沉默STAT3基因对人卵巢癌化疗敏感性的影响

Inhibition of STAT3 expression on chemosensitivity of ovarian cancer cell line SKOV3

背景与目的:信号转导因子与转录激活因子3(STAT3)是近期研究较多的一种基因,在多种实体瘤如乳腺癌、胃癌和卵巢癌等中均有异常表达和活性增强。本研究旨在探讨STAT3短发夹RNA(short hairpin RNA,shRNA)真核表达载体对卵巢癌细胞SKOV3化疗敏感性的作用。方法:构建STAT3基因shRNA真核表达质粒,并转染卵巢癌SKOV3细胞。试验分为SKOV3、SKOV3NS和SKOV3siRNA3组,以RT-PCR及免疫蛋白印迹法分别检测各组STAT3基因的mRNA及蛋白表达水平。细胞经20μmol/L顺铂(DDP)作用后,MTT法检测细胞生长抑制率,流式细胞术(FCS)检测细胞凋亡率。结果:MTT法检测肿瘤细胞的生长抑制率,SKOV3组、SKOV3NS组和SKOV3siRNA组细胞抑制率分别为0.46±0.13、0.44±0.11和0.71±0.12。与SKOV3组、SKOV3NS组相比,SKOV3siRNA组细胞抑制率明显增加,差异均有统计学意义(P<0.05);而SKOV3NS组与SKOV3组比较,细胞抑制率差异无统计学意义(P>0.05)。SKOV3组、SKOV3NS组和SKOV3siRNA组细胞凋亡率分别为18±4、17±3和35±4。与SKOV3组、SKOV3NS组比较,SKOV3siRNA组细胞凋亡率明显增加,差异均有统计学意义(P<0.05);而SKOV3NS组与SKOV3组比较,细胞凋亡率差异无统计学意义(P>0.05)。SKOV3组、SKOV3NS组和SKOV3siRNA组细胞STAT3mRNA检测结果分别为0.50±0.08、0.48±0.07和0.31±0.09。与SKOV3组、SKOV3NS组比较,SKOV3siRNA组细胞STAT3mRNA表达水平明显降低,差异均有统计学意义(P<0.05);而SKOV3NS组与SKOV3组细胞比较,STAT3mRNA表达水平差异无统计学意义(P>0.05)。SKOV3组、SKOV3NS组和SKOV3siRNA组细胞STAT3蛋白检测结果分别为0.54±0.09、0.56±0.08和0.32±0.09。与SKOV3组、SKOV3NS组比较,SKOV3siRNA组细胞STAT3蛋白表达明显降低,差异均有统计学意义(P<0.05);而SKOV3NS组与SKOV3组比较,细胞STAT3蛋白表达差异无统计学意义(P>0.05)。结论:针对STAT3基因的shRNA真核表达载体能有效地抑制卵巢癌SKOV3细胞STAT3基因表达,增强其对化疗药物DDP的敏感性。

Background and purpose：The signal transducers factor and activator in transcription 3（STAT3） is a recently studied gene from a variety of solid tumors such as breast, stomach, and ovarian cancer, in which the increase of abnormal expression and activity are accompanied.The aim of this study was to investigate the effects of eukaryotic vectors that express short hairpin RNA（shRNA） in signal transducers and activators of STAT3 on chemosensitivity of human ovarian cancer SKOV3 cells.Methods：Vectors containing shRNA targeting STAT3（pSTAT3-siRNA） were constructed and transfected into SKOV3 cells.These vectors were then divided into 3 groups：SKOV3, SKOV3NS, and SKOV3siRNA.The mRNA and protein expressions of STAT3 were determined by RT-PCR and Western blot, respectively.The growth inhibition and apoptosis rates of the different group cells under chemotherapeutic agents such as cisplatin（20 μmol/L） were measured by MTT assay and flow cytometry（FCM）.Results：MTT assay growth inhibition rates in the tumor cells of the SKOV3 group, SKOV3NS group and SKOV3siRNA group had inhibition rates of 0.46±0.13, 0.44±0.11 and 0.71±0.12.Compared with the SKOV3, SKOV3NS and SKOV3siRNA group, there was a marked increase of SKOV3siRNA group in inhibition rate of cells.The differences were also statistically significant（P0.05）, whereas when the SKOV3NS group was compared with the SKOV3 group, the cell inhibition rate had no significant difference（P0.05）.The SKOV3 group, SKOV3NS group and SKOV3siRNA group＇s cell apoptosis rates were 18±4, 18±3 and 35±4, respectively.However, the SKOV3 group, SKOV3NS group, and SKOV3siRNA group cell apoptosis were significantly increased and the differences were statistically significant（P0.05） whereas in the SKOV3NS group and SKOV3 group, the apoptotic rate had no significant difference（P0.05）.The results for the SKOV3 group, SKOV3NS group and SKOV3siRNA in cell STAT3 mRNA were 0.50±0.08, 0.48±0.07 and 0.31±0.09.With the SKOV3 group, SKOV3NS group, SKOV3siRNA group of cells in STAT3 mRNA, its expression in the lungs were significantly lower and the differences were statistically significant（P0.05）.When the SKOV3NS group was compared with the SKOV3 group of cells, STAT3 mRNA expression levels of difference were not statistically signif icant（P0.05）.SKOV3 group, SKOV3NS group and SKOV3siRNA group of cells checked the results of STAT3 protein were 0.54±0.09, 0.56±0.08 and 0.32±0.09, respectively.The SKOV3 group, SKOV3NS group, and SKOV3siRNA group in STAT3 protein expression was significantly lower and the differences were statistically significant（P0.05） whereas when the SKOV3NS group was compared with the SKOV3 cells, STAT3 protein expression had no significant difference（P0.05）.Conclusion：The STAT3 specific shRNA expression vector could effectively suppress the expression level of STAT3 gene in SKOV3 cells as well as enhance their sensitivity to cisplatin.