CN116712548A - Use of co-inhibition of EZH2 and ATX-LPA-LPA2 axis in colorectal cancer treatment - Google Patents
Use of co-inhibition of EZH2 and ATX-LPA-LPA2 axis in colorectal cancer treatment Download PDFInfo
- Publication number
- CN116712548A CN116712548A CN202310562558.8A CN202310562558A CN116712548A CN 116712548 A CN116712548 A CN 116712548A CN 202310562558 A CN202310562558 A CN 202310562558A CN 116712548 A CN116712548 A CN 116712548A
- Authority
- CN
- China
- Prior art keywords
- atx
- lpa
- ezh2
- substance
- lpa2
- 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.)
- Pending
Links
- 101000882127 Homo sapiens Histone-lysine N-methyltransferase EZH2 Proteins 0.000 title claims abstract description 157
- 102100038970 Histone-lysine N-methyltransferase EZH2 Human genes 0.000 title claims abstract description 155
- 206010009944 Colon cancer Diseases 0.000 title claims abstract description 88
- 208000001333 Colorectal Neoplasms Diseases 0.000 title claims abstract description 88
- 238000011282 treatment Methods 0.000 title abstract description 61
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 115
- 239000003112 inhibitor Substances 0.000 claims abstract description 87
- 101001038001 Homo sapiens Lysophosphatidic acid receptor 2 Proteins 0.000 claims abstract description 53
- 102100040387 Lysophosphatidic acid receptor 2 Human genes 0.000 claims abstract description 53
- 229940122849 Autotaxin inhibitor Drugs 0.000 claims abstract description 20
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 10
- 108020004459 Small interfering RNA Proteins 0.000 claims description 79
- 230000014509 gene expression Effects 0.000 claims description 75
- FKSFKBQGSFSOSM-QFIPXVFZSA-N 1-[(2S)-butan-2-yl]-N-[(4,6-dimethyl-2-oxo-1H-pyridin-3-yl)methyl]-3-methyl-6-[6-(1-piperazinyl)-3-pyridinyl]-4-indolecarboxamide Chemical group C1=C2N([C@@H](C)CC)C=C(C)C2=C(C(=O)NCC=2C(NC(C)=CC=2C)=O)C=C1C(C=N1)=CC=C1N1CCNCC1 FKSFKBQGSFSOSM-QFIPXVFZSA-N 0.000 claims description 61
- 101100465401 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) SCL1 gene Proteins 0.000 claims description 47
- 239000000126 substance Substances 0.000 claims description 44
- 108090000623 proteins and genes Proteins 0.000 claims description 40
- 102000005962 receptors Human genes 0.000 claims description 31
- 108020003175 receptors Proteins 0.000 claims description 31
- 230000000694 effects Effects 0.000 claims description 25
- 101000584499 Homo sapiens Polycomb protein SUZ12 Proteins 0.000 claims description 18
- 102100030702 Polycomb protein SUZ12 Human genes 0.000 claims description 16
- 230000002401 inhibitory effect Effects 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 101150096980 LPA2 gene Proteins 0.000 claims description 10
- 102000004137 Lysophosphatidic Acid Receptors Human genes 0.000 claims description 9
- 108090000642 Lysophosphatidic Acid Receptors Proteins 0.000 claims description 9
- 101150090105 Ezh2 gene Proteins 0.000 claims description 8
- 230000036961 partial effect Effects 0.000 claims description 6
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 claims description 5
- 101000966782 Homo sapiens Lysophosphatidic acid receptor 1 Proteins 0.000 claims description 4
- 101001038006 Homo sapiens Lysophosphatidic acid receptor 3 Proteins 0.000 claims description 4
- 101001038043 Homo sapiens Lysophosphatidic acid receptor 4 Proteins 0.000 claims description 4
- 101001038037 Homo sapiens Lysophosphatidic acid receptor 5 Proteins 0.000 claims description 4
- 101100097224 Homo sapiens SUZ12 gene Proteins 0.000 claims description 4
- 102100040607 Lysophosphatidic acid receptor 1 Human genes 0.000 claims description 4
- 102100040388 Lysophosphatidic acid receptor 3 Human genes 0.000 claims description 4
- 102100040405 Lysophosphatidic acid receptor 4 Human genes 0.000 claims description 4
- 102100040404 Lysophosphatidic acid receptor 5 Human genes 0.000 claims description 4
- 101150078910 SUZ12 gene Proteins 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 229940125507 complex inhibitor Drugs 0.000 claims description 2
- 230000002708 enhancing effect Effects 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 230000000069 prophylactic effect Effects 0.000 claims description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 25
- 230000005764 inhibitory process Effects 0.000 abstract description 22
- 238000000338 in vitro Methods 0.000 abstract description 21
- 238000001727 in vivo Methods 0.000 abstract description 14
- 230000012010 growth Effects 0.000 abstract description 13
- 230000035755 proliferation Effects 0.000 abstract description 13
- 230000008685 targeting Effects 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000011269 treatment regimen Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 161
- 108050004000 Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 Proteins 0.000 description 114
- 102100021977 Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 Human genes 0.000 description 92
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 75
- 210000001519 tissue Anatomy 0.000 description 55
- 101150042537 dld1 gene Proteins 0.000 description 42
- 101000967087 Homo sapiens Metal-response element-binding transcription factor 2 Proteins 0.000 description 37
- 102100040632 Metal-response element-binding transcription factor 2 Human genes 0.000 description 37
- 239000003814 drug Substances 0.000 description 22
- 238000003197 gene knockdown Methods 0.000 description 22
- 241000699670 Mus sp. Species 0.000 description 21
- 229940079593 drug Drugs 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 18
- 108020004999 messenger RNA Proteins 0.000 description 18
- 102000007469 Actins Human genes 0.000 description 16
- 108010085238 Actins Proteins 0.000 description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 15
- 238000011529 RT qPCR Methods 0.000 description 15
- 230000002829 reductive effect Effects 0.000 description 15
- 238000001514 detection method Methods 0.000 description 14
- 238000003119 immunoblot Methods 0.000 description 14
- 239000002904 solvent Substances 0.000 description 13
- 108010077544 Chromatin Proteins 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 12
- 210000003483 chromatin Anatomy 0.000 description 12
- 239000002299 complementary DNA Substances 0.000 description 12
- WRGQSWVCFNIUNZ-GDCKJWNLSA-N 1-oleoyl-sn-glycerol 3-phosphate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)COP(O)(O)=O WRGQSWVCFNIUNZ-GDCKJWNLSA-N 0.000 description 11
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 11
- 239000012096 transfection reagent Substances 0.000 description 11
- 239000012098 Lipofectamine RNAiMAX Substances 0.000 description 10
- 238000002487 chromatin immunoprecipitation Methods 0.000 description 10
- 101150112014 Gapdh gene Proteins 0.000 description 8
- 230000004663 cell proliferation Effects 0.000 description 8
- 239000006166 lysate Substances 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 238000011278 co-treatment Methods 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000002195 synergetic effect Effects 0.000 description 7
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 6
- 229920002556 Polyethylene Glycol 300 Polymers 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 239000002504 physiological saline solution Substances 0.000 description 6
- 230000007115 recruitment Effects 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 241000699660 Mus musculus Species 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 239000002773 nucleotide Substances 0.000 description 5
- 125000003729 nucleotide group Chemical group 0.000 description 5
- 238000011580 nude mouse model Methods 0.000 description 5
- 238000007920 subcutaneous administration Methods 0.000 description 5
- 230000005757 colony formation Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 229960001570 ademetionine Drugs 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000002648 combination therapy Methods 0.000 description 3
- 238000011284 combination treatment Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000002596 correlated effect Effects 0.000 description 3
- 239000003684 drug solvent Substances 0.000 description 3
- 230000001973 epigenetic effect Effects 0.000 description 3
- 230000006718 epigenetic regulation Effects 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 238000011729 BALB/c nude mouse Methods 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 102100023584 Histone-binding protein RBBP7 Human genes 0.000 description 2
- 101000897035 Homo sapiens Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 Proteins 0.000 description 2
- 101000866766 Homo sapiens Polycomb protein EED Proteins 0.000 description 2
- 101150015362 LPAR2 gene Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102100031338 Polycomb protein EED Human genes 0.000 description 2
- 101150050921 RBBP7 gene Proteins 0.000 description 2
- ZJUKTBDSGOFHSH-WFMPWKQPSA-N S-Adenosylhomocysteine Chemical compound O[C@@H]1[C@H](O)[C@@H](CSCC[C@H](N)C(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZJUKTBDSGOFHSH-WFMPWKQPSA-N 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- 108010022198 alkylglycerophosphoethanolamine phosphodiesterase Proteins 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010219 correlation analysis Methods 0.000 description 2
- 230000000875 corresponding effect Effects 0.000 description 2
- 238000003235 crystal violet staining Methods 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000001394 metastastic effect Effects 0.000 description 2
- 206010061289 metastatic neoplasm Diseases 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000000474 nursing effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 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 2
- 239000000758 substrate Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 108091029523 CpG island Proteins 0.000 description 1
- 101150081946 ENPP2 gene Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108091006096 Gα12 Proteins 0.000 description 1
- 102100025190 Histone-binding protein RBBP4 Human genes 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 102000006947 Histones Human genes 0.000 description 1
- 101100075164 Homo sapiens LPAR4 gene Proteins 0.000 description 1
- 101001038034 Homo sapiens Lysophosphatidic acid receptor 6 Proteins 0.000 description 1
- 101150086964 LPAR1 gene Proteins 0.000 description 1
- 101150081221 LPAR3 gene Proteins 0.000 description 1
- 101150068138 LPAR5 gene Proteins 0.000 description 1
- 101150011404 LPAR6 gene Proteins 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102100040406 Lysophosphatidic acid receptor 6 Human genes 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 1
- 102000009609 Pyrophosphatases Human genes 0.000 description 1
- 108010009413 Pyrophosphatases Proteins 0.000 description 1
- 101150118761 RBBP4 gene Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000001035 methylating effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000030363 nerve development Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000037426 transcriptional repression Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
-
- 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
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses application of EZH2 and ATX-LPA-LPA2 shaft co-inhibition in colorectal cancer treatment. The invention discovers that: the combination of an ATX inhibitor and an EZH2 inhibitor can significantly inhibit the growth and proliferation of colorectal cancer tumor cells in vivo and in vitro; inhibition of LPA2 can enhance the sensitivity of in vitro or in vivo tumor cells to EZH2 inhibitors and enhance the tumor therapeutic effect of EZH2 inhibitors. The co-inhibition of the EZH2 and the ATX-LPA-LPA2 axis can further inhibit the growth and proliferation of colorectal cancer tumor cells, and lays a theoretical foundation for the combined use of the ATX inhibitor and the EZH2 inhibitor in colorectal cancer treatment and the combined treatment strategy of targeting the EZH2 and the ATX-LPA-LPA 2.
