The Experimental Study Of The Lentiviral-mediated HER2-shRNA On Chemosensitivity Of Ovarian Cancer SKOV3

Objective: We investigated that HER2-short hairpin(sh) RNA(HER2-short hairpin RNA, HER2-sh RNA) lentiviral vectors infected ovarian cancer SKOV3 in vitro to establish stable cell lines,and to study changes in their biological behavior.Experimental study of tumors after transplantation in vivo by targeting the human epidermal growth factor receptor silencing 2(HER2) gene chemosensitivity of ovarian cancer was explored, to explore RNA interference(RNA interference, RNAi) technical feasibility in terms of ovarian cancer treatment.Methods: This laboratory saved HER2-sh RNA lentivirus vectors and negative control sequence lentiviral vector targeting HER2 gene infected ovarian cancer SKOV3; we used puromycin pressure screening, and established a stable expression of HER2-sh RNA SKOV3 cell lines( KD) became KD group,and a negative control cell lines(NC) became NC group, uninfected cells SKOV3 was CON group.The expression of three groups of cells was detected of HER2 m RNA and protein by Real time PCR and Western blot in vitro; Transwell cell invasion assay detected the invasion ability of three groups of cells;CCK-8 assay was used to detect the chemosensitivity change of 3 cells with different concentrations of cisplatin(0, 0.312, 0.625, 1.25, 2.5, 5, 10, 20, 40 μg / m L); each experimental group were seeded in nude mice which were injected intravenously injection of cisplatin(DDP), and detected each group nude mice tumor formation rate, time to tumor, tumor size; immunohistochemical determination of each HER2 protein expression in the experimental group.Results: Effective interference and negative control sequences experiment cell lines of stably expressing were obtained by puromycin pressure screening sequences. Fluorescence microscopy and flow cytometry were used to detect fluorescence expression rates of NC group and KD group of more than 96%. Real time PCR showed HER2 m RNA relative content(HER2 / β-actin) of CON、NC and KD groups were 1.002±0.078、0.956±0.314、0.047±0.016, HER2 m RNA levels of KD group compared with CON and NC groups was significantly lower and was difference statistically significant(P<0.01). Western blot showed that the relative HER2 protein expression of the CON group, NC group and KD groups was(0.487±0.012)、(0.447±0.021) and(0.124±0.013),KD group was significantly lower than the NC and the CON groups(P<0.01). Transwell invasion assay results showed KD cells decreased the ability of cell invasion significantly.With increasing concentrations of cisplatin time, the three groups had received inhibition of cell growth, but the growth rate of the cells in the KD group cisplatin ≧ concentration 2.5μg / m L was significantly higher than the CON group and NC groups slower(P<0.05), CON and NC cells were no significant difference(P>0.05). Animal experiments showed that the average tumor volume and tumor weight of KD ovarian cancer xenografts significantly reduced by cisplatin treatment than the other group(P<0.05); immunohistochemical findings also showed that the expression of HER2 protein of KD + DDP group reduced significantly.Conclusion: The lentiviral-mediated HER2-sh RNA can effectively inhibit the expression of the HER2 gene, increase the chemosensitivity to cisplatin in ovarian cancer.