFarlane, L.-A. & Murphy, P. R. MicroRNA: Biogenesis, Function and Role in Cancer. Current Genomics 11, 537-561, 2010, Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-33, 2009). miRNAs are recorded in the genomes of higher plant and animal cells and are known to play a key role in regulating cell metabolism and functions, including cell production, growth, differentiation and death. To date, about 2,000 types of miRNAs have been found in the human genome, the function of many of these miRNAs are still unknown.
- miRNAs are transcribed from the genomes into RNAs by an RNA polymerase called “Pol II”, the initial length of which is too variable to be specified (Carthew, R. W. & Sontheimer, E. J. Origins and Mechanisms of miRNAs and siRNAs. Cell 136, 642-55, 2009, Brodersen, P. & Voinnet, O. Revisiting the principles of microRNA target recognition and mode of action. Nat Rev
Mol Cell Biol 10, 141-148, 2009). This is due to the diversity of the location of the miRNA in the genome. That is, miRNA is produced in many ways, for example, miRNA is located in an intron, which is a part not involved in the protein production of mRNAs, and is transcribed at the same time as in production of mRNAs, or miRNA is located in the intergenic region on the genome and is transcribed independently (Malone, C. D. & Hannon, G. J. Small RNAs as guardians of the genome. Cell 136, 656-68, 2009). miRNA produced at an early stage is called “primary microRNA”, and the primary miR is edited into the precursor miRNA (precursor miRNA, pre-miR) by an RNA-cleaving enzyme (RNase) called “Drosha” in the nucleus (Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-33, 2009). Pre-miR has an RNA hairpin structure consisting of approximately 70 to 80 nucleotides. Pre-miR inside the cell nucleus is transported from the nucleus to the cytoplasm by exportin proteins, and is secondarily processed by another RNA-cleaving enzyme (RNase) called “Dicer” in the cytoplasm to produce double-stranded mature microRNAs (hereinafter referred to as “miRs” without any other modifiers) composed of 16 to 27 nucleotides. RNA of one strand of the double-stranded miRs is selectively determined, has activity by binding to the ribonucleoprotein complex, RISC, and binds to the target mRNA using the miR sequence. - In general, mRNAs can be divided into three parts based on involvement in protein production. That is, mRNAs can be divided into a coding region having protein translation information, and the 5′ and 3′ parts of the coding region having no protein translation information, respectively, 5′-UTR (untranslated region) and 3′-UTR. siRNAs, which cause degradation of target mRNAs using complementary sequences, act regardless of the 5′-UTR, 3′-UTR and coding parts of the mRNA, whereas miRs bind primarily to the 3′-UTR ((Carthew, R. W. & Sontheimer, E. J. Origins and Mechanisms of miRNAs and siRNAs. Cell 136, 642-55, 2009., Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-33, 2009).
- The unique features of miRNAs distinguished from siRNAs, in addition to their binding sites to the mRNAs, are that the siRNAs bind primarily to mRNAs containing sequences complementary to the entire siRNA sequence, while, regarding the miRNAs, seed region sequences with a limited size, located at nucleotides at positions of 2 to 8 from the 5′ end, are mainly used to recognize target mRNAs, so the sequence of the entire miRNA does not have a perfectly complementary sequence with the target gene, and although it contains even a part of the non-complementary sequence, it does not affect miRNA activity (Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-33, 2009). Since the sequence size of the seed region is 6 to 8 nucleotides, there are various mRNA types having the sequence complementary thereto in the 3′ UTR. For this reason, it is possible to simultaneously control several types of mRNAs with one type of miRNA. The properties of these miRNAs impart, to miRNAs, functions as efficient regulators that are involved in controlling various cell physiological properties that entail cell division, growth, differentiation and death. In addition, the functions of miRNAs as regulators are advantageous in achieving effective anticancer effects because siRNAs target the suppression of single gene expression, while miRNAs simultaneously inhibit the expression of many cancer-inducing genes.
- A large number of mRNAs contain portions of the 3′ UTR to which one or more types of miRNAs are likely to bind, and one bioinformatic calculation shows that protein production of approximately 30% of the total mRNA is regulated by miRNAs.
- The fact that miRNAs act as major regulators in signaling pathways of mRNAs can be identified in that they play a key role in major diseases including cancer (MacFarlane, L.-A. & Murphy, P. R. MicroRNA: Biogenesis, Function and Role in Cancer. Current Genomics 11, 537-561. 2010, Malone, C. D. & Hannon, G. J. Small RNAs as guardians of the genome. Cell 136, 656-68. 2009, Nicoloso, M. S., Spizzo, R., Shimizu, M., Rossi, S. & Calin, G.A. MicroRNAs—the micro steering wheel of tumour metastases. Nat Rev Cancer 9, 293-302. 2009, Landi, D., Gemignani, F. & Landi, S. Role of variations within microRNA-binding sites in cancer. Mutagenesis 27, 205-10. 2012). In fact, several studies have shown that expression patterns of miRNAs in cancer cells differ greatly from the expression patterns of miRNAs in normal cells. In addition, there is a great difference in miRNA expression patterns depending on the primary organs in which cancer occurs. Various types of cancers such as lung cancer, liver cancer, skin cancer and blood cancer have unique miRNA expression patterns, which indicates that miRNAs have a pivotal role in cancer biology. In particular, the levels of miRNAs expressed in carcinomas are known to be generally lower than levels of miRNAs expressed in normal cells.
- Recently, attempts have been made to use miRNAs as therapeutic agents based on the aforementioned close correlation of miRNAs with cancer. For example, clinical testing is underway to identify the ability of miRNAs called “miR-34a” to inhibit proliferation of cancer cells and induce apoptosis (Wiggins, J. F. et al. Development of a lung cancer therapeutic based on the tumor suppressor microRNA-34. Cancer Res 70, 5923-30. 2010, Bader, A. G. et al. miR-34 Regulated Genes and Pathways as Targets for Therapeutic Intervention. Google Patents, 2009, Hermeking, H. The miR-34 family in cancer and apoptosis. Cell Death Differ 17, 193-9. 2010, Chang, T. C. et al. Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. Mol Cell 26, 745-52. 2007).
- In order to use miRNA as an anticancer agent, there is a need for an effective method for transferring miRNAs injected from the outside of the living body to pathological tissues without degradation of miRNAs in the living body. For this purpose, RNA oligo structures containing miRNA sequences can be used. It is known that a high efficiency can be induced in vivo by linking a chemical substance or the like to the terminal site of RNA oligos so as to provide enhanced pharmacokinetic characteristics (Nature 11; 432(7014):173-8, 2004). The stability of the RNA oligos depends on the nature of chemical substance bound to the ends of sense (passenger) or antisense (guide) strands of RNA oligos. For example, an RNA oligo, which is conjugated with a polymer compound such as polyethylene glycol (PEG), interacts with an anionic phosphate group of oligo in the presence of a cationic substance to form a complex, which can become a carrier with improved stability (J Control Release 129 (2): 107-16, 2008). In particular, micelles composed of polymer complexes are extremely small in size, very uniform in distribution and are spontaneously formed, as compared with other systems used as drug delivery carriers, such as microspheres or nanoparticles, thus having advantages of easy quality control of preparations and reproducibility.
- Further, in order to improve intracellular delivery efficiency of RNA oligos, techniques for securing the stability of oligos and for securing efficient cell membrane permeability through oligo conjugates wherein RNA oligos are simply bonded to a hydrophilic substance (polyethylene glycol (PEG)) as a biocompatible polymer via a simple covalent bond or a linker-mediated covalent bond have been developed (Korean Patent No. 10-0883471). However, chemical modification of oligo and conjugation of polyethylene glycol (PEG) still do not solve the drawbacks of low in vivo stability and non-favorable delivery to target organs. In order to solve these drawbacks, double-stranded oligo RNA structures in which hydrophilic and hydrophobic substances are bonded to double-stranded oligo RNAs have been developed. These structures form self-assembled nanoparticles called “SANiRNATM” (self-assembled micelle inhibitory RNAs) via hydrophobic interaction of hydrophobic substances (See Korean Patent No. 10-1224828), and this SAMiRNA™ technology has advantages of obtaining much smaller and more homogenous nanoparticles than conventional delivery technologies.
- Under these technical backgrounds, as a result of efforts to find miRNAs with excellent efficacies to inhibit proliferation of cancer cells and induce death of cancer cells, the present inventors found miR-3670, miR-4477a and miR-3477a with excellent anticancer efficacy, and identified that these miRNAs and double-stranded oligo RNA structures containing the same effectively inhibited the expression of a number of genes known as cancer-inducing genes to achieve anticancer effects, thus completing the present invention.
- It is an object of the present invention to find miRNAs with excellent efficacies to inhibit proliferation of cancer cells and induce death of cancer cells, and provide double-stranded oligo RNA structures containing the same and a composition for preventing or treating cancer containing the same as an active ingredient.
