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目的构建携带信号转导与转录激活子3(STAT3)基因的短发夹RNA(shRNA)真核表达载体,观察pGPU6/GFP/NeoSTAT3重组质粒对HCT116细胞顺铂化疗敏感性的影响。方法设计并构建稳定转录shRNA STAT3的质粒,采用脂质体法转染结肠癌HCT116细胞,Western blot法检测转染后STAT3蛋白表达变化,MTT法检测细胞增殖变化。重组质粒联合顺铂作用于HCT116细胞后,MTT法检测细胞存活率。结果成功构建了pGPU6/GFP/Neo-STAT3重组质粒,测序证实重组质粒构建正确。重组质粒转染HCT116细胞后,细胞增殖明显受抑制,STAT3蛋白表达降低。重组质粒联合顺铂治疗后,细胞增殖活性显著降低。结论 shRNA STAT3重组质粒能明显降低HCT116细胞中STAT3蛋白的表达,抑制细胞增殖,提高结肠癌细胞对顺铂的敏感性。
Objective To construct short hairpin RNA (shRNA) eukaryotic expression vector carrying signal transducers and activator of transcription 3 (STAT3) gene and investigate the effect of pGPU6 / GFP / NeoSTAT3 recombinant plasmid on cisplatin chemosensitivity of HCT116 cells. Methods Plasmid stably transfected with shRNA STAT3 was designed and constructed and transfected into HCT116 cells by lipofectamine. The expression of STAT3 protein was detected by Western blot and the cell proliferation was detected by MTT assay. The recombinant plasmids combined with cisplatin on HCT116 cells, MTT assay cell viability. Results The recombinant plasmid pGPU6 / GFP / Neo-STAT3 was successfully constructed. The sequencing confirmed that the recombinant plasmid was constructed correctly. After transfection with HCT116 cells, the cell proliferation was significantly inhibited and STAT3 protein expression was decreased. Recombinant plasmid combined with cisplatin treatment, cell proliferation activity was significantly reduced. Conclusion shRNA STAT3 recombinant plasmid can significantly reduce the STAT3 protein expression in HCT116 cells, inhibit cell proliferation and improve the sensitivity of colon cancer cells to cisplatin.