Treatment Of Ovarian Cancer And The Regulation To Expression Of HK10by Pre-let-7d Transfection In Vitro

Objective:Human pre-let-7d was transfected into human ovarian caner cells to explore a novel microRNA therapy for the malignancies and to identify the effects on hK10expression induced by pre-let-7d.Methods:A vector was constructed by cloning the sequence of human microRNA let-7d precurcer, the pre-let-7d into the plasmid. Upon amplification and extraction, the plasmids were transfected into human ovarian cancer cell lines SKOV3and HO8910by means of Lipofectamine2000respectively. The SKOV3and HO8910cells were randomly devided into grous, including the groups where the cells were transfected only by the transfction reagent as a blank control, the groups where the cells were transfected by the pre-let-7d plasmid as the experimental group, and the control groups where the cells were transfected by the plasmid consisted of a scrambled sequence. Both fluorescence microscopy and flow cytometry were used to determine the transfection efficiency by counting the cells undergo fluorescence. MTT, SRB and cell counting were employed in testing the inhibitory effects on proliferation in the transfected cells. The cells were lablled by a kit of Annexin V-PE and PI for apoptosis assay in the cells by flow cytometry. Ferthurmore, Hoechst33258staining was utilized to identify the induction of apoptosis. The expression of hK10mRNA was deteced by RT-PCR. Protein level of hK10, moreover, was measured by ELISA assay.Results:The transfection rates in all the cells were approximately60%at24h、48h and72h post-transfetion by3μg plasmid DNA per well. The proliferation inhibition rates, tested by MTTand SRB, in the human ovarian cancer cells increased in a time-dependent manner. Based on cell counting, the cell grow curves were made. The peaks of proliferation inhibition, set at the checkpoint of72h post-transfection, where the inhibition rates were80%approximately, and hereafter the inhibition rates did not increase significantly. Apoptotic cells induced by pre-let-7d, lablled by Annexin V-PE/PI and tested by flow cytometry, accounted for about68.54%、75.58%、81.26%after the transfection24h、48h and72h respectively. While the apoptotic cells were about49%,55%and61%by Hoechst33258staining and by fluorescence microscopy at the same time points. By RT-PCT, hK10mRNA was down-regulated by pre-let-7d. However, hK10protein levels tsted by ELISA were not down-regulated accordingly.Conclusion:1. The proliferation of human ovarian cancer cell was inhibited by pre-let-7d transfection.2. The human ovarian cancer cell was induced apoptosis by pre-let-7d transfection.3. hK10mRNA was down-regulated and the protein levels were not down-regulated accordingly by pre-let-7d transfection. It can be concluded that microRNA let-7d can be used as a target to designing novel therapeutics for human ovarian cancer by down-regulating hK10expression.