CN114606233A - 靶向长链非编码RNA HOXD-AS2的siRNA及其在肝癌治疗中的应用 - Google Patents
靶向长链非编码RNA HOXD-AS2的siRNA及其在肝癌治疗中的应用 Download PDFInfo
- Publication number
- CN114606233A CN114606233A CN202210278276.0A CN202210278276A CN114606233A CN 114606233 A CN114606233 A CN 114606233A CN 202210278276 A CN202210278276 A CN 202210278276A CN 114606233 A CN114606233 A CN 114606233A
- Authority
- CN
- China
- Prior art keywords
- sirna
- hoxd
- liver cancer
- coding rna
- cells
- 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.)
- Granted
Links
- 201000007270 liver cancer Diseases 0.000 title claims abstract description 76
- 208000014018 liver neoplasm Diseases 0.000 title claims abstract description 76
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 69
- 108091027963 non-coding RNA Proteins 0.000 title claims abstract description 9
- 102000042567 non-coding RNA Human genes 0.000 title claims abstract description 9
- 230000014509 gene expression Effects 0.000 claims abstract description 35
- 238000013508 migration Methods 0.000 claims abstract description 15
- 230000009545 invasion Effects 0.000 claims abstract description 10
- 230000005012 migration Effects 0.000 claims abstract description 10
- 230000022131 cell cycle Effects 0.000 claims abstract description 9
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 9
- 230000035755 proliferation Effects 0.000 claims abstract description 9
- 239000003814 drug Substances 0.000 claims abstract description 8
- 230000018199 S phase Effects 0.000 claims abstract description 5
- 230000001939 inductive effect Effects 0.000 claims abstract 2
- 108091046869 Telomeric non-coding RNA Proteins 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 238000012986 modification Methods 0.000 claims description 6
- 230000004048 modification Effects 0.000 claims description 6
- 150000007523 nucleic acids Chemical group 0.000 claims description 6
- 239000003607 modifier Substances 0.000 claims description 5
- 238000003753 real-time PCR Methods 0.000 claims description 5
- 230000025084 cell cycle arrest Effects 0.000 claims description 4
- 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 4
- 239000002502 liposome Substances 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 2
- 241000700605 Viruses Species 0.000 claims description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 2
- 235000012000 cholesterol Nutrition 0.000 claims description 2
- 239000002105 nanoparticle Substances 0.000 claims description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 1
- 239000000825 pharmaceutical preparation Substances 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 7
- 230000002194 synthesizing effect Effects 0.000 abstract description 3
- 238000012827 research and development Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 85
- 239000004055 small Interfering RNA Substances 0.000 description 43
