AKT in Differential miRNA Processing in Prostate Carcinoma

Our original hypothesis was AKT regulates the RNA-induced silencing Complex (RISC) activity through the phosphorylation of AGO proteins in Prostate carcinoma cells . Objectives: To test the hypothesis following specific aims were proposed. Aim 1: To show that AKT regulates the miRNA expression profile in prostate carcinoma cells. Aim 2: To show that AKT mediated phosphorylation of AGO proteins regulates the activity of RISC complex. Aim 3: To show that abrogation of AKT mediated phosphorylation of AGO proteins sensitizes prostate carcinoma cells to chemotherapy. For Aim 1, we initially tried studying the interaction of Ago (pan) with 14-3-3. We could see interaction between Ago(pan) and (tau) isoform of 14-3-3. We treated prostate carcinoma cells (PC3) with 40 M AKT inhibitor V, Triciribine.Total RNA was isolated and miRNA and mRNA expression analysis in prostate carcinoma cells (PC3) was carried out to identify the miRNA-mRNA correlation matrices using Pearson Correlation in response to cells treated with AKT inhibitor (Triciribine). We also identified the miRNA-mRNA correlation matrices using Pearson Correlation in response to cells treated with AKT inhibitor (Triciribine)These matrices were further classified into four groups viz., I. miRNA up mRNA down. II miRNA down mRNA up. III. Both miRNA and mRNA up. IV. Both miRNA and mRNA down..Using the GeneGo analysis we identified TGF- dependent induction of Epithelial Mesenchymal Transition (EMT) via SMADs as its target. For aim 2, in order to identify the miRNA associated with specific AGO proteins, Immunoprecipitation was carried out.Since, the quality of miRNA isolated was poor, we could not proceed further on this aim. Once the miRNA-mRNA pair regulated by PI3K/AKT/14-3-3 is identified, we could then test the hypothesis that AKT regulates the RNA-induced silencing Complex (RISC) activity through the phosphorylation of KSRP protein in Prostate carcinoma cells .