Figure 4

miR-299-3p expression reduced expression of AR, PSA and VEGFA, and increased miRNA/mRNA interaction in RISC. All data show the mean ± SD of at least 3 independent experiments. (A) Immunoblot analysis showing reduced expression of full length AR and AR-V7 in lysates from induced C4-2B-299 and 22Rv-1-299 cells overexpressing miR-299-3p compared to uninduced cells. Alpha tubulin was used as the internal control. (B) Densitometric analysis of full-length AR and AR-v-7 protein expression normalized to internal controls. (C) Immunoblot analysis showing reduced expression of VEGFA in lysates of induced C4-2B-299 and PC-3-299 cells compared to uninduced or induced Scr RNA expressing PC-3 cells. GAPDH and α-tubulin were used as the internal controls. (D) Densitometric analysis of VEGFA protein expression normalized to internal controls. (E,F) Quantitative RT-PCR of miR-299-3p, AR, PSA and VEGFA in RNA extracted from the induced C4-2B-299 cells transfected with miR-299-3p inhibitor or control inhibitor. (E) Average fold-change in expression of miR-299-3p and AR showing reduced expression of miR-299-3p and increased expression of AR mRNA in induced C4-2B-299 cells upon transfection of miR-299-3p inhibitor compared to the control inhibitor. Decreased expression of AR mRNA could be noted in the untransfected induced C4-2B-299 cells compared to the uninduced cells. (F) Average fold-change in expression of PSA and VEGFA mRNAs showing rescue effect upon transfection of miRNA inhibitor compared to the control inhibitor. Reduced expression of PSA and VEGFA mRNAs could be noted in the untransfected but induced C4-2B-299 cells. (G) Quantitative RT-PCR of PSA and VEGFA in RNA extracted from the induced C4-2B-299 cells transfected with miR-299-3p inhibitor or control inhibitor followed by treatment with and without dihydrotestosterone (DHT). Average fold change in expression showing increased expression of PSA and VEGFA mRNAs in the absence of DHT treatment in cells transfected with miR-299-3pi. Treatment with DHT showed greater increase in PSA and VEGFA mRNA expressions in miR-299-3pi transfected cells compared to control inhibitor. (H–J) RNA-immunoprecipitation studies with the pan-AGO antibody was used in the pulldown of interacting miRNAs-mRNAs complexes. Total RNA extraction from pulldown samples followed by qRT-PCR analysis revealed enrichment of miR-299-3p (3.3-fold), AR mRNA (2.6-fold) and VEGFA mRNA (1.96-fold) in the induced C4-2B stable line expressing miR-299-3p compared to the uninduced control. Quantitative RT-PCR of miR-299-3p, AR and VEGFA in the input RNA from induced C4-2B-299 cells showing increased miR-299-3p, and decreased AR and VEGFA mRNAs compared to uninduced cells.