Identification of Novel MiR-122 Targets with Potential Role in Hepatocellular Cancer
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Molecular Genetics Undergraduate Research Theses; 2016
MicroRNA 122 (MiR-122) is an abundant liver-specific miRNA which serves to regulate hepatic metabolism, and functions as a tumor suppressor. We are determining how a tiny, twenty-two nucleotide microRNA maintains homeostatic liver function. Loss of hepatic phenotype, metastasis, and poor prognosis are characteristics of down regulation of miR-122 expression. Spontaneous Hepatocellular Carcinoma (HCC) development has been observed in liver-specific (LKO) and germ-line (KO) knockout miR-122 mice. Results of Ago-HITS-CLIP analysis conducted in livers of 6 week-old wild type and KO mice confirmed ~1800 miR-122 binding sites present in wild-type, but absent in KO livers. MiR-122 binding sites were identified on the 3’ untranslated regions (3’-UTR) of the snail family zinc finger 2 (Snai2) gene, TIMP metallopeptidase inhibitor 2 (Timp2) gene, LIM domain kinase 1 (Limk1) gene, Hepatocyte Nuclear Factor 4 alpha (Hnf4a) and Pygopus Family PHD Finger 2 (Pygo2). The encoded protein of Snai2 is involved in epithelial mesenchymal transitions, and has the ability to enable the cell to resist apoptotic signals. Timp2 functions as a metastasis suppressor. Limk1 regulates actin polymerization through phosphorylation and subsequent inactivation of cofilin, while Hnf4a regulates liver development. Pygo2 is involved in signal transduction of the Wnt and GPCR pathways. The presence of these discovered binding sites highly suggests targeting and regulation by miR-122. These genes are potentially involved in liver pathogenesis, and could be crucial with respect to determining HCC progression. To explore the possibility of targeting and regulation by miR-122, real-time reverse-transcription polymerase chain reaction (qRT-PCR) was conducted. We hypothesized that in the absence of miR-122, expression levels of these genes would be increased. These five targets were found to be highly expressed in miR-122 knockout mice. RNA-sequencing analysis suggests that these genes are undergoing regulation by miR-122. Snai2 has been validated as a direct miR-122 target using luciferase assay technology with hepatoma (Hepa) cells. Snai2 has been further validated using site-directed mutagenesis in Hepa cells. Broader implications of this study include miR-122 itself or Snai2 being targeted for HCC therapy in human patients. .
Academic Major: Molecular Genetics
Second-year Transformational Experience Program (STEP) Funding
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