化学合成siRNA对卵巢癌SKOV3细胞中STAT3基因表达的抑制作用

目的:观察小干扰RNA(small interference RNA,siRNA)对卵巢癌SKOV3细胞STAT3表达的抑制作用及其诱导凋亡作用的机制。方法:将STAT3 siRNA经LipofectamineTM2000脂质体介导的方法将其转染到卵巢癌细胞株SKOV3。实验分为以下3组:空白对照组(未经转染的人卵巢癌SKOV3细胞)、阴性对照转染组(阴性干扰STAT3)及特异性转染组(干扰STAT3)。用MTT法检测siRNA转染后SKOV3细胞增殖活性;流式细胞仪检测其对细胞凋亡的影响,免疫荧光法检测STAT3蛋白水平的变化,qRT-PCR和Western blot方法分别检测STAT3 mRNA和蛋白表达水平的变化。结果:成功转染STAT3干扰片段,转染率达90%。siRNA干扰STAT3组明显抑制卵巢癌细胞增殖活性,细胞凋亡率33.7%,并且下调STAT3蛋白在SKOV3细胞中的表达。RT-PCR和Western blot结果显示:siRNA STAT3组明显抑制人卵巢癌细胞SKOV3中STAT3 mRNA和蛋白的表达,与对照组相比,差异有统计学意义(P<0.05)。结论:特异性转染组48h后能有效地抑制SK-OV3细胞内STAT3的表达、抑制细胞增殖,其抑制卵巢癌细胞的机制可能与抑制了STAT3信号通路,促进细胞凋亡有关,为卵巢癌的生物治疗提供了一定的依据。

Inhibiting effect of chemically synthesized siRNA interference on STAT3 gene expression in human ovarian cancer cell SKOV3

Objective:To observe the inhibition of small interfering RNA(small interference RNA,siRNA) in ovarian cancer SKOV3 cells and STAT3 expression in the mechanism of apoptosis.Methods:STAT3 siRNA was transfected into ovarian cancer cell SKOV3 by LipofectamineTM 2000 liposome-mediated method,there were groups in this experiment: control group(human ovarian cancer SKOV3 cells),negative control transfected group(negative interferenced with STAT3) and special transfection group(interference with STAT3),the siRNA STAT3 gene inhibiting cell proliferation was mearsured by MTT;its effect on ovarian cancer cells was examined by flow cytometry;the detection of STAT3 protein levels was checked by immunofluorescence,STAT3 mRNA and changes of protein expression level were detected by qRT-PCR and Western blot.Results:STAT3 interference fragment was transfected successfully,transfection rate was 90%,in the special transfection group,siRNA significantly inhibited the proliferation of ovarian cancer cell,the apoptosis rate was 33.7%.At the same time,SKOV3 downward protein could be found in STAT3 cells.RT-PCR and Western blot results showed that siRNA STAT3 could significantly inhibite the STAT3 mRNA and protein in SKOV3 from human varian cancer cells,compared with the control group,the difference was statistically significant(P<0.05).Conclusion:Special transfection group can inhibit the expression and proliferation of STAT3 in SKOV3 cells effectively after 48h,its mechanism of inhibition of ovarian cancer cells may be the inhibition of STAT3 signaling pathway and the promotion of apoptosis,which provides us some new clues for the treatment of ovarian cancer.