- To accomplish the object, the present invention provides a double-stranded oligo RNA structure comprising the following Formula (1):
-
A-X-R-Y-B Formula (1) - wherein A represents a hydrophilic material, B represents a hydrophobic material, X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond, and R represents at least one miRNA selected from the group consisting of miR-3670, miR-4477a, and miR-8078.
- The present invention also provides a composition for preventing or treating cancer comprising the oligonucleotide structure.
-
FIG. 1 is a graph showing test results regarding inhibitory activities against protein expression by miR-34a, miR-100 and miR-125b, which are representatively selected from the entire screening library, and eachmRNA 3′ UTR whose expression is known to be inhibited by miR-34a, miR-100 and miR-125b is inserted into the 3′ UTR of the luciferase expression vector, in order to identify the activity of the miRNAs; -
FIG. 2 is a graph showing results, when treating NCI-H460 lung cancer cell lines with a screening library composed of 1,700 kinds of miRNAs, quantifying cell growth using Resazurin reagent and then converting the resulting value into a relative growth value; -
FIG. 3 is a graph showing results when selecting about 50 kinds of miRNAs showing excellent efficacy in NCI-H460 cell lines and measuring relative inhibitory activity against cancer cell growth with WST-1 reagent using the miRNAs; -
FIG. 4 shows results, when injecting miR-34a, miR-3670, miR-8078 and miR-4477a by transfection into cells in order to measure apoptotic effects in lung cancer cell lines, staining Annexin V labeled with FITC dye and then analyzing the degree of cell death with a flow cytometer; -
FIG. 5 shows measurement results regarding effects of miRNAs on clustering ability of lung cancer cell lines, after injecting each miRNA by transfection into lung cancer cell lines and culturing in soft agar for 2 weeks; -
FIG. 6 shows cell death performance represented by Z-score when selecting target candidate groups using the miRNA target prediction software, TargetScan, and reducing the intracellular content using siRNAs targeting the target candidate groups; -
FIG. 7 shows results of qPCR analysis indicating the degree of inhibition of expression levels of genes identified inFIG. 6 by miRNA; -
FIG. 8 shows the degree of inhibition against the expression of luciferase proteins by miRNAs by cloning the genes identified inFIG. 6 on the 3′ UTR of luciferase; and -
FIGS. 9A and 9B are images showing comparison in induction of apoptosis mechanisms, when treating lung cancer cell lines with an oligo RNA structure containing a miRNA sequence, and then staining with Annexin V. - Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those appreciated by those skilled in the field to which the present invention pertains. In general, nomenclature used herein is well-known in the art and is ordinarily used.
- According to the present invention, miRNAs having better efficacies than miR-34a known to have anticancer effects are found and anti-cancer effects thereof are identified.
- According to the present invention, 1,700 types of miRNA screening libraries are synthesized (Example 1), NCI-H460 (lung cancer cell line) cells are treated with the miRNA screening libraries, and the ability to suppress growth of cancer cells is measured, as a result, miR-3670, miR-4477a and miR-8078 having the following base sequences (Table 3) have found to have better efficacies than miR-34a, so that it is identifed that miR-3670, miR-4477a and miR-8078 have excellent anticancer efficacies (
FIGS. 4 to 8 ). - Accordingly, the present invention is directed to a double-stranded oligo RNA structure that comprises at least one miRNA selected from the group consisting of miR-3670, miR-4477a and miR-8078, and the structure represented by the following Formula (1):
-
A-X-R-Y-B Formula (1) - wherein A represents a hydrophilic material, B represents a hydrophobic material, X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond, and R represents at least one miRNA selected from the group consisting of miR-3670, miR-4477a, and miR-8078.
- According to the present invention, miR-3670 may be a double stranded RNA comprising a base sequence of SEQ ID NO. 35; and a base sequence of SEQ ID NO. 36 or SEQ ID NO. 67.
- According to the present invention, miR-4477a may be a double stranded RNA comprising a base sequence of SEQ ID NO. 43; and a base sequence of SEQ ID NO. 44 or SEQ ID NO. 68.
- According to the present invention, miR-8078 may be a double stranded RNA comprising a base sequence of SEQ ID NO. 65; and a base sequence of SEQ ID NO. 66 or SEQ ID NO. 69.
- That is, the template strand of miR-3670 may be represented by SEQ ID NO. 35.
-
(MIMAT0018093. SEQ ID NO. 35) 5′-AGAGCUCACAGCUGUCCUUCUCUA-3′ - miRNAs are ultimately active in vivo as single-strands, but should be fed into cells in the form of double strands with a base sequence having a similar base size for binding to RISC. At this time, the antisense binding to the active sequence has a complementary sequence to the active sequence. The complementary sequence has a perfect complementary sequence or an in vivo endogenous sequence. All sequences having double strands or bases located at 3′ of one strand may not have a base bond with an opposite (the other) sequence, which is referred to as a 3′ overhang. That is, the perfect complementary sequence of miR-3670 is represented by SEQ ID NO. 36.
-
(SEQ ID NO. 36) 5′-GAGAAGGACAGCUGUGAGCUCUUU-3′ - In addition, the endogenous complementary sequence of miR-3670 may be represented by SEQ ID NO. 67.
-
(SEQ ID NO. 67) 5′-GACUGGUAUAGCUGCUUUUGGAGCCUCA-3′ - As described in the Background, the seed region corresponding from the 2nd base to the 8th-9th bases of miRNA active sequences are major active factors, and long double strands containing the same may be produced and used upon the production of double stranded RNAs.
- Like miR-3670, active sequences of miR-4477a and miR-8078 and complementary sequences that form double strands with the active sequences are represented as follows. As described above, these double strands may include a 3′ overhang and may be double strands with a long sequence including the seed region.
-
miR-4477a (MIMAT0019004 SEQ ID NO. 43) 5′-CUAUUAAGGACAUUUGUGAUUC-3′ Perfect complementary sequence of miR-4477a (SEQ ID NO. 44) 5′-AUCACAAAUGUCCUUAAUAGUU-3′ Endogenous complementary sequence of miR-4477a (SEQ ID NO. 68) 5′-AUCACAAAUGUCCUUAAUGGCA-3′ miR-8078 (MIMAT0031005, SEQ ID NO. 65) 5′-GGUCUAGGCCCGGUGAGAGACUC Perfect complementary sequence of miR-8078 (SEQ ID NO. 66) 5′-GUCUCUCACCGGGCCUAGACCUU Endogenous complementary sequence of miR-8078 (SEQ ID NO. 69) 5′-CUCCACCGGGCUGACCGGCCUG-3′ - The miRNAs discovered through the library screening according to the present invention were found to provide anticancer efficacies by controlling genes commonly known to play a key role in the induction, production and growth of cancer. The feature of miRNAs is that one type of miRNA can simultaneously control expression of a plurality of mRNAs. This feature can be identified by the present invention as well and is useful for the development of oligo-based anti-cancer drugs.
- The miR-3670 of the present invention simultaneously inhibits expressions of CBX4, NRAS, CASR, TXLNA, SNIP1, HNF1A, FZD4, TRIB1, ADMA19 and CKAP5, miR-8078 inhibits expressions of GREB1, HECTD3 and RIPK4, and miR-4477a simultaneously inhibits expressions of STIL, KIF11, AKAP11, and FAM120A (
FIG. 6 ). - The target genes inhibited by miRNAs of the present invention are known to have the following functions.
- CBX4 (polycomb chromobox 4) is involved in angiogenesis of tumors and facilitation of metastasis thereof, and NRAS is known to play a key role in tumor growth and cell division (Orouji, E. et al. MAP Kinase pathway gene copy alterations in NRAS/BRAF wild-type advanced melanoma. Int J Cancer (2015); Zheng, C. et al. MicroRNA-195 functions as a tumor suppressor by inhibiting CBX4 in hepatocellular carcinoma. Oncol Rep 33, 1115-22 (2015); Jiao, H. K. et al. Prognostic significance of Cbx4 expression and its beneficial effect for transarterial chemoembolization in hepatocellular carcinoma.
Cell Death Dis 6, e1689 (2015); Ohashi, K. et al. Characteristics of lung cancers harboring NRAS mutations. Clin Cancer Res 19, 2584-91 (2013)). - CASR is found to be over-expressed in tumors and is required for metastasis of tumors, and TXLNA is known to be involved in the growth and metastasis of tumors. Clinical results have been reported that the survival rate of patients with high expression rates of TXLNA is low (Mashidori, T., Shirataki, H., Kamai, T., Nakamura, F. & Yoshida, K. Increased alpha-taxilin protein expression is associated with the metastatic and invasive potential of renal cell cancer. Biomed Res 32, 103-10 (2011); Tennakoon, S., Aggarwal, A. & Kallay, E. The calcium-sensing receptor and the hallmarks of cancer. Biochim Biophys Acta (2015); Ohtomo, N. et al. Expression of alpha-taxilin in hepatocellular carcinoma correlates with growth activity and malignant potential of the tumor. Int J Oncol 37, 1417-23 (2010)).