- 108020005198 Long Noncoding RNA Proteins 0.000 description 39
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 22
- 210000001519 tissue Anatomy 0.000 description 20
- 206010028980 Neoplasm Diseases 0.000 description 18
- 230000000694 effects Effects 0.000 description 16
- 238000002474 experimental method Methods 0.000 description 16
- 230000008685 targeting Effects 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 13
- 238000001890 transfection Methods 0.000 description 13
- 239000000243 solution Substances 0.000 description 12
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 201000011510 cancer Diseases 0.000 description 9
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 238000010609 cell counting kit-8 assay Methods 0.000 description 5
- 230000004709 cell invasion Effects 0.000 description 5
- 230000012292 cell migration Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 108010085238 Actins Proteins 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 108010087230 Sincalide Proteins 0.000 description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 description 4
- 239000006143 cell culture medium Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000003021 clonogenic effect Effects 0.000 description 4
- 206010017758 gastric cancer Diseases 0.000 description 4
- 210000003494 hepatocyte Anatomy 0.000 description 4
- 210000005229 liver cell Anatomy 0.000 description 4
- 238000011580 nude mouse model Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000010839 reverse transcription Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 201000011549 stomach cancer Diseases 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 108091092584 GDNA Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 230000030279 gene silencing Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000002271 resection Methods 0.000 description 3
- 238000002626 targeted therapy Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 239000012096 transfection reagent Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000010242 baoji Substances 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- MLDQJTXFUGDVEO-UHFFFAOYSA-N BAY-43-9006 Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=CC(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 MLDQJTXFUGDVEO-UHFFFAOYSA-N 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108091007703 DDX11-AS1 Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 239000005511 L01XE05 - Sorafenib Substances 0.000 description 1
- 239000002138 L01XE21 - Regorafenib Substances 0.000 description 1
- 239000002176 L01XE26 - Cabozantinib Substances 0.000 description 1
- 108700019961 Neoplasm Genes Proteins 0.000 description 1
- 102000048850 Neoplasm Genes Human genes 0.000 description 1
- 206010061309 Neoplasm progression Diseases 0.000 description 1