Description
Technical Field
The present invention relates to the use of co-inhibition of EZH2 with the ATX-LPA 2 axis in the biomedical field for the treatment of colorectal cancer.
Background
Colorectal cancer is one of the most common cancers in men and women, and the discovery of early lesions and effective therapeutic intervention in the late stages is a great challenge in current colorectal cancer treatments. Conventional enteroscopy and surgical excision are the first choice for treating primary colorectal cancer, and the treatment of chemotherapeutic drugs such as 5-FU and the like also has certain curative effects. However, colorectal cancer patients have a poor prognosis and a low five-year survival rate due to high metastatic, high recurrent and drug resistance. For metastatic patients, the survival rate is less than 20% in 5 years. Therefore, developing new colorectal cancer therapeutic targets and adopting multi-drug combined therapeutic strategies is a problem to be solved in colorectal cancer treatment.
The PRC2 complex consists of four core subunits, EZH2, SUZ12, EED, RBAP46/RBAP48, which can bind to genomic CpG islands and catalyze the trimethylation of lysine 27 (H3K 27me 3) of histone H3, thereby participating in the transcriptional inhibition of genes, embryo development and various cancer processes. EZH2 is a key subunit of PRC2 that plays a catalytic role, and is converted into S-adenosyl-homocysteine (SAH) by transferring a methyl group from S-adenosyl-methionine (SAM), thereby methylating H3 and inhibiting gene expression. Dysfunctions of PRC2 are associated with carcinogenesis, tumor immunity and drug resistance, and EZH2 has been reported to be highly expressed or mutated in a variety of tumors. A variety of EZH2 inhibitors have been used for clinical trials and exhibit potent antitumor effects, however, EZH2 inhibitors are mostly used for the treatment of lymphoma and hematological-related cancers, with limited inhibition of solid tumors.
GSK126 is an S-adenosyl-methionine competitive EZH2 inhibitor, and can inhibit both wild type and Y641 mutant EZH2, and can be used in cell experiments and animal in vivo experiments. The structure is shown as formula I.
Autotaxin (ATX) is a secreted glycoprotein whose coding gene is ENPP2, belonging to the family of exonucleotide pyrophosphatases/phosphodiesterases (ENPP). ATX has lysophospholipase D (lysoPLD) activity and can catalyze the production of lysophosphatidic acid (lysophosphatidic acid, LPA) using lysophosphatidic choline (LPC) as a substrate. LPA is a small lipid molecule that is found in almost all mammalian cells and is capable of activating downstream pathways and exerting important biological effects through the surface of the cell membrane by 6 LPA receptors (LPA 1-6, encoding genes LPA1-6, respectively). LPA2 is coupled to Gαi/0, Gαq/11 and Gα12/13 to activate downstream signaling pathways Ras, rac, P13K, MAPK, rho, etc., thereby promoting cell migration and survival.
Research in recent years shows that the ATX-LPA-LPA receptor signal shaft has wide biological functions, and not only participates in angiogenesis and nerve development, but also plays an important role in obesity-related diseases and immune regulation.
PF8380 is a potent ATX inhibitor that inhibits ATX enzyme activity by mimicking the occupation of the ATX active site by LPC substrates. In vitro enzyme experiments and human whole blood cell experiments, the IC50 is 2.8nM and 101nM respectively, and the kit can be used for cell experiments and animal in-vivo experiments, and has the structure shown in the formula II.
Disclosure of Invention
The technical problem to be solved by the invention is how to treat tumors, especially colorectal cancer.
In order to solve the technical problems, the invention firstly provides the application of the substance in preparing a product for treating or assisting in treating tumor;
the substance is a substance 1 and/or a substance 2, and the substance 1 is a substance which inhibits the activity of the PRC2 complex or the activity of all or part of the components thereof, reduces the activity of the PRC2 complex or the content of all or part of the components thereof, inhibits the expression of all or part of the genes of the PRC2 complex, or knocks out all or part of the genes of the PRC2 complex;
the substance 2 is a substance which blocks the ATX-LPA-LPA receptor axis, or inhibits the activity of all or part of the components of the ATX-LPA-LPA receptor axis, reduces the content of all or part of the components of the ATX-LPA-LPA receptor axis, inhibits the expression of all or part of the genes of the ATX-LPA-LPA receptor axis, or knocks out all or part of the genes of the ATX-LPA-LPA receptor axis.
Wherein, the ATX-LPA-LPA receptor axis (namely ATX-LPA-LPA receptor signal path) consists of ATX (Autotaxin gene name is ENPP 2), LPA (lysophosphatidic acid) and LPA receptor (LPA receptors 1-6, and the encoding genes are LPARs 1-6 respectively).
The PRC2 complex consists of four core subunits, EZH2, SUZ12, EED, RBAP46/RBAP 48.
In the above application, a part of the components of the PRC2 complex may be EZH2 and/or SUZ12, and a part of the genes of the PRC2 complex may be EZH2 genes and/or SUZ12 genes;
the LPA receptor can be LPA2, LPA1, LPA3, LPA4, LPA5, and/or LPA6.
In the above application, the substance that inhibits the activity of the PRC2 complex or the activity of all or part of the component thereof may be a PRC2 complex inhibitor or a component inhibitor thereof, and the substance that inhibits the expression of all or part of the gene of the PRC2 complex may be a specific siRNA of all or part of the gene of the PRC2 complex;
the substance inhibiting the activity of all or part of the components of the ATX-LPA-LPA receptor axis can be an inhibitor of all or part of the components of the ATX-LPA-LPA receptor axis, the substance inhibiting the expression of all or part of the genes of the ATX-LPA-LPA receptor axis can be a specific siRNA of all or part of the genes of the ATX-LPA-LPA receptor axis, and the substance knocking out all or part of the genes of the ATX-LPA-LPA receptor axis can be a substance knocking out all or part of the genes of the ATX-LPA-LPA receptor axis by using a CRISPR-Cas9 method.
In the above application, the PRC2 complex component inhibitor may be an EZH2 inhibitor;
the specific siRNA of the PRC2 complex part gene can be siRNA specifically recognizing the EZH2 gene and/or the SUZ12 gene;
the ATX-LPA receptor shaft moiety inhibitor can be an ATX inhibitor;
the specific siRNA of all or part of the ATX-LPA-LPA receptor axis genes can be siRNA specifically recognizing LPA 2;
the substance that knocks out all or part of the ATX-LPA-LPA receptor axis gene by using CRISPR-Cas9 method can be the substance that knocks out LPA2 gene.
In one embodiment of the invention, the partial sequence of the second exon of the LPA2 gene is compiled from 5'-ATGGTCATCATGGGCCAGTGCTACTACAACGAGACCATCGGCTTCTTCTATAACAA-3': 5'-ATGGTCATCATGGGCCAGTGCTACTACAACGAGACCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACCATCG GCTTCTTCTATAACAA-3', or, by
5'-GGGCTTGCTGGACACAAGCCTCACTGCGTCGGTGGCCACACTGCTGGCCATCGCCGTGGAGCGGCACCGCAGTGT GATGGCCGTGCAGCTGCACAGCCGC-3' is compiled as:
5’-GGGCTTGCTGGAGCGGCACCGCAGTGTGATGGCCGTGCAGCTGCACAGCCGC-3’。
in the above application, the EZH2 inhibitor may be GSK126;
the ATX inhibitor may be PF8380;
The siRNA specifically recognizing the EZH2 gene can be shown as SEQ ID No.1, and the siRNA specifically recognizing the SUZ12 gene can be shown as SEQ ID No. 2;
the siRNA specifically recognizing the LPA2 gene can be shown as SEQ ID No.5 or 6.
