Establishment of hsa-mir-122 stably transfected-HepG2 cell line and its lipid metabolism characteristics
10.3724/SP.J.1008.2009.00825
- Author:
Ya-Nan ZHANG
1
Author Information
1. Department of Medical Genetics
- Publication Type:Journal Article
- Keywords:
Hepg2 cell;
Hsa-mir-122;
Lipid metabolism;
Puromycin;
Stable transfection
- From:
Academic Journal of Second Military Medical University
2010;30(7):825-829
- CountryChina
- Language:Chinese
-
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 Lipofectamine™. 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.