- SNIP1, known as a transcriptional coactivator, promotes the expression of cyclin D1, which is essential for cell growth and division. It is known that the anticancer prognosis of patients with a high expression level of SNIP1 is bad. In addition, SNIP1 is known to function to promote tumor growth in combination with c-Myc, which acts as a major regulator of cell proliferation (Li, Q. et al. SNIP1: a new activator of HSE signaling pathway. Mol Cell Biochem 362, 1-6 (2012); Fujii, M. et al. SNIP1 is a candidate modifier of the transcriptional activity of c-Myc on E box-dependent target genes. Mol Cell 24, 771-83 (2006); Roche, K. C., Rocha, S., Bracken, C. P. & Perkins, N. D. Regulation of ATR-dependent pathways by the FHA domain containing protein SNIP1. Oncogene 26, 4523-30 (2007); Jeon, H. S. et al. High expression of SNIP1 correlates with poor prognosis in non-small cell lung cancer and SNIP1 interferes with the recruitment of HDAC1 to RB in vitro. Lung Cancer 82, 24-30 (2013); Liang, X. et al. Hypoxia-inducible factor-1 alpha, in association with TWIST2 and SNIP1, is a critical prognostic factor in patients with tongue squamous cell carcinoma. Oral Oncol 47, 92-7 (2011)).
- HNF1A and FZD4 are constituent factors of the Wnt signaling system that is deeply involved in the growth and survival of tumors. The Wnt signaling system has been intensively researched in tumor biology and its importance is widely known. TRIB1 is known to play a role in growth and metastasis of tumor cells and inhibition of cell apoptosis, and is known as a factor regulating the MAPK signaling system which is one of the main signaling pathways of tumor growth (Pecina-Slaus, N. et al. Wnt signaling transcription factors TCF-1 and LEF-1 are upregulated in malignant astrocytic brain tumors. Histol Histopathol 29, 1557-64 (2014); Ueno, K. et al. Tumor suppressor microRNA-493 decreases cell motility and migration ability in human bladder cancer cells by downregulating RhoC and FZD4. Mol Cancer Ther 11, 244-53 (2012); Lin, Z.Y. et al. MicroRNA-224 inhibits progression of human prostate cancer by downregulating TRIB1. Int J Cancer 135, 541-50 (2014); Soubeyrand, S., Naing, T., Martinuk, A. & McPherson, R. ERK1/2 regulates hepatocyte Trib1 in response to mitochondrial dysfunction. Biochim Biophys Acta 1833, 3405-14 (2013)).
- ADAM19 is a protein distributed on the cell membrane, which is known to play diverse biological roles including cell-cell contact and cell-extracellular matrix contact. It is known in tumor biology that ADAM19 is strongly correlated with growth and metastasis of tumors. The CKAP5 gene, which is known to play a key role in the survival of tumors through functional genetic screens, is also controlled by the miRNA, which is identified by the present invention. GREB1, which is a gene related to signaling pathways of hormone-responsive tissues or tumors, is known to be over-expressed in various types of tumors to promote cell growth (Zhang, Q. et al. Role of microRNA-30c targeting ADAM19 in colorectal cancer.
PLoS One 10, e0120698 (2015); Shan, N., Shen, L., Wang, J., He, D. & Duan, C. MiR-153 inhibits migration and invasion of human non-small-cell lung cancer by targeting ADAM19. Biochem Biophys Res Commun 456, 385-91 (2015); Martens-de Kemp, S. R. et al. Functional genetic screens identify genes essential for tumor cell survival in head and neck and lung cancer. Clin Cancer Res 19, 1994-2003 (2013); Rae, J. M. et al. GREB1 is a novel androgen-regulated gene required for prostate cancer growth. Prostate 66, 886-94 (2006); Zhang, L. et al. Development of transcriptomic biomarker signature in human saliva to detect lung cancer. Cell Mol Life Sci 69, 3341-50 (2012); Laviolette, L. A., Hodgkinson, K. M., Minhas, N., Perez-Iratxeta, C. & Vanderhyden, B. C. 17beta-estradiol upregulates GREB1 and accelerates ovarian tumor progression in vivo. Int J Cancer 135, 1072-84 (2014)). - HECTD3 is known as an E3 ubiquitin ligase that inhibits tumor death by inducing degradation of caspase-8 by attaching polyubiquitin to caspase-8 which facilitates cell apoptosis and increases drug resistance to cisplatin anticancer drugs by stabilizing MALT1 proteins. RIPK4 has been reported as receptor-interacting
protein kinase 4 to induce accumulation of β-catenin, which is a cell growth signaling factor, and to activate the Wnt signaling system. It has been found that artificial elimination of RIPK4 can inhibit tumor growth in tumor animal models (Li, Y. et al. The HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by promoting K63-linked polyubiquitination of caspase-8.Cell Death Dis 4, e935 (2013); Li, Y. et al. The HECTD3 E3 ubiquitin ligase suppresses cisplatin-induced apoptosis via stabilizing MALT1.Neoplasia 15, 39-48 (2013); Huang, X. et al. Phosphorylation of Dishevelled by protein kinase RIPK4 regulates Wnt signaling. Science 339, 1441-5 (2013)). - The STIL gene as an essential element in the transition from the G2 phase to the M phase during the cell cycle is observed to be highly expressed in various types of cancer and is known to be necessary for tumor proliferation and survival. KIF11 is also reported to be one of factors necessary for the growth and metastasis of tumor cells and is known to inhibit growth of tumors by inhibiting the activity of KIF11 (Erez, A. et al. Sil overexpression in lung cancer characterizes tumors with increased mitotic activity. Oncogene 23, 5371-7 (2004); Erez, A. et al. The SIL gene is essential for mitotic entry and survival of cancer cells. Cancer Res 67, 4022-7 (2007); Tang, Y., Orth, J.D., Xie, T. & Mitchison, T.J. Rapid induction of apoptosis during Kinesin-5 inhibitor-induced mitotic arrest in HL60 cells. Cancer Lett 310, 15-24 (2011); Venere, M. et al. The mitotic kinesin KIF11 is a driver of invasion, proliferation, and self-renewal in glioblastoma. Sci Transl Med 7, 304ra143 (2015)).
- AKAP11, which binds to protein kinase A (PKA) to increase PKA activity, simultaneously binds to GSK-3beta as well to promote phosphorylation of GSK-3beta by PKA. Phosphorylated GSK-3beta loses its activity, which is recognized as a signal that stimulates growth in cells and is one of the major mechanisms leading to tumor growth. Tumor cells are exposed to a variety of stress conditions such as acidic conditions and oxygen deficiency conditions. Under these severe conditions, the mechanism of tumor cell death is maintained. Among them, FAM120A, which is an RNA binding protein, is known to activate a kinase such as Src, thereby increasing inhibition of cell death and resistance to drugs (Logue, J. S. et al. AKAP220 protein organizes signaling elements that impact cell migration. J Biol Chem 286, 39269-81 (2011); Whiting, J. L. et al. Protein Kinase A Opposes the Phosphorylation-dependent Recruitment of Glycogen Synthase Kinase 3beta to A-kinase Anchoring Protein 220. J Biol Chem 290, 19445-57 (2015); Tanji, C. et al. A-kinase anchoring protein AKAP220 binds to glycogen synthase kinase-3beta (GSK-3beta) and mediates protein kinase A-dependent inhibition of GSK-3beta. J Biol Chem 277, 36955-61 (2002); Tanaka, M. et al. A novel RNA-binding protein, Ossa/C9orf10, regulates activity of Src kinases to protect cells from oxidative stress-induced apoptosis. Mol Cell Biol 29, 402-13 (2009); Bartolome, R. A. et al. IL13 Receptor alpha2 Signaling Requires a Scaffold Protein, FAM120A, to Activate the FAK and PI3K Pathways in Colon Cancer Metastasis. Cancer Res 75, 2434-44 (2015)).
