The Molecular Mechanism Of MicroRNA-31-5p Regulating 14-3-3? On The Proliferation And Apoptosis Of Prostate Cancer Cells

Prostate cancer(PCa)is one of the most common malignancies and the second most prevalent cancers in men worldwide.Almost all patients with advanced PCa will develop into castration-resistant prostate cancer(CRPC)after receiving endocrine therapy.Although the androgen receptor(AR)is an effective way to treat metastatic castrate-resistant prostate cancer(mCRPC),29,430 people still die of PCa in the USA in 2018.Recent studies on the 14-3-3 protein family revealed that the family member 14-3-3? plays important roles in the occurrence and development of PCa and may serve as molecular targets for the diagnosis and treatment of PCa.Meanwhile,a large number of studies has confirmed that miRNAs also play crucial roles in the occurrence and development of PCa,and possess broad application prospects in the molecular diagnosis and targeted therapy of PCa.However,the molecular mechanism of miRNAs regulating 14-3-3? in the occurrence and development of PCa remains unclear.In this study,UALCAN-TCGA online database combined with qRT-PCR,CCK-8,cell scratched,Transwell,double luciferase system,flow cytometry and other experimental methods were used to clarify the molecular mechanism of miRNAs regulating 14-3-3? on the proliferation and apoptosis of PCa cells.This study will provide new insight into molecular diagnosis of PCa and development of targeted anticancer drugs.The main results are as following:1.Expression of 14-3-3? in PCa tissues and cells and its effect on proliferation of 22Rv1 cellsThe results of UALCAN-TCGA database analysis showed that different subtypes of 14-3-3 protein had different expression patterns in PCa tissues and paracancerous tissues.The expression of 14-3-3?/YWHAE,14-3-3?/YWHAG and 14-3-3?/YWHAQ were up-regulated in PCa tissues,while 14-3-3?/YWHAH,14-3-3?/ YWHAB and 14-3-3?/YWHAZ were down-regulated in PCa tissues.Meanwhile,the expression pattern of 14-3-3 ? in different PCa cells,such as PC3,22Rv1 and LNCaP,was similar to that in PCa tissues.The results of CCK8 cell proliferation test,cell scratch test and Transwell test showed that the interference of 14-3-3? expression obviously affected the proliferation,migration and invasion of 22Rv1 cells(P < 0.01).These results suggested that 14-3-3? might play a role of proto-oncogene in PCa.2.Screening and functional verification of microRNAs regulating 14-3-3?According to the prediction of online softwares including Targetscan,mi RSystem,miRanda and PicTar,miR-155-5p,miR-31-5p,miR-29b-3p,miR-29a-3p and miR-29c-3p were the five mi RNAs possessing the highest score of 14-3-3 ?.The results of qRTPCR,double luciferase test and CCK-8 test showed that mi R-31-5p and 14-3-3 ? were inversely expressed in 22Rv1 cells,and miR-31-5p was the most efficient miRNA in regulating the expression of 14-3-3?.The wild-type and mutant luciferase reporter vectors experiments showed that the 7 base sites of "UCUUGCC" in 14-3-3? 3'UTR had specific functional targets for miR-31-5p to regulate the expression of 14-3-3?.The deletion of these sites remarkably affected the regulatory ability of miR-31-5p on 14-3-3? gene.3.Effect of 14-3-3? regulated by miR-31-5p on phenotype of 22Rv1 cellsThe results of CCK-8,cell scratch and Transwell experiments showed that miR-31-5p overexpression seriously affected the proliferation,migration and invasion of 22Rv1 cells.Flow cytometry showed that the upregulation of mi R-31-5p directly led to the arrest of 22Rv1 cells in G1 phase and indirectly inhibited the proliferation of 22Rv1 cells.The results of apoptosis assay showed that the upregulation of mi R-31-5p promoted the early apoptosis and necrosis of 22Rv1 cells.The results of compensation test showed that miR-31-5p and 14-3-3? cotransfection partially reversed the proliferation,migration and invasion ability of mi R-315 p on 22Rv1 cells,and also partially reversed the inhibition of mi R-31-5p on 22Rv1 cell cycle and the proapoptotic ability of 22Rv1 cells.4.Effect of 14-3-3 ? regulated by mi R-31-5p on PI3K/Akt/Bcl-2 signaling pathwayThe results of signal pathway about the effects of miR-31-5p regulating 14-3-3 ? on 22Rv1 proliferation and apoptosis showed that mi R-31-5p upregulated the ratio of p-PI3K/ PI3 K and p-AKT/AKT,increased the expression levels of Bad and Bax/Bcl-2,and activated the cascade reactions related to apoptosis such as caspase-9 and caspase-3.However,co-transfection of miR-31-5p and 14-3-3? partially reversed this effect.These results suggestted that mi R-31-5p could inhibit the proliferation and promote apoptosis of 22Rv1 cells by regulating PI3K/Akt/Bcl-2 signaling pathway.In summary,this study demonstrated the significant increase of 14-3-3? protein in PCa tissues and cells,and found that miR-31-5p was the best miRNAs for targeted regulation of 14-3-3? expression,which made the association between miR-31-5p targeted regulation of 14-3-3? and apoptosis in 22Rv1 cells,and clarified the possible molecular mechanism for the effect of miR-31-5p regulating 14-3-3? gene on the proliferation and apoptosis of 22Rv1 cells.These findings provide a new theoretical basis for the combined application of miR-31-5p and 14-3-3? in CRPC precision therapy.