| Objective:Prostate cancer is the second most common malignancy in men and the second most common cause of cancer death in men,second only to lung cancer.In recent years,with the change of Chinese lifestyle,the incidence of prostate cancer has also been rising rapidly,but the specific pathogenesis and etiology of prostate cancer have still not been clearly explained to a large extent.As a member of the RP gene family,RPS15a has been increasingly studied in the regulation of biological functions of tumor cells,and it has been confirmed that RPS 15a is highly expressed in most tumor tissues and cells,and plays a key role in the occurrence and development of tumors.However,the expression and role of RPS 15a in prostate cancer have not been reported.Therefore,the purpose of this study was to investigate the expression of RPS 15a in prostate cancer and normal prostate tissues and its correlation with clinicopathological data.Secondly,the effect of RPS15A gene expression on the biological function of prostate cancer DU 145 and PC-3 cells was investigated.Methods:1.The expression of RPS15AmRNA in prostate cancer tissues and normal tissues was analyzed by The Cancer Genome Atlas(TCGA)database;2.The expression of RPS15A protein in tissue microarray(including 159 prostate cancer tissues and 80 normal prostate tissues)was detected by immunohistochemical assay,and the correlation between RPS 15 A and age,distant metastasis,PSA value,T stage,lymph node metastasis,clinical stage and Gleason grade was analyzed.3.According to the design principles of RNA interference sequence and the evaluation of the design software,three groups of sequence shRPS15A-1,shRPS15A-2 and shRPS15A-3 were selected as interference series,and the prostate cancer DU 145 cells were respectively transfected with lentiviruses containing three groups of different RNA interference sequences.The most effective interference series of RPS15A gene were screened by qPCR.4.The selected RNA interference series were transfected into DU145 and PC-3 cells,and the cell transfection efficiency was monitored by fluorescence microscopy;The transfection efficiency of DU145 and PC-3 cells was verified by qPCR and WB at RNA and protein levels,respectively.5.The effect of RPS15A on the proliferation of prostate cancer cells was detected by cell clone formation assay and Celigo cell count assay;6.The effect of RPS15A on the migration and invasion of prostate cancer cells in vitro was detected by scratch assay and Transwell invasion assay;7.The effect of RPS15A on apoptosis of prostate cancer cells was detected by flow cytometry.Results:1.Gene expression profile analysis was based on RNAseq data from 498 prostate cancer tissues and 52 normal prostate tissues from TCGA database.Results showed that the median,25%and 75%of RPS15A expression levels in prostate cancer tissues were higher than those in normal prostate tissues.2.The expression of RPS15A protein in tissue microarray was detected by immunohistochemical assay,which indicated that the expression of RPS15A protein in prostate cancer tissues was significantly higher than that in normal prostate tissues(P<0.001).Statistical analysis showed that the expression of RPS15A protein was significantly different with the depth of tumor invasion(T stage)(P<0.05),and there was no significant difference between the expression of RPS15A protein and age,distant metastasis,PSA value,lymph node metastasis,clinical stage and Gleason grade(P>0.05).Meanwhile,correlation analysis showed that the expression of RPS15A protein was positively correlated with the depth of tumor invasion(T stage).3.The effective interference series results of qPCR screening showed that the RPS15A gene knockdown in the shrpsl5a-2 and shrpsl5a-3 groups had no significant changes compared with the control group when the lentivirus containing three groups of different RNA interference sequences was transfected into prostate cancer cell DU145.The RPS15A gene knockout efficiency of the shrpsl5a-1 group was 83.6%(P<0.001),so lentiviruses containing the interference series of the shrpsl5a-1 group were used for the subsequent experiment.4.72h after the infection of prostate cancer DU 145 and PC-3 cells with lentivirus,the observation results under fluorescence microscope showed that the infection efficiency of the experimental group(shRPS 15A)and the control group(shCtrl)cells reached more than 80%,and the cells grew well.qPCR and WB validation showed that the knockout efficiency of RPS15A gene in DU145 and PC-3 cells infected with lentivirus was 51.9%compared with control cells(P<0.001)and 91.8%(P<0.01),and the expression of RPS15A protein was down-regulated;5.Clonal formation experiments showed that the number of clones of DU 145 and PC-3 cells decreased after being infected by lentivirus(P<0.01 and P<0.001,respectively).Celigo cell count assay showed that the cell count of PC-3 cells and DU145 cells increased slowly and cell proliferation was significantly inhibited after lentivirus infection(P<0.001).6.The results of scratch experiment showed that the migration rate of PC-3 cells of DU 145 cells was reduced by 27%(P<0.001)and 53%(P<0.01)compared with the control group after infection with lentivirus.The results of in vitro invasion assay showed that the cell metastasis rate of PC-3 cells of DU 145 cells was reduced by 56%(P<0.001)and 85%(P<0.001)compared with the control group after infection with lentivirus.7.Flow cytometry results showed that apoptosis of DU145 cells and PC-3 cells was increased compared with the control group after lentivirus infection(P<0.001).Conclusion:1.RPS15 A gene expression in the tissue of prostate cancer is significantly higher than normal prostate tissue,the expression was positively correlated with tumor infiltration depth,with the patients with tumor infiltration degree deepening,RPS 15 A gene expression increased,prompt RPS 15 A gene may be associated with the development of prostate cancer,is expected to be as a potential new targets for diagnosis and treatment of prostate cancer.2.RPS15A gene can significantly promote the proliferation,invasion and migration of prostate cancer cells,and inhibit the apoptosis of prostate cancer cells. |
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