RNAi沉默STAT3基因对人宫颈癌HeLa细胞增殖和凋亡的影响

Effects of RNA interference targeting STAT3 gene on proliferation and apoptosis of HeLa cells

【目的】探讨信号转导与激活因子3(signal transducers and activators of transcription 3,STAT3)短发夹RNA(short hairpin RNA,shRNA)真核表达载体对宫颈癌HeLa细胞增殖和凋亡的作用。【方法】根据siRNA(small inter-ference RNA,siRNA)设计原则,结合pSilencer2.1-U6-neo质粒特点,针对STAT3基因设计并合成两条寡聚DNA片段,退火后克隆入pSilencer2.1-U6-neo质粒,脂质体法将重组质粒转染人宫颈癌HeLa细胞。RT-PCR和Western blotting检测HeLa细胞STAT3基因mRNA和蛋白表达水平;MTT法和流式细胞仪检测细胞的增殖和凋亡。【结果】成功构建了STAT3基因shRNA真核表达载体,转染人宫颈癌HeLa细胞。非特异siRNA组细胞STAT3蛋白、mRNA、细胞增殖率及凋亡率分别为0.55±0.08、0.49±0.05、(4.12±0.45)%和(11.31±1.69)%,而目的siRNA组细胞STAT3蛋白、mRNA、细胞增殖率及凋亡率分别为0.36±0.06、0.32±0.05、(2.51±0.48)%和(17.39±1.58)%。与非特异siRNA组细胞相比,目的siRNA组细胞STAT3蛋白及mRNA表达下降;同时HeLa细胞增殖能力下降,细胞凋亡率增加。【结论】构建的STAT3基因shRNA真核表达载体能够通过下调STAT3基因表达,抑制宫颈癌细胞的增殖能力,诱导细胞凋亡。

[ Objective] To construct eukaryotic vector expressing short hairpin RNA （shRNA） of signal transducers and activators of transcription 3 （STAT3）, and to observe its effects on proliferation and apoptosis of HeLa cells. [Methods] shRNA templates was designed based on STAT3 gene sequence and was cloned into pSilencer2.1-U6-neo vector. The resultant plasmid was trans- fected into HeLa cells with Lipofectamine 2000. The STAT3 protein and mRNA were detected by Western blotting and RT-PCR, respectively. The cellular growth activity was assayed by MTT, and the apoptosis was tested by flow cytometry. [Results] The plasmid pgileneer2. I-UO-neo-STAT3 wag successfully comtrueted and transfected into HeLa cell. The protein of STAT3, the mRNA of STAT3, the ratio of cellular growth activity, and the ratio of ceU apoptosis in nonspecific siRNA group were 0.55 ± 0.08, 0.49±0.05, （4.12±0.45）%, and （11.31±1.69）%, The protein ofSTAT3, themRNA of STAT3, the ratio ofcellular growth activity, and the ratio of cell apoptosis in target siRNA group were 0.36±0.06, 0.32 ±0.05, （2.51 ±0.48） %, and （17.39±1.58）%, Contrasted with nonspecific siRNA group, the expressions of STAT3 in target siRNA group decreased, The cellular growth activity decree, and cell apoptosis Incrceased. [ Conclusions] The sRNA expressing plasmld psilencer 2. 1-U6- neo-STAT3 could inhibit the cellular proliferation and induce apoptosis of cervical cancer cells through suppressing the expression of STAT3.