Abstract
S 20TH ANNUAL CONFERENCE OF INASL, MARCH 2–4, 2012 S26 © 2012, INASL A b tra ts 44 OVER-EXPRESSION OF MICRORNA-122 (MIR-122) INDUCES APOPTOSIS BY TARGETING CYCLING1 AND BCL-W GENES IN VITRO S Kumar1, YK Chawla2, S Ghosh1, A Chakraborti1 Departments of 1Experimental Medicine and Biotechnology, 2Hepatology, Postgraduate Institute of Medical Education and Research, Chandigarh Background: MicroRNA-122 (miR-122), liver-specific microRNA, plays an important role in many physiological and pathological conditions of liver, such as stress response, lipid metabolism and hepatocellular carcinoma (HCC). It has been reported that, miR-122 is frequently downregulated in HCC; in contrast, its target genes cyclinG1 and bcl-w is up-regulated. Aim: To study the pro-apoptotic and tumor suppressor function of miR-122. Method: Initially, HeLa (cervical epithelial carcinoma) and HepG2 (human hepatoblastoma) cell lines, which lack miR-122 expression, were transfected with pEGPmmu-mir-122 expression vector by lipofectamine. After transfection, miR-122 vector expression was confirmed by fluorescence microscopy, fluorescent assisted cell sorter (FACS) analysis and quantitatively by real-time PCR (qRTPCR). The cytopathic changes were analyzed at different time intervals (24 h, 48 h, 72 h, and 96 h post-transfection) by light microscopy, propidium iodide (PI) staining and ethidium bromide/acridine orange (EtBr/AO) staining. Subsequently, the expression of cyclinG1 and bcl-w genes was quantified by qRT-PCR. Result: The qRT-PCR analysis determined the threefold higher expression of miR-122 in transfected cells as compared to control. Light microscopy showed altered cell morphology, and a consistent increase in population of dead cells was visible after 96 hours post-transfection by PI staining. Also, the transfected cells showed the higher apoptotic index as compared to the control by EtBr/AO staining. Furthermore, the miR-122 target genes cyclinG1 and bclw showed approximately fivefold down-regulation during overexpression of miR-122. Conclusion: This study shed light on the functions of miR-122, which may act as an endogenous apoptosis regulator, and thus negatively regulates tumourigenesis. HEPATOCELLULAR CARCINOMA AND OTHER TUMORS
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