| Objective(s):Prostate cancer(Prostate cancer,PCa)is one of the most common malignant tumors in male cancer diseases,and its incidence is the first highest in elderly male urogenital malignant tumors and the second highest in male systemic malignant tumors in the world.In China,the incidence and mortality of PCa have increased significantly in recent years,whose main reasons include the aging population and the lack of early diagnosis and effective treatment of PCa.Many PCa patients have found metastasis at the first visit.At present,the treatment of late metastatic PCa is mainly new endocrine therapy,but endocrine therapy will eventually lead to castration resistance of PCa,and the treatment effect is greatly reduced.Chemistry therapy is an indispensable therapy for patients with castration-resistant prostate cancer(Castration resistant prostate cancer,CRPC),but most PCa patients develop drug resistance in the later stage of treatment,leading to chemotherapy failure.Therefore,investigating methods to overcome resistance to chemotherapeutic drugs is important for patients with CRPC.RPS15A belongs to the family of ribosomal proteins(RPs),which are involved in the development of many tumors.Numerous studies have found that many RPs family proteins can affect drug resistance in tumors.Cisplatin(DDP)mainly acts by inducing DNA damage,and the RPs family has extra-ribosomal functions such as DNA repair.Many studies have shown that many RPs family proteins are associated with DDP resistance in tumor cells.Previous studies of our research group have found that RPS15 A knockdown can inhibit the proliferation and invasion of DU145 in PCa cells and promote cell apoptosis.Therefore,this topic intends to examine the effect of RPS15 A overexpression on the biological function of DU145 in PCa cells and explore the correlation of RPS15 A and DDP resistance in PCa.Methods:1.The RPS15 A knockdown and overexpressed PCa cell lines DU145 stable strains shRPS15A-DU145 and oeRPS15A-DU145,and the lentiviral transfection efficiency was observed by fluorescence microscopy,and the RNA and protein expression levels of RPS15 A in the transfected cells were measured by fluorescence quantitative PCR and WB experiments,respectively;2.The effect of RPS15 A overexpression on the proliferation,migration and invasion ability of DU145 cells was tested by plate clone formation assay,cell scratch assay and Transwell invasion assay;the apoptosis of RPS15 A overexpressing DU145 cells was determined by flow cytometry;3.The cell survival rate of shRPS15A-DU145,oeRPS15A-DU145 and normal DU145 cells treated with DDP was measured by CCK-8 method,calculate the half inhibition concentration and drug resistance index,and evaluate the difference of DDP resistance;4.The effect of DU145 cells on DDP resistance after RPS15 A overexpression and knockdown was determined by plate clone formation assay and flow cytometry;5.The expression of apoptotic proteins in shRPS15A-DU145,oeRPS15A-DU145,and DU145 normal cells was determined by western blot(WB).Results:1.The PCa cell line DU145 at 72 h after infection with an RNA lentiviral vector,Fluorescence microscopy showed the infection efficiency of shRPS15A-DU145,oeRPS15A-DU145 and no-load groups of more than 80%,Good cell growth;The results of quantitative PCR showed,The m RNA expression of RPS15 A in the shRPS15A-DU145 group was significantly lower than that in the sh Ctrl-DU145 group,The m RNA expression of RPS15 A in the oeRPS15A-DU145 group was significantly higher than that in the oe Vec-DU145 group(P<0.01);The results of the WB experiment showed,The protein expression level of RPS15 A in the shRPS15A-DU145 group compared to the sh Ctrl-DU145 group,The protein expression level of RPS15 A was increased in the oeRPS15A-DU145 group as compared with the oe Vec-DU145 group;2.The results of the plate clone formation assay showed,After the overexpression of RPS15 A in DU145 cells,The number of cell clones and the clone formation rate increased(P <0.05);The results of the cell scratch assay showed that,After the overexpression of RPS15 A in DU145 cells,Compared with the control group,Cell migration rate increased by 9%(P <0.01);The results of Transwell invasion assay showed that,After the overexpression of RPS15 A in DU145 cells,Compared with the control group,The cell transfer rate was increased by 16%(P<0.01);The results of flow cytometry showed that,After the overexpression of RPS15 A in DU145 cells,Compared to the control group,Apoptosis was decreased(P <0.01);3.The results of CCK-8 method showed that after shRPS15A-DU145,oeRPS15A-DU145,DU145 and normal DU145 cells were treated with DDP,the cell survival rate of shRPS15A-DU145 group was significantly reduced at the same concentration,and the half inhibitory concentration and drug resistance index were also significantly lower than that of oeRPS15A-DU145 and normal DU 145 group(P<0.01);4.The results of plate clone formation assay showed that after treatment with DDP,the number and size of shRPS15A-DU145 cells were significantly inferior to oeRPS15A-DU145 and DU145 cells(P<0.01);5.The results of flow cytometry showed that after treatment with DDP,early and late apoptosis in shRPS15A-DU145 cells increased significantly compared with normal DU145 cells(P<0.001),and early and late apoptosis in oeRPS15A-DU145 cells decreased compared with normal DU145 cells(P<0.01);6.The results of WB experiments showed that after treatment with DDP,the expression of apoptosis-related proteins,but slightly decreased apoptosis in oeRPS15A-DU145.Conclusion(s):1.The overexpression of RPS15 A gene can promote the proliferation,invasion and migration of PCa cell line DU145 and inhibit cell apoptosis;suggesting that RPS15 A gene may be a potential target for the diagnosis,treatment and prognosis of PCa.2.Inhibition of RPS15 A gene expression increased the drug sensitivity of PCa cell line DU145 to DDP and decreased its resistance to DDP,while overexpression of RPS15 A gene decreased the drug sensitivity of PCa cell line DU145 to DDP and enhanced its resistance to DDP.It suggests that the RPS15 A gene is associated with chemosensitivity in PCa cells,and that the knockdown of RPS15 A is beneficial to strengthen the killing effect of DDP on PCa and reduce drug resistance. |
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