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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。
Objective: To investigate the effect of short hairpin RNA (shRNA) eukaryotic expression vector of signal transducer and activator of transcription 3 on proliferation and apoptosis of cervical cancer SiHa cells. Methods: According to the principle of siRNA design, combined with the characteristics of pSilencer2.1-U6-neo plasmid, two oligo DNA fragments were designed and synthesized for STAT3 gene. After annealing, the two oligo DNA fragments were cloned into pSilencer2.1-U6-neo plasmid. Plasmid-transfected human cervical cancer SiHa cells. The expression of STAT3 mRNA and protein in SiHa cells was detected by RT-PCR and Western blot. The proliferation and apoptosis of cells were detected by MTT assay and flow cytometry (FCM). Results: STAT3 gene shRNA eukaryotic expression vector was successfully constructed and transfected into human cervical cancer SiHa cells. The expression of STAT3 protein and mRNA in the cells decreased; meanwhile, the proliferation ability of SiHa cells decreased and apoptosis increased. CONCLUSION: The constructed shRNA eukaryotic expression vector of STAT3 gene can induce cell apoptosis by down-regulating STAT3 gene expression, inhibiting the proliferation of cervical cancer SiHa cells.