CN116603065B - Application of PCBP2 inhibitor in preparation of medicine for promoting simulated fasted diet to treat gallbladder cancer - Google Patents
Application of PCBP2 inhibitor in preparation of medicine for promoting simulated fasted diet to treat gallbladder cancer Download PDFInfo
- Publication number
- CN116603065B CN116603065B CN202310585279.3A CN202310585279A CN116603065B CN 116603065 B CN116603065 B CN 116603065B CN 202310585279 A CN202310585279 A CN 202310585279A CN 116603065 B CN116603065 B CN 116603065B
- Authority
- CN
- China
- Prior art keywords
- pcbp2
- gallbladder cancer
- diet
- cancer cells
- fasted
- 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.)
- Active
Links
- 101000735358 Homo sapiens Poly(rC)-binding protein 2 Proteins 0.000 title claims abstract description 143
- 102100034961 Poly(rC)-binding protein 2 Human genes 0.000 title claims abstract description 140
- 201000010175 gallbladder cancer Diseases 0.000 title claims abstract description 127
- 208000022072 Gallbladder Neoplasms Diseases 0.000 title claims abstract description 123
- 235000005911 diet Nutrition 0.000 title claims abstract description 56
- 230000037213 diet Effects 0.000 title claims abstract description 56
- 239000003112 inhibitor Substances 0.000 title claims abstract description 12
- 239000003814 drug Substances 0.000 title claims abstract description 8
- 230000001737 promoting effect Effects 0.000 title claims abstract description 8
- 238000002360 preparation method Methods 0.000 title description 3
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 5
- 239000004055 small Interfering RNA Substances 0.000 claims description 5
- 101100462954 Homo sapiens PCBP2 gene Proteins 0.000 claims description 2
- 101150100247 PCBP2 gene Proteins 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 48
- 230000014509 gene expression Effects 0.000 abstract description 46
- 230000005012 migration Effects 0.000 abstract description 33
- 238000013508 migration Methods 0.000 abstract description 33
- 230000009545 invasion Effects 0.000 abstract description 27
- 230000035755 proliferation Effects 0.000 abstract description 22
- 230000001105 regulatory effect Effects 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 13
- 230000034659 glycolysis Effects 0.000 abstract description 13
- 230000005764 inhibitory process Effects 0.000 abstract description 6
- 238000001727 in vivo Methods 0.000 abstract description 5
- 230000008827 biological function Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 230000001093 anti-cancer Effects 0.000 abstract 1
- 230000001276 controlling effect Effects 0.000 abstract 1
- 230000006870 function Effects 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 101
- 101150054149 ANGPTL4 gene Proteins 0.000 description 57
- 102000045205 Angiopoietin-Like Protein 4 Human genes 0.000 description 57
- 108700042530 Angiopoietin-Like Protein 4 Proteins 0.000 description 57
- 230000002018 overexpression Effects 0.000 description 22
- 206010028980 Neoplasm Diseases 0.000 description 20
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 18
- 239000008103 glucose Substances 0.000 description 18
- 230000002829 reductive effect Effects 0.000 description 17
- 210000001519 tissue Anatomy 0.000 description 17
- 238000010521 absorption reaction Methods 0.000 description 16
- 238000003197 gene knockdown Methods 0.000 description 16
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 12
- 238000011529 RT qPCR Methods 0.000 description 10
- 238000003119 immunoblot Methods 0.000 description 10
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 10
- 108020004999 messenger RNA Proteins 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 230000002414 glycolytic effect Effects 0.000 description 9
- 230000036284 oxygen consumption Effects 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 230000004907 flux Effects 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 206010027476 Metastases Diseases 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 235000013305 food Nutrition 0.000 description 7
- 230000009401 metastasis Effects 0.000 description 7
- 238000002054 transplantation Methods 0.000 description 7
- 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 6
- 101001091538 Homo sapiens Pyruvate kinase PKM Proteins 0.000 description 5
- 102100034911 Pyruvate kinase PKM Human genes 0.000 description 5
- 108010087230 Sincalide Proteins 0.000 description 5
- 210000003445 biliary tract Anatomy 0.000 description 5
- 238000010609 cell counting kit-8 assay Methods 0.000 description 5
- 230000005757 colony formation Effects 0.000 description 5
- 235000014655 lactic acid Nutrition 0.000 description 5
- 239000004310 lactic acid Substances 0.000 description 5
- 101001117144 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) [Pyruvate dehydrogenase (acetyl-transferring)] kinase 1, mitochondrial Proteins 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 201000007487 gallbladder carcinoma Diseases 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 230000035899 viability Effects 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 230000012292 cell migration Effects 0.000 description 3
- 230000003828 downregulation Effects 0.000 description 3
- 230000004190 glucose uptake Effects 0.000 description 3
- 238000012151 immunohistochemical method Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 230000004709 cell invasion Effects 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000000749 co-immunoprecipitation Methods 0.000 description 2
- 238000007398 colorimetric assay Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011580 nude mouse model Methods 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- 230000004614 tumor growth Effects 0.000 description 2
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 244000064895 Cucumis melo subsp melo Species 0.000 description 1
- 235000009847 Cucumis melo var cantalupensis Nutrition 0.000 description 1
- 101000600756 Homo sapiens 3-phosphoinositide-dependent protein kinase 1 Proteins 0.000 description 1
- 101001117146 Homo sapiens [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 1, mitochondrial Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 102100040160 Rabankyrin-5 Human genes 0.000 description 1
- 101710086049 Rabankyrin-5 Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 102100024148 [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 1, mitochondrial Human genes 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 230000001617 migratory effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 201000008129 pancreatic ductal adenocarcinoma Diseases 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000002924 silencing RNA Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
Abstract
The invention discloses application of a PCBP2 inhibitor in preparing a medicament for promoting simulated fasted diet to treat gallbladder cancer. According to the invention, in vivo functional experiments show that the simulated fasted diet can inhibit biological functions such as proliferation, migration, invasion, glycolysis and the like of gallbladder cancer cells; in-depth researches on the fasting related gene PCBP2 show that the expression level of the PCBP2 in the gallbladder cancer tissue is obviously up-regulated, and in-vivo functional experiments show that after the PCBP2 is knocked down, the proliferation of gallbladder cancer cells is obviously weakened; then, in-vivo and in-vitro function experiments show that the inhibition effect of simulated fasted diet on proliferation, migration and invasion of gall bladder cancer cells after knocking down PCBP2 is obviously enhanced, and the anticancer effect of simulated fasted diet is obviously inhibited after the PCBP2 is overexpressed. The PCBP2 provided by the invention is an important factor for regulating and controlling the simulated fasted diet to treat the gallbladder cancer, and is expected to become a potential molecular target for treating the gallbladder cancer.
Description
Technical Field
The invention relates to application of a PCBP2 inhibitor in preparing a medicament for promoting simulated fasted diet to treat gallbladder cancer, and belongs to the technical field of biological medicines.
Background
Gallbladder cancer is a common malignant tumor of biliary tract, rank 5 th in digestive tract tumor, it is difficult to diagnose in early stage, there is no obvious specific symptom, the discovery is already in late stage at most, the radical rate of operation is low, easy to appear metastasis, prognosis is extremely bad, survival rate is less than 10% in 5 years. Fasted or simulated fasted diets are currently being used as a strategy for treating various digestive tract tumors, and the lack of nutrients can lead to changes in reverse growth factors and metabolic substances of muskmelon, thereby reducing proliferation, invasion and metastasis of tumor cells. Although simulated fasted diets have made some progress in tumor treatment, there is still a lack of research into gallbladder cancer. There is a need to further explore molecular mechanisms such as gallbladder carcinogenesis and invasion and metastasis.
PCBP2 is a currently known fasted regulated gene, and has also been found to be involved in promoting tumorigenesis, proliferation, metastasis, etc., and in prognosis of malignant tumor associated with metabolic diseases, such as hepatocellular carcinoma, gastric cancer, pancreatic ductal adenocarcinoma, etc. However, there is no report on PCBP2 as a therapeutic target for inhibiting gallbladder cancer.
Disclosure of Invention
The purpose of the invention is that: in order to solve the technical problems of low radical cure rate, easy occurrence of metastasis and extremely poor prognosis in the existing treatment of the gallbladder cancer, the invention provides the application of the PCBP2 inhibitor in preparing the medicament for promoting the simulation of the fasted diet treatment of the gallbladder cancer, the PCBP2 can simulate the fasted diet to inhibit the occurrence, the development and the invasion and the metastasis of the gallbladder cancer, and a new treatment target is provided for clinically treating the gallbladder cancer.
In order to achieve the above object, the present invention provides the use of a PCBP2 inhibitor for the preparation of a medicament for promoting the treatment of gallbladder cancer in a simulated fasted diet.
Wherein the PCBP2 inhibitor can regulate and control the inhibition effect of simulated fasted diet on proliferation, migration and invasion of gallbladder cancer and glycolysis.
The PCBP2 plays a role in treating gallbladder cancer by targeting ANGPTL4 and regulating and simulating fasted diet.
Preferably, the PCBP2 inhibitor comprises RNAi, microRNA, shRNA, siRNA specific to the PCBP2 gene, or an antibody, activity inhibitor of the PCBP2 protein.
Preferably, the PCBP2 inhibitor is a shRNA specific for PCBP2 genes.
Preferably, the shRNA has a sequence as set forth in SEQ ID NO:3 and/or SEQ ID NO: 4.
Compared with the prior art, the invention has the beneficial effects that:
The invention discovers that PCBP2 can regulate and control the generation, development and invasion and metastasis of the simulated fasted diet to inhibit the gallbladder cancer in the gallbladder cancer cells for the first time, and confirms that the PCBP2/ANGPTL4 signal axis exists for the first time, thereby being a potential treatment target in the field of gallbladder cancer treatment, providing a theoretical basis for the subsequent mechanism research of treating the gallbladder cancer through the simulated fasted diet, and simultaneously, the PCBP2 has potential application value as the treatment target of the gallbladder cancer.
Drawings
FIG. 1 illustrates that a simulated fasting diet can inhibit proliferation, migration and invasion of gallbladder cancer cells, glycolysis; wherein, A is CCK-8 experiment to prove that the simulated fasted diet can reduce the cell viability of gallbladder cancer cells GBC-SD and SGC-996; b is a colony formation experiment, which proves that the simulated fasted diet can inhibit the proliferation activity of gallbladder cancer cells GBC-SD and SGC-996; c is invasion and migration capacity of gallbladder cancer cells GBC-SD after simulated fasted diet detected by a Transwell experiment; d is invasion and migration capacity of gallbladder cancer cells SGC-996 after simulated fasted diet detected by a Transwell experiment; e is the migration ability of the gallbladder cancer cells GBC-SD after simulated fasted diet detected by a scratch experiment; f is the migration capacity of the gallbladder cancer cells SGC-996 after the scratch test to simulate the fasted diet; g is a glucose absorption detection experiment, and the simulated fasted diet is proved to be capable of obviously reducing the glucose absorption level of the gallbladder cancer cells GBC-SD and SGC-996; the colorimetric detection experiment of the lactic acid by H proves that the simulated fasted diet can obviously reduce the lactic acid generation level of the gallbladder cancer cells GBC-SD and SGC-996; i is that the oxygen consumption rate of the gallbladder cancer cells GBC-SD is obviously increased after the extracellular flux analyzer detects that the gallbladder cancer cells are fasted; j is that the oxygen consumption rate of the gallbladder cancer cells SGC-996 is obviously increased after the extracellular flux analyzer detects that the gallbladder cancer cells are fasted; k is Western blotting to confirm that the simulated fasted diet can significantly reduce the protein expression level of each glycolytic rate-limiting enzyme (PDK 1, PKM2 and HK-2) in gallbladder cancer cells GBC-SD and SGC-996;
FIG. 2 shows that PCBP2 is up-regulated in gallbladder cancer tissue and promotes proliferation of gallbladder cancer; wherein, A is the up-regulation of PCBP2 expression in biliary tract tumor tissues is confirmed by a TCGA public database; b is 36 pairs of gallbladder cancer and other tissues beside the cancer, and the qRT-PCR method proves that the mRNA level of PCBP2 is up-regulated in tumor tissues; c is an immunohistochemical method to confirm that the protein level of PCBP2 is up-regulated in tumor tissues; d is the gene expression condition of PCBP2 in each gallbladder cancer cell line (EH-GB 1, NOZ, GBC-SD, SGC-996); e is a transfection efficiency map of constructing knockdown PCBP2 on gall bladder cancer cells SGC-996 and over-expressing PCBP2 on GBC-SD; f is CCK-8 experiment, and it is proved that the knock-down PCBP2 can reduce the activity of SGC-996 of gallbladder cancer cells, and the over-expression of PCBP2 can promote the cell activity of GBC-SD of gallbladder cancer cells; g is a colony formation experiment, and the knock-down PCBP2 can reduce the proliferation capability of gall bladder cancer cells SGC-996, and the overexpression of PCBP2 can promote the proliferation capability of gall bladder cancer cells GBC-SD; h is a nucleoplasm separation experiment, and proves that PCBP2 is expressed in cell nucleuses and cytoplasm of gall bladder cancer cells SGC-996 and GBC-SD;
FIG. 3 shows the effect of PCBP2 in regulating simulated fasted diet in inhibiting proliferation of gallbladder cancer; wherein, A is that the expression level of PCBP2 protein and mRNA in gall bladder cancer cells SGC-996 and GBC-SD after simulated fasted diet treatment is proved to be reduced by immunoblotting experiments and qRT-PCR experiments; b is a test of immunoblotting and qRT-PCR, which proves that the simulated fasted diet can further inhibit the PCBP2 protein and mRNA level in the SGC-996 of the gallbladder cancer cells of PCBP2 after knockdown or over-expression; c is a simulation fasting diet which can further inhibit PCBP2 protein and mRNA level in the gallbladder cancer cells GBC-SD of the PCBP2 after knockdown or over-expression through immunoblotting experiments and qRT-PCR experiments; d is CCK-8 experiment, which proves that knocking down PCBP2 can reduce the vitality of SGC-996 and GBC-SD of gallbladder cancer cells more than single fasted food, and over-expression of PCBP2 can increase the vitality of the cells, but the fasted food reduces the influence of the over-expression of PCBP2 on the vitality of the gallbladder cancer cells; e is a colony forming experiment, and the experiment proves that knocking down PCBP2 can reduce the proliferation capacity of gall bladder cancer cells SGC-996 and GBC-SD more than single fasted food, the overexpression of PCBP2 can increase the proliferation capacity of cells, but the fasted food reduces the influence of the overexpression of PCBP2 on the proliferation capacity of gall bladder cancer cells; f, constructing a nude mouse subcutaneous transplantation tumor model by using a gall bladder cancer cell SGC-996 and a cell strain of the knocked-down PCBP2, and carrying out experiments to prove that the size and the volume of transplantation tumor can be inhibited by combining the knocked-down PCBP2 with a simulated fasted diet; g is a nude mice subcutaneous transplantation tumor model constructed by using gallbladder cancer cells GBC-SD and cell strains which overexpress PCBP2, and experiments prove that the overexpression of PCBP2 can promote the size and the volume of transplantation tumor, but the simulated fasted diet can inhibit the size and the volume of transplantation tumor;
FIG. 4 shows the effect of PCBP2 in regulating simulated fasted diet in inhibiting gallbladder cancer migration and invasion and glycolysis; wherein, A is Transwell experiment, which proves that over-expression of PCBP2 can obviously promote the invasion and migration capacity of gallbladder cancer cells, but the fasted food can reduce the level of cell migration and invasion; b is a Transwell experiment, and the knock-down PCBP2 can obviously inhibit the invasion and migration capacity of gallbladder cancer cells and can be further reduced through fasted food; c is a scratch experiment, and proves that the over-expression of PCBP2 can obviously promote the migration capacity of gallbladder cancer cells, but the fasting can reduce the level of cell migration; d is a scratch experiment, and the knock-down PCBP2 can obviously inhibit the migration capacity of gallbladder cancer cells and can be further reduced through fasted; e is glucose absorption detection experiments, and the knock-down PCBP2 and fasting can continuously reduce the glucose absorption level of gall bladder cancer cells SGC-996, while the over-expression of PCBP2 obviously increases the glucose absorption level; f is lactic acid colorimetric detection experiments prove that the lactic acid generation level of the SGC-996 of the gallbladder cancer cells can be continuously reduced by knocking down PCBP2 and fasting, and the lactic acid generation level is obviously increased by over-expressing PCBP 2; g is the oxygen consumption rate of the gallbladder cancer cells which can be continuously increased by knocking down PCBP2 and fasted after the extracellular flux analyzer detects the gallbladder cancer cells GBC-SD and SGC-996 and differentially expresses PCBP2 and fasted; h is Western blotting method to confirm that knocking down PCBP2 and simulating fasted diet can obviously reduce protein expression level of glycolytic rate-limiting enzymes (PDK 1, PKM2 and HK-2) in gallbladder cancer cells GBC-SD and SGC-996;
FIG. 5 shows the interaction of PCBP2 with ANGPTL4 and the negative regulation of ANGPTL4 expression by PCBP 2; wherein, A is the interaction of ANGPTL4 and PCBP2 in the gallbladder cancer cell GBC-SD proved by the co-immunoprecipitation technology; b is an immune coprecipitation technology to prove that the ANGPTL4 and PCBP2 interact in a gall bladder cancer cell SGC-996; c is immunoblotting and qRT-PCR method to confirm that the expression level of the protein and mRNA of the ANGPTL4 is increased after knocking down PCBP2, and the expression of the ANGPTL4 can be reduced by over-expressing PCBP 2; d is immunoblotting and qRT-PCR methods to confirm that protein and mRNA expression levels of ANGPTL4 after differential expression of PCBP2 and simulated fasted diet, fasted can significantly increase expression of ANGPTL4, over-expression of PCBP2 can reduce expression of ANGPTL4 during fasted, and inhibition of PCBP2 can enhance expression of ANGPTL4 during fasted; e is the down regulation of the expression of the ANGPTL4 in biliary tract tumor tissues through TCGA public database; f is 36 pairs of gallbladder cancer and other tissues beside the cancer, and the qRT-PCR method proves that the mRNA level of the ANGPTL4 is down-regulated in the tumor tissues; g is an immunohistochemical method to confirm that the protein level of ANGPTL4 is down-regulated in tumor tissues; h is the gene expression condition of the ANGPTL4 in each gallbladder cancer cell line (EH-GB 1, NOZ, GBC-SD, SGC-996); i is a transfection efficiency diagram for constructing knockdown ANGPTL4 on a gall bladder cancer cell SGC-996 and over-expressing ANGPTL4 on GBC-SD;
FIG. 6 shows that PCBP2 modulates the migration, invasion and glycolytic capacity of gallbladder cancer cells by targeting ANGPTL 4; wherein, A is a result of Transwell experiments, when PCBP2 is over-expressed, the migration and invasion level of the gallbladder cancer cells GBC-SD are obviously increased, and when ANGPTL4 is over-expressed, the migration and invasion level is inhibited; b is a result of Transwell experiments, wherein when PCBP2 is knocked down, the migration and invasion level of gall bladder cancer cells SGC-996 is obviously reduced, and when ANGPTL4 is knockdown, the migration and invasion level is promoted; c is a scratch experiment, and proves that the migration level of the gallbladder cancer cells GBC-SD is obviously increased when the PCBP2 is over-expressed, and the migration level is inhibited after the ANGPTL4 is over-expressed; d is a scratch experiment, which proves that when PCBP2 is knocked down, the migration level of the gall bladder cancer cell SGC-996 is obviously reduced, and when ANGPTL4 is knocked down, the migration level is promoted; e is glucose absorption detection experiment, which proves that when PCBP2 is over-expressed, the relative glucose absorption level is highest, when ANGPTL4 is over-expressed, the relative glucose absorption level is lowest, and when PCBP2 and ANGPTL4 are double over-expressed or double knocked down, the relative glucose absorption level is similar to that of a control group; f is a lactate colorimetric assay experiment demonstrating that relative lactate production levels are highest when PCBP2 is overexpressed, lowest when ANGPTL4 is overexpressed, and similar to the control when PCBP2 and ANGPTL4 are double overexpressed or double knocked down; g is the oxygen consumption rate is higher when the extracellular flux analyzer detects that the gallbladder cancer cell GBC-SD is over-expressed by the ANGPTL4, and is reduced when PCBP2 is over-expressed, and the over-expression of PCBP2 and ANGPTL4 is similar to that of a control group; h is the lowest oxygen consumption rate when the extracellular flux analyzer detects that the gallbladder cancer cells SGC-996 are knocked down by ANGPTL4, and the rise is obvious when PCBP2 is knocked down, and the common knockdown of PCBP2 and ANGPTL4 is similar to that of a control group; i is a western blot method for confirming that knock-down and over-expression of PCBP2 and ANGPTL4 significantly regulate the protein expression levels of glycolytic rate-limiting enzymes (PDK 1, PKM2 and HK-2) of gallbladder cancer cells GBC-SD and SGC-996;
in the above figures, P <0.05, P <0.01, P <0.001, indicating that there was a significant difference between the two groups through statistical analysis.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
Example 1: the simulated fasted diet can inhibit proliferation, migration, invasion and glycolysis of gallbladder cancer cells
This example first examined the cell functions of GBC-SD and SGC-996 of two gallbladder cancer cells cultured by a simulated fasting diet (cells were cultured in DMEM medium (glucose: 0.5g/L, FBS 1%) for 24 hours and 48 hours), and CCK8 proliferation and colony formation experiments showed that the viability of gallbladder cancer cells could be significantly reduced after 24 hours and 48 hours of the simulated fasting diet treatment (FIG. 1A), and cell proliferation was inhibited (FIG. 1B). Scratch and Transwell experiments showed that the ability to significantly inhibit invasion and migration of gallbladder cancer cells after 24 hours and 48 hours of simulated fasted diet treatment (fig. 1C-F). The above results indicate that the simulated fasting diet can inhibit the proliferation, migration and other biological functions of gallbladder cancer cells.
Subsequently, the glucose absorption and lactate production levels of the gallbladder cancer cells were analyzed by the glucose absorption test and the lactate colorimetric test, and the results showed that the glucose absorption and lactate production levels were significantly reduced after the simulated fasting diet treatment (fig. 1g, h), while the oxygen consumption rate was significantly increased after the fasting treatment of the gallbladder cancer cells detected by the extracellular flux analyzer (fig. 1i, j). Further Western blotting was used to detect the expression of each glycolytic rate-limiting enzyme (PDK 1, PKM2 and HK-2) in gallbladder carcinoma cells, indicating a significant decrease in expression of each glycolytic rate-limiting enzyme after simulated fasting diet treatment (FIG. 1K). The above results show that the simulated fasting diet can inhibit glycolysis of gallbladder cancer cells.
Example 2: PCBP2 expression was up-regulated in gallbladder cancer tissues, and in view of the fact that PCBP2 was closely related to fasted food, PCBP2 expression was up-regulated in biliary tract tumor tissues (fig. 2A) as confirmed by TCGA public database, 36 mRNA expression levels and protein expression levels in gallbladder cancer tissues and paracancerous tissues were detected by qRT-PCR and immunohistochemical methods, and PCBP2 was found to be significantly up-regulated in gallbladder cancer tissues. And the qRT-PCR method is used for detecting that the expression of PCBP2 in a gallbladder cancer cell line is also obviously increased (figure 2D). Further constructing a gallbladder cancer cell strain which knocks down and overexpresses PCBP2, carrying out plasmid transfection on the cell strain GBC by using GENEPHARMA company (China, shanghai), constructing an overexpressed PCBP2 plasmid (a control group pcDNA and an overexpressed group pcDNA-PCBP2 are arranged, the primer sequences of the overexpressed plasmids are shown as SEQ ID NO: 1-2, namely PCBP2-F:5'-AGGCAGGTTACCATCACTGG-3', PCBP2-R: 5'-CATTGTTCTAGCTGCTCCCC-3'), and constructing an overexpressed PCBP2 gallbladder cancer cell strain by using Lipo2000 (Thermo FISHER SCIENTIFIC); the sh-RNA PCBP2 (comprising shPCBP2-1#, the sequence of which is shown as SEQ ID NO: 3: 5'-ACCGGGCATTCCACAATCCATCATTGCTCGAGCAATGATGGATTGT GGAATGCTTTTT-3'; shPCBP2-2#, the sequence of which is shown as SEQ ID NO: 4: 5'-ACCGGCCATGATCCATCTGTGTAGTTCTCGAGAACTACACAGATGGATCA TGGTTTTT-3'; and the control group sh-NC) is transfected into the GBC cell strain by Lipo-RNAiMax (U.S. A.Thermo FISHER SCIENTIFIC) to obtain the gallbladder cancer cell strain with the knocked-down PCBP 2.
CCK-8, colony formation experiments showed that after knocking down PCBP2 expression, the vitality and proliferation ability of gallbladder cancer cells were inhibited, while after over-expressing PCBP2, the vitality and proliferation ability of gallbladder cancer cells were significantly increased (FIG. 2F, G), and nuclear mass separation experiments showed that PCBP2 was expressed in the nuclei and cytoplasm of both gallbladder cancer cells SGC-996 and GBC-SD (FIG. 2H).
Example 3: PCBP2 can regulate and control inhibition of simulated fasted diet on proliferation, migration, invasion and glycolysis of gallbladder cancer
After confirming that both the simulated fasting diet and the fasting related gene PCBP2 had an effect on the gallbladder cancer cells, the immunoblotting experiments in this example revealed that the expression levels of PCBP2 protein and mRNA in the gallbladder cancer cells after the simulated fasting diet treatment (24 hours and 48 hours of culturing the cells with DMEM medium (glucose: 0.5g/L, FBS 1%) were reduced (fig. 3A). The effect of the simulated fasting diet on PCBP2 was then again examined using immunoblotting experiments in gallbladder cancer cells that differentially expressed PCBP2, and it was found that the simulated fasting diet further inhibited PCBP2 expression after knockdown or overexpression (fig. 3b, c). The effect of PCBP2 differential expression and fasting on the viability and proliferative capacity of gallbladder cancer cells was examined by CCK-8, colony formation experiments, and it was found that knocking down PCBP2 more reduced the viability and proliferation of GBC cells than fasting alone, and that over-expressing PCBP2 increased the cell viability and proliferation level, but fasting reduced the effect of PCBP2 overexpression on the viability and proliferative capacity of GBC cells (fig. 3d, e). Subcutaneous transplantation tumor experiments were subsequently performed in vivo animal experiments, and it was found that PCBP2 knockout, simulated fasting diet, and PCBP2 knockout in combination with simulated fasting diet significantly inhibited the size and volume of the transplantation tumor (fig. 3f, g). While simulated fasted diet can inhibit tumor growth, knockdown of PCBP2 expression can further reduce tumor growth in vivo.
Next, PCBP2 differential expression and the effect of simulated fasted diet on invasion and migration of gallbladder cancer cells were again examined using a Transwell experiment. The results showed that over-expression of PCBP2 significantly promoted the invasive migration ability of gallbladder cancer cells, but fasting reduced the level of cell migration and invasion (fig. 4A). In contrast to PCBP2 overexpression, knockdown of PCBP2 significantly inhibited the invasive migratory capacity of gallbladder cancer cells and was further reduced by fasting (fig. 4B). The scratch experiments also confirmed the above results (fig. 4c, d). To determine whether these changes in cell biological function were affected by glycolysis, the glucose absorption and lactate production levels of the mock fasted diet-treated glucose absorption and lactate production levels of the differentially expressed PCBP2 gallbladder cancer cells were again analyzed by glucose absorption detection experiments and lactate colorimetric detection experiments, and it was found that knocking down PCBP2 and fasted continuously reduced the glucose absorption and lactate production levels of the gallbladder cancer cells, while overexpression of PCBP2 significantly increased glucose uptake and lactate production (fig. 4e, f), suggesting that PCBP2 was involved in glycolysis of the gallbladder cancer cells. While the oxygen consumption rate was measured by an extracellular flux analyzer and the expression of each glycolytic rate-limiting enzyme was measured by an immunoblotting method, the results showed that knocking down PCBP2 and fasting could continuously increase the oxygen consumption of gallbladder cancer cells and decrease the expression levels of PDK1, PKM2 and HK-2, which also confirmed that PCBP2 was involved in glycolysis of gallbladder cancer cells (FIGS. 4G, H).
Example 4: PCBP2 negatively regulates migration, invasion and glycolysis of gallbladder cancer cells by targeting ANGPTL4
To further elucidate the regulatory mechanism of PCBP2 for gallbladder cancer, the present example found that ANGPTL4 interacted with PCBP2 in gallbladder cancer cells using co-immunoprecipitation (fig. 5a, b). And the expression level of the protein and mRNA of the ANGPTL4 is detected in the gallbladder cancer cells with the differential expression of PCBP2 by using an immunoblotting and qRT-PCR method, which shows that after the PCBP2 is knocked down, the expression of the ANGPTL4 is increased, and the expression of the ANGPTL4 can be reduced by over-expressing the PCBP2 (figure 5C). Furthermore, expression of ANGPTL4 was again examined after gallbladder cancer cells differentially expressing PCBP2 by simulated fasted diet treatment, and it was found that fasted can significantly increase expression of ANGPTL4, over-expression of PCBP2 can reduce expression of ANGPTL4 during fasted, and inhibition of PCBP2 can enhance ANGPTL4 expression during fasted (fig. 5D). The down-regulation of ANGPTL4 expression in biliary tract tumor tissues was subsequently confirmed by TCGA public database (fig. 5E), and the expression of ANGPTL4 was examined by immunohistochemistry in 36 pairs of gallbladder carcinoma and paracancerous tissues and found to be down-regulated in gallbladder carcinoma tissues (fig. 5f, g). In addition, the gene expression of ANGPTL4 in each gallbladder cancer cell line (EH-GB 1, NOZ, GBC-SD, SGC-996) was examined (FIG. 5H); knock-down ANGPTL4 was constructed on gallbladder carcinoma cells SGC-996, ANGPTL4 was overexpressed on GBC-SD and transfection efficiency was examined (fig. 5I). These results indicate that there is a correlation between PCBP2 and ANGPTL4 expression, PCBP2 interacts with ANGPTL4 and PCBP2 negatively regulates ANGPTL4 expression.
To further examine the interaction between PCBP2 and ANGPTL4, a double over-expressed and double knocked down gallbladder cancer cell line of PCBP2/ANGPTL4 was constructed using a co-transfection technique, and it was found through transwell experiments that when PCBP2 was over-expressed or ANGPTL4 was knocked down, the migration and invasion of gallbladder cancer cells was at the highest level, and when ANGPTL4 was over-expressed or PCBP2 was knocked down, the migration and invasion levels were the lowest. Whereas when PCBP2 and ANGPTL4 were double overexpressed or double knocked down, migration and invasion levels were similar to control levels (fig. 6a, b). Scratch assays also confirmed this result (fig. 6c, d). Finally, using the glucose uptake assay and lactate colorimetric assay analysis, relative glucose uptake and lactate production levels were highest when PCBP2 was overexpressed, lowest when ANGPTL4 was overexpressed, and similar to the control when PCBP2 and ANGPTL4 were double overexpressed or double knocked down (fig. 6e, f). The rate of oxygen consumption was measured by an extracellular flux analyzer and the expression of each glycolytic rate-limiting enzyme was measured by immunoblotting, and it was found that glycolysis levels were higher when PCBP2 was overexpressed and glycolysis was lower when ANGPTL4 was overexpressed, and that the overexpression or inhibition of PCBP2 and ANGPTL4 was similar to the control (fig. 6G-I). These results indicate that PCBP2 regulates the migration, invasion and glycolytic capacity of gallbladder cancer cells by targeting ANGPTL 4.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be limiting in any way and in nature, and it should be noted that several modifications and additions may be made to those skilled in the art without departing from the invention, which modifications and additions are also intended to be construed as within the scope of the invention.
Claims (1)
- Application of PCBP2 inhibitor in preparing medicine for promoting simulated fasted diet to treat gallbladder cancer is characterized in that the PCBP2 inhibitor is shRNA specific to PCBP2 gene;The sequence of the shRNA is shown as SEQ ID NO:3 and/or SEQ ID NO: 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310585279.3A CN116603065B (en) | 2023-05-23 | Application of PCBP2 inhibitor in preparation of medicine for promoting simulated fasted diet to treat gallbladder cancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310585279.3A CN116603065B (en) | 2023-05-23 | Application of PCBP2 inhibitor in preparation of medicine for promoting simulated fasted diet to treat gallbladder cancer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116603065A CN116603065A (en) | 2023-08-18 |
| CN116603065B true CN116603065B (en) | 2024-06-28 |
Family
ID=
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115927627A (en) * | 2022-09-30 | 2023-04-07 | 杭州添帆生物科技有限公司 | Gallbladder cancer biomarker and application thereof |
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115927627A (en) * | 2022-09-30 | 2023-04-07 | 杭州添帆生物科技有限公司 | Gallbladder cancer biomarker and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| B型超声观察长期禁食患者的胆囊改变;邱洁;上海医学影像;20051231;第14卷(第4期);第302-303页 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhu et al. | miR-155-5p inhibition promotes the transition of bone marrow mesenchymal stem cells to gastric cancer tissue derived MSC-like cells via NF-κB p65 activation | |
| Zhang et al. | RETRACTED ARTICLE: MicroRNA-155-3p promotes breast cancer progression through down-regulating CADM1 | |
| WO2016145974A1 (en) | Use of gene engineering bacteria vpn 20009-m in preparation of medicaments for preventing and treating cancer metastasis | |
| Wu et al. | Effect of hypoxia-inducible factor 1-α on Survivin in colorectal cancer | |
| CN103656641B (en) | Transcribe mediator Med23 subunit as target spot for prevention or Therapeutic cancer | |
| Liu et al. | Soybean-derived gma-miR159a alleviates colon tumorigenesis by suppressing TCF7/MYC in mice | |
| Chen et al. | Knockdown of POLDIP2 suppresses tumor growth and invasion capacity and is linked to unfavorable transformation ability and metastatic feature in non-small cell lung cancer | |
| CN112043837A (en) | Application of MiR-205-5p in preparation of angiogenesis agent | |
| Yao et al. | Glypican‐3 Enhances Reprogramming of Glucose Metabolism in Liver Cancer Cells | |
| CN116603065B (en) | Application of PCBP2 inhibitor in preparation of medicine for promoting simulated fasted diet to treat gallbladder cancer | |
| Jiang et al. | Lipopolysaccharide affects the proliferation and glucose metabolism of cervical cancer cells through the FRA1/MDM2/p53 pathway | |
| CN106474489A (en) | Application on preparation treatment liver-cancer medicine for the attenuated salmonella typhimurium genetic engineering bacterium | |
| CN116603065A (en) | Application of PCBP2 inhibitor in preparation of medicine for promoting simulated fasted diet to treat gallbladder cancer | |
| US9284557B2 (en) | Double-stranded nucleic acid molecule, cancer cell proliferation inhibitor and pharmaceutical agent suitable for prevention or treatment of cancer | |
| CN112175997B (en) | Application of targeting exosome PKM2 in improvement of cisplatin resistance of non-small cell lung cancer | |
| CN115990191A (en) | Antitumor pharmaceutical composition | |
| Huang et al. | Dextran sulfate effects EMT of human gastric cancer cells by reducing HIF-1α/TGF-β | |
| CN104436216B (en) | The Expression modulation agent of transcription factor Yin Yang 1 is used as glycometabolism adjusting control agent | |
| CN111358951A (en) | Method for converting mature right ventricular cardiomyocytes into immature cardiomyocytes | |
| CN105671195B (en) | Purposes, pharmaceutical composition and the kit of miR-520c nucleotide | |
| US10835551B2 (en) | Double-stranded nucleic acid molecule, DNA, vector, cancer cell growth inhibitor, cancer cell migration inhibitor, and drug | |
| CN109939122B (en) | Application of substance for regulating and controlling one-carbon metabolism to influence dryness of tumor stem cells | |
| Fu et al. | Circular RNA Ubiquitin-associated protein 2 silencing suppresses bladder cancer progression by downregulating DNA topoisomerase 2-alpha through sponging miR-496 | |
| CN107582525A (en) | TRIM31 inhibitor magnetic target drug bearing microspheres are preparing the application in suppressing PDAC multiplication capacity medicines | |
| Sun et al. | MicroRNA-32-5p promotes the proliferation and metastasis of gastric cancer cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |