| BackgroundOvarian cancer is the deadliest gynecologic malignancy.Although cytoreductive surgery combined with platinum-based chemotherapy has significantly improved the therapeutic effect of ovarian cancer in recent years,most patients relapse within 2-3 years after treatment and develop resistance to platinum-based chemotherapy.The overall five-year survival rate of the patients is only 40%.Therefore,it is particularly vital to clarify the mechanism of platinum-resistance.Autophagy is the process in which damaged,denatured or senescent proteins and organelles are transported to lysosomes for digestion and degradation.Tumor cells maintain cellular growth through autophagy so that can resist chemotherapeutic drugs.Thioredoxin-associated protein14(Thioredoxin-related protein of 14 kDa,TRP14),a disulphide reductase discovered in 2004,is widely expressed in tissues and involved in the regulation of TNF-α(Tumor necrosis factor-α)signal pathway and autophagy.However,it still remains unclear whether TRP14 can be involved in platinum-resistance of ovarian cancer by regulating autophagy.ObjectiveThis study aims to investigate the expression of TRP14 in ovarian carcinoma tissues and its correlativity with the expression level of Beclin 1,to investigate the role of autophagy in platinum-resistance of ovarian cancer and to elucidate the role and molecular mechanism of TRP14 in the regulation of platinum-resistance through autophagy.The principal objective of this study is to provide a theoretical basis for TRP14 and Beclin 1 being predictors of chemotherapy response and prognosis among ovarian cancer patients,and to serve as an experimental underpinning for TRP14 being a potential therapeutic target in platinum-resistant ovarian cancer.Methods 1.The expression of TRP14 and Beclin l in ovarian carcinoma tissues and their correlationThe expression of TRP14 mRNA in ovarian cancer was detected with Oncomine database.Kaplan-Meier-Plotter database was used to analyze the expression of TRP14 mRNA and autophagy regulatory protein Beclin 1mRNA in ovarian carcinoma tissues,as well as the correlation with ovarian cancer prognosis and chemotherapy resistance.Immunohistochemical staining was utilized to observe TRP14 and Beclin 1 expression in ovarian carcinoma and paracancerous tissues,and their correlation.2.The effect of autophagy on platinum-resistance of ovarian carcinoma cellsThe parental cell lines SKOV3 and A2780,the platinum-resistant SKOV3/DDP,A2780/DDP of ovarian cancer were cultured in vitro.The platinum-resistance of SKOV3/DDP and A2780/DDP was examined with CCK-8 assay.The expression of autophagy related proteins was measured by Western blotting.The formation of autophagy vesicles was observed using MDC staining with laser confocal fluorescence microscope.The cell model of RFP-LC3 was constructed by transfecting RFP-LC3 plasmid into the cell.The amount of positive vesicles in cells was observed and evaluated under a laser confocal fluorescence microscope,and the ultrastructure of intracellular autophagosomes was observed with a transmission electron microscope.The above experiments evaluated the level of autophagy in platinumresistant ovarian carcinoma cells.After SKOV3/DDP and A2780/DDP cells were processed with autophagy inhibitor 3MA,the expression of autophagy-related proteins was detected with Western blotting.CCK-8 assay was used to analyze the platinum-resistance of cells.ATG5 expressions in SKOV3/DDP and A2780/DDP cells were down-regulated by gene silencing,and the changes of platinum-resistance in SKOV3/DDP and A2780/DDP were assayed by CCK-8.SKOV3 and A2780 cells were subjected to autophagy activator Rapamyci(RAPA).Western blotting was utilized to assess the expression of autophagy-associated proteins.MDC staining was adopted to observe the formation of autophagy vesicles.This study also applied the laser confocal fluorescence microscope to observe the amount of positive vesicles after the transfection of plasmid RFP-LC3.The transmission electron microscope was used to observe the ultrastructure of autophagosomes,and the drug-resistance of cells was assessed by CCK-8.Through the above experiments,the impact of autophagy on platinum-resistance in ovarian carcinoma cells was evaluated.3.The regulatory of TRP14 on autophagy and its effect on platinum-resistance of ovarian carcinoma cellsThe parental strains SKOV3,A2780,and platinum-resistant strains SKOV3/DDP and A2780/DDP of ovarian cancer cells were cultured in vitro.The protein expression of TRP14 was analyzed by Western blotting.The expression of TRP14 in SKOV3/DDP and A2780/DDP cells was down-regulated by gene silencing,and the expression of TRP14 in SKOV3 and A2780 cells was up-regulated by transfection of the over-expression plasmid pcDNA3.1(+)-TRP14.Western blotting was utilized to assess the expression of autophagy-associated proteins.The formation of autophagy vesicle was observed using MDC staining.Laser confocal fluorescence microscope was used to observe the amount of positive vesicles after the transfection of RFP-LC3 plasmid.Furthermore,the ultrastructure of autophagosomes was detected under a transmission electron microscope.Through the above experiments,the effects of TRP14 on autophagy of ovarian cancer parental strains and drug-resistant strains were evaluated respectively.After down-regulating the expression of TRP14 in SKOV3/DDP and A2780/DDP,CCK-8 assay was used to evaluate the platinum-sensitivity.SKOV3/DDP and A2780/DDP with down-regulated TRP14 expression were processed with the X autophagy activator RAPA,and the platinum-sensitivity was assayed by CCK-8.After the over-expression of TRP14,the effect of platinum drugs on the cellular activity of SKOV3 and A2780 was assayed with CCK-8.In the overexpressed TRP14 parental cells,the platinum-resistance of gene silenced autophagy regulatory protein ATG5 was detected via CCK-8.Through the above experiments,the effect of autophagy regulated by TRP14 on platinum-resistance of ovarian cancer cells was determined.The expressions and phosphorylation levels of AMPK,mTOR,P70S6 K in SKOV3/DDP and A2780/DDP cells were analyzed by Western blotting.Plasmid transfection was applied to up-regulate TRP14 expression in SKOV3 and A2780,and gene silencing was used to down-regulate the expression of TRP14 in SKOV3/DDP and A2780/DDP.Moreover,the effects of TRP14 on the expression and phosphorylation level of AMPK,mTOR,and P70S6 K were detected.After treating SKOV3/DDP and A2780/DDP with AMPK inhibitor Compound C,the effect of platinum on cellular viability was assayed by CCK-8.Through the above experiments,the molecular mechanism of TRP14 affecting platinum-resistance by regulating autophagy in ovarian carcinoma cells was investigated.Results 1.The expression of TRP14 and Beclin l in ovarian carcinoma tissues and their correlationThe Bioinformatics analysis of this study suggested that compared with healthy ovarian tissues,the expression of TRP14 mRNA was up-regulated in many subtypes of ovarian cancer tissues.The mRNA levels of TRP14 and Beclin 1 were negatively correlated with the survival time of patients with ovarian cancer.The results of immunohistochemical staining showed that in comparison with the adjacent tissues para-tumorous tissues,the protein expression of TRP14 and Beclin 1was up-regulated in ovarian cancer tissues,and their expression levels were positively correlated.2.The effect of autophagy on platinum-resistance of ovarian carcinoma cellsTreated with the same dose of cisplatin,the cell viability of the drug-resistant strain cells SKOV3/DDP and A2780/DDP was significantly increased compared with the parental strain cells SKOV3 and A2780.The protein expressions of Beclin 1,LC3 II and ATG5 in the autophagy regulatory protein were significantly up-regulated.The amount of autophagy vesicles and RFP-LC3 positive vesicles increased significantly,and the structures of typical autophagosomes and autophagy lysosomes were clearly observed.It is suggested that the autophagy levels of drug-resistant cells SKOV3/DDP and A2780/DDP was increased.In order to verify the above results,after SKOV3/DDP and A2780/DDP were subjected to autophagy inhibitor 3MA,the expression of autophagy-related proteins Beclin 1,LC3 II and ATG5 were significantly down-regulated,and the platinum-sensitivity increased.The platinum-sensitivity of SKOV3/DDP and A2780/DDP cells increased significantly followed by the gene silencing of ATG5.Autophagy occurred in parental SKOV3 and A2780 after the intervention with autophagy activator RAPA while platinum-sensitivity decreased.It showed that autophagy is one of the essential mechanisms of platinum-resistance in ovarian cancer.3.The regulation of TRP14 on autophagy and its effect on platinum-resistance of ovarian cancerCompared with parental SKOV3 and A2780 cells,the expressions of TRP14 in platinum-resistant cell lines SKOV3/DDP and A2780/DDP were significantly up-regulated,suggesting that TRP14 might be involved in the regulation of cell resistance.The expressions of autophagy regulatory proteins Beclin 1,LC3 II,and ATG5 in drug-resistant SKOV3/DDP and A2780/DDP were significantly down-regulated after gene silencing TRP14.The numbers of autophagy vesicles and RFP-LC3 positive vesicles were significantly reduced,while the intracellular autophagosome and autophagolysosome structure decreased.After over-expressing of XII TRP14,the expressions of autophagy regulatory proteins Beclin 1,LC3 II,and ATG5 in parental SKOV3 and A2780 cells were significantly up-regulated,while the amount of autophagy vesicles and RFP-LC3 positive vesicles increased distinctly.The structure of intracellular autophagosome and autophagy-lysosome increased.These results indicated that TRP14 was involved in the regulation of autophagy in ovarian cancer.After gene silencing TRP14,the sensitivity of platinum-resistant SKOV3/DDP and A2780/DDP increased.This process could be reversed by autophagy activator RAPA.After over-expressing TRP14,the platinum-sensitivity of parental cell lines SKOV3 and A2780 were decreased,which could be reversed by ATG5 gene silencing.It showed that TRP14 is one of the key proteins of autophagy in the regulation of drug resistance in ovarian cancer cells.The Western blotting analysis showed that compared with the parental cell lines SKOV3 and A2780,the phosphorylation level of AMPK in drug-resistant SKOV3/DDP and A2780/DDP cells was up-regulated.While the phosphorylation level of mTOR and P70S6 K was down-regulated.After overexpressing TRP14 in parental cell lines SKOV3 and A2780,the phosphorylation level of AMPK was up-regulated,and the phosphorylation level of mTOR and P70S6 K was down-regulated.After Silencing TRP14 in SKOV3/DDP and A2780/DDP,phosphorylation of AMPK was down-regulated and phosphorylation of mTOR and P70S6 K was up-regulated.It is suggested that TRP14 induced autophagy through the AMPK/mTOR/P70S6 K signal pathway,thereby increasing platinum-resistance in ovarian carcinoma cells.Conclusion1.TRP14 is highly expressed in ovarian cancer;its expression level is positively correlated with Beclin 1 expression and negatively correlated with the survival time of patients.2.Platinum drugs promote autophagy in ovarian cancer cells,thereby induce ovarian cancer cells become platinum-resistant.3.TRP14 changes the autophagy level of ovarian cancer cells and leads to platinum-resistance.4.TRP14 affects drug-resistance by regulating autophagy through the AMPK/mTOR/P70S6 K signal pathway. |
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