Abstract 5281: Insulin-mediated regulation of the miR132/212 cluster and miR 122/181a expression through PI3K, Akt, mTOR signaling in primary cultured rat hepatocytes.

Abstract

Abstract Background and Objective: Several microRNAs were selected for characterization of their response to insulin signaling based on previous reports implicating their role in the regulation of hepatic metabolism, diabetes, hepatocellular carcinoma, and predictions of targeting CYP2E1. CYP2E1 catalyzes the metabolic activation of carcinogens, increases cellular oxidative stress, and is elevated in diabetes and at low insulin levels (0.1nM) in primary cultured hepatocytes. In addition, aberrant miR-122 expression was reported to be associated with hepatocellular carcinoma in rats, as well as in human tissue, and may be involved with hepatocarcinogenesis. In view of the critical role of the PI3K, Akt and mTOR signaling pathway in pathogenesis, mechanistic studies were conducted to examine the role of this pathway in regulation miR 132/212, 122 and 181a expression. Methods: Insulin effects on miRNA (miRs) levels were monitored using qPCR, and mechanistic studies employing inhibitors of PI3K, Akt, and mTOR were used to examine the role of this insulin signaling pathway on miR expression. The results were compared to miR let-7a, which is not implicated in the regulation of hepatic metabolism or diabetes and does not target CYP2E1 mRNA. Results: Insulin at concentrations of 0.1, 1.0, and 10 nM increased the expression of miRNA-132 and 212 ∼2- and 1.8-fold respectively, whereas expression of miRNAs 181a and 122 increased ∼1.6- and 1.4-fold, respectively. In contrast, insulin failed to significantly alter the expression of miRNA let-7a. Inhibition of PI3K with LY294002, Akt with A-443654 and mTOR with rapamycin resulted in significant suppression of the insulin-mediated elevation of miR-132, miR-212, and miR-122 levels monitored over the insulin concentrations examined. Inhibition of PI3K with LY294002, or Akt with A-443654 decreased the insulin-mediated increase in miR-181a levels by ∼20 to 50% relative to the respective insulin-concentration. Rapamycin inhibition of mTOR produced a marginal (<10%) decrease in miR-181a levels. Inhibition of PI3K or Akt did not show any significant effect on miR let-7a. In summary, seminal evidence is provided for insulin regulation of microRNA expression in primary cultured hepatocytes through PI3K, Akt and mTOR (TORC1). Citation Format: Upasana Shukla, Nithin Tumma, Theresa Gratsch, Alan Dombkowski, Raymond Novak. Insulin-mediated regulation of the miR132/212 cluster and miR 122/181a expression through PI3K, Akt, mTOR signaling in primary cultured rat hepatocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5281. doi:10.1158/1538-7445.AM2013-5281