The Role And Mechanism Of RTN4 In The Development Of Human Prostate Cancer

Objective:Reticulon-4(RTN4),a reticulon family protein localized in the endoplasmic reticulum,is reported to be involved in multiple physiological processes like neuroendocrine secretion and membrane trafficking in neuroendocrine cells.Several studies had demonstrated that RTN4 plays an important role in regulating apoptosis via interaction with Bcl-2 and Bcl-XL-like family.Although it seems that RTN4 is involved in development of multiple types of cancer including in colorectal cancer,hepatocellular carcinoma,renal cell carcinoma,glioma,bladder cancer,and so on,its role in prostate cancer remains largely unknown.The purpose of this study was to explore the role and mechanism of RTN4 in the development of human prostate cancer,and to provide a possibly promising target for prostate cancer therapy.Methods:1.TCGA data mining was used to analyze the DNA copy number and expression profile of RTN4 in prostate cancer and normal prostate tissue.2.MicroRNA binding sites in 3 '-UTR of specific genes were predicted by TargetScan Human 7.0 and miRDB.Differentially expressed miRNA lists in human prostate cancers were queried at dbDEMC 2.0.Coexpression profiles of miRNAs were obtained at miRTarBase and dual-luciferase reporter gene assay was applied to validate target genes of miRNAs.3.The expression levels of RTN4 mRNA in PC-3,DU 145 and LNCaP cell lines were detected by quantitative real-time polymerase chain reaction(qRT-PCR).4.The expression levels of RTN4 protein in PC-3,DU 145 and LNCaP cell lines were detected by Western Blot.5.Recombinant lentiviral system for gene overexpression was constructed applied for introduction of overexpression of RTN4 in DU 145 and LNCaP cells.The expression levels of RTN4 protein in DU145 and LNCaP cells after transfection were detected by Western Blot.6.The proliferation of DU145 and LNCaP cells after transfection was detected by MTT assay,and the proliferation curve was drawn for comparative analysis.The survival of DU145 and LNCaP cells after transfection was detected by single colony formation assay.7.Cell cycle and cell senescence of transfected DU 145 cells were analyzed by flow cytometry and SA-?-gal staining.8.The phosphorylation of RTN4 and its sites were analyzed by database mining.Results:1.Database mining showed that RTN4 DNA copy numbers were higher,while both RTN4 mRNA and protein expression levels were relatively lower in prostate cancer than in normal prostate gland.2.Database mining,MicroRNA.Cog prediction and dual-luciferase analysis showed that chromosomal neighbor gene EML6 had similar expression patterns with RTN4 in prostate cancer tissues and cell lines,and further analysis revealed that both RTN4 and EML6 could be targeted by miR-148a-3p.3.qRT-PCR result showed that the mRNA levels of RTN4 was significantly higher in PC-3 cells as compared with DU145 and LNCaP cells.4.The different protein expression levels of RTN4 in PC-3,DU145 and LNCaP cell lines were showed by Western Blot results.As compared with DU 145 and LNCaP cells,the RTN4 was shown to be highly expressed in PC-3 cells.5.The overexpression of RTN4 protein in DU145 and LNCaP cells mediated by lentiviral system was verified by Western Blot analysis.6.MTT assay and single colony formation assay results showed that as compared with the control group,the cell proliferation rate was significantly lower,and the colony number of cell was also significantly fewer in DU 145 and LNCaP cells with overexpression of RTN4.7.The results of flow cytometry assay showed that 40.2%of RTN4 overexpressed DU 145 cells were in G0/G1 phase,whereas the percentage of which was 44.6%in control cells.Besides,28.5%of RTN4 overexpressed cells were in G2/M phase but only 22.4%of control cells were in this stage,suggesting that cell cycle was blocked in G2/M phase(P<0.05).SA-?-gal staining showed that cells with overexpression of RTN4 were showed to develop cellular senescence spontaneously.8.Database mining of the interaction network between normal prostate and tumor tissue shows that RTN4 could be phosphorylated at site amino acid residue tyrosine 591 and serine 107 by MAPKAPK2 and FYN,respectively.Conclusion:1.Both mRNA and protein expression levels of RTN4 are significantly down-regulated in prostate cancer as compared with normal prostate tissue.2.Overexpression of RTN4 inhibits the proliferation of prostate cancer cells.3.RTN4 may act as a potential target for development of new therapy against prostate cancer.