The use of said substance 1 and/or said substance 2 for the preparation of a product for the prevention or co-prevention of tumors also falls within the scope of the present invention.
The application of the substance 2 in preparing a product for enhancing the tumor treatment effect of the substance 1 also belongs to the protection scope of the invention.
The invention also provides a therapeutic or co-therapeutic tumour product or a prophylactic or co-prophylactic tumour product, said product being (or its active ingredient being) said substance 1 and/or said substance 2.
In the present invention, the tumor may be a solid tumor or a liquid tumor. The solid tumor may be colorectal cancer.
Wherein, EZH2: genBank:2146, update date: 2023, 5, 9; SUZ12: genBank:23512, update date: 2023, 4 months and 4 days; ENPP2: genBank:5168, update date: 2023, 5, 9; LPAR2: genBank:9170, update date: 2023, 4, 17; LPAR1: genBank:1902, update date: 2023, 3, 29; LPAR3: genBank:23566, update date: 2023, 3, 29; LPAR4: genBank:2846, update date: 2023, 3, 29; LPAR5: genBank:57121, update date: 2023, 5, 9; LPAR6: genBank:10161, update date: 2023 month 29.
The invention discovers that: (1) the expression of EZH2 in colorectal cancer tissue and colorectal cancer cells is inversely related to ATX; (2) the EZH2/PRC2 complex inhibits expression of the ATX gene in colorectal cancer cells at the epigenetic level by adding an H3K27me3 modification to the ATX promoter; (3) MTF2 helps PRC2 recruit to the promoter region of ATX gene to exert transcription inhibition by forming complex with EZH 2; (4) the use of the ATX inhibitor PF8380 in combination with the EZH2 inhibitor GSK126 significantly inhibited the growth and proliferation of colorectal cancer tumor cells in vivo and in vitro; (5) LPA2 is highly expressed in colorectal cancer tumor tissues, and inhibition of LPA2 can enhance the sensitivity of in vitro or in vivo tumor cells to EZH2 inhibitor GSK126 and enhance the tumor treatment effect of EZH2 inhibitor GSK 126.
The invention not only explains the molecular mechanism of epigenetic regulation of ATX gene in colorectal cancer tumor cells for the first time, but also discovers a novel strategy aiming at colorectal cancer tumor intervention and combined therapy. EZH2 inhibitors have been used in clinical trials to treat a variety of lymphoid-related or advanced cancers, but have limited therapeutic effects in colorectal cancer, the invention reveals the role of EZH2 in ATX epigenetic regulation, and the co-inhibition of EZH2 with the ATX-LPA-LPA2 axis further inhibits the growth and proliferation of colorectal cancer tumor cells, laying a theoretical foundation for the combined use of ATX inhibitors and EZH2 inhibitors in colorectal cancer treatment and combined therapeutic strategies targeting EZH2 and ATX-LPA-LPA 2.
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
Drawings
FIG. 1 ATX and EZH2 expression in colorectal cancer tumor tissue appear to be inversely correlated.
Figure 2 prc2 complex inhibited expression of ATX in colorectal cancer tumor cells.
FIG. 3 EZH2/PRC2 mediated inhibition of ATX expression in colorectal cancer tumor cells by H3K27me 3.
FIG. 4 MTF2 recruits PRC2 to the ATX gene promoter in colorectal cancer tumor cells.
Fig. 5. Co-inhibition of ezh2 and ATX significantly inhibited growth and proliferation of colorectal cancer tumor cells in vitro. G126 represents GSK126, P8 represents PF8380.
Fig. 6. Co-inhibition of ezh2 and ATX has synergistic anti-tumor effects in vivo. T1-T6 represents 1-6 of six tumor tissues per group.
Fig. 7. Knockdown of LPA2 enhances the sensitivity of colorectal cancer tumor cells to EZH2 inhibitors in vitro.
Fig. 8.knockout of lpa2 enhances the tumor therapeutic effect of the EZH2 inhibitor GSK 126.
FIG. 9 is a schematic diagram of the modulation of ATX expression by PRC2 at the epigenetic level and a novel colorectal cancer treatment strategy.
In the figure, p < 0.05, p < 0.01, p < 0.001, p < 0.0001, ns, no significant difference.
Detailed Description
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were repeated three times and more, and the results were averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
TABLE 1 reagents and antibodies
TABLE 2 primer sequences
TABLE 3 siRNA sequences (synthesized by Suzhou Ji Ma Co., ltd., all purified by HPLC)
| Name of the name | Sequence (5 '-3') |
| EZH2 siRNA | GCUCUAGACAACAAACCUUTT(SEQ ID No.1) |
| SUZ12 siRNA | GAACAGCAAAGAACAUAUATT(SEQ ID No.2) |
| MTF2#1 siRNA | GCAGUUCUGGACCAGAAUATT(SEQ ID No.3) |
| MTF2#2 siRNA | GCAUGUUCUGGAGGCAUUATT(SEQ ID No.4) |
| LPA2#1siRNA | GGUCAAUGCUGCUGUGUACTT(SEQ ID No.5) |
| LPA2#2siRNA | GCCUGGUCAAGACUGUUGUTT(SEQ ID No.6) |
| NC siRNA | UUCUCCGAACGUGUCACGUTT |
TABLE 4 CHIP-qPCR primer sequences
The HT29-LPA2 knockout cell lines HT29-LPA2 KO#1, HT29-LPA2 KO#2 were constructed as follows:
1. CRISPR-Cas9 plasmid construction
1) Two sgRNA sequences targeting the second exon of the LPA2 gene were designed using CRISPOR (http:// crispor.tefor.net/crispor.py) and the following sgRNA sequences were synthesized. Wherein the method comprises the steps of
LPA2-sg#1：5’-TGCTACTACAACGAGACCAT-3’；
LPA2-sg#2：5’-CACAAGCCTCACTGCGTCGG-3’。
2) The annealed sgrnas were cloned into vectors using PX458 (adedge, plasmid # 48138) Plasmid as vector, constituting PX458-LPA2-sgRNA Plasmid.
2. Construction of HT29-LPA2 knockout cell line
1) PX458-LPA2-sgRNA was transfected into HT29 cells using Lipo3000 transfection reagent (Thermo Fisher Scientific).
2) After 48-72 h of transfection, the transfection efficiency was observed with a fluorescence microscope, GFP positive cells were sorted into 96-well plates by flow cytometry (BD Biosciences) to ensure the monoclonality of the cells, and the cells were continued to be cultured until cell line construction was completed.
3) All knockdown cell lines were identified by genomic PCR and sequencing and verified at the protein level by Western blot.
Knockout cell line HT29-LPA2 KO#1: the LPA2 gene in this cell line is inserted 113 nucleotides in the second exon relative to the wild type. The sequence of the second exon part before LPA2 gene knockout is as follows: 5'-ATGGTCATCATGGGCCAGTGCTACTACAACGAGACCATCGGCTTCTTCTATAACAA-3', the partial sequence is gene-edited after LPA2 knockout: 5'-ATGGTCATCATGGGCCAGTGCTACTACAACGAGACCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACCATCG GCTTCTTCTATAACAA-3'.
Knockout cell line HT29-LPA2 KO#2: the LPA2 gene in this cell line was deleted for 48 nucleotides in the second exon relative to the wild type. The sequence of the second exon part before LPA2 gene knockout is as follows: 5'-GGGCTTGCTGGACACAAGCCTCACTGCGTCGGTGGCCACACTGCTGGCCATCGCCGTGGAGCGGCACCGCAGTGT GATGGCCGTGCAGCTGCACAGCCGC-3', the partial sequence is gene-edited after LPA2 knockout: 5'-GGGCTTGCTGGAGCGGCACCGCAGTGTGATGGCCGTGCAGCTGCACAGCCGC-3'.
Example 1 ATX and EZH2 expression in colorectal cancer clinical tissue samples were negatively correlated
1. Analysis of ATX and EZH2 expression in colorectal cancer tumor tissue clinical samples.
1. Analysis of ATX expression in colorectal cancer tumor tissue samples
To investigate the ATX expression level in colorectal cancer tumor tissues, ENPP2 gene (encoding ATX protein) (GenBank: 5168, update date: 2023, day 5, month 9) expression profile (TCGA) in colorectal cancer tumor tissues and normal tissues was analyzed using the GEPIA database (http:// GEPIA. Cancer-pku. Cn). It was found that ATX expression was significantly reduced in colorectal cancer tumor tissue (n=275) compared to adjacent normal tissue (n=349) (FIG. 1A).
