MiR-15a/16 Regulates Invasion Of Prostate Cancer LNCaP Cells Through Targeting TGF-? Signaling Pathway

Objective:Prostate cancer is one of the most common malignant tumors of the male genitourinary system.The incidence of prostate cancer ranks the third in China.In recent years,the incidence of prostate cancer has increased rapidly,and the age of onset has become gradually younger.As the main biological characteristics of malignant cancer,metastasis is the most important factor affecting the recurrence and prognosis of tumor.For example,as the most common way of metastasis in prostate cancer,bone metastasis occurred at least 65%-75% of prostate cancer patients,and the metastasis rate is as high as 85% to 100% in those who die of prostate cancer.To clarify the molecular biological mechanism for metastasis of prostate cancer will be meaningful and provide theoretical basis for the treatment of prostate cancer.Micro RNA(micro RNA,miRNA)is an important regulatory molecule of gene at post-transcriptional level.The miR-15a/16 cluster is located in 13q14.2,and its deficiency occurs frequently in prostate cancer.Recently,miR-15a/16 is proved to act as a tumor suppressor gene,and plays a vital function in the process of tumorigenesis and progression.In prostate cancer,13q14.2 microdeletion leads to loss of heterozygosity of micro RNAs-15a/16 clusters,especially in metastatic prostate cancer,suggesting that deletion of micro RNAs-15a/16 clusters may be closely related to metastasis of prostate cancer.TGF-beta signaling pathway is one of the hotspots in the study of metastasis mechanism of prostate cancer.Typical pathway is TGF-beta/Smads signaling pathway.In addition,signal molecules such as Smads,BMP and Activin in the TGF-beta superfamily play important roles in tumor metastasis,and may be potential targets for the regulation of micro RNA-15a/16.Nowadays,it is not clear whether miR-15a/16 can regulate the metastasis of prostate cancer by regulating related signaling molecules of TGF-beta signaling pathway.In this study,bioinformatics prediction,reporter gene assay,real-time PCR and Western blot were used to confirm the mechanism of micro RNAs-15a/16 regulating metastasis of prostate cancer by targeting molecules related to TGF-beta signaling pathway.Materials and MethodsMaterials:1.Human prostate cancer cell line LNCa P and Cell line HEK2932.Reagents for gent cloning.3.Reagents for luciferase assay4.Reagents for Real-time PCR and Western blot 5.Reagents for Transwell assayMethods: cell culture,bioinformatics prediction,transient gene transfection method,dual-luciferase reporter gene assay,Real-time PCR assay,Western blot detection,Transwell assay,statistical analysis.Results:1.Bioinformatics analysis showed putative miR-15a/16 binding sites at the 3?-UTR of Smad3,Smad5,BMPR1 A,ACVR2A and ACVR2 B.2.Over-expression of miR-15a/16 decreased reporter activity of Smad3,ACVR2A3?-UTR construct significantly;and inhibiting of miR-15a/16 with miR-15a/16 inhibitor increased the reporter activity.3.Western Blot and Real-time PCR results showed that Smad3 and ACVR2 A protein level were decreased significantly after transfection of miR-15a/16,but endogenous Smad3 and ACVR2 A m RNA level didn't change markedly.In addition,Overexpression of miR-15a/16 decreased the protein expression of p-smad3 and MMP2,the m RNA expression of Snail1 and Twist1.4.Transwell assay showed that over-expression of miR-15a/16 suppressed the invasion of LNCa P cell.Conclusions:1.miR-15a/16 suppresses the Smad3 and ACVR2 A expression at the post-transcriptional level by binding to the 3?-UTR of Smad3 and ACVR2 A.2.miR-15a/16 regulates the invasion of LNCa P cell though Activin/smad signaling pathway.