A2M And CRABP2 Reverse Ovarian Cancer Platinum Resistance Via Fas Apoptosis Signaling Pathway

Objective On the basis of our previous studies, the first objective of the experiment is respectively importing the genes A2M and CRABP2 to the TCGA data base which offers 499 cases of ovarian cancer patients genome-wide expression patterns to analyze the correlation between them and the platinum chemotherapy resistance, the clinical prognosis of the ovarian cancer patients, in order to provide clinical evidence for regarding A2M and CRABP2 as the potential upstream regulatory molecules of ovarian cancer platinum resistance signaling pathway. In addition, the other purpose of this study is to make further functional verification of the genes A2M and CRABP2 both on the gene and protein levels on the basis of the above research results, to explore the influence on the downstream cisplatin-resistance-associated signaling pathways after their imbalance expression, and to lay foundation for searching out new cisplatin-resistance-reverse targets of ovarian cancer.Methods The gene expression profiles of the ovarian cancer patients in the TCGA database were used to analyze the expression differences of gene A2M and CRABP2 in the drug sensitive and drug resistance patients. ROC curve analysis is used to analyze the relationship between them and platinum resistance. Pearson correlation analysis is taken to discuss the relationship between the expression levels of A2M and CRABP2 and clinical drug resistance and clinical outcomes of ovarian cancer patients (including the patient’s overall survival, progression-free survival and interval of chemotherapy survival). The sensitivity of A2M and CRABP2 in the diagnosis of ovarian cancer patients with drug resistance is taken by Cox regression analysis. Finally the expression spectrum data of all the 283 cases of ovarian cancer patients in the TCGA basis are divided into two groups according to high and low expression of A2M and CRABP2 expression and Kaplan-Meierw analysis is applied to compare the differences of the patients’platinum chemotherapy interval between A2M and CRABP2 high and low expressed groups. In addition, the green fluorescent sensitive SKOV3 ovarian cancer cells (SKOV3-GFP) and cisplatin resistant cells (SKOV3-GFP/DDPⅡ) we have built are inoculated subcutaneously into nude mice to develop implantation tumor. Real-time PCR and immunoblotting are respectively taken to detect the mRNA and protein expression level of A2M and CRABP2 in the tumor tissues with different times of cisplatin intervention. Then we use both real-time fluorescent quantitative PCR and Western Blotting to detect the expression level of the important cell apoptosis-associated node genes on the Fas pathway in the above tumor tissues. On the other hand, we plan to use the PWPI and pSico slow virus expression system and the established cisplatin acquired drug resistant ovarian cancer cell lines SKOV3-GFP/DDPⅡ to build a CRABP2 over-expressed cisplatin resistance cell line SKOV3-GFP/DDPⅡ-CRABP2, which is marked by the mcherry fluorescent tags. Then the changes of the cells’biological behaviors such as IC50 and resistance index are determined by CCK kit and with the purpose of verifying the function of CRABP2, Western Blot is taken to detect the protein expression changes of the key genes on the Fas apoptosis signal pathway and the important genes that are associated with the cell cycle reglation.Result A2M and CRABP2 are relevant with the clinical drug resistance of ovarian cancer patients and relative to the sensitive cases, they expressed lower in the patients with platinum resistance(P<0.05, P<0.05), which is highly correlated with the Overall Survival, the Platinum Free Interval and the Progression Free Survival of ovarian cancer patients (P<0.00, P<0.00), the higher they expressed, the longer patients survival. Cox regression analysis showed that the genes can be regarded as the independent prognostic indicators(Figure 3).Receiver operating characteristic (ROC curve) analysis showed that the expression levels of A2M and CRABP2 are relevant with drug resistance(Area=0.969,0.980). In addition, Kaplan-Meier analysis showed that there were significant differences in the patients’platinum chemotherapy survival time between the two groups (P<0.00). The genes A2M, CRABP2, the cell apoptosis related important genes Fas, FADD, and the key apoptosis effect factors Caspase10, Caspase9 and Caspase3 are relatively lower express in the transplantation tumor of resistant cells along with the cisplatin injection (P<0.00), while its downstream target gene PARP1, which is very important to cell DNA repair, is relatively high expresses in the resistant transplantation tumor tissues (P<0.05). The protein expressions of all these genes in the transplanted tumor tissues show roughly consistent trends with their mRNA expression. There are linear correlations between the expressions of the genes that may be associated with platinum mentioned above and the node genes on the Fas signal pathway respectively (P<0.05, P<0.00).IC50 with cisplatin and the resistant index of the SKOV3-GFP/DDP II-CRABP2 cell line are significantly lower relative to the SKOV3-GFP/DDP II and the control SKOV3 GFP/DDP Ⅱ-Blank cell lines (P<0.00). The cell cycle of the SKOV3-GFP/DDP Ⅱ-CRABP2 cell line was significantly suppressed while the cell apoptosis was activated. The protein expressions of the cell cycle dependent protein kinase CDK2, CDK4, CDK6 and the cell cycle protein cyclinDl in the SKOV3-GFP/DDP Ⅱ-CRABP2 cell line was significantly decreased (P<0.00), while the expression of the cell cycle blocker protein p27 kip1 was significantly enhanced (P<0.00). The protein expressions of the nodes genes Fas, FADD, Caspase10, Caspase9 and Caspase3 which are on the Fas apoptosis signaling pathway were significantly increased (P<0.00), while the downstream gene PARP1 which was related to DNA repair was reduced (P<0.05). Namely the sensitivity to cisplatin of the cisplatin resistant ovarian cancer cells was recoverd after CRABP2 overexpression.Conclusion A2M and CRABP2 are associated with the platinum resistance of ovarian cancer and they express lower in the platinum resistant ovarian cancer cells and tissues. As potential upstream molecules of the platinum resistance associated critical signaling pathways, both of them may maintain the sensitivity of the ovarian cancer cells to platinum drugs by activating the Fas apoptosis signaling pathways. And their overexpression may bring up the reactivity recovery of the resistant ovarian cancer cells to cisplatin, which provides new tagets and theoretical basis for further study on chemoresistant reversal.