2. EZH2 expression analysis in colorectal cancer tumor tissue samples
To investigate the EZH2 expression levels in colorectal cancer tumor tissues, the expression profile (TCGA) of the EZH2 gene (GenBank: 2146, update date: 2023, 5, 9) in colorectal cancer tumor tissues and normal tissues was analyzed using the GEPIA database (http:// GEPIA. Cancer-pku. Cn). It was found that the expression of EZH2 was significantly increased in colorectal cancer tumor tissue (n=275) compared to adjacent normal tissue (n=349) (B in fig. 1).
2. Correlation analysis of ATX and EZH2 expression in colorectal cancer tumor tissue clinical samples.
Further correlation analysis of ATX and EZH2 expression in colorectal cancer showed that ATX and EZH2 expression in colorectal cancer tissues were inversely correlated (C in fig. 1). These results indicate that low expression of ATX in colorectal cancer tumor tissue may be associated with EZH2 and PRC2 mediated epigenetic regulation.
Example 2 EZH2/PRC2 is involved in the inhibition of expression of ATX in colorectal cancer tumor cells
1. ATX and EZH2 mRNA levels were measured in various colorectal cancer tumor cells.
1. Detection of ATX Gene expression in different colorectal cancer cell lines
Total RNAs of human colorectal cancer tumor cell lines HCT116, HT29, SW480 and DLD1 were extracted respectively, and RT-qPCR detection was performed using the total RNAs as templates with ATX primers (Table 2), and the expression levels of ATX mRNA in different cells were detected using GAPDH gene as an internal reference.
2. Detection of EZH2 Gene expression in different colorectal cancer cell lines
Total RNAs of human colorectal cancer tumor cell lines HCT116, HT29, SW480, DLD1 were extracted, respectively, and RT-qPCR was performed using the total RNAs as templates with EZH2 primers (table 2), and the expression levels of EZH2 mRNA in different cells were detected using GAPDH gene as an internal reference.
The results are shown in figure 2 a, where mRNA expression of ATX was at lower levels in all four different colorectal cancer tumor cells, while mRNA expression of EZH2 was higher, consistent with colorectal cancer tumor tissue samples (figure 1).
2. EZH2 inhibitor treatment and use of specific siRNA to knock down PRC2 key subunit EZH2 or SUZ12 to promote ATX expression in colorectal cancer cells
1. EZH2 inhibitor treatment
HCT116, HT29, SW480, DLD1 cells were treated with EZH2 inhibitor GSK126 (final concentration 5 μm) or an equal volume of DMSO, respectively, dissolved in DMSO for 48 hours; total RNAs of cells of the GSK126 treated group and DMSO control group were extracted, respectively, qPCR analysis was performed using the inverted cDNA as a template and ATX primers (table 2), and expression levels of ATX mRNA in the cells were detected using GAPDH gene as an internal reference.
Lysates of cells of an EZH2 inhibitor GSK126 treated group and a DMSO control group in HCT116, HT29, SW480, DLD1 cells were prepared, respectively; the levels of total H3K27me3 and beta-actin in the cells were detected by immunoblotting (western blot), respectively. The immunoblotting detection primary antibody is an anti-H3K 27me3 or beta-actin antibody respectively.
The results are shown in fig. 2B, where the expression level of ATX mRNA in the EZH2 inhibitor GSK 126-treated cells was significantly increased and the level of H3K27me3 was significantly decreased compared to the DMSO control group. I.e. EZH2 inhibitors promote expression of the ATX gene in different colorectal cancer cells.
2. EZH2 siRNA treatment
And (3) respectively transfecting NC siRNA or EZH2 siRNA into HCT116, HT29, SW480 and DLD1 cells by using a Lipofectamine RNAiMAX transfection reagent, respectively extracting total RNA of the EZH2 siRNA treatment group cells and NC siRNA control group cells after 48 hours, reversing the total RNA into cDNA, carrying out qPCR amplification by taking the cDNA as a template and ATX or EZH2 primers, taking GAPDH genes as an internal reference, and detecting the expression level of ATX and EZH2 mRNA in the cells.
Preparing lysate of HCT116, HT29, SW480, DLD1 cell EZH2 siRNA treatment group and NC siRNA control group respectively; the levels of EZH2, H3K27me3 and β -actin in the cells were detected separately by immunoblotting. The immunoblotting detection primary antibodies are anti-EZH 2, H3K27me3 or beta-actin antibodies respectively.
As shown in fig. 2C, the expression level of ATX mRNA was significantly increased in the cells of the EZH2 siRNA treated group (siEZH 2) and the levels of EZH2 and H3K27me3 were significantly decreased compared to the NC siRNA control group (siNC). That is, knockdown of EZH2 in different colorectal cancer cells significantly promoted expression of ATX gene.
3. SUZ12 siRNA treatment
NC siRNA or SUZ12 siRNA was transfected into HCT116, HT29, SW480, DLD1 cells using Lipofectamine RNAiMAX transfection reagent, total RNA of the above SUZ12 siRNA treated cells and NC siRNA control cells was extracted respectively after 48 hours, inverted into cDNA, qPCR amplified using cDNA as a template and ATX or SUZ12 primers (table 2) and GAPDH gene as an internal control, and expression levels of ATX and SUZ12mRNA in the cells were detected.
Preparing lysate of HCT116, HT29, SW480, DLD1 cell SUZ12 siRNA treatment group and NC siRNA control group respectively; SUZ12 (GenBank: 23512, date of renewal: 2023, 4 th month) H3K27me3 and β -actin levels were measured separately in cells by immunoblotting. The immunoblotting detection primary antibodies are anti-SUZ 12, H3K27me3 or beta-actin antibodies respectively.
Results as shown in fig. 2D, the expression level of ATX mRNA was significantly increased in cells of the SUZ12 siRNA treated group (siSUZ 12) and the levels of SUZ12 and H3K27me3 were significantly decreased compared to NC siRNA control group (siNC). That is, knocking down SUZ12 in different colorectal cancer cells significantly promoted ATX gene expression.
The above results indicate that the PRC2 complex inhibits expression of the ATX gene in colorectal cancer cells.
3. EZH2 inhibitor treatment and use of specific siRNA to knock down EZH2 to reduce H3K27me3 levels on ATX gene promoters in colorectal cancer tumor cells
1. EZH2 inhibitor treatment
To investigate the effect of EZH2 inhibition on the extent of enrichment of H3K27me3 on the ATX gene promoter, HT29, DLD1 cells were treated with the EZH2 inhibitor GSK126 (final concentration 5 μm) dissolved in DMSO or an equal volume of DMSO, respectively, for 48 hours; collecting cell precipitates of the GSK126 treatment group and the DMSO control group respectively, and enriching chromatin DNA interacted with H3K27me3 through chromatin immunoprecipitation; using the obtained chromatin DNA as a template, respectively using 4 pairs of primers (A in FIG. 3 and Table 4) on the ATX promoter as detection primers, and analyzing the enrichment degree of H3K27me3 on the ATX promoter by qPCR; the antibody used for chromatin immunoprecipitation was an anti-H3K 27me3 antibody, and an IgG antibody was used as a negative control.
As shown in fig. 3B, the enrichment of H3K27me3 on the ATX promoter was significantly reduced in the EZH2 inhibitor GSK126 treated cells compared to the DMSO control, i.e., the EZH2 inhibitor treatment reduced the level of H3K27me3 on the ATX gene promoter in HT29 and DLD1 cells.
2. EZH2 siRNA treatment
To explore the effect of EZH2 knockdown on the degree of enrichment of H3K27me3 on the ATX gene promoter, NC siRNA or EZH2 siRNA was transfected into HT29, DLD1 cells, respectively, using Lipofectamine RNAiMAX transfection reagents; after 48 hours, respectively collecting the sediment of the EZH2 siRNA treatment group and NC siRNA control group cells, and enriching the chromatin DNA interacted with H3K27me3 through chromatin immunoprecipitation; using the obtained chromatin DNA as a template, respectively using 4 pairs of primers (A in FIG. 3 and Table 4) on the ATX promoter as detection primers, and analyzing the enrichment degree of H3K27me3 on the ATX promoter by qPCR; the antibody used for chromatin immunoprecipitation was an anti-H3K 27me3 antibody, and an IgG antibody was used as a negative control.
The results are shown in figure 3C, with a significant decrease in the enrichment of H3K27me3 on the ATX promoter in EZH2 siRNA treated (siEZH 2) cells compared to NC siRNA treated control (siNC), i.e. EZH2 knockdown reduced the level of H3K27me3 on the ATX gene promoter in HT29 and DLD1 cells.
The above results indicate that PRC2 complex inhibits expression of ATX gene in colorectal cancer cells at epigenetic level by H3K27me 3.