- 238000010806 PrimeScriptTM RT Reagent kit Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 229960003982 apatinib Drugs 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960001292 cabozantinib Drugs 0.000 description 1
- ONIQOQHATWINJY-UHFFFAOYSA-N cabozantinib Chemical compound C=12C=C(OC)C(OC)=CC2=NC=CC=1OC(C=C1)=CC=C1NC(=O)C1(C(=O)NC=2C=CC(F)=CC=2)CC1 ONIQOQHATWINJY-UHFFFAOYSA-N 0.000 description 1
- 238000003783 cell cycle assay Methods 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000009643 clonogenic assay Methods 0.000 description 1
- 231100000096 clonogenic assay Toxicity 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000005206 flow analysis Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000001638 lipofection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000011880 melting curve analysis Methods 0.000 description 1
- WPEWQEMJFLWMLV-UHFFFAOYSA-N n-[4-(1-cyanocyclopentyl)phenyl]-2-(pyridin-4-ylmethylamino)pyridine-3-carboxamide Chemical compound C=1C=CN=C(NCC=2C=CN=CC=2)C=1C(=O)NC(C=C1)=CC=C1C1(C#N)CCCC1 WPEWQEMJFLWMLV-UHFFFAOYSA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000001915 proofreading effect Effects 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 229960002633 ramucirumab Drugs 0.000 description 1
- -1 ranvatinib Chemical compound 0.000 description 1
- 229960004836 regorafenib Drugs 0.000 description 1
- FNHKPVJBJVTLMP-UHFFFAOYSA-N regorafenib Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=C(F)C(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 FNHKPVJBJVTLMP-UHFFFAOYSA-N 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000001743 silencing effect Effects 0.000 description 1
- 229960003787 sorafenib Drugs 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000000107 tumor biomarker Substances 0.000 description 1
- 230000005751 tumor progression Effects 0.000 description 1
- 230000000007 visual effect Effects 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
- 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
-
- 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
- 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
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- 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
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Oncology (AREA)
- Pharmacology & Pharmacy (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Hospice & Palliative Care (AREA)
- Plant Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种靶向长链非编码RNA HOXD‑AS2的siRNA及其在肝癌治疗中的应用。通过设计并合成靶向HOXD‑AS2的siRNA,将其转染肝癌细胞系,证实利用siRNA靶向抑制HOXD‑AS2的表达可显著抑制肝癌细胞的增殖、侵袭、迁移的能力并诱导肝癌细胞周期阻滞于S期。本发明为肝癌治疗药物的研发提供了新靶点和有效途径。
Description
技术领域
本发明属于生物医药领域,具体涉及靶向长链非编码RNA HOXD-AS2(HOXDCluster Antisense RNA 2)的小干扰RNA(Small interfering RNA,siRNA)及其在肝癌治疗中的应用。
背景技术
