Poster abstracts

Poster number 75 submitted by Lyudmyla Khrapenko

Identification of cancer-related protein targets regulated by Microrna-21 in Human Hepatocellular Carcinoma

Lyudmyla Khrapenko (CCC, OSU), Luke Meng (CCC, OSU), Chiara Braconi (CCC, OSU), Erica Swenson (CCC, OSU), Fanyin Meng (CCC, OSU), Tushar Patel (CCC, OSU)

Abstract:
MicroRNAs are short noncoding RNAs that can regulate gene expression. We have previously shown that miR-21 is over-expressed in human hepatocellular carcinoma (HCC) and contributes to tumor growth. However, the specific protein targets involved in tumor growth are not well established. To identify candidate target proteins of miR-21 we performed semi-quantitative proteomic analysis using HPLC-Chip Cube MS.

Proteomic screening identified and quantified 715 proteins in HCC cells, of which 206 proteins were differentially regulated in HepG2 cells by at least 2-fold after transfection with anti-miR-21. Of these 206 proteins, 117 were up-regulated and 89 down-regulated in HepG2 cells. Amongst these a 2.1-fold increase in Tropomyosin 1 (TM 1) was identified in response to anti-miR-21 by semi-quantitative MS proteomic analysis. TM 1 is predicted as a miR-21 target by several target prediction databases including TargetScan, Sanger, miRanda and PicTar. Up-regulation of TM 1 expression by anti-miR-21 was further confirmed by western blot (2.83 ± 0.74; p < 0.01). TM 1 is an actin-binding protein that is associated with stability of actin filaments and cell motility. Expression of tropomyosin is decreased in some types of tumors including human HCC. Silencing of TM 1 has been shown to alter TGF-β tumor suppressor function and contribute to metastatic properties in tumor cells. Consistent with these findings, anti-miR-21 treatment significantly reduced motility and invasive potential by 46.5 ± 8.7% and 41.4 ± 6.2% (both p < 0.01) in HepG2 cells. Interestingly, after anti-miR-21 treatment, expression of beta-catenin, an onco-protein associated with HCC, was reduced to 0.45-fold by semi-quantitative MS analysis and 0.41 ± 0.07-fold by quantitative immunoblot analysis. In conclusion, the tumor suppressor gene TM1 was identified as a target for miR-21, and shown to be involved in miR-21 dependent effects on tumor cell migration and invasion.

Proteomic analyses using HPLC/MS is an effective tool to identify microRNA dependent protein targets involved in tumor growth. Functional and expression proteomics can play important complementary roles in the identification of molecular targets involved in miR-21 mediated tumorigenesis.

Keywords: miR-21, human hepatocellular carcinoma, Proteomic analyses