Example 3 MTF2 is involved in the recruitment of PRC2 to the ATX promoter in colorectal cancer tumor cells
1. Knock-down of MTF2 using specific siRNA to promote ATX expression in colorectal cancer cells
1. siRNA knockdown of MTF2 promotes ATX expression in HT29 cells
NC siRNA or MTF 2-targeted siRNA (simtf2#1 or simtf2#2) was transfected into HT29 cells using Lipofectamine RNAiMAX transfection reagent, total RNA of the MTF2 siRNA treated cells and NC siRNA control cells described above was extracted after 48 hours, inverted into cDNA, qPCR amplified with ATX or MTF2 primer (table 2) using cDNA as a template, and expression levels of ATX and MTF2 mRNA in the cells were detected using GAPDH gene as an internal reference.
Preparing lysate of MTF2 siRNA treatment group and NC siRNA control group cell respectively; MTF2 (GenBank: 22823, date of renewal: 2023, month 3, 29) and beta-actin levels in the cells were separately examined by immunoblotting. The immunoblotting detection primary antibodies are anti-MTF 2 or beta-actin antibodies respectively.
As a result, as shown in fig. 4 a and B, the expression level of ATX mRNA in HT29 cells of mtf2#1siRNA treated group (simtf2#1) or mtf2#2siRNA treated group (simtf2#2) was significantly increased and the level of MTF2 protein was significantly decreased compared to NC siRNA control group (siNC). That is, knocking down MTF2 in HT29 cells significantly promoted ATX gene expression.
2. siRNA knockdown of MTF2 promotes ATX expression in DLD1 cells
NC siRNA or MTF 2-targeted siRNA (simtf2#1 or simtf2#2) was transfected into DLD1 cells using Lipofectamine RNAiMAX transfection reagent, total RNA of the MTF 2siRNA treated cells and NC siRNA control cells was extracted after 48 hours, respectively, inverted into cDNA, qPCR amplified with ATX or MTF2 primer (table 2) using cDNA as a template, and expression levels of ATX and MTF2 mRNA in the cells were detected using GAPDH gene as an internal reference.
Preparing lysate of MTF 2siRNA treatment group and NC siRNA control group cell respectively; the levels of MTF2 and beta-actin in the cells were separately detected by immunoblotting. The immunoblotting detection primary antibodies are anti-MTF 2 or beta-actin antibodies respectively.
As shown in fig. 4E and F, the expression level of ATX mRNA in DLD1 cells of mtf2#1siRNA treated group (simtf2#1) or mtf2#2siRNA treated group (simtf2#2) was significantly increased and the level of MTF2 protein was significantly decreased compared to NC siRNA control group (siNC). That is, knocking down MTF2 in DLD1 cells significantly promoted ATX gene expression.
2. Inhibiting enrichment of H3K27me3 on ATX gene promoter and occupancy of EZH2 in colorectal cancer cells using specific siRNA knockdown MTF2
1. siRNA knockdown of MTF2 inhibits enrichment of H3K27me3 on ATX gene promoter and occupancy of EZH2 in HT29 cells
Transfecting NC siRNA or MTF2#2siRNA into HT29 cells by using Lipofectamine RNAiMAX transfection reagent, collecting the precipitate of the MTF2#2siRNA treatment group and NC siRNA control group cells after 48 hours, and enriching the chromatin DNA interacted with H3K27me3 or EZH2 by chromatin immunoprecipitation; using the obtained chromatin DNA as a template, respectively using 4 pairs of primers (A in FIG. 3 and Table 4) on the ATX promoter as detection primers, and analyzing the enrichment degree of H3K27me3 and the occupation condition of EZH2 on the ATX promoter by qPCR; the antibody used for chromatin immunoprecipitation is an anti-H3K 27me3 antibody or an EZH2 antibody, and an IgG antibody is used as a negative control.
As shown in fig. 4C and D, the enrichment of H3K27me3 on the ATX promoter in HT29 cells was significantly reduced and the occupancy of EZH2 was significantly reduced in mtf2#2siRNA treated group compared to NC siRNA treated control group, i.e. MTF2 knockdown inhibited H3K27me3 levels and occupancy of EZH2 on the ATX gene promoter in HT29 cells; MTF2 is involved in the process of recruitment of PRC2 to the ATX gene promoter in HT29 cells.
2. siRNA knockdown MTF2 inhibits enrichment of H3K27me3 on ATX gene promoter and occupation of EZH2 in DLD1 cells
Transfecting NC siRNA or MTF2#2siRNA into DLD1 cells by using a Lipofectamine RNAiMAX transfection reagent, collecting precipitates of the MTF2#2siRNA treatment group and NC siRNA control group cells respectively after 48 hours, and enriching chromatin DNA interacted with H3K27me3 or EZH2 by chromatin immunoprecipitation; using the obtained chromatin DNA as a template, respectively using 4 pairs of primers (A in FIG. 3 and Table 4) on the ATX promoter as detection primers, and analyzing the enrichment degree of H3K27me3 and the occupation condition of EZH2 on the ATX promoter by qPCR; the antibody used for chromatin immunoprecipitation is an anti-H3K 27me3 antibody or an EZH2 antibody, and an IgG antibody is used as a negative control.
As shown in G and H in fig. 4, the enrichment of H3K27me3 on the ATX promoter in DLD1 cells was significantly reduced and the occupancy of EZH2 was reduced compared to NC siRNA treated control group, i.e. MTF2 knockdown inhibited the level of H3K27me3 and occupancy of EZH2 on the ATX gene promoter in DLD1 cells; MTF2 is involved in the process of PRC2 recruitment to the ATX gene promoter in DLD1 cells.
3. MTF2 and EZH2 exist on ATX gene promoter in the form of complex
As shown on the left in fig. 4I, HT29 cell pellets were collected, and chromatin fragments that interacted with EZH2 were enriched by one-time chromatin immunoprecipitation, using anti-EZH 2 antibodies; the eluted chromatin fragments were subjected to a second chromatin immunoprecipitation to enrich for chromatin DNA interacting with MTF2, and the antibody used was an anti-MTF 2 antibody. The occupancy of EZH2-MTF2 on the ATX promoter was analyzed by qPCR using the obtained chromatin DNA as a template and the ATX promoter site primer 2 and primer 3 (A in FIG. 3, table 4) as detection primers, respectively.
The results are shown on right in fig. 4, where the occupancy level of EZH2-MTF2 on the ATX promoter is significantly increased compared to the negative control IgG, indicating that MTF2 is able to form a complex with EZH2 in HT29 cells, co-existing on the ATX promoter. MTF2 was further demonstrated to be involved in the recruitment of PRC2 to the ATX promoter in colorectal cancer tumor cells.
The above results indicate that MTF2 recruits PRC2 to the ATX promoter to exert transcriptional repression in colorectal cancer tumor cells by forming complexes with EZH 2.
Example 4 Co-inhibition of EZH2 and ATX has synergistic anti-tumor effects
1. Co-inhibition of EZH2 and ATX has synergistic inhibitory effect on colorectal cancer tumor cells in vitro
1. Co-treatment with EZH2 inhibitors and ATX inhibitors inhibits colorectal cancer cell viability
HCT116, HT29, SW480, DLD1 cells were seeded in 96-well plates at a density of 3000 cells per 100 μl/well, and treated with equal volumes of DMSO and EZH2 inhibitor GSK126 (final concentration 5 μΜ) and ATX inhibitor PF8380 (final concentration 5 μΜ) dissolved in DMSO, respectively, individually or together; after 72 hours, 10. Mu.L of CCK-8 solution was added to each well and incubated in a cell incubator at 37℃for 1-4 hours; detecting the absorbance at 450nm by using an enzyme-labeled instrument; and cell viability was calculated.
Results as shown in fig. 5 a, the co-treatment of EZH2 inhibitor GSK126 and ATX inhibitor PF8380 significantly inhibited the viability of HCT116, HT29, SW480, DLD1 cells compared to the EZH2 inhibitor GSK126 or ATX inhibitor PF8380 alone treatment group.
2. Co-treatment with EZH2 inhibitors and ATX inhibitors inhibits colorectal cancer cell colony formation
HT29 or DLD1 cells were seeded in 6 well plates at 1000 cells/well, and treated with equal volumes of DMSO and either individually or together with the EZH2 inhibitor GSK126 (final concentration 2.5. Mu.M) and the ATX inhibitor PF8380 (final concentration 2.5. Mu.M or 5. Mu.M) dissolved in DMSO, respectively; after 7 days, cell colony counts were performed by crystal violet staining.
As shown in fig. 5B and D, the co-treatment of EZH2 inhibitor GSK126 and ATX inhibitor PF8380 significantly inhibited the size and number of colony formation of HT29 and DLD1 cells compared to the EZH2 inhibitor GSK126 or ATX inhibitor PF8380 alone treated group.
3. Co-treatment of EZH2 inhibitors and ATX inhibitors inhibits proliferation of colorectal cancer cells
HT29 or DLD1 cells were seeded in 96-well plates at 3000 cells/well, treated with equal volumes of DMSO and EZH2 inhibitor GSK126 (final concentration 5. Mu.M) and ATX inhibitor PF8380 (final concentration 5. Mu.M) dissolved in DMSO, individually or together, and transferred to an incubator of the IncuCyte S3 platform for culture and imaging. The different areas of each well were photographed at 2h intervals using a 10X objective and analyzed for cell proliferation using an IncuCyte S3 image after 72 h.