肝癌是最常见的消化系统恶性肿瘤之一,为全球第四大致死性肿瘤,对人类健康构成严重威胁,尤其在东亚、非洲和南欧地区发病率较高。由于发病机制复杂、早期病程隐匿、发病率高、预后差以及术后高复发与高转移等特点,特别是缺乏有效的早期诊断方法和有效的治疗手段,导致患者病死率居高不下。目前,早期手术切除是肝癌最主要和最有效的治疗手段,但大部分患者初次诊断时肝癌就已进入中晚期,极大限制了手术治疗,且手术复发率高。此外,局部消融、放化疗、介入治疗及肝移植等治疗方法,由于在临床应用中受到众多禁忌症的限制,其总体疗效仍然有限。
近年来,肿瘤分子靶向治疗作为一种新型疗法,已逐步成为临床肿瘤治疗的重要手段。由于分子靶向治疗是通过特异性分子干预(封闭或抑制)肿瘤发生关键基因及信号传导通路等分子靶点,从而抑制肿瘤细胞的生长、转移或诱导其凋亡,因此与传统治疗手段相比,具有更好的精准性,能选择性地杀伤肿瘤细胞,对正常组织损伤较低或无损伤、副作用小,并且不易产生耐药性。目前,临床上应用于肝癌治疗的分子靶向药主要有索拉非尼、仑伐替尼、瑞戈非尼、卡博替尼、雷莫芦单抗和阿帕替尼,可见用于肝癌治疗的分子靶向药物非常有限和匮乏,其关键原因在于有效的分子靶点数量不足。因此目前迫切需要开发新的有效分子靶点。
长链非编码RNA(long noncoding RNA,lncRNA)是一类长度超过200个核苷酸但缺乏蛋白编码能力的RNA分子,大量研究表明许多lncRNA在肿瘤中表达失调,可在表观遗传、转录以及转录后水平调控基因表达,进而参与肿瘤的发生发展,已成为肿瘤生物标志物和治疗靶点研发的重要分子靶点类型。但获得可以高效(抑制率大于50%)降低lncRNA表达水平的siRNA仍是较为困难的,例如中国专利CN108546702A中就需要利用多条siRNA降低长链非编码RNA DDX11-AS1的表达,而且这一类专利公开的siRNA临床应用极少,主要原因包括其在动物个体上对肝癌的治疗效果并未证实,客观反映出相应lncRNA作为靶点在肝癌治疗潜力上的不足。
LncRNA HOXD-AS2为一条转录于2号染色体q31.1区域HOXD基因簇的非编码转录本。研究发现,lncRNA HOXD-AS2在胶质母细胞瘤中上调表达,其可通过促进细胞增殖、迁移和侵袭促进肿瘤进展,提示lncRNA HOXD-AS2发挥促癌作用;而最近一项在胃癌中的研究发现,lncRNA HOXD-AS2在胃癌中呈现低表达趋势,其过表达可抑制胃癌进展,提示lncRNAHOXD-AS2在胃癌中发挥抑癌作用,可见lncRNA HOXD-AS2在不同肿瘤中的生物学功能存在差异。LncRNA HOXD-AS2在肝癌中的表达情况、对肝癌的发生发展的影响,以及有作为肝癌治疗靶点的潜力目前均不清楚。
发明内容
本发明的目的在于提供一种靶向长链非编码RNA HOXD-AS2的siRNA及其在肝癌治疗中的应用。该siRNA可以高效抑制LncRNA HOXD-AS2表达水平,应用于肝癌治疗药物开发。
为了达到上述目的,本发明采用了以下技术方案:
本发明首先采用荧光定量PCR的方法对临床肝癌组织/癌旁组织、肝癌细胞系/正常肝细胞中的lncRNA HOXD-AS2表达水平进行了检测,发现与癌旁组织和正常肝细胞相比,LncRNA HOXD-AS2在肝癌组织和肝癌细胞系中高表达;其次,针对lncRNA HOXD-AS2基因序列设计并合成多条特异靶向lncRNA HOXD-AS2的siRNA,采用脂质体介导的方法转染肝癌细胞后,通过荧光定量PCR的方法检测siRNA沉默lncRNA HOXD-AS2的效率,结果筛选出了可以通过联合转染对lncRNA HOXD-AS2具有较高的沉默效率的siRNA。
优选的,本发明提供的能高效抑制LncRNA HOXD-AS2表达的siRNA,为序列分别如SEQ.ID.NO.1至SEQ.ID.NO.6所示siRNA中3~6条siRNA的混合物(其中6条siRNA联合转染后lncRNA HOXD-AS2表达水平下调70%以上)。
本发明提供的能高效抑制LncRNA HOXD-AS2表达的siRNA可以在制备治疗肝癌的药物中应用。
优选的,通过采用CCK-8、PI染色流式分析、Transwell等实验方法检测siRNA对细胞增殖、周期、侵袭、迁移的影响,结果表明本发明提供的能高效抑制LncRNA HOXD-AS2表达的siRNA,可以在显著抑制肝癌细胞的增殖、迁移和侵袭的同时诱导细胞周期阻滞在S期(即显著抑制肝癌细胞进展),结合动物实验明确了上述能高效抑制LncRNA HOXD-AS2表达的siRNA达到治疗肝癌的目的。
本发明提供的用于治疗肝癌的药物制剂,包括上述能高效抑制LncRNA HOXD-AS2表达的siRNA或其核酸序列修饰物及药学上接受的载体。
优选的,所述核酸序列修饰物为通过对靶向LncRNA HOXD-AS2的siRNA(例如SEQ.ID.NO.1至SEQ.ID.NO.6中任意一个或多个序列所示的siRNA)进行任意核苷酸的核糖修饰、碱基修饰和磷酸骨架修饰中的一种或多种修饰后获得的核酸序列修饰物。
优选的,所述载体选自病毒、纳米颗粒、胆固醇或脂质体。
本发明还提供一种用于诊断肝癌或检测细胞中LncRNA HOXD-AS2的表达水平的试剂盒,该试剂盒包括用于采用实时荧光定量PCR方法检测LncRNA HOXD-AS2的表达量的引物对。
本发明的有益效果体现在:
本发明通过靶向lncRNA HOXD-AS2的siRNA,降低lncRNA HOXD-AS2表达水平,能够有效抑制肝癌细胞的增殖、迁移和侵袭能力,同时有效诱导肝癌细胞周期阻滞在S期,结合动物实验结果表明lncRNA HOXD-AS2作为肝癌治疗靶点,对于开发新的抗肝癌基因药物和提高肝癌的治疗效果有重要的意义,具有显著的应用前景和经济价值。
进一步的,本发明所提供的siRNA能高效抑制lncRNA HOXD-AS2的表达,抑制率可达70%以上。
附图说明
图1为lncRNA HOXD-AS2在人肝细胞癌及癌旁组织(A)、人肝细胞癌细胞系及正常肝细胞株(B)中的表达,其中:*p<0.05,**p<0.01,差异具有统计学意义。
图2为靶向lncRNA HOXD-AS2的siRNA对肝癌细胞Bel-7402和SMMC-7721中lncRNAHOXD-AS2的抑制效率,其中:**p<0.01,差异具有统计学意义。
图3为靶向lncRNA HOXD-AS2的siRNA对肝癌细胞增殖(A)和克隆形成(B)的影响,其中:**p<0.01,差异具有统计学意义。
图4为靶向lncRNA HOXD-AS2的siRNA对肝癌细胞周期的影响,其中:**p<0.01,差异具有统计学意义。
图5为靶向lncRNA HOXD-AS2的siRNA对肝癌细胞迁移(Migration)能力的影响,其中:**p<0.01,差异具有统计学意义。
图6为靶向lncRNA HOXD-AS2的siRNA对肝癌细胞侵袭(Invasion)能力的影响,其中:**p<0.01,差异具有统计学意义。
