STAT3短发夹RNA表达载体的构建及对SiHa细胞增殖和凋亡的作用

Construction of RNA recombinant vector expressing STAT3 short hairpin and its effects on proliferation and apoptosis of SiHa cells

目的:探讨信号转导子与转录激活子3短发夹RNA(shRNA)真核表达载体对宫颈癌SiHa细胞增殖和凋亡的作用。方法:根据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SiHa细胞。RT-PCR和Western blot检测SiHa细胞STAT3基因mRNA和蛋白表达水平;MTT法和流式细胞术(FCM)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SiHa细胞。细胞STAT3蛋白及mRNA表达下降;同时SiHa细胞增殖能力下降,细胞凋亡增加。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SiHa细胞的增殖能力,诱导细胞凋亡。

AIM：To investigate the effects of eukaryotic vector expressing short hairpin RNA（shRNA）of signal transducers and activators of transcription 3（STAT3）on the proliferation and apoptosis of SiHa cells.METHODS：shRNA templates were designed based on STAT3 gene sequence and cloned into pSilencer2.1-U6-neo vector.The resultant plasmid was transfected into SiHa cells with Lipofectamine 2000.The STAT3 protein and mRNA were detected by Western blot and RT-PCR,respectively.The cellular growth activity was assayed by MTT and the apoptosis was tested by flow cytometry.RESULTS：The plasmid pSilencer2.1-U6-neo-STAT3 was successfully constructed and transfected into SiHa cells.The expression of STAT3 in SiHa cells decreased,the cellular growth activity decreased,and the cell apoptosis increased.CONCLUSION：The siRNA expressing plasmid pSilencer2.1-U6-neo-STAT3 can inhibit cellular proliferation and induce the apoptosis of cervical cancer cells by suppressing the expression of STAT3.