Abstract C16: miR-148a regulates the expression of PTEN in hepatitis B associated hepatocellular carcinoma

Abstract

Abstract Previous work has shown that hepatitis B virus encoded X antigen (HBx) is a trans-regulatory protein that alters the activity of selected transcription factors and cytoplasmic signal transduction pathways. The resulting changes in host gene expression are thought to contribute importantly to the development and progression of hepatocellular carcinoma (HCC). In this context, HBx up-regulates the expression of a unique gene, URG11, which in turn up-regulates β-catenin, thereby contributing importantly to hepatocarcinogenesis. Changes in the expression levels of microRNAs (miRNAs) are also characteristic of many tumors, including HCC. To determine whether altered miRNA expression contributes to the mechanism whereby HBx and URG11 contribute to HCC, recombinant retroviruses encoding HBx, URG11, or the bacterial chloramphenicol acetyltransferase (CAT) gene (the latter used as a negative control) were used to infect separate cultures of HepG2 and Hep3B cells. When these cells were compared by miRNA array analysis, both HBx and URG11 were shown to promote the expression of miR-148a. This was confirmed by quantitative (q)RT/PCR analysis. Elevated levels of miR-148a strongly correlated with HBx positive staining in liver samples from infected patients by qRT/PCR (P< 0.001). To study the function of miR-148a, anti-miR-148a was introduced into HepG2 and Hep3B cells stably expressing HBx or stably over-expressing URG11. Anti-miR-148a suppressed cell proliferation (P < 0.01), cell cycle progression (P < 0.01), cell migration (P < 0.01), anchorage independent growth in soft agar (P < 0.01) and subcutaneous tumor formation in SCID mice (P < 0.05). Introduction of anti-miR-148a also increased PTEN mRNA and protein expression, suggesting that PTEN was targeted by miR-148a. This was verified in experiments where anti-miR-148a failed to suppress PTEN expression when co-transfected with reporter gene mutants of the 3′UTR of PTEN mRNA. When HBx positive HepG2 and Hep3B cells were transiently transfected with URG11 specific siRNA, both URG11 and miR-148a levels were suppressed, suggesting that up-regulated expression of miR-148a in HBx positive cells was URG11 dependent. Finally, anti-miR-148a blocked the stimulation of Akt signaling by HBx and URG11, resulting in decreased expression of β-catenin (P < 0.05). Thus, miR-148a appears to contribute a central role to HBx/URG11 mediated HCC, and may be an early diagnostic marker and/or therapeutic target associated with this tumor type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C16.