- It was identified by the present invention that, when contents of genes in cells were reduced using siRNAs, the growth of the cells was decreased as in the case of using miR-3670, miR4477a and miR-8078 (
FIG. 6 ). qPCR results showed that, when miR-3670, miR-4477a and miR-8078 were delivered into lung cancer cells, the mRNA expression of the genes was decreased (FIG. 7 ). In addition, luciferase results identified that the genes were direct targets of miR-3670, miR-4477a and miR-8078 (FIG. 8 ). This indicates that miR-3670, miR-4477a and miR-8078 induce death of tumor cells by directly and simultaneously inhibiting the expression of several genes important for growth and survival of tumor cells. - Among the miRNAs, miR-3670 simultaneously inhibits expression of CBX4, NRAS, CASR, TXLNA, SNIP1, HNF1A, FZD4, TRIB1, ADMA19 and CKAP5, miR-8078 inhibits expression of GREB1, HECTD3 and RIPK4, and miR-4477a simultaneously inhibits expression of STIL, KIF11, AKAP11 and FAM120A. For this reason, the mRNAs of the genes include a perfect match wherein the corresponding miRNA regions are complementary 100% to base sequences, as well as mismatch wherein the corresponding miRNA regions are inconsistent with some base sequences. For these miRNAs, the match of the seed regions is the most important, and miRNAs preferably have at least 70%, more preferably at least 80%, even more preferably at least 90%, still even more preferably 95% or more, and most preferably 100%, of an identity with a part of mRNA sequences of the corresponding genes.
- Such miRNAs may be duplexes, include single molecule polynucleotide, and be antisense oligonucleotides or microRNAs (miRNAs), but are not limited thereto.
- With regard to the oligo conjugates, wherein hydrophilic and hydrophobic materials are bound to RNA oligos, according to the present invention, the conjugates, wherein hydrophilic and hydrophobic materials are bound to both ends of RNA oligos, can efficiently deliver RNA oligos in vivo and improve stability.
- Self-assembled nanoparticles are formed through hydrophobic interactions of hydrophobic materials. These nanoparticles have extremely excellent in vivo delivery efficiency and in vivo stability, and the improvement in structures provides very uniform particle sizes and easy quality control (QC), thus having an advantage of simple drug manufacturing process.
- In one embodiment, the hydrophilic material in the double-stranded oligo RNA structures comprising miRNAs according to the present invention is represented by (A)n, (Am-J)n or (J-Am)n, wherein A represents a hydrophilic material monomer, n represents 1 to 200, m represents 1 to 15, and J represents a linker that links m hydrophilic material monomers to one another, or links m hydrophilic material monomers to oligonucleotides.
- When the hydrophilic substance is (A)n, the double-stranded oligo RNA structure according to the present invention has the structure represented by the following Formula (1′):
-
A-X-S-Y-B -
AS [Formula (1′)] - wherein A, B, X and Y are as defined in Formula (1), S represents a sense strand of specific miRNA regarding the corresponding gene and AS represents an antisense strand of specific miRNA regarding the corresponding gene.
- In one embodiment, the double-stranded oligo RNA structure comprising miRNAs according to the present invention may be a double-stranded oligo RNA structure comprising the structure represented by the following Formula (2):
-
A-X-5′ R 3′ Y-B [Formula (2)] - wherein A, B, X, Y and R are as defined in Formula (1).
- More preferably, the double-stranded oligo RNA structure has a structure represented by the following Formula (2′):
-
A-X-5═S 3′-Y-B -
AS [Formula (2′)] - In one embodiment, the hydrophilic material may be a cationic or non-ionic polymer material having a molecular weight of 200 to 10,000, preferably a non-ionic polymer substance having a molecular weight of 1,000 to 2,000. As the hydrophilic material, a non-ionic hydrophilic polymer compound, for example, polyethylene glycol, polyvinylpyrrolidone or polyoxazoline is preferably used, but the present invention is not limited thereto.
- In another embodiment, when the hydrophilic substance is (Am-J)n or (J-Am)n, the double-stranded oligo RNA structure according to the present invention has a structure represented by the following Formula (3) or Formula (4):
-
(Am-J)n-X-R-Y-B [Formula (3)] -
(J-Am)n-X-R-Y-B [Formula (4)] - In Formula (3) and Formula (4), A represents a hydrophilic material monomer, n represents 1 to 200, represents 1 to 15, J represents a linker that links m hydrophilic material monomers to one another, or links m hydrophilic material monomer to oligonucleotide, X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond, and R represents specific miRNA according to the present invention. More preferably, the double-stranded oligo RNA structure comprising miRNA according to the present invention may have a structure represented by Formula (3′):
-
(Am-J)n-X-S-Y-B -
AS [Formula (3′)] - wherein A, B, J, m, n, X and Y are as defined in Formula (3), S represents a sense strand of specific miRNA regarding the corresponding gene, and AS represents an antisense strand of specific miRNA regarding the corresponding gene.
- More preferably, the double-stranded oligo RNA structure comprising miRNAs according to the present invention has a structure represented by the following Formula (4′):
-
(J-Am)n-X-S-Y-B AS [Formula (4′)] - wherein A, B, J, m, n, X and Y are as defined in Formula (4), S represents a sense strand of specific miRNA regarding the corresponding gene, and AS represents an antisense strand of specific miRNA regarding the corresponding gene.
- Any monomer of non-ionic hydrophilic polymers may be used as the hydrophilic material monomer (A) in Formula (3) and Formula (4) without particular limitation so long as it satisfies the objects of the present invention. Preferred is a monomer selected from Compounds (1) to (3) shown in Table 1, and more preferred is a monomer of Compound (1). G in Compound (1) is preferably selected from CH2, O, S and NH.
- In particular, among the hydrophilic material monomers, the monomer represented by compound (1) has advantages of having a variety of functional groups which would be introduced, exhibiting excellent bio-compatibility, for example, providing better in vivo affinity and inducing less immune reactions, and improving in vivo stability and delivery efficiency of oligonucleotide contained in the structure according to Formula (3) and Formula (4), thus being very suitable for the manufacture of the structure according to the present invention.
- The hydrophilic material in Formula (3) and Formula (4) preferably has a total molecular weight of 1,000 to 2,000. Accordingly, for example, wherein hexaethylene glycol in Compound (1), that is, G is 0, and m is 6, is used in Formula (3) and Formula (4), the molecular weight of the hexaethylene glycol spacer is 344 and thus the number of repeats (n) is preferably 3 to 5. According to the present invention, in Formula (3) and Formula (4), a repeat unit represented by (Am-J) or (J-Am), that is, a hydrophilic material block may be used as an appropriate number represented by “n”. A, the hydrophilic material monomer and J, the linker, contained in each hydrophilic material block, may be independently identical or different between the hydrophilic material blocks. That is, when three hydrophilic material blocks are used (n=3), different hydrophilic material monomers may be used for all the hydrophilic material blocks, for example, the hydrophilic material monomer according to Compound (1) may be used for the first block, the hydrophilic material monomer according to Compound (2) may be used for the second block, and the hydrophilic material monomer according to Compound (3) may be used for the third block. Alternatively, any one hydrophilic material monomer selected from hydrophilic material monomers according to Compounds (1) to (3) may be used for all hydrophilic mateiral blocks. Similarly, the linker, which mediates the bond between the hydrophilic material monomers, used for respective hydrophilic material blocks, may be identical or different. In addition, m, the number of the hydrophilic material monomers, may be identical or different for the respective hydrophilic material blocks. That is, different numbers of hydrophilic material monomers may be used, for example, for the first hydrophilic material block, three hydrophilic material monomers (m=3) are connected, for second hydrophilic mateiral blocks, five hydrophilic material monomers (m=5) are connected, and for the third hydrophilic material block, four hydrophilic material monomers (m=4) are connected. Alternatively, the same number of hydrophilic material monomers may be used for all hydrophilic material blocks.
- According to the present invention, the linker (J) is preferably selected from the group consisting of PO3 −, SO3 and CO2, but is not limited thereto. Depending on the monomer of the used hydrophilic material or the like, any linker may be used so long as it satisfies the object of the present invention and is obvious to those skilled in the art.
- The entirety or part of the hydrophilic material monomer may be modified to have a functional group required for bonding to other substances such as a target specific ligand.
- In some cases, one to three phosphate groups may be bonded to the 5′ end of the antisense strand of a double-stranded oligo RNA structure comprising specific miRNA for the gene.
- For example, the double-stranded oligo RNA structure comprising miRNA has a structure represented by the following Formula (3″) or Formula (4″):
-
(Am-J)n-X-5′ S 3′-Y-B -
3′ AS 5′-PO4 [Formula (3″)] -
(J-Am)n-X-5′ S 3′-Y-B -
3′ AS 5′-PO4 [Formula (4″)] - The hydrophobic material (B) functions to form nanoparticles having an oligonucleotide structure represented by Formula (1) through hydrophobic interaction.
- The hydrophobic material preferably has a molecular weight of 250 to 1,000, and may be a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, C12 to C50 unsaturated or saturated hydrocarbon, diacyl-phosphatidylcholine, fatty acid, phospholipid, lipopolyamine or the like, but is not limited thereto. Any hydrophobic material may be used without limitation so long as it satisfies the objects of the present invention, which is obvious to those skilled in the art to which the present invention pertains.
- The steroid derivative may be selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate and cholestanylamine, and the glyceride derivative may be selected from mono-, di- and tri-glyceride and the like, wherein the fatty acid of glyceride is preferably a C12 to C50 unsaturated or saturated fatty acid.
- In particular, among the hydrophobic mateirals, saturated or unsaturated hydrocarbon or cholesterol is preferred for easy binding to oligonucleotide during synthesis of the oligonucleotide structures according to the present invention.
- The hydrophobic material is bonded to the distal end of the hydrophilic mateiral and may be bonded to the sense strand or antisense strand of miRNAs.
- According to the present invention, the hydrophilic material, the hydrophilic material block or the hydrophobic material is bonded to oligonucleotide through a simple covalent bond or linker-mediated covalent bond (X or Y). The covalent bond may be a non-degradable or degradable bond. Examples of the non-degradable bond include an amide bond or a phosphoryl bond, and the degradable bond includes a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, an enzyme-degradable bond or the like, but is not limited thereto.
- The miRNA oligo structures according to the present invention were produced and in vitro treated with lung cancer cell lines, and the cell lines were stained with Annexin V and were analyzed by flow cytometry. As can be seen from
FIGS. 9A and 9B , when nanoparticles were used to improve in vivo stability using the RNA structure, apoptosis of cell lines can be concentration-dependently induced. - Based on this, the present invention relates to a composition for preventing or treating cancer comprising the oligonucleotide structure. The present invention also relates to a method for preventing or treating cancer comprising a step of administering the oligonucleotide structure.
- According to the present invention, the cancer is at least one selected from the group consisting of primary cancer such as lung cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, gall bladder cancer, biliary tract cancer, breast cancer, leukemia, esophageal cancer, non-Hodgkin's lymphoma, thyroid cancer, cervical cancer and skin cancer, and metastatic cancer caused by metastasis from primary cancer to other organs, and tumor-associated cell diseases caused by promotion of abnormal excessive cell division, but the present invention is not limited thereto.
- The miRNA sequence that can be used as an active ingredient of the composition for treating cancer provided by the present invention is a sequence derived from the human genome, however, a miRNA sequence obtained from the genome derived from another animal also can be used without limiting the genome of the miRNA
- The miRNA can be used in the form of various miRNA derivatives (miRNA mimics) that generate the bioequivalence efficacy of miRNAs, and can be modified miRNAs containing miRNA sequences containing the same seed region. At this time, the length of
sequence 1 orsequence 2 can be reduced, and a short derivative consisting of 15 nucleotides can also be used. - The miRNA derivatives for miRNAs may partially include a phosphorothiolate structure in which O in the RNA phosphate backbone structure is replaced with another element such as sulfur, and can be used in the forms wherein DNA, PNA (peptide nucleic acid) and LNA (locked nucleic acid) molecules are entirely or partially replaced with RNA and can be used in the forms wherein the 2′ hydroxyl group of RNA sugar is replaced with various functional structures, and examples of such modifications include, but are not limited to, methylation, methoxylation, fluorination and the like.
- The miRNA is not limited to mature miRNA and the double stranded RNA of the miRNA derivative derived therefrom, and can be used in the form of a miRNA precursor and, for the miRNA precursor, aforementioned partial or entire replacement of the RNA phosphate backbone structure and RNA nucleic acid with DNA, PNA, LNA and the like, and modification of the 2′ hydroxyl group of the RNA sugar molecule are possible.
- The miRNA can be used in the form of precursor miRNA or primary miRNA (pri-miRNA) and can be synthesized by a chemical method or delivered in the form of a plasmid to cells which express the same.
- According to the present invention, methods for delivering miRNAs to cells cultured on culture dishes include, but are not limited to, mixing with cationic lipids, using electrical stimulation, and using viruses.
- The composition for treating cancer comprising the miRNA as an active ingredient may be a pharmaceutical composition further containing a pharmaceutically acceptable carrier and may be formulated together with a carrier.
- The term “pharmaceutically acceptable carrier” as used herein refers to a carrier or diluent that does not impair biological activities or properties of an administered compound without stimulating an organism. Acceptable pharmaceutical carriers for compositions, which are formulated into liquid solutions, are sterilized and biocompatible and examples thereof include saline, sterile water, Ringer's solution, buffered saline, albumin injection solutions, dextrose solutions, maltodextrin solutions, glycerol, ethanol and mixtures thereof. If necessary, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate injectable solutions such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.
- The composition for preventing or treating cancer comprising the miRNA and the pharmaceutically acceptable carrier can be applied to any formulation containing the same as an active ingredient and can be prepared for oral or parenteral formulation. The pharmaceutical formulation may include formulations suitable for oral, rectal, nasal, topical (including under the cheek and tongue), subcutaneous, vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration, or inhalation or insufflation. Examples of formulations for oral administration containing the composition of the present invention as an active ingredient include tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. In order to prepare formulations such as tablets and capsules, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrating agent such as corn starch or sweet potato starch, a lubricant such as calcium stearate, sodium stearyl fumarate or polyethyleneglycol wax can be incorporated, and capsule formulations may further contain a liquid carrier such as a fatty oil, in addition to the above-mentioned ingredients.
- Examples of the formulations for parenteral administration containing the composition of the present invention as an active ingredient include injection forms such as subcutaneous injection, intravenous injection or intramuscular injection, suppository or spray forms such as aerosols inhalable through a breathing apparatus. For preparation into injectable formulations, the compositions of the present invention can be mixed in water with stabilizers or buffers to prepare solutions or suspensions and the solutions or suspensions can be formulated on the basis of an ampule or vial unit for administration. For suppository injection, compositions for rectal administration such as suppositories containing a conventional suppository base such as cocoa butter or other glycerides or enema preparations can be formulated. For spray formulation such as an aerosol, an additive such as a propellant may be mixed such that a water-dispersed concentrate or wet powder is dispersed.
- Hereinafter, the present invention will be described in more detail with reference to examples. However, it is obvious to those skilled in the art that these examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.
- As 21-version human miRNA sequences provided from the miRNA database, miRBase (www.mirbase.org), double-stranded sequences of miRNA were synthesized by a solid synthesis method used for common synthesis of oligo from 1,700 miRNA screening libraries, based on the stem-loop structure. Each strand of the synthesized miRNA was purified by reverse phase separation using a C18 resin. Whether or not the intended sequence was synthesized for all synthesized miRNA strands was detected and identified with a MALDI-TOF mass spectrometer. In order to prepare double-stranded miRNAs, the synthesized miRNA strands and the corresponding complementary strands were heated in the presence of a salt at 95° C. for 2 minutes and then slowly cooled.
Sequences 1 of respective tested double-stranded miRNAs are represented by SEQ ID NO. 70 to SEQ ID NO. 1797 in this order andSequences 2 thereof are represented by SEQ ID NO. 1798 to 3525 in this order. - In order to identify whether or not miRNA double strands synthesized in Example 1 were active, miR-34a, miR-100 and miR-125b were selectively selected from about 1,700 types of miRNA screening libraries. miRNAs have been selected based on the large number of studies previously performed on the types of target mRNAs that control functions and expression and the binding sites that bind to each
mRNA 3′ UTR. The 3′ UTR sites of Bcl2, mTOR and Lin28b mRNAs, which are known to be regulated by miR-34a, miR-100 and miR-125b, respectively, are replaced with the 3′ UTR sites of the firefly luciferase vector to produce vectors corresponding to respective miRNAs. The HEK-293T cell lines were co-transfected with each vector and miR control group, or miR-34a, miR-100 and miR-125b corresponding to each vector, using an intracellular delivery reagent of oligo, Lipofectamine 2000 (Invitrogen) (three replicate samples) and cultured at 37° C. and 5% (v/v) carbon dioxide for 24 hours. The activity of luciferase was measured using a luminometer (Thermo Scientific) to identify the activity of the synthesized miRNA (FIG. 1 ). - 3,000 to 10,000 NCI-H460 cells were seeded on a 96-well plate and cultured at 37° C., and 5% (v/v) carbon dioxide for 24 hours. Each miRNA of the miRNA library was transfected using RNAiMAX reagent (Invitrogen) to a final concentration of 100 nM. Each miRNA was transfected three times, which means that three 96-well plates for each miRNA library stored on a 96-well plate were prepared. The cells were further cultured for 24 hours under the same conditions as the cell culture conditions described above, and the fluorescence value generated by adding Resazurin reagent (Promega) was measured using a fluorescence meter (Fluoremeter, Tecan). In order to comparatively evaluate the ability of each miRNA to inhibit cell proliferation, a mean value and a standard deviation of 96 values measured on the 96-well plate were determined. The difference according to standard deviation multiple between the measured value in each miRNA-containing well and the mean value was calculated in accordance with the following (Z-score) formula:
-
- wherein xi is the measured value of each well, μ is the mean value of the whole well of the plate, and σ is the standard deviation. The standard deviation multiple, zi, of each well was the mean value obtained from the three plate replicates, and was used to select 50 primary candidate miRNAs having a z value less than −2 (
FIG. 2 , Table 2). -
TABLE 2 Primarily-screened miRNA sequences SEQ ID NO. hsa-miR- 23c strand 1 AUCACAUUGCCAGUGAUUACCC 1 SEQ ID NO. hsa-miR- 23c strand 2 GUAAUCACUGGCAAUGUGAUUU 2 SEQ ID NO. hsa-miR- 219b strand 1 AGAUGUCCAGCCACAAUUCUCG 3 SEQ ID NO. hsa-miR- 219b strand 2 AGAAUUGCGUUUGGACAAUCAGU 4 SEQ ID NO. hsa-miR- 378c strand 1 ACUGGACUUGGAGUCAGAAGAGUGG 5 SEQ ID NO. hsa-miR- 378c strand 2 ACUCUUCUGACUCCAAGUCCAGUUU 6 SEQ ID NO. hsa-miR- 548aa strand 1 AAAAACCACAAUUACUUUUGCACCA 7 SEQ ID NO. hsa-miR- 548aa strand 2 GUGCAAAAGUAAUUGUGGUUUUUUU 8 SEQ ID NO. hsa-miR- 548u strand 1 CAAAGACUGCAAUUACUUUUGCG 9 SEQ ID NO. hsa-miR- 548u strand 2 CAAAAGUAAUUGCAGUCUUUGUU 10 SEQ ID NO. hsa-miR-571 strand 1UGAGUUGGCCAUCUGAGUGAG 11 SEQ ID NO. hsa-miR-571 strand 2CACUCAGAUGGCCAACUCAUU 12 SEQ ID NO. hsa-miR-641 strand 1AAAGACAUAGGAUAGAGUCACCUC 13 SEQ ID NO. hsa-miR-641 strand 2GGUGACUCUAUCCUAUGUCUUUUU 14 SEQ ID NO. hsa-miR-1244 strand 1AAGUAGUUGGUUUGUAUGAGAUGGUU 15 SEQ ID NO. hsa-miR-1244 strand 2CCAUCUCAUACAAACCAACUACUUUU 16 SEQ ID NO. hsa-miR-1248 strand 1ACCUUCUUGUAUAAGCACUGUGCUAAA 17 SEQ ID NO. hsa-miR-1248 strand 2UAGCACAGUGCUUAUACAAGAAGGUUU 18 SEQ ID NO. hsa-miR-1298 strand 1CAUCUGGGCAACUGACUGAAC 19 SEQ ID NO. hsa-miR-1298 strand 2UUCAUUCGGCUGUCCAGAUGUA 20 SEQ ID NO. hsa-miR-2392 strand 1UAGGAUGGGGGUGAGAGGUG 21 SEQ ID NO. hsa-miR-2392 strand 2CCUCUCACCCCCAUCCUAUU 22 SEQ ID NO. hsa-miR-3119 strand 1UGGCUUUUAACUUUGAUGGC 23 SEQ ID NO. hsa-miR-3119 strand 2CAUCAAAGUUAAAAGCCAUU 24 SEQ ID NO. hsa-miR-3164 strand 1UGUGACUUUAAGGGAAAUGGCG 25 SEQ ID NO. hsa-miR-3164 strand 2CCAUUUCCCUUAAAGUCACAUU 26 SEQ ID NO. hsa-miR-3188 strand 1AGAGGCUUUGUGCGGAUACGGGG 27 SEQ ID NO. hsa-miR-3188 strand 2CCGUAUCCGCACAAAGCCUCUUU 28 SEQ ID NO. hsa-miR-3609 strand 1CAAAGUGAUGAGUAAUACUGGCUG 29 SEQ ID NO. hsa-miR-3609 strand 2GCCAGUAUUACUCAUCACUUUGUU 30 SEQ ID NO. hsa-miR-3612 strand 1AGGAGGCAUCUUGAGAAAUGGA 31 SEQ ID NO. hsa-miR-3612 strand 2CAUUUCUCAAGAUGCCUCCUUU 32 SEQ ID NO. hsa-miR-3662 strand 1GAAAAUGAUGAGUAGUGACUGAUG 33 SEQ ID NO. hsa-miR-3662 strand 2UCAGUCACUACUCAUCAUUUUCUU 34 SEQ ID NO. hsa-miR-3670 strand 1AGAGCUCACAGCUGUCCUUCUCUA 35 SEQ ID NO. hsa-miR-3670 strand 2GAGAAGGACAGCUGUGAGCUCUUU 36 SEQ ID NO. hsa-miR-3943 strand 1UAGCCCCCAGGCUUCACUUGGCG 37 SEQ ID NO. hsa-miR-3943 strand 2CCAAGUGAAGCCUGGGGGCUAUU 38 SEQ ID NO. hsa-miR-4424 strand 1AGAGUUAACUCAAAAUGGACUA 39 SEQ ID NO. hsa-miR-4424 strand 2GUCCAUUUUGAGUUAACUCUUU 40 SEQ ID NO. hsa-miR-4461 strand 1GAUUGAGACUAGUAGGGCUAGGC 41 SEQ ID NO. hsa-miR-4461 strand 2CUAGCCCUACUAGUCUCAAUCUU 42 SEQ ID NO. hsa-miR- 4477a strand 1 CUAUUAAGGACAUUUGUGAUUC 43 SEQ ID NO. hsa-miR- 4477a strand 2 AUCACAAAUGUCCUUAAUAGUU 44 SEQ ID NO. hsa-miR- 4477b strand 1 AUUAAGGACAUUUGUGAUUGAU 45 SEQ ID NO. hsa-miR- 4477b strand 2 CAAUCACAAAUGUCCUUAAUUU 46 SEQ ID NO. hsa-miR-4765 strand 1UGAGUGAUUGAUAGCUAUGUUC 47 SEQ ID NO. hsa-miR-4765 strand 2ACAUAGCUAUCAAUCACUCAUU 48 SEQ ID NO. hsa-miR-4773 strand 1CAGAACAGGAGCAUAGAAAGGC 49 SEQ ID NO. hsa-miR-4773 strand 2CUUUCUAUGCUCCUGUUCUGUU 50 SEQ ID NO. hsa-miR-4776 strand 1GUGGACCAGGAUGGCAAGGGCU 51 SEQ ID NO. hsa-miR-4776 strand 2CUUGCCAUCCUGGUCCACUGCAU 52 SEQ ID NO. hsa-miR-4999 strand 1UGCUGUAUUGUCAGGUAGUGA 53 SEQ ID NO. hsa-miR-4999 strand 2UCACUACCUGACAAUACAGU 54 SEQ ID NO. hsa-miR-5096 strand 1GUUUCACCAUGUUGGUCAGGC 55 SEQ ID NO. hsa-miR-5096 strand 2CUGACCAACAUGGUGAAACUU 56 SEQ ID NO. hsa-miR-5697 strand 1UCAAGUAGUUUCAUGAUAAAGG 57 SEQ ID NO. hsa-miR-5697 strand 2UUUAUCAUGAAACUACUUGAUU 58 SEQ ID NO. hsa-miR-5705 strand 1UGUUUCGGGGCUCAUGGCCUGUG 59 SEQ ID NO. hsa-miR-5705 strand 2CAGGCCAUGAGCCCCGAAACAUU 60 SEQ ID NO. hsa-miR-5707 strand 1ACGUUUGAAUGCUGUACAAGGC 61 SEQ ID NO. hsa-miR-5707 strand 2CUUGUACAGCAUUCAAACGUUU 62 SEQ ID NO. hsa-miR-8053 strand 1UGGCGAUUUUGGAACUCAAUGGCA 63 SEQ ID NO. hsa-miR-8053 strand 2CCAUUGAGUUCCAAAAUCGCCAUU 64 SEQ ID NO. hsa-miR-8078 strand 1GGUCUAGGCCCGGUGAGAGACUC 65 SEQ ID NO. hsa-miR-8078 strand 2GUCUCUCACCGGGCCUAGACCUU 66 - Secondary screening was carried out by improving the measurement accuracy using 50 miRNA candidates group obtained by primary screening. The test conditions were the same as the primary screening conditions, except that WST-1 reagent (Roche) was used as a reagent for measuring cell proliferation ability, instead of resazurin. WST-1 was used because of the advantage of the capability of measuring the intensity of signal more quantitatively than resazurin. Measured values of cell inhibitory ability by each miRNA are shown relative to the control in
FIG. 3 , and miR-34a was also included as a positive control. cl EXAMPLE 5 - The method used for screening is to measure the degree of relative inhibition of cell proliferation by measuring the number of cells in a quantitative sense. The mechanisms that inhibit cell proliferation include a method of reducing the cell cycle rate and a method of inducing apoptosis. In order to analyze the mechanisms of inhibitory activity of miRNAs found in the present invention against cell proliferation, the degree of apoptosis was analyzed by flow cytometry (fluorescence activated cell sorter (FACS)). For this purpose, cells were seeded on a 6-well plate, and the miRNA was injected into the cells using RNAiMAX reagent. Then, cells were cultured under the conditions described above for 48 hours. Then, the cells were treated with annexin V labeled with FIT-C fluorescent dye and analyzed by flow cytometry (
FIG. 4 ). Analysis results showed that most of the cells treated with miR-3670, miR-4477a and miR-8078 were killed. This indicates that inhibition of tumor growth of the miRNA identified in the screening is caused by inducing apoptosis (Table 3). -
TABLE 3 Finally-screened miRNA sequences SEQ ID NO. hsa-miR-3670 strand 1AGAGCUCACAGCUGUCCUUCUCUA 35 SEQ ID NO. hsa-miR-3670 strand 2GAGAAGGACAGCUGUGAGCUCUUU 36 SEQ ID NO. miR-3670-IC IC GACUGGUAUAGCUGCUUUUGGAGCCUCA 67 SEQ ID NO. hsa-miR- 4477a strand 1 CUAUUAAGGACAUUUGUGAUUC 43 SEQ ID NO. hsa-miR- 4477a strand 2 AUCACAAAUGUCCUUAAUAGUU 44 SEQ ID NO. miR-4477a-IC IC AUCACAAAUGUCCUUAAUGGCA 68 SEQ ID NO. hsa-miR-8078 strand 1GGUCUAGGCCCGGUGAGAGACUC 65 SEQ ID NO. hsa-miR-8078 strand 2GUCUCUCACCGGGCCUAGACCUU 66 SEQ ID NO. miR-8078-IC IC CUCCACCGGGCUGACCGGCCUG 69 - By culturing tumor cells using soft agar, the characteristics of the tumor cells can be measured. Normal cells require a support such as a culture dish to grow, whereas tumor cells tend to grow under an environment free from a physically rigid support such as soft agar. Cell clustering ability in soft agar was determined using these tumor specific properties. NCI-H460 lung cancer cell lines were treated with control miRNA, miR-34a, miR-8078, miR-3670, miR-4477a and miR-4765, cultured for 24 hours, mixed with soft agar, and cultured on a 6 well-plate for 2 weeks. The cells were stained with a crystal violet dye and the numbers of clusters were counted (
FIG. 5 ). Results showed that the cells treated with miR-8078 and miR-3670 formed almost no clusters, and that the cells treated with miR-4477a showed about 30% of cluster formation ability as compared with the control. - Target mRNAs, whose protein expression is controlled by miRNA, have a sequence partially complementary to the sequence of the miRNAs. In order to inhibit expression of mRNAs, the sequence of the seed region for miRNAs is particularly important, because it binds to mRNA having a sequence complementary to the seed region sequence to inhibit gene expression. However, because the seed region sequence is relatively short, i.e., 8 to 9 bases, the mRNA targeted by the miRNA is estimated using software. However, even when software is used, it is known that only some of the estimated targets are actual targets. In order to solve this difficulty, the target genes predicted through software were treated with siRNA to reduce intracellular content and were selected by determining whether or not cell growth was inhibited. TargetScan was used as a target prediction software generally used in the art in order to predict the target mRNA of miRNA, and the total 600 types of genes were selected as estimated targets of miR-3670, miR-4477a and miR-8078. Three siRNAs were synthesized for each gene selected, and the same experiment was conducted as in Example 3 using the siRNAs. Cells were seeded on 96-well plates, treated with each siRNA and cultured for 48 hours, and then cell proliferation was measured using a resazurin reagent. The Z-score of each gene was calculated from the mean of the measurement values of a total of about 1,800 (600 genes×3 kinds of siRNAs) in the same manner as in Example 3 and shown in
FIG. 6 . - The action mode of miRNAs deteriorates production of proteins from mRNAs and, at the same time, causes the degradation of most of the target mRNAs. Accordingly, miRNAs are injected into cells and the contents of mRNAs, which are the targets of miRNAs, are analyzed using qPCR, and the decrease in the content is measured, which can be used as a criterion for determining the target mRNA of miRNAs. In order to determine whether the target mRNAs were substantially degraded, when the miRNAs were transferred into cells, while targeting the target genes of miRNAs identified in Example 7, miR-3677, miR-4477a and miR-8078 were transfected into lung cancer cell line and then cultured for 48 hours, and RNAs were extracted from respective cells to quantitatively measure RNA contents (
FIG. 7 ). Results showed that the predicted target mRNA contents of miR-3670, miR-4477a and miR-8078 were remarkably lowered. - Because miRNAs inhibit production of proteins from target mRNAs by binding to the 3′ UTR (untranslated region) of target mRNAs, luciferase assay is commonly used as a method for directly measuring the relationships between miRNAs and target mRNAs. The TargetScan software provides the 3′ UTR sequence containing the miRNA-binding sequence. The 3′ UTR sequence was inserted by gene cloning into the 3′ UTR of firefly luciferase as described in Example 2, and the vector produced in this manner was transfected into human embryonic kidney (HEK) cells simultaneously with the corresponding miRNA to measure the expression level of luciferase in the vector. At this time, renilla luciferase was also transfected simultaneously to calibrate the transfection efficiency. miRNA, firefly luciferase and Renilla luciferase were simultaneously injected and cultured for 48 hours and then measured with a luminometer (
FIG. 8 ). Results showed that each target mRNA is directly controlled by the corresponding miRNA. - The double-stranded oligo RNA structure produced according to the present invention has a structure represented by the following Formula (5):
-
(ethyleneglycol6-PO3 −)4-5′ S 3′-C6—S—S—C 18 3′ AS 5′-PO4 [Formula (5)] - wherein S represents a sense strand of miRNA, AS represents an anti-sense strand of miRNA, PO4 represents a phosphate group, ethylene glycol is a hydrophilic material monomer, and hexaethylene glycols are bonded to through a phosphate group (PO3 −) as a linker (J), C24 represents a tetradocosane containing a disulfide bond as a hydrophobic material, and 5′ and 3′ represent the end directions of double-stranded oligo RNA.
- Regarding the sense strand of miRNA in Formula (5), a phosphodiester bond constituting an RNA backbone structure using β-cyanoethyl phosphoamidite and DMT-hexaethyl glycol-CPG as a support is linked, so an oligo RNA-hydrophilic material structure containing the sense strand wherein hexaethylene glycol is bonded to the 3′ end is synthesized, and then tetradodecanoic acid containing a disulfide bond is bonded at the 5′ end, to form a desired RNA-polymer structure of the sense strand. In the case of the antisense strand to be annealed with the strand, an antisense strand of the sequence complementary to the sense strand was prepared through the aforementioned reaction.
- The RNA oligo structure was prepared by the method in accordance with Example 10 in order to ensure in vivo stability of the miRNA selected through Examples described above. In order to evaluate whether or not nanoparticles produced in this manner induced apoptosis in lung cancer cell lines, the lung cancer cell lines were seeded and cultured on a 6-well plate. The nanoparticles with different concentrations for respective wells were added to a culture medium. 48 hours after addition of the nanoparticles, the cells were stained with Annexin V labeled with an FIT-C fluorescent dye, and the degree of apoptosis was analyzed by flow cytometry. As can be seen from
FIGS. 9A and 9B , miRNAs as the RNA structure kill cells dependent upon the concentration of treated miRNAs. - The double-stranded oligo RNA structure and the composition for treating cancer containing the same according to the present invention include at least one miRNA selected from the group consisting of miR-3670, miR-4477a and miR-8078, thereby being widely used as an anti-cancer therapeutic agent because of improved anti-cancer effects, as compared to the pharmaceutical composition for treating cancer containing miR-34a and other miRNAs as an active ingredient.
- Although specific configurations of the present invention has been described in detail, those skilled in the art will appreciate that this description is provided as preferred embodiments for illustrative purposes and should not be construed as limiting the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the accompanying claims and equivalents thereto.
Claims (15)
1. A method for treating a lung cancer, comprising administering to a subject in need thereof a double-stranded oligo RNA structure comprising the following Formula (1):
A-X-R-Y-B Formula (1)
A-X-R-Y-B Formula (1)
wherein A represents a hydrophilic material, B represents a hydrophobic material, X and Y each independently represent a simple covalent bond or a linker-mediated covalent bond, and R represents an miRNA, wherein said miRNA is miR-8078.
2. The method of claim 1 , wherein the miRNA treats cancer by inducing apoptosis of cancer cell.
3. The method of claim 1 , wherein the hydrophilic material is represented by (A)n, (Am-J)n or (J-Am)n, wherein A represents a hydrophilic material monomer, n represents 1 to 200, m represents 1 to 15, and J represents a linker that links m hydrophilic material monomers to one another, or links m hydrophilic material monomers to oligonucleotides.