图7为靶向lncRNA HOXD-AS2的siRNA对肝癌细胞在裸鼠体内生长的影响,其中:*p<0.05,差异具有统计学意义。
具体实施方式
下面结合附图和实施例对本发明做进一步详细说明。
本发明为了明确lncRNA HOXD-AS2是否参与肝癌的发生发展,首先通过实时荧光定量PCR(qRT-PCR)的方法对HOXD-AS2在临床肝癌组织和肝癌细胞系中的表达水平进行了检测,发现lncRNA HOXD-AS2在肝癌组织和肝癌细胞系中高表达;其次,针对lncRNA HOXD-AS2基因序列设计并合成多条特异靶向lncRNA HOXD-AS2的siRNA,采用脂质体介导的方法将不同siRNA的混合物转染肝癌细胞Bel-7402和SMMC-7721以沉默其lncRNA HOXD-AS2的表达,并观察对细胞增殖、凋亡、侵袭、迁移的影响。最终对筛选得到一组siRNA中全部6条siRNA通过动物实验进行了治疗肝癌的在体验证。
实验1、LncRNA HOXD-AS2在人肝细胞癌细胞系及正常肝细胞株、肝细胞癌及癌旁组织中的表达
1、材料
细胞:人肝细胞癌细胞系HepG2、SMMC-7721、MHCC97H、MHCC97L、Bel-7402、Bel-7404及正常肝细胞株L-O2,均购自中国科学院上海生命科学研究院细胞资源中心。
试剂:DMEM高糖型细胞培养液、青霉素、链霉素购自Hyclone公司,胎牛血清购自Gibco公司,Trizol购自Invitrogen公司,反转录试剂盒PrimeScriptTM RT reagent Kitwith gDNA Eraser(Perfect Real Time)和实时荧光定量试剂TBPremix Ex TaqTMII购自TaKaRa公司。qRT-PCR特异性引物由苏州金唯智生物科技有限公司合成。
2、方法
2.1、标本来源
收集西安交通大学第二附属医院和宝鸡市中心医院2016年1月~2017年12月手术切除肝癌标本20例,手术切除后30min内取材,每例分别取癌组织和癌旁组织(距肿瘤边缘>2cm),立即投入液氮保存。所有肿瘤标本经病理证实为肝细胞癌。
2.2、细胞培养
人肝细胞癌细胞系HepG2、SMMC-7721、MHCC97H、MHCC97L、Bel-7402、Bel-7404和正常肝细胞株L-O2用高糖型DMEM细胞培养液(加入10%胎牛血清、100U/mL青霉素链霉素双抗),置于37℃、5%CO2的恒温培养箱中培养。
2.3、细胞及组织中总RNA的提取
对于人肝癌细胞及正常肝细胞、肝癌组织及癌旁组织,使用Trizol试剂提取细胞和组织总RNA。利用Nanodrop 2000分光光度法(Thermo Fisher Scientific,USA)和琼脂糖凝胶电泳评估RNA的浓度和质量。总RNA提取方法具体如下:
①将6孔板中培养的细胞去掉培养液,用预冷的PBS洗涤2次,加入1mL Trizol试剂裂解细胞,并移至无酶EP管中;或取100mg组织置入1mL Trizol,匀浆器匀浆,并移至无酶EP管中;
②向无酶EP管中加入200μL氯仿,充分震荡混匀15s,室温静置5min后置于4℃超速离心机,12000rpm离心15min;
③将上层含有RNA的水相移入新的无酶EP管中,作好相应标记,加入500μL异丙醇,振荡器上充分震荡混匀,置于4℃超速离心机,12000rpm离心10min;
④完全弃除上清,沉淀即为RNA,然后加入提前配制的1mL 75%乙醇,振荡器上充分混匀后于4℃超速离心机,7500rpm离心5min;
⑤完全弃除上清,风干沉淀后加入无酶水20μL,并测定RNA浓度及纯度,标记好后置于-80℃超低温冰箱保存。
2.4、反转录
采用TAKARA公司的PrimeScript RT reagent Kit with gDNA Eraser(PerfectReal Time)试剂盒,并按说明书操作,分为两步:
第一步去除提取的总RNA中的基因组DNA,体系如下:提取的总RNA1μg、5×gDNAEraser Buffer 2μL,及gDNA Eraser 1μL,无RNA酶的ddH2O补到10μL。置于PCR仪中42℃孵育2min,得反应液。
第二步进行反转录反应,体系如下:上一步的反应液10μL、5×PrimescriptBuffer2(for Real Time)4μL、Primescript RT Enzyme Mix I 1μL、RT Primer Mix 1μL,及ddH2O4μL,总体积20μL。置于PCR仪中37℃孵育15min,85℃孵育5s灭活逆转录酶,得到cDNA。
2.5、qRT-PCR
采用TAKARA公司TBPremix Ex TaqTM II试剂盒,其反应体系如下:SYBR10μL、lncRNA-HOXD-AS2正向引物(5`-AACTGCTCTGGTGAACTCC-3`)0.4μL、lncRNA-HOXD-AS2反向引物(5`-TTCTTGTGTCCTCTGCTTCC-3`)0.4μL、cDNA 2μL、ROX Reference Dye II 0.4μL,及ddH2O 6.8μL,总体积20μL。
内参基因β-Actin的正向引物为:5`-TGGCACCCAGCACAATGAA-3`,反向引物为:5`-CTAAGTCATAGTCCGCCTAGAAGCA-3`。
反应条件如下:95℃30s预变性;95℃5s变性,60℃34s退火并延伸,40个循环。熔解曲线分析:60~95℃,每0.4℃读1次。同时以内参β-Actin基因作为校对,使用ABI7500 fast进行qRT-PCR和数据收集,采用2-ΔΔCt进行数据分析。
3、结果
LncRNA HOXD-AS2在癌旁组织中表达水平较低,而在肝癌组织中呈现高表达,并达到显著差异(p<0.05、图1A)。LncRNA HOXD-AS2在HCC细胞系(HepG2、SMMC-7721、MHCC97H、MHCC97L、Bel-7402、Bel-7404)中表达显著高于正常肝细胞柱LO-2(图1B)。这些结果表明lncRNA HOXD-AS2在HCC组织和细胞中异常上调表达,提示其可能具有促癌作用。
实验2、siRNA抑制LncRNA HOXD-AS2表达后对肝癌细胞生物学功能的影响
1材料
细胞:人肝细胞癌细胞系Bel-7402和SMMC-7721,购自中国科学院上海生命科学研究院细胞资源中心。
试剂:转染试剂Lipofectamine 3000购自Thermo Fisher Scientific公司,CCK-8试剂购自日本DOJINDO公司,细胞周期与细胞凋亡检测试剂盒购自上海碧云天生物技术有限公司,Transwell小室购自美国Corning公司。
2、方法
2.1细胞培养
同实验1。
2.2靶向lncRNA HOXD-AS2的siRNA序列的设计与合成
在NCBI获得lncRNA HOXD-AS2基因序列(NR_038435.1),然后利用BLOCK-iT RNAiDesigner软件(Thermo Fisher Scientific)设计特异靶向HOXD-AS2的siRNA(siHOXD-AS2),从结果中选择不同siRNA的序列进行合成并制成siRNA混合物(等摩尔比),以下展示了最终通过动物实验验证的6条siRNA的序列(于2016年03月完成设计):
siRNA1:5`-CGCTCATGTTGGTGAAGAA-3`
siRNA2:5`-ACAAGAAGCTTGGATGTGA -3`
siRNA3:5`-CCACCTCTGCAGAGACAAA-3`
siRNA4:5`-GCGATTCTTACCCGAAGGCT-3`
siRNA5:5`-AGGAACTGCTCTGGTGAACT-3`
siRNA6:5`-ATGCAGCCTTCAGAACCTTC-3`
上述6条靶向lncRNA HOXD-AS2的siRNA由广州锐博生物科技有限公司合成,其混合物称为siHOXD-AS2。
实验用到的阴性对照序列(siNC,在人基因组上无作用靶点)购自广州锐博生物科技有限公司。
2.3细胞转染
将人肝细胞癌细胞系Bel-7402和SMMC-7721接种于6孔板中,37℃、5%CO2培养过夜,在转染前生长至汇合度30~50%,参照Lipofectamine 3000(Thermo FisherScientific)说明书进行转染48h。具体步骤如下:
a)将5μL siHOXD-AS2/siNC(终浓度50nm)和3.75μL lipo3000转染试剂分别加入至125μL无血清DMEM培养液中,分别混匀后,将含有siRNA的DMEM培养液加入至含有lipo3000转染试剂的DMEM培养液中,小心混匀,静置5min,得转染液;
c)转染:将上述转染液加入上述6孔板(含有2mL培养液)中,置于37℃、5%CO2培养箱孵育;
d)转染后收集细胞,采用qRT-PCR检测siRNA对lncRNA HOXD-AS2的干扰效果,或进行细胞增殖、周期、侵袭与迁移实验。
2.4qRT-PCR检测siRNA对lncRNA HOXD-AS2的干扰效果
转染48h后,收集转染后的实验组细胞(转染siHOXD-AS2的肝癌细胞)和对照组细胞(转染siNC的肝癌细胞),进行细胞总RNA提取,反转录及qRT-PCR,方法同实验1。
2.5细胞增殖实验
采用CCK-8实验和平板克隆形成实验检测细胞增殖活性。
CCK-8实验具体操作方法:收集转染siRNA 24h后的实验组和对照组细胞,加入完全培养液重悬,细胞计数,以3000个/孔密度接种于96孔板中,实验组和对照组各设置5行,每行4个复孔,37℃、5%CO2培养。设置24h、48h、72h及96h 4个时间点,检测时每孔加入10μLCCK-8试剂并在37℃培养箱中孵育1小时30分钟后,通过酶标仪检测450nm处各孔的光密度(OD)。在无细胞孔中加入完全培养基作为调零孔。
克隆形成实验具体操作方法:收集转染siRNA 24h后的实验组和对照组细胞,加入完全培养液重悬,细胞计数,以1000个/孔密度接种于6孔板,实验组和对照组各3孔,培养7天后吸去各孔培养基,PBS洗涤2次,每孔加1mL 4%多聚甲醛固定20min,吸去多聚甲醛,PBS洗涤2次,每孔加入1mL结晶紫染色液,20min后吸去,自来水下冲洗6孔板,晾干后计算集落。
2.6细胞周期实验
根据试剂盒说明书,细胞接种于6孔板中,收集转染siRNA 24h后的实验组和对照组细胞,并用预冷的70%乙醇于4℃下固定过夜,用PBS洗涤细胞后,用碘化丙啶(PI)染色溶液(含RNase A)重悬细胞沉淀并在37℃避光孵育30分钟,通过流式细胞仪(BD FACSCantoII)测定细胞DNA含量,并用FlowJo软件(Treestar,USA)根据DNA含量进行各阶段细胞百分数分析。
2.7细胞侵袭与迁移实验
收集转染siRNA 24h后的实验组和对照组细胞,加入完全培养液重悬,细胞计数。在未铺和预铺基质胶的Transwell小室内加入100μL细胞悬液(含1%胎牛血清的DMEM高糖细胞培养液,每孔约4×104个细胞),将小室放入24孔板细胞培养板内,在下层中加入600μL含20%胎牛血清的DMEM高糖细胞培养液,37℃、5%CO2浓度、饱和湿度培养细胞24~48h。取出小室,弃除24孔板内的培养液,加入600μL 90%乙醇固定10min,用无菌棉签轻轻拭去小室内部残留的乙醇和细胞,待风干后加入600μL0.1%结晶紫染液,染色10min。使用倒置显微镜,于低倍镜下,每个小室随机选取5~8个视野进行观察并拍照、计数。
3结果
3.1siHOXD-AS2对肝癌细胞lncRNA HOXD-AS2的抑制效率
如图2所示,与转染siNC的对照组相比,肝癌细胞系Bel-7402和SMMC-7721转染siHOXD-AS2后,沉默效果显著,Bel-7402和SMMC-7721细胞中LncRNA HOXD-AS2的表达水平分别下调了71%和77%,表明siHOXD-AS2转染肝癌细胞可显著抑制lncRNA HOXD-AS2的表达水平。
3.2siHOXD-AS2对肝癌细胞的增殖和克隆形成能力的影响
如图3A所示,与siNC对照组相比,转染siHOXD-AS2可明显抑制肝癌细胞的增殖能力,且平板克隆形成实验结果与CCK8实验结果一致,转染siHOXD-AS2后肝癌细胞的平板克隆形成能力显著减弱(图3B),表明siHOXD-AS2可抑制肝癌细胞的增殖和克隆形成能力。
3.3siHOXD-AS2对肝癌细胞周期的影响
如图4所示,在肝癌细胞系Bel-7402和SMMC-7721中转染靶向lncRNA HOXD-AS2的siRNA(即siHOXD-AS2)后,细胞周期阻滞在S期,表明siHOXD-AS2导致肝癌细胞周期发生阻滞。
3.4siHOXD-AS2对肝癌细胞的迁移与侵袭的影响
如图5、图6所示,与转染siNC的对照组相比,肝癌细胞系Bel-7402和SMMC-7721中转染靶向lncRNA HOXD-AS2的siRNA(即siHOXD-AS2),发生迁移和侵袭的细胞数量显著减少,提示siHOXD-AS2可显著抑制肝癌细胞的迁移与侵袭能力。
上述结果表明本发明提供的靶向lncRNA HOXD-AS2的siRNA,具有良好的lncRNAHOXD-AS2表达抑制效果,转染后可显著抑制肝癌细胞的增殖、侵袭和迁移能力,同时可诱导肝癌细胞周期阻滞。
实验3、siHOXD-AS2对肝癌细胞在裸鼠体内生长的影响
细胞:人肝细胞癌细胞系Bel-7402,购自中国科学院上海生命科学研究院细胞资源中心。
利用脂质体将siHOXD-AS2/siNC(工作浓度均为50nM)转染至肝癌细胞Bel-7402后,按5×106个细胞/只的剂量将肝癌细胞移植至裸鼠皮下,并观测皮下荷瘤组织的生长情况(体积和重量)。
如图7所示,与siNC对照组相比,siHOXD-AS2可显著抑制肝癌细胞Bel-7402在裸鼠皮下生长能力。
总之,本发明首次提供了对HOXD-AS2具有较高的沉默效率的siRNA,可显著抑制肝癌细胞的增殖、侵袭、迁移能力,以及诱导细胞周期阻滞,肝癌的治疗提供了有效的药物靶点,从而使得靶向lncRNA HOXD-AS2的siRNA可明确用于开发新的肝癌治疗药物,具有良好的应用前景。
<110> 西安交通大学
<120> 靶向长链非编码RNA HOXD-AS2的siRNA及其在肝癌治疗中的应用
<160> 10
<210> 1
<211> 19
<212> DNA
<213> siRNA1
<400> 1
cgctcatgtt ggtgaagaa 19
<210> 2
<211> 19
<212> DNA
<213> siRNA2
<400> 2
acaagaagct tggatgtga 19
<210> 3
<211> 19
<212> DNA
<213> siRNA3
<400> 3
ccacctctgc agagacaaa 19
<210> 4
<211> 20
<212> DNA
<213> siRNA4
<400> 4
gcgattctta cccgaaggct 20
<210> 5
<211> 20
<212> DNA
<213> siRNA5
<400> 5
aggaactgct ctggtgaact 20
<210> 6
<211> 20
<212> DNA
<213> siRNA6
<400> 6
atgcagcctt cagaaccttc 20
<210> 7
<211> 19
<212> DNA
<213> lncRNA-HOXD-AS2正向引物
<400> 7
aactgctctg gtgaactcc 19
<210> 8
<211> 20
<212> DNA
<213> lncRNA-HOXD-AS2反向引物
<400> 8
ttcttgtgtc ctctgcttcc 20
<210> 9
<211> 19
<212> DNA
<213>内参基因β-Actin正向引物
<400> 9
tggcacccag cacaatgaa 19
<210> 10
<211> 25
<212> DNA
<213>内参基因β-Actin反向引物
<400> 10
ctaagtcata gtccgcctag aagca 25
Claims (10)
1.一种siRNA,其特征在于:该siRNA抑制长链非编码RNA HOXD-AS2的表达。
2.根据权利要求1所述的siRNA,其特征在于:所述siRNA使肝癌细胞中长链非编码RNAHOXD-AS2的表达水平下调70%以上。
3.根据权利要求1所述的siRNA,其特征在于:所述长链非编码RNA HOXD-AS2上的siRNA靶点个数为3~6个。
4.一种靶向长链非编码RNA HOXD-AS2的siRNA在制备用于治疗肝癌的药物中的应用。
5.根据权利要求4所述的应用,其特征在于:所述siRNA选自SEQ.ID.NO.1~6中的三种以上。
6.根据权利要求4所述的应用,其特征在于:所述siRNA通过抑制肝癌细胞的增殖、迁移和侵袭并诱导细胞周期阻滞治疗肝癌。
7.根据权利要求6所述的应用,其特征在于:所述siRNA使肝癌细胞周期阻滞在S期。
8.一种用于治疗肝癌的药物制剂,其特征在于:该药物制剂包括siRNA或其核酸序列修饰物及载体,所述siRNA或其核酸序列修饰物抑制长链非编码RNA HOXD-AS2的表达。
9.根据权利要求8所述的药物制剂,其特征在于:所述核酸序列修饰物是通过对所述siRNA进行任意核苷酸的核糖修饰、碱基修饰和磷酸骨架修饰中的一种或多种修饰而获得的;所述载体选自病毒、纳米颗粒、胆固醇或脂质体。
10.一种用于诊断肝癌或检测细胞中长链非编码RNA HOXD-AS2的表达水平的试剂盒,其特征在于:该试剂盒包括用于采用实时荧光定量PCR方法检测长链非编码RNA HOXD-AS2的表达量的引物对。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210278276.0A CN114606233B (zh) | 2022-03-21 | 2022-03-21 | 靶向长链非编码RNA HOXD-AS2的siRNA及其在肝癌治疗中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210278276.0A CN114606233B (zh) | 2022-03-21 | 2022-03-21 | 靶向长链非编码RNA HOXD-AS2的siRNA及其在肝癌治疗中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114606233A true CN114606233A (zh) | 2022-06-10 |
CN114606233B CN114606233B (zh) | 2024-05-28 |
Family
ID=81864595
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210278276.0A Active CN114606233B (zh) | 2022-03-21 | 2022-03-21 | 靶向长链非编码RNA HOXD-AS2的siRNA及其在肝癌治疗中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114606233B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170260592A1 (en) * | 2016-03-11 | 2017-09-14 | University Of Southern California | Next generation rna-sequencing and long non-coding rna in glioblastoma multiforme |
CN108546702A (zh) * | 2018-04-10 | 2018-09-18 | 西安交通大学 | 靶向长链非编码RNA DDX11-AS1的siRNA及其在肝癌治疗中的应用 |
-
2022
