干扰转录因子信号转导与转录激活因子-3之间的表达对胆管细胞癌生物功能的影响

Effect of interference with singal transducer and activator of transcription 3 expression on biological function of cholangiocarcinoma

目的观察通过小干扰RNA(siRNA)干扰信号转导与转录激活因子-3(STAT3)的表达对胆管细胞癌生物功能的影响。方法培养胆管细胞癌细胞系人胆管上皮癌细胞(HUCCT1)和人胆管癌细胞(RBE),构建siRNA-710、siRNA-551和siRNA-2021这3个siRNA,使用脂质体3000(lipo3000)转染试剂将siRNA转染胆管细胞癌细胞,然后通过聚合酶链反应(PCR)和蛋白质印迹法(Western blot)检测siRNA对STAT3的表达的干扰效果后选择siRNA-551进行后续实验。siRNA-551转染胆管细胞癌细胞48 h后通过细胞计数试剂盒(CCK-8)检测细胞增殖、通过流式细胞术检测细胞凋亡率、通过Western blot检测细胞中白细胞介素-6(IL-6)和血管内皮生长因子(VEGF)的表达水平,并采用单因素方差分析实验结果。结果siRNA-551能够显著降低胆管细胞癌细胞中STAT3的mRNA(Messenger RNA)和蛋白质表达。HUCCT1和REB胆管细胞癌细胞转染siRNA-551后,STAT3干扰组胆管细胞癌细胞增殖水平[(74.49±0.96)%、(91.08±5.32)%]低于对照组[(100.00±5.52)%、(100.00±2.82)%,F=62.20、6.57,P<0.05]和NC组[(90.99±1.87)%、(99.74±4.71)%,F=184.57、4.45,P<0.05],差异有统计学意义;干扰组细胞凋亡率[(15.68±1.79)%、(11.30±0.67)%]高于对照组[(6.97±0.76)%、(6.12±0.37)%,F=60.01、138.40,P<0.05]和NC组[(9.21±0.63)%、(6.22±0.21)%,F=34.77、155.96,P<0.05],差异有统计学意义。HUCCT1细胞转染siRNA-551的胆管细胞癌细胞中IL-6和VEGF蛋白相对表达量(0.23±0.07、0.16±0.02)低于对照组(0.62±0.34、0.35±0.15,F=3.78、4.90,P<0.05)和NC组(0.59±0.07、0.47±0.39,F=37.46、2.11,P<0.05),差异有统计学意义;REB细胞转染siRNA-551的胆管细胞癌细胞中IL-6和VEGF蛋白相对表达量(0.22±0.11、0.80±0.08)低于对照组(0.51±0.11、1.89±0.13,F=11.39、151.69,P<0.05)和NC组(0.60±0.34、1.46±0.48,F=3.39、140.20,P<0.05),差异有统计学意义。结论通过siRNA干扰胆管细胞癌细胞中的STAT3表达具有抑癌的效果。

Objective To explore the effect of interference on expression of signal transducer and activator of transcription 3(STAT3)by small interfering RNA(siRNA)on biological function of cholangiocarcinoma.Methods Cholangiocarcinoma cell lines human cholangiocarcinoma cells1(HUCCT1)and human cholangiocarcinoma cell(RBE)were cultured,siRNA-710,siRNA-551 and siRNA-2021 were constructed,Lipo 3000 transfection reagent was used to transfect siRNA into cholangiocarcinoma cells.The interference effect of siRNA on STAT3 expression was detected by polymerase chain reaction(PCR)and Western blotting,and siRNA-551 was selected for subsequent experiments.After 48 h of siRNA-551 transfection on cholangiocarcinoma cells,cell proliferation was tested by cell counting kit-8(CCK-8)assay,apoptosis rate examined by flow cytometry,and the expression levels of interleukin-6(IL-6)and vascular endothelial growth factor(VEGF)were detected by Western blotting.One-way ANOVA of variance was used to analyze experimental results.Results SiRNA-551 can significantly reduce the expression of STAT3 mRNA and protein in cholangiocarcinoma cells.After HUTT1 and RBE cholangiocarcinoma cells transfection with siRNA-551,the proliferation of cholangiocarcinoma cells in the STAT3 interference group[(74.49±0.96)%,(91.08±5.32)%]was lower than that of the blank control group[(100.00±5.52)%,(100.00±2.82)%,F=62.20,6.57,P<0.05]and NC group[(90.99±1.87)%,(99.74±4.71)%,F=184.57,4.45,P<0.05],The difference was statistically significant;the apoptosis rate of the interference group[(15.68±1.79)%,(11.30±0.67)%]was higher than that of the blank control group[(6.97±0.76)%,(6.12±0.37)%,F=60.01,138.40,P<0.05]and NC group[(9.21±0.63)%,(6.22±0.21)%,F=34.77,155.96,P<0.05],The difference was statistically significant;After HUCCT1 cholangiocarcinoma cells transfection with siRNA-551,The expression of IL-6 and VEGF protein in the interference group(0.23±0.07,0.16±0.02)was low in the blank control group(0.62±0.34,0.35±0.15,F=3.78,4.90,P<0.05)and NC group(0.59±0.07,0.47±0.39,F=37.46,2.11,P<0.05),The difference was statistically significant;After RBE cholangiocarcinoma cells transfection with siRNA-551,the proliferation of cholangiocarcinoma cells in the STAT3 interference group(0.22±0.11,0.80±0.08)was lower than that of the blank control group(0.51±0.11,1.89±0.13,F=11.39,151.69,P<0.05)and NC group(0.60±0.34,1.46±0.48,F=3.39,140.20,P<0.05),The difference was statistically significant.Conclusion Interfering the expression of STAT3 in cholangiocarcinoma cells by siRNA has a tumor suppressor effect.