As shown in fig. 5C and E, the co-treatment of EZH2 inhibitor GSK126 and ATX inhibitor PF8380 significantly inhibited proliferation of HT29 and DLD1 cells compared to the EZH2 inhibitor GSK126 or ATX inhibitor PF8380 alone treatment groups.
The above results indicate that in vitro colorectal cancer tumor cell models, the combination of an EZH2 inhibitor and an ATX inhibitor can further inhibit cell growth and proliferation, i.e., co-inhibition of EZH2 and ATX has a synergistic inhibitory effect on colorectal cancer cells in vivo.
2. Co-inhibition of EZH2 and ATX has synergistic inhibitory effect on colorectal carcinoma tumors in vivo
1. Combination treatment of EZH2 inhibitors and ATX inhibitors significantly inhibited tumor volume, growth rate and weight
The BALB/c male nude mice with the age of 6-8 weeks are selected as experimental mice, the feeding period of the mice is followed by 12 hours of day and night feeding period, the daily nursing of the mice is carried out according to relevant criteria, and the experimental mice are subjected to experiments to obtain the permissions of the experimental animal ethical examination meeting of Beijing university.
Experimental procedure As shown in FIG. 6A, HT29 cells were cultured to appropriate density, cells were digested and resuspended to a density of 5X10 using serum free medium 6 And 200. Mu.L. 200 mu L of a total of 5X10 was inoculated in the armpit of BALB/c nude mice 6 HT29 of individualsAnd (3) cells. Four days after inoculation, until the underarm tumor can be observed visually, measurement of tumor length and width using a ruler was started, measurement was performed every two days and tumor volume was calculated from length×width×width/2. The average volume of the tumor is 50mm 3 Mice were randomized into the following four groups and treated with drug injections, six in each group:
control group (ctrl.): the solvent control (prepared from 50% PEG300+50% physiological saline) was injected once daily for 8 days in the same volume as the drug injected.
PF8380 treatment group (PF 8380): calculating the injection quantity of the medicine according to the weight of the mice; PF8380 drug solvent (prepared from 50% PEG300+50% physiological saline) was injected at 10mg/kg, once daily for 8 days.
GSK126 treatment group (GSK 126): calculating the injection quantity of the medicine according to the weight of the mice; GSK126 drug solvent (prepared from 50% PEG300+50% physiological saline) was injected at 50mg/kg, once daily for 8 days.
GSK126 and PF8380 combination treatment group (gsk126+pf 8380): calculating the injection quantity of the medicine according to the weight of the mice; according to GSK12650 mg/kg, PF838010mg/kg was injected with the drug solvent (the drug was prepared from 50% PEG300+50% physiological saline) once daily for 8 days.
The length and width of the tumor were measured every two days simultaneously, and the tumor volume was calculated from length×width×width/2. After 8 days of drug treatment, mice were euthanized, the subcutaneous tumor tissue was dissected and weighed and photographed.
As shown in figure 6, B, C, D, PF8380 and GSK126 alone drug treated groups partially inhibited tumor size, tumor growth rate and tumor weight compared to the solvent treated group; in contrast, the combination treatment of GSK126 and PF8380 further inhibited tumor size, growth rate, and tumor weight compared to the drug alone treatment. The research results show that EZH2 and ATX inhibit the colorectal cancer tumor in vivo together and have synergistic anti-tumor effect.
2. Treatment with EZH2 inhibitors inhibits H3K27me3 levels and promotes ATX expression in tumor tissue
The allogenic tumor transplantation experiment and the drug treatment experiment of the nude mice were performed according to step 1 (experimental flow chart 6A), and after 8 days of drug treatment, the mice were euthanized and the subcutaneous tumor tissues were peeled off. Lysates of tumor tissues of different drug treatment groups are prepared respectively, protein levels of H3K27me3 and beta-actin in the tumor tissues of each group of mice are detected by an immunoblotting method, and antibodies against the H3K27me3 and the beta-actin are used as primary antibodies.
As shown in fig. 6E, protein levels of H3K27me3 were significantly reduced in tumor tissues of GSK126 treated group (GSK 126) and GSK126 and PF8380 combined treated group (GSK 126+pf 8380) compared to control group (ctrl.); whereas there was no significant change in protein levels of H3K27me3 in tumor tissue of the PF8380 treatment group (PF 8380). This suggests that treatment with GSK126 may significantly down-regulate the level of H3K27me3 in tumor tissue.
Extracting total RNA of each group of tumor tissues respectively, using cDNA as a template and ATX primers for amplification by using an RT-qPCR method, and detecting the expression level of ATX mRNA in different tumor tissues. The GAPDH gene is used as an internal reference.
Results as shown in fig. 6F, mRNA levels of ATX were significantly up-regulated in tumor tissues of GSK 126-treated group (GSK 126) compared to control group (ctrl). This suggests that treatment of GSK126 promotes expression of ATX in tumor tissue.
3. Combination therapy of EZH2 inhibitor and ATX inhibitor significantly inhibits the expression of tumor proliferation marker Ki67
The allogenic tumor transplantation experiment and the drug treatment experiment of the nude mice were performed according to step 1 (experimental flow chart 6A), and after 8 days of drug treatment, the mice were euthanized and the subcutaneous tumor tissues were peeled off. And respectively taking the four groups of mouse tumor tissues, and carrying out tissue embedding, slicing and immunohistochemical analysis on the expression level of a proliferation related marker Ki67 in the tumor tissues after formaldehyde fixation, wherein the antibody is a Ki67 antibody.
As shown in fig. 6G, the protein expression level of Ki67 was partially down-regulated in tumor tissues of the GSK 126-injected treatment group (GSK 126) and the PF 8380-injected treatment group (PF 8380) compared to the control group (ctrl.) tumor tissues; whereas the expression level of Ki67 in tumor tissue of the GSK126 and PF8380 combination therapy group (gsk126+pf 8380) was further down-regulated. The results show that the co-treatment with the two inhibitors can significantly inhibit the growth and proliferation of colorectal cancer tumors, namely the co-inhibition of EZH2 and ATX has a synergistic inhibition effect on colorectal cancer tumors in vivo treatment.
Example 5 inhibition of LPA2 enhances the tumor therapeutic Effect of EZH2 inhibitors
1. Knock-down of LPA2 enhances the inhibitory effect of in vitro EZH2 inhibitors on colorectal cancer cells
1. Analysis of LPA2 expression in colorectal cancer tumor tissue samples
To investigate the expression level of LPAR2 gene (encoding LPA2 protein) in colorectal cancer tumor tissue (GenBank: 9170, update date: 2023, 4 months, 17 days), gene expression profile (TCGA) in colorectal cancer tumor tissue and normal tissue was analyzed using the GEPIA database (http:// GEPIA. Cancer-pku. Cn). LPA2 expression was found to be significantly elevated in colorectal cancer tumor tissue (n=275) compared to adjacent normal tissue (n=349) (a in fig. 7).
2. Analysis of LPA receptor expression in colorectal cancer cells HT29 and DLD1
Total RNA of HT29 and DLD1 cells was extracted, inverted into cDNA, and PCR amplification was performed using cDNA as a template, LPAR1 gene (encoding LPA1 protein) (GenBank: 1902, date of renewal: 2023, 3 months, 29), LPAR2 gene (encoding LPA2 protein) (GenBank: 9170, date of renewal: 2023, 4, 17 days), LPAR3 gene (encoding LPA3 protein) (GenBank: 23566, date of renewal: 2023, 3 months, 29), LPAR4 gene (encoding LPA4 protein) (GenBank: 2846, date of renewal: 2023, 3 months, 29), LPAR5 gene (encoding LPA5 protein) (GenBank: 57121, date of renewal: 2023, 5 months, 9), LPAR6 gene (encoding LPA6 protein) (GenBank: 10161, date of renewal: 2023, 3, month, 29), and GAPDH as primers (Table 2), to detect the expression levels of various LPA receptors in the cells.
As shown in FIGS. 7B and C, the expression level of LPA2 receptor in HT29 and DLD1 cells was high, which is consistent with the high expression level of LPA2 in colorectal cancer tissues.
3. Knock-down LPA2 enhances the inhibition of HT29 and DLD1 cell survival by in vitro EZH2 inhibitors
NC, lpa2#1, lpa2#2siRNA were transfected into HT29 or DLD1 cells using Lipofectamine RNAiMAX transfection reagent; HT29 or DLD1 cells transfected with NC, LPA2#1, LPA2#2siRNA as described above were seeded in 96-well plates at a density of 3000 cells/100. Mu.L/well, respectively; cells were treated with the EZH2 inhibitor GSK126 (final concentration 5 μm) or an equal volume of DMSO, respectively, dissolved in DMSO; after 72 hours, 10. Mu. LCCK-8 solution was added to each well and incubated in a cell incubator at 37℃for 1-4 hours; detecting the absorbance at 450nm by using an enzyme-labeled instrument; and cell viability was calculated.
As shown in fig. 7D and G, the viability of HT29 cells (D in fig. 7) or DLD1 cells (G in fig. 7) was significantly inhibited by siRNA knockdown LPA2 under GSK 126-treated conditions compared to NC control. I.e., knockdown of LPA2 enhances the inhibition of HT29 and DLD1 cell survival by in vitro EZH2 inhibitors.
4. Knock-down LPA2 enhances the inhibition of HT29 and DLD1 cell proliferation by in vitro EZH2 inhibitors
NC, lpa2#1, lpa2#2siRNA were transfected into HT29 or DLD1 cells using Lipofectamine RNAiMAX transfection reagent; HT29 and DLD1 cells transfected with NC, LPA2#1 and LPA2#2siRNA as described above were seeded in 96-well plates at a density of 3000 cells/100. Mu.L/well, respectively; cells were treated with the EZH2 inhibitor GSK126 (final concentration 5 μm) dissolved in DMSO or an equal volume of DMSO, respectively, and transferred to an incubator of the IncuCyte S3 platform for culture and imaging; different areas of each well were photographed at 2h intervals using a 10X objective and analyzed for cell proliferation using an IncuCyte S3 image.
As shown in fig. 7E and H, the proliferation of HT29 cells (E in fig. 7) or DLD1 cells (H in fig. 7) was further inhibited by siRNA knockdown LPA2 under GSK 126-treated conditions compared to NC control. I.e., knockdown of LPA2 enhances the inhibition of HT29 and DLD1 cell proliferation by in vitro EZH2 inhibitors.
5. LPA 2siRNA treatment and LPA2 mRNA expression analysis
NC, lpa2#1, lpa2#2siRNA were transfected into HT29 or DLD1 cells using Lipofectamine RNAiMAX transfection reagent; after 48 hours, total RNAs of LPA2#1 and LPA2#2siRNA treated cells and NC siRNA control cells were extracted, respectively, and were inverted into cDNAs, and qPCR amplification was performed using the cDNAs as templates and LPA2 as primers (Table 2), to thereby detect the expression level of LPA2 mRNA in the cells. The GAPDH gene is used as an internal reference.
The results are shown in fig. 7F and I, and LPA2mRNA expression levels in HT29 cells (F in fig. 7) or DLD1 cells (I in fig. 7) were significantly reduced in lpa2#1 or lpa2#2siRNA treated groups compared to NC siRNA control groups.
2. Inhibition of colorectal cancer cells by knockout of LPA2 to enhance in vitro EZH2 inhibitors
1. Identification of HT29-LPA2 KO cell lines
Lysates of HT29-WT (i.e., HT29 cell line), HT29-LPA2 KO#1, HT29-LPA2 KO#2 cells were prepared, respectively; the levels of LPA2 and beta-actin in the cells were separately detected by immunoblotting. The immunoblotting detection primary antibodies are respectively anti-LPA 2 or beta-actin antibodies.
As shown in FIG. 8A, the LPA2 protein levels in HT29-LPA2 KO#1 or HT29-LPA2 KO#2 cells were significantly reduced compared to HT29-WT cells. I.e. the knockout of LPA2 was achieved in HT29 cells.
2. Knockout of LPA2 enhances inhibition of HT29 cell survival by in vitro EZH2 inhibitors
HT29-WT, HT29-LPA2 KO#1, HT29-LPA2 KO#2 cells were seeded in 96-well plates at a density of 3000 cells/100. Mu.L/well, respectively; treating the cells with the EZH2 inhibitor GSK126 (final concentration 5 μm) or an equal volume of DMSO, respectively, dissolved in DMSO; after 72 hours, 10. Mu.L of CCK-8 solution was added to each well and incubated in a cell incubator at 37℃for 1-4 hours; detecting the absorbance at 450nm by using an enzyme-labeled instrument; and cell viability was calculated.
As shown in FIG. 8B, the viability of HT29-LPA2 KO#1 or HT29-LPA2KO#2 cells was further inhibited under the conditions of treatment with the EZH2 inhibitor GSK126, i.e., inhibition of HT29 cell viability by the EZH2 inhibitor in vitro was enhanced upon LPA2 knockout, as compared to the HT29-WT control.
3. Knockout of LPA2 enhances inhibition of HT29 cell proliferation by in vitro EZH2 inhibitors
Respectively inoculating HT29-WT, HT29-LPA2 KO#1 and HT29-LPA2KO#2 cells into 96-well plates according to a density of 3000 cells/100 μl/well; cells were treated with the EZH2 inhibitor GSK126 (final concentration 5 μm) dissolved in DMSO or an equal volume of DMSO, respectively, and transferred to an incubator of the IncuCyte S3 platform for culture and imaging; different areas of each well were photographed at 2h intervals using a 10X objective and analyzed for cell proliferation using an IncuCyte S3 image.
As a result, as shown in FIG. 8C, proliferation rate of HT29-LPA2 KO#1 or HT29-LPA2KO#2 cells was further inhibited when EZH2 inhibitor GSK126 was treated alone, compared to control HT 29-WT. Namely, the knockout of LPA2 enhances the inhibition of HT29 cell proliferation by in vitro EZH2 inhibitors.
4. Knockout of LPA2 enhances inhibition of HT29 cell colony formation by in vitro EZH2 inhibitors
HT29-WT, HT29-LPA2 KO#1, HT29-LPA2 KO#2 cells were seeded in a 6 well plate at 1000 cells/well and treated with the EZH2 inhibitor GSK126 (final concentration 2.5. Mu.M) dissolved in DMSO or an equal volume of DMSO, respectively; after 7 days, cell colony counts were performed by crystal violet staining.
As a result, as shown in FIG. 8D, when EZH2 inhibitor GSK126 was treated alone, the size and number of cell colony formation of HT29-LPA2 KO#1 or HT29-LPA2 KO#2 was further reduced as compared with control HT 29-WT. I.e., knockout of LPA2 enhances the in vitro inhibition of the colony forming ability of HT29 cells by EZH2 inhibitors.
3. Knocking out LPA2 improves the tumor treatment effect of in-vivo EZH2 inhibitor
1. The LPA2 is knocked out to enhance the inhibiting effect of EZH2 inhibitor on tumor size, tumor growth speed and tumor weight in vivo
The BALB/c male nude mice with the age of 6-8 weeks are selected as experimental mice, the feeding period of the mice is followed by 12 hours of day and night feeding period, the daily nursing of the mice is carried out according to relevant criteria, and the experimental mice are subjected to experiments to obtain the permissions of the experimental animal ethical examination meeting of Beijing university.
Experimental procedure As shown in FIG. 8E, HT29-WT, HT29-LPA2 KO #1 cells were cultured to appropriate density, the cells were digested and resuspended to a density of 5X 10 using serum free medium 6 Personal-200. Mu.L. 200 mu L total of 5×10 are inoculated on the armpits of the left side and the right side of the BALB/c nude mice respectively 6 HT29-WT or HT29-LPA2 KO#1 cells. When the tumor in the armpit was visually observed, the length and width of the tumor were measured using a ruler. The average volume of the tumor is 50mm 3 The mice were randomized into the following two groups and treated with drug injection:
solvent control group (solvent): the solvent control (prepared from 50% PEG300+50% physiological saline) was injected once daily for 8 days in the same volume as the injected drug group.
GSK126 treatment group (GSK 126): calculating the injection quantity of the medicine according to the weight of the mice; GSK126 drug (prepared from 50% PEG300+50% physiological saline) was injected per kg, once daily for 8 days.
The length and width of the tumor were measured every two days and tumor volume was calculated from length x width/2. After 8 days of drug treatment, mice were euthanized, the subcutaneous tumor tissue was dissected and weighed and photographed.
As a result, as shown in FIG. 8, F, G, H, in the case of the solvent treatment, the LPA2 knockout cell HT29-LPA2 KO#1 (LPA 2 KO#1+ solvent) formed smaller tumor, slower growth rate of tumor, and smaller weight of tumor than HT29-WT (WT+ solvent). In the GSK126 treated group, the LPA2 knockout cells HT29-LPA2 KO#1 (LPA 2 KO#1+GSK126) formed smaller tumors, slower growth rates of tumors, and less weight of tumors than HT29-WT (WT+GSK126). And the tumor size, growth rate and weight were partially reduced in the wt+gsk126 group or lpa2ko#1+solvent group compared to the wt+solvent control group, while the tumor size, growth rate and weight were further reduced in the lpa2ko#1+gsk126 group.
The above study results indicate that there is a partial anti-tumor effect in the treatment of the EZH2 inhibitor GSK126 alone or in the knockout of LPA2, but the knockout of LPA2 can further enhance the sensitivity of the tumor in vivo to the treatment of the EZH2 inhibitor GSK126, i.e., the knockout of LPA2 can further enhance the tumor treatment effect of the EZH2 inhibitor.
2. Treatment with EZH2 inhibitors inhibits H3K27me3 levels in tumor tissue
The allogenic tumor transplantation experiment and the drug treatment experiment of the nude mice were performed according to E in the experimental flow chart 8, and after 8 days of drug treatment, the mice were euthanized and subcutaneous tumor tissues were peeled off. Lysates of tumor tissues of a control group and an experimental group are prepared respectively, protein levels of H3K27me3 and beta-actin in the tumor tissues of each group of mice are detected by an immunoblotting method, and antibodies against the H3K27me3 and the beta-actin are used as primary antibodies.
As shown in fig. 8I, protein levels of H3K27me3 were significantly reduced in tumor tissue in GSK126 treated group (wt+gsk126 or LPA2 ko#1+gsk126) compared to solvent control group (wt+solvent or LPA2 ko#1+solvent). This suggests that treatment with GSK126 may significantly down-regulate the level of H3K27me3 in tumor tissue.
In conclusion, the experiments of the invention prove that in colorectal cancer tumor cells, EZH2/PRC2 inhibits the expression of ATX gene by catalyzing the formation of H3K27me3 on an ATX gene promoter; MTF2 acts as a recruitment factor to aid in the recruitment of EZH2/PRC2 to the ATX promoter. Inhibition of increased expression of ATX and activation of the ATX-LPA pathway following EZH2 may exert effects that are partially resistant to tumor therapy by promoting growth and proliferation of tumor cells, thus targeting co-inhibition of EZH2 and ATX-LPA-LPA2 axis as a novel colorectal cancer treatment strategy, i.e., co-inhibition of ATX inhibitor and EZH2 inhibitor in combination or targeting EZH2 and ATX-LPA-LPA2 axis for colorectal cancer treatment (FIG. 9).
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. The application of the substance in preparing a product for treating or assisting in treating tumor;
the substance is a substance 1 and/or a substance 2, and the substance 1 is a substance which inhibits the activity of the PRC2 complex or the activity of all or part of the components thereof, reduces the activity of the PRC2 complex or the content of all or part of the components thereof, inhibits the expression of all or part of the genes of the PRC2 complex, or knocks out all or part of the genes of the PRC2 complex;
the substance 2 is a substance which blocks the ATX-LPA-LPA receptor axis, or inhibits the activity of all or part of the components of the ATX-LPA-LPA receptor axis, reduces the content of all or part of the components of the ATX-LPA-LPA receptor axis, inhibits the expression of all or part of the genes of the ATX-LPA-LPA receptor axis, or knocks out all or part of the genes of the ATX-LPA-LPA receptor axis.
2. The use according to claim 1, characterized in that: the partial components of the PRC2 complex are EZH2 and/or SUZ12, and the partial genes of the PRC2 complex are EZH2 genes and/or SUZ12 genes;
the LPA receptor is LPA2, LPA1, LPA3, LPA4, LPA5 and/or LPA6.
3. Use according to claim 1 or 2, characterized in that: the substance inhibiting the activity of the PRC2 complex or all or part of the component activity of the PRC2 complex is a PRC2 complex inhibitor or a component inhibitor of the PRC2 complex, and the substance inhibiting the expression of all or part of the genes of the PRC2 complex is a specific siRNA of all or part of the genes of the PRC2 complex;
the substance for inhibiting the activity of all or part of the components of the ATX-LPA-LPA receptor axis is an ATX-LPA-LPA receptor axis all or part of component inhibitor, the substance for inhibiting the expression of all or part of the genes of the ATX-LPA-LPA receptor axis is a specific siRNA of all or part of the genes of the ATX-LPA-LPA receptor axis, and the substance for knocking out all or part of the genes of the ATX-LPA-LPA receptor axis is a substance for knocking out all or part of the genes of the ATX-LPA-LPA receptor axis by using a CRISPR-Cas9 method.
4. A use according to claim 3, characterized in that: the PRC2 complex component inhibitor is an EZH2 inhibitor;
The specific siRNA of the PRC2 complex part gene is siRNA for specifically recognizing the EZH2 gene and/or the SUZ12 gene;
the ATX-LPA-LPA receptor shaft portion grouping inhibitor is an ATX inhibitor;
the specific siRNA of all or part of the ATX-LPA-LPA receptor axis genes is siRNA specifically recognizing LPA 2;
the substance for knocking out all or part of the ATX-LPA-LPA receptor axis gene by using the CRISPR-Cas9 method is a substance for knocking out LPA2 gene.
5. The use according to claim 4, characterized in that: the EZH2 inhibitor is GSK126;
the ATX inhibitor is PF8380;
the siRNA for specifically recognizing the EZH2 gene is shown as SEQ ID No.1, and the siRNA for specifically recognizing the SUZ12 gene is shown as SEQ ID No. 2;
the siRNA specifically recognizing the LPA2 gene is shown as SEQ ID No.5 or 6.
6. Use according to any one of claims 1-5, characterized in that: the tumor is a solid tumor or a liquid tumor.
7. The use according to claim 6, characterized in that: the solid tumor is colorectal cancer.
8. Use of the substance 1 and/or the substance 2 according to any one of claims 1 to 5 for the preparation of a product for the prevention or co-prevention of tumors.
9. Use of the substance 2 according to any one of claims 1-5 for the preparation of a product enhancing the therapeutic effect of said substance 1 on tumors.
10. A therapeutic or co-therapeutic tumor product or a prophylactic or co-prophylactic tumor product, being substance 1 and/or substance 2 according to any of claims 1 to 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310562558.8A CN116712548A (en) | 2023-05-18 | 2023-05-18 | Use of co-inhibition of EZH2 and ATX-LPA-LPA2 axis in colorectal cancer treatment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310562558.8A CN116712548A (en) | 2023-05-18 | 2023-05-18 | Use of co-inhibition of EZH2 and ATX-LPA-LPA2 axis in colorectal cancer treatment |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116712548A true CN116712548A (en) | 2023-09-08 |
Family
ID=87870589
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310562558.8A Pending CN116712548A (en) | 2023-05-18 | 2023-05-18 | Use of co-inhibition of EZH2 and ATX-LPA-LPA2 axis in colorectal cancer treatment |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116712548A (en) |
-
2023
- 2023-05-18 CN CN202310562558.8A patent/CN116712548A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Penta et al. | Mitochondrial DNA in human malignancy | |
| US20160367572A1 (en) | Methods and compositions for inhibiting the growth and/or proliferation of myc-driven tumor cells | |
| Shao et al. | MicroRNA‐133a alleviates airway remodeling in asthtama through PI3K/AKT/mTOR signaling pathway by targeting IGF1R | |
| Hou et al. | mTOR inhibitor rapamycin alone or combined with cisplatin inhibits growth of esophageal squamous cell carcinoma in nude mice | |
| Doghish et al. | The potential role of miRNAs in the pathogenesis of testicular germ cell tumors-A Focus on signaling pathways interplay | |
| Xie et al. | Elevated expression of APE1/Ref-1 and its regulation on IL-6 and IL-8 in bone marrow stromal cells of multiple myeloma | |
| Brenet et al. | Akt phosphorylation of La regulates specific mRNA translation in glial progenitors | |
| CN111956658B (en) | Application of miRNA148 cluster as marker for diagnosing and/or treating cognitive disorder related diseases | |
| Liu et al. | MELK accelerates the progression of colorectal cancer via activating the FAK/Src pathway | |
| CN111518886A (en) | MicroRNA related to sorafenib drug resistance of tumor cells and application thereof | |
| Wu et al. | Genomic and GeneChip expression profiling reveals the inhibitory effects of Amorphophalli Rhizoma in TNBC cells | |
| Zhou et al. | Acetyl-coenzyme A synthetase 2 potentiates macropinocytosis and muscle wasting through metabolic reprogramming in pancreatic cancer | |
| CN108888620B (en) | Novel application of compound KNK437 | |
| CN116712548A (en) | Use of co-inhibition of EZH2 and ATX-LPA-LPA2 axis in colorectal cancer treatment | |
| CN114306611B (en) | ABHD2 gene expression inhibitor, application and medicine thereof | |
| CN113564252B (en) | New use of methylase METTL3 | |
| Chen et al. | Celastrol inhibits the proliferation and migration of MCF-7 cells through the leptin-triggered PI3K/AKT pathway | |
| CN109224076B (en) | Gene miR-140-3P related to lung cancer diagnosis and treatment, and mimics and application thereof | |
| Duan et al. | ULK1 Depletion Protects Mice from Diethylnitrosamine-Induced Hepatocarcinogenesis by Promoting Apoptosis and Inhibiting Autophagy | |
| US11642362B2 (en) | Methods of inhibiting cell proliferation and METTL8 activity | |
| US9737515B2 (en) | Compositions and methods for inhibiting tumor growth | |
| CN113521291B (en) | Application of ZNF143-MDIG-CDC6 axis in hepatocellular carcinoma | |
| CN113440511B (en) | HOTAIR-PRC2 blocker and application of compound preparation thereof in preparation of endometrial cancer treatment drug | |
| Song et al. | Downregulation of miR‑7 and miR‑153 is involved in Helicobacter pylori CagA induced gastric carcinogenesis and progression | |
| CN114917217B (en) | Application of phosphodiesterase 4 inhibitor ZL-n-91 in preparation of medicine for treating triple negative breast cancer |
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 |