- 2022-03-21 CN CN202210278276.0A patent/CN114606233B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170260592A1 (en) * | 2016-03-11 | 2017-09-14 | University Of Southern California | Next generation rna-sequencing and long non-coding rna in glioblastoma multiforme |
CN108546702A (zh) * | 2018-04-10 | 2018-09-18 | 西安交通大学 | 靶向长链非编码RNA DDX11-AS1的siRNA及其在肝癌治疗中的应用 |
Non-Patent Citations (4)
Title |
---|
JIN SUN 等: "OE-0641 (PE-0416) Knockdown of long non-coding RNA HOXD-AS2 inhibits the proliferation, migration, and invasion of hepatocellular carcinoma cells via MEK/ERK pathway", 《JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY》, vol. 33, no. 4, pages 418 * |
JIN SUN 等: "Silencing of long noncoding RNA HOXD‐AS1 inhibits proliferation, cell cycle progression, migration and invasion of hepatocellular carcinoma cells through MEK/ERK pathway", 《J CELL BIOCHEM》, vol. 121, pages 443, XP071665697, DOI: 10.1002/jcb.29206 * |
XINGMING ZHONG 等: "Long non-coding RNA (lncRNA) HOXD-AS2 promotes glioblastoma cell proliferation, migration and invasion by regulating the miR-3681-5p/MALT1 signaling pathway", 《BIOENGINEERED》, vol. 12, no. 2, pages 9115 * |
ZHONG X 等: "NR_038435.1 Homo sapiens HOXD cluster antisense RNA 2 (HOXD-AS2), long noncoding RNA", 《GENBANK》, pages 1 - 2 * |
Also Published As
Publication number | Publication date |
---|---|
CN114606233B (zh) | 2024-05-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108546702B (zh) | 靶向长链非编码RNA DDX11-AS1的siRNA及其在肝癌治疗中的应用 | |
CN110804613B (zh) | 一种靶向抑制lncRNA-00861基因表达的siRNA在肝癌治疗中的应用 | |
CN108179194B (zh) | 一种肿瘤分子标志物circBIRC6及其抑制剂和用途 | |
Han et al. | MicroRNA-1251-5p promotes tumor growth and metastasis of hepatocellular carcinoma by targeting AKAP12 | |
WO2021022888A1 (zh) | 靶向长链非编码rna ddx11-as1的aso、试剂盒及在肝癌治疗中的应用 | |
CN111500734A (zh) | 一种肝癌诊断标志物及其应用 | |
CN107760784A (zh) | 环状RNA circ‑FOXP1的用途 | |
CN107828888A (zh) | 环状RNA circ‑PTPRA的用途 | |
CN110201172B (zh) | Yy1表达抑制剂在制备治疗乳腺癌药物中的应用 | |
CN109481685B (zh) | Cd317抑制剂在制备治疗肝癌的药物中的应用 | |
CN116024211A (zh) | tRNA衍生物tRF-His-008在诊断、治疗肾癌中的应用 | |
CN107893115B (zh) | Alkbh1基因及其表达产物在制备用于诊断肿瘤的试剂盒、治疗肿瘤的药物中的用途 | |
CN111172290B (zh) | 肝细胞癌诊断和治疗的miRNA | |
CN110129319B (zh) | 一种PRALR的siRNA及其用途 | |
CN108192977B (zh) | 一种与胃癌发生发展相关的分子标志物 | |
CN114457158B (zh) | Hsa_circ_0006867作为食管癌分子靶标在制备药物和试剂盒中的应用 | |
CN113667733B (zh) | circRNA PRDM5在钙化性主动脉瓣疾病的诊断试剂盒及治疗药物开发中的应用 | |
CN114606233B (zh) | 靶向长链非编码RNA HOXD-AS2的siRNA及其在肝癌治疗中的应用 | |
CN110577952B (zh) | 干扰长非编码RNA的siRNA在制备治疗乳腺癌药物中的应用 | |
CN109224076B (zh) | 与肺癌诊疗相关的基因miR-140-3P及其mimics和应用 | |
CN108642176B (zh) | Part1作为乳腺癌诊断、治疗以及预后标志物的应用 | |
CN113789340B (zh) | 环状RNA hsa_circ_0001741的表达载体、重组工程菌及其应用 | |
CN111808954B (zh) | lncRNA及其在疾病中的应用 | |
WO2021037265A1 (zh) | 一种抑制MCM7基因表达的siRNA、组合物及其应用 | |
WO2021037264A1 (zh) | 抑制MCM7基因表达的siRNA、组合物及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |