| CHAPTER 1 SCREENING OF POSITIVE SELECTION SITES FOR MULTIDRUG RESISTANCE IN OVARIAN CANCEROBJECTIVE: To synthesize the positive selection sites in genes associated with multidrug resistance in ovarian cancer using a combination of bioinformatics methods and previous studies.Methods: From the perspective of bioinformatics,this study included 30 species of metazoan from the GenBank,Ensembl,and UniProt databases(ABCA1,ABCB11,ABCC8,ABCC11,ABCD2,TAP1,COL4A2,CTNNB1,EGFR,FYN,IGF1 R.,ITGB4,LAMA2,LAMA4,MRLC2,PAK7,PARVA,PDGFC,PIK3R1,PPP1 CA,PRKCA,PTK2,RAGEFI1,SHC4,TLN1,VAV2)and ovarian cancer resistance-related gene sequence information,using positive selection bits that have been developed so far Point analysis,molecular evolution analysis,protein structure,and functional site analysis were used to analyze the adaptive evolution of these genes using site model analysis.The positive selection sites were converted into SNP sites,and the positive selection gene was predicted by phosphorylation sites.The SNPs that overlap the positive selection site with the phosphorylation site were selected to detect SNP mutations using the Sequenom MassARRAY method.Samples of platinum-sensitive ovarian cancer patients and platinum-resistant ovarian cancer patients were taken from fresh frozen tissue samples for DNA detection.RESULTS: A total of 13 genes(ABCA1,ABCB11,ABCC8,ABCC11,ABCD2,COL4A2,EGFR,IGF1 R,ITGB4,LAMA2,PIK3R1,RAPGEF1,VAV2)were positively selected by the locus model,and each of them had an unequal number of positive Select sites.Among the SNPs in which the positive selection site coincides with the predicted phosphorylation site are four ITGB4-rs368526254,RAPGEF1-rs41297229,RAPGEF1-rs202232900,and VAV2-rs756680997.SNP genotyping revealed that none of the above four sites had mutations in ovarian cancer patients.CONCLUSION: ABCA1,ABCB11,ABCC8,ABCC11,ABCD2,COL4A2,EGFR,IGF1 R,ITGB4,LAMA2,PIK3R1,RAPGEF1,and VAV2 genes are positively selected during species evolution.Among them,there were 4 positive selection sites coincided with the phosphorylation prediction sites,but no mutations occurred in sensitive and resistant patients with ovarian cancer,and there was no significant correlation with drug resistance to ovarian cancer.CHAPTER 2 VALIDATION OF CLINICAL SAMPLES OF POSITIVE SELECTION SITESOBJECTIVE: To screen positive selection sites associated with multidrug resistance in ovarian cancer and analyze its relationship with ovarian cancer drug resistance,clinical features,and prognosis.METHODS: All positively selected amino acid sites were converted to DNA SNPs,and SNPs with MAF > 0.05 and predicted functionalities were used for SNP genotyping.A total of 199 cases of ovarian cancer tissue wax and fresh frozen tissue DNA were extracted,including 62 cases in the resistant group and 137 cases in the sensitive group.The AgneMassArray system was used to genotype SNPs.And analyze its relationship with drug resistance,clinical pathology,and prognosis.RESULTS:(1)Comparison of the distribution of different clinical indicators between the two groups was performed by Chi-square test.The differences in staging,surgical satisfaction(residual diameter),lymph node metastasis,and celiac metastasis between sensitive and resistant groups were found.Statistically significant(p<0.05)(2)LAMA2 site rs1027199 was found to be associated with drug resistance by binary logistic regression analysis.Homozygous mutants had a lower incidence of drug resistance than wild-type(p=0.049,adjusted OR = 0.343,95% CI: 0.118-0.997).After calibration,it remained statistically significant(p=0.035,adjusted OR=0.255,95%CI: 0.072-0.908).(3)The binary logistic regression analysis showed that the positive selection site rs757110 of ABCC8 was related to the stage of onset,and the heterozygous mutant decreased the stage of onset.(OR=0.280,95%CI0.097-0.809,P=0.019)(4)Kaplan-Meier method showed that FIGO stage,residual tumor diameter,lymph node metastasis,and peritoneal metastasis had effects on PFS and OS(p<0.05).The positive selection site of LAMA2,rs3816665,was associated with prognosis.GA mutation significantly decreased the PFS time compared with GG wild type(mutant: mean 17.874±3.452,95%CI11.109-24.640,wild type: mean 65.066±6.690,95.%CI 51.953-78.179,P=0.049),also significantly decreased overall survival(mutant: mean 32.101±6.270,95% CI 19.813-44.390,wild type: mean 76.054±11.864,95% CI 52.801-99.308,P=0.014).COX risk proportional regression model analysis showed that residual tumor diameter is an independent factor that influences PFS in patients with ovarian cancer.Rs3816665 genotype and residual tumor diameter are independent risk factors for OS in ovarian cancer patients.CONCLUSIONS: The rs1027199 locus of LAMA2 is associated with drug resistance,and homozygous mutants have a lower incidence of resistance than wild-type ones.Another positive selection site for LAMA2,rs3816665,is associated with prognosis.The mutant OS is significantly lower than the wild type.The positive selection site rs757110 of ABCC8 is associated with the stage of onset,and heterozygous mutations reduce the stage of onset.CHAPTER III BIOLOGICAL FUNCTION OF POSITIVELY SELECTED GENES IN OVARIAN CANCER AND DRUG RESISTANT CELLSOBJECTIVE: To study the biological function of the 13 positively selected genes among the ovarian cancer parental and drug-resistant cells.Knockout and overexpression of lentiviral vectors were constructed and identified,and changes in cell biological function following changes in gene expression were studied.Methods:(1)m RNA was extracted from SKOV3,SKOV3/DDP,A2780 and A2780/DDP cells.The positive expression of 13 positive selection genes was determined by 2-△ △ Ct method.(2)The ITGB4 knock-out and over-expression ovarian cancer cell lines were constructed using CRISP/Cas9 technology,and the transfection efficiency was identified by QRT-PCR and WB methods.(3)The CCK8 method was used to detect the IC50 and growth curve of the cells transfected with lentivirus.(4)Flow cytometry was used to detect the changes of cell cycle under different concentrations of cisplatin in cells transfected with lentivirus.(5)The transwell assay was used to detect the migration and invasion of the cells after transfection of lentivirus.RESULTS:(1)ITGB4 had the highest relative expression in the SKOV3/DDP and A2780/DDP resistant cells compared to the correspondingparental cells,especially in A2780/DDP cells.The relative expression level was 37.27 times.(2)Successfully using the CRISP/Cas9 method to construct ITGB4 knockout and overexpression ovarian cancer cell lines,QRT-PCR results show that ITGB4-sgRNA(02447-1)has the highest knock-out efficiency(99.4% relative to the negative control group)),ITGB4-sgRNA(02458-1)had the highest efficiency of overexpression of lentivirus(relative expression level was 16.99 times relative to the negative control group).The WB results are consistent with the QRT-PCR results.(3)The IC50 of A2780/DDP cells that down-regulated ITGB4 gene to cisplatin was significantly decreased(P<0.05),but the difference between the blank control group and the negative virus group was not significant(P>0.05).The IC50 of A2780 cells up-regulated ITGB4 gene to cisplatin was significantly higher(P<0.05),but the difference between the blank control group and the negative virus group was not significant(P>0.05).CCK8 assay was used to detect the growth curves of the six groups of cells for 7 days.The results showed that the cell proliferation ability of A2780/DDP cells after ITG4 gene down-regulation was significantly weakened from the 4th day(P<0.05),and A2780 cells up-regulated ITGB4 gene from the 4rd day.The cell proliferation ability was significantly increased(P<0.05).(4)In the cell cycle assay by flow cytometry,cisplatin induced an increase in S and G2 cells in each group compared with the group without DDP.When the DDP concentration reached 1 μg/ml,the ITGB4 knockout strain was more than two.In the control group,cells in the S/G2 phase were significantly decreased(P<0.05),and ITG4 overexpressing cells significantly increased in cells arrested in the S/G2 phase(P<0.05).(5)In the transwell experiment,ITGB4 knockout strains had lower migration and invasive ability than the other two control groups(P<0.05),and ITGB4 overexpression strains showed greater migration and invasion than theother two control groups(P<0.05).CONCLUSION: ITGB4 down-regulation attenuates the resistance,proliferation,migration,and invasion ability of ovarian cancer A2780/DDP cells.Cisplatin down-regulates the cell line arrest at S/G2 phase and the cisplatin sensitivity increases.ITGB4 up-regulation attenuated the resistance,proliferation,migration and invasive ability of ovarian cancer A2780 cells.In response to cisplatin,ITGB4 up-regulation of cell lines increased at the S/G2 phase and cisplatin sensitivity decreased.CHAPTER 4 EFFECTS AND MECHANISM OF ITGB4 ON AUTOPHAGY AND APOPTOSIS IN MULTIDRUG RESISTANCE OF A2780/DDP CELLSOBJECTIVE: To investigate whether the effect of ITGB4 gene on the resistance of ovarian cancer cells is related to autophagy and apoptosis and which pathway to regulate.METHODS:(1)4μg/ml cisplatin were treated with A2780/DDP scramble control cells and ITGB4 knockout cells for 24 hours,respectively,and the formation of autophagosomes was observed by transmission electron microscopy.(2)A2780/DDP scramble control cells and ITGB4 knockout cells were treated with 4μg/ml cisplatin for 24 hours,and the aggregation and distribution of LC3 protein in the two groups were observed by laser confocal immunofluorescence.(3)A2780/DDP scramble control cells and ITGB4 knockout cells were treated with 4 μg/ml cisplatin for 24 hours,and the expression of LC3-II and p62 protein was detected by Western blot.(4)A2780,A2780-NC,A2780-ITGB4 overexpressing cells and A2780/DDP,A2780/DDP-NC,A2780/DDP-ITGB4 knockout cells were treated with 4 μg/ml cisplatin for 24 hours,and Autophagy and expression of apoptosis-relatedproteins was examined by Western blotting in each group.RESULTS:(1)After cisplatin treatment,TEM showed that autophagy bodies were formed in the A2780/DDP scramble control group,and A2780/DDP-ITGB4 knockout cells had no autophagy body formation.(2)After treatment with cisplatin,the LC3 protein in the control group and the experimental group began to agglomerate and distributed in the cytoplasm by immunofluorescence assay.Among them,the phenomenon of LC3 aggregation in the experimental group was weaker than that in the control group.(3)After cisplatin treatment,the expression of LC3-II protein in control group and experiment increased.The expression of LC3-II protein in the experimental group was lower than that in the control group(p<0.05).The expression level of p62 was higher than that in the control group(p<0.05).The degree of autophagy in the experimental group may be considered weaker than that in the control group.(4)After treatment with 4 μg/ml cisplatin,compared with the control group,the expression of ERK1/2,AMPK,PTEN,AKT1,p-AKT1,p-AMPK,m TOR,and p-mTOR protein in the over-expression group cells was no significant difference.In the ITGB4 overexpression group,the expression of p53 protein was increased,but there was no statistical significance(p>0.05).In the ITGB4 knockout group,p53 protein expression was significantly downregulated(p<0.05).CONCLUSION: Knocking down ITGB4 gene reduced the autophagy induced by cisplatin in drug resistant ovarian cancer cells.ITGB4 may enhance the resistance of ovarian cancer cells through autophagy and may be through p53 mediated autophagy pathway. |
PDF Full Text Request