[Construction of let-7d expression vector and its inhibitory effect on HMGA2 and ras expression in human ovarian cancer cells in vitro]

Haiyan Ye, Jianguo Chen, Xiaosui Huang, Ailin Guo, Peipei Hao
Nan Fang Yi Ke da Xue Xue Bao, Journal of Southern Medical University 2012, 32 (12): 1752-7

OBJECTIVE: To elucidate the role of let-7d in regulating the biological behavior of ovarian cancer cells and their expressions of HMGA2 and ras proteins.

METHODS: The 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.

RESULTS: The 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.

CONCLUSION: Let-7d plays an important role in altering the malignant cell phenotype of ovarian cancer IGROV1 cells by regulating the expression of HMGA2.

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