4. The method of claim 1 , wherein the hydrophilic material has a molecular weight of 200 to 10,000.
5. The method of claim 1 , wherein the hydrophilic material is polyethylene glycol (PEG).
7. The method of claim 3 , wherein the linker (J) is selected from the group consisting of PO3 −, SO3, and CO2.
8. The method of claim 1 , wherein the hydrophobic material has a molecular weight of 250 to 1,000.
9. The method of claim 1 , wherein the hydrophobic material is selected from the group consisting of a steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, C12 to C50 unsaturated or saturated hydrocarbon, diacyl-phosphatidylcholine, fatty acid, phospholipid, and lipopolyamine.
10. The method of claim 9 , wherein the steroid derivative is selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate and cholestanylamine.
11. The method of claim 9 , wherein the glyceride derivative is selected from mono-, di- and tri-glyceride.
12. The method of claim 1 , wherein the covalent bond represented by X and Y is a non-degradable or degradable bond.
13. The method of claim 12 , wherein the non-degradable bond is an amide bond or a phosphoryl bond.
14. The method of claim 12 , wherein the degradable bond is a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzyme-degradable bond.
15. The method of claim 1 , wherein miR-8078 comprises a double stranded RNA comprising a base sequence of SEQ ID NO. 65; and a base sequence of SEQ ID NO. 66 or SEQ ID NO. 69.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/402,081 US20210369758A1 (en) | 2016-08-24 | 2021-08-13 | DOUBLE-STRANDED OLIGO RNA STRUCTURE COMPRISING miRNA |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2016-0107937 | 2016-08-24 | ||
KR1020160107937A KR101861738B1 (en) | 2016-08-24 | 2016-08-24 | Double Stranded Oligo RNA Structure Comprising miRNA |
PCT/KR2017/009271 WO2018038558A1 (en) | 2016-08-24 | 2017-08-24 | Double-stranded oligo rna structure comprising microrna |
US201816302670A | 2018-11-18 | 2018-11-18 | |
US17/402,081 US20210369758A1 (en) | 2016-08-24 | 2021-08-13 | DOUBLE-STRANDED OLIGO RNA STRUCTURE COMPRISING miRNA |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/302,670 Division US11123361B2 (en) | 2016-08-24 | 2017-08-24 | Double-stranded oligo RNA structure comprising miRNA |
PCT/KR2017/009271 Division WO2018038558A1 (en) | 2016-08-24 | 2017-08-24 | Double-stranded oligo rna structure comprising microrna |
Publications (1)
Publication Number | Publication Date |
---|---|
US20210369758A1 true US20210369758A1 (en) | 2021-12-02 |
Family
ID=61245104
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/302,670 Active US11123361B2 (en) | 2016-08-24 | 2017-08-24 | Double-stranded oligo RNA structure comprising miRNA |
US17/402,081 Abandoned US20210369758A1 (en) | 2016-08-24 | 2021-08-13 | DOUBLE-STRANDED OLIGO RNA STRUCTURE COMPRISING miRNA |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/302,670 Active US11123361B2 (en) | 2016-08-24 | 2017-08-24 | Double-stranded oligo RNA structure comprising miRNA |
Country Status (4)
Country | Link |
---|---|
US (2) | US11123361B2 (en) |
EP (1) | EP3505630A4 (en) |
KR (1) | KR101861738B1 (en) |
WO (1) | WO2018038558A1 (en) |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2006280600B2 (en) | 2005-08-17 | 2012-01-19 | Bioneer Corporation | Sirna-hydrophilic polymer conjugates for intracellular delivery of siRNA and method thereof |
CN101801419A (en) | 2007-06-08 | 2010-08-11 | 米尔纳疗法公司 | Gene and path as the miR-34 regulation and control for the treatment of the target of intervening |
KR101325186B1 (en) | 2008-01-23 | 2013-11-07 | (주)바이오니아 | An Anticancer Agent Comprising Double-stranded miRNAs as an Active Ingredient |
KR101224828B1 (en) | 2009-05-14 | 2013-01-22 | (주)바이오니아 | SiRNA conjugate and preparing method thereof |
US9326941B2 (en) * | 2012-01-05 | 2016-05-03 | Bioneer Corporation | High-efficiency nanoparticle-type double-helical oligo-RNA structure and method for preparing same |
WO2014100252A1 (en) * | 2012-12-18 | 2014-06-26 | University Of Washington Through Its Center For Commercialization | Methods and compositions to modulate rna processing |
MX2016000020A (en) * | 2013-07-05 | 2016-08-18 | Bioneer Corp | Improved nanoparticle type oligonucleotide structure having high efficiency and method for preparing same. |
US20160122764A1 (en) * | 2013-07-05 | 2016-05-05 | Bioneer Corporation | Respiratory disease-related gene specific sirna, double-helical oligo rna structure containing sirna, compositon containing same for preventing or treating respiratory disease |
KR20150006743A (en) * | 2013-07-09 | 2015-01-19 | (주)바이오니아 | Liver cancer related genes-specific siRNA, double-stranded oligo RNA molecules comprising the siRNA, and composition for the prevention or treatment of cancer comprising the same |
SE539156C2 (en) | 2014-02-18 | 2017-04-18 | Scania Cv Ab | Map structure in a vehicle |
CN112159807A (en) * | 2014-04-04 | 2021-01-01 | 柏业公司 | Novel double-stranded oligo-RNA and pharmaceutical composition for preventing or treating fibrosis or respiratory disease comprising the same |
RU2686313C2 (en) * | 2015-02-25 | 2019-04-25 | Байонир Корпорейшн | PHARMACEUTICAL COMPOSITION FOR CANCER TREATMENT CONTAINING microRNA AS AN ACTIVE INGREDIENT |
-
2016
- 2016-08-24 KR KR1020160107937A patent/KR101861738B1/en active IP Right Grant
-
2017
- 2017-08-24 WO PCT/KR2017/009271 patent/WO2018038558A1/en unknown
- 2017-08-24 EP EP17843978.2A patent/EP3505630A4/en not_active Withdrawn
- 2017-08-24 US US16/302,670 patent/US11123361B2/en active Active
-
2021
- 2021-08-13 US US17/402,081 patent/US20210369758A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
KR101861738B1 (en) | 2018-05-29 |
KR20180022468A (en) | 2018-03-06 |
EP3505630A1 (en) | 2019-07-03 |
US20200179432A1 (en) | 2020-06-11 |
WO2018038558A1 (en) | 2018-03-01 |
EP3505630A4 (en) | 2020-04-01 |
US11123361B2 (en) | 2021-09-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6538183B2 (en) | Pharmaceutical composition for cancer treatment containing microRNA as active ingredient | |
US20180207191A1 (en) | Human cancer micro-rna expression profiles predictive of chemo-response | |
Hernando | microRNAs and cancer: role in tumorigenesis, patient classification and therapy | |
Iorio et al. | Breast cancer and microRNAs: therapeutic impact | |
Garofalo et al. | MicroRNAs as anti-cancer therapy | |
US20150329858A1 (en) | Long non-coding rna used for anticancer therapy | |
WO2009015071A1 (en) | Screening of micro-rna cluster inhibitor pools | |
US20160024597A1 (en) | miRNAs AS THERAPEUTIC TARGETS IN CANCER | |
JP2008239596A (en) | Tumor proliferation inhibitor containing micro-rna as active ingredient and medicinal composition for cancer treatment | |
Wu et al. | MiRNAs in human cancers: the diagnostic and therapeutic implications | |
Sekar et al. | Therapeutic evaluation of microRNAs by molecular imaging | |
Cao et al. | Emerging roles and potential clinical applications of noncoding RNAs in hepatocellular carcinoma | |
JP2011093892A (en) | Tumor proliferation inhibitor containing cancer-inhibitive micro-rna | |
US8980854B2 (en) | miRNA compounds for treatment of prostate carcinoma | |
WO2011111715A1 (en) | Nucleic acid capable of regulating cell cycle | |
US20210369758A1 (en) | DOUBLE-STRANDED OLIGO RNA STRUCTURE COMPRISING miRNA | |
KR101993894B1 (en) | Double Stranded Oligo RNA Structure Comprising miRNA | |
Joshi et al. | Noncoding RNA landscape and their emerging roles as biomarkers and therapeutic targets in meningioma | |
Qattan | Gene silencing agents in breast cancer | |
US11820984B2 (en) | Double-helix oligonucleotide construct comprising double-stranded miRNA and use thereof | |
KR101862247B1 (en) | Pharmaceutical Composition for Treating Cancer Comprising miRNA having Drug Response to serpinb5 and Application Thereof | |
HUSSEIN MOHAMMED AHMED ALI | A novel in vivo nanoparticle-mediated delivery for microRNA in a CLL mouse model: identification of miR-26a as a potential therapeutic agent | |
Sharma et al. | Clinical Implications of miRNAs in Human Diseases |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |