Construction of let-7d expression vector and its inhibitory effect on HMGA2 and ras expression in human ovarian cancer cells in vitro.
- Author:
Haiyan YE
1
;
Jianguo CHEN
;
Xiaosui HUANG
;
Ailin GUO
;
Peipei HAO
Author Information
- Publication Type:Journal Article
- MeSH: Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; HMGA2 Protein; genetics; metabolism; Humans; MicroRNAs; genetics; Ovarian Neoplasms; genetics; metabolism; pathology; Plasmids; ras Proteins; genetics; metabolism
- From: Journal of Southern Medical University 2012;32(12):1752-1757
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo elucidate the role of let-7d in regulating the biological behavior of ovarian cancer cells and their expressions of HMGA2 and ras proteins.
METHODSThe pre-let-7d sequence was synthesized and inserted into pcDNA6.2GW/EmGFPmiR and transfected into ovarian cancer IGROV1 cells to cause pre-let-7d overexpression. Real-time quantitative RT-PCR was employed to examine the expression levels of let-7d miRNA and HMGA2 mRNA, and Western blotting was performed to detect the expressions of HMGA2 and ras protein in the transfected cells. The effect of pcDNA6.2GW-let-7d transfection on IGROV1 cell proliferation was determined using MTT assay and the cell apoptosis rate was measured using flow cytometry.
RESULTSThe eukaryotic expression vector containing the target gene let-7d was successfully constructed and transfected into IGROV1 cells. The transfected cells showed a marked reduction of HMGA2 expression but a less obvious down-regulation of ras expression. Transfection with pcDNA6.2GW-let-7d to suppress the expression of HMGA2 caused alterations of the phenotype of IGROV1 cells shown by a reduced proliferative activity and increased cell apoptosis.
CONCLUSIONLet-7d plays an important role in altering the malignant cell phenotype of ovarian cancer IGROV1 cells by regulating the expression of HMGA2.