Establishment of hsa-mir-122 stably transfected-HepG2 cell line and its lipid metabolism characteristics

Abstract

Objective:To establish a HepG2 cell line stably overexpressing hsa-mir-122 by transfecting the HepG2 cells with pEZX-hsa-mir-122 vectors,so as to observe the influence of miR-122 on the lipid metabolism in the liver cells.Methods:The constructed pEZX-hsa-mir-122 and pEZX-mir-control plasmids were identified by restriction enzyme digestion and the right clones by sequencing were subjected to sequencing.And the right clones by sequencing were transfected by LipofectamineTM.The transfection efficiency was assessed by green fluorescent protein expression.The stably transfected cell lines were screened by Puromycin.The expression of miR-122 was examined by real-time PCR.The inhibitory function of miR-122 was testified by Western blotting assay.Nile red was used to observe the content of lipid in HepG2 cells stably overexpressing hsa-mir-122.Results:HepG2 cells were successfully transfected with pEZX-hsa-mir-122 and pEZX-mir-control plasmids.The precursor miRNA sequences were integrated into the genome of HepG2 cells stably overexpressing miR-122.Compared with the control HepG2 cells,the HepG2 cells overexpressing miR-122 contained more lipid in the cytoplasm.Conclusion:The miR-122 precursor sequence can intergrate into the genome of HepG2 cells.The increase of miR-122 level in HepG2 cells can result in abnormal metabolism of lipid.