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探讨小干扰RNA(siRNA)对人皮肤鳞状细胞癌A431细胞株生存素基因表达的抑制作用及对细胞凋亡、增殖和细胞周期的影响。将已构建的靶向生存素的siRNA表达质粒采用脂质体法转染A431细胞株。应用RT-PCR检测A431细胞生存素mRNA的表达,Western blot检测生存素蛋白的表达,流式细胞术检测Annexin标记的凋亡细胞和分析细胞周期,噻唑蓝(MTT)法分析细胞增殖。结果显示,与未处理的A431细胞株和转染空载体的A431细胞株比较,转染siRNA表达质粒可以明显抑制生存素基因在转录和翻译上的表达;siRNA表达质粒组的凋亡率明显增加达到21.3%;24、48、72和96h的细胞增殖抑制率也明显增加,并随着时间抑制效率变高;细胞周期分析发现抑制生存素表达使得A431细胞株出现明显的G2/M期阻滞现象。因此,靶向生存素的siRNA表达质粒可以特异性抑制生存素的表达,显著抑制肿瘤细胞增殖并诱导细胞凋亡。靶向生存素的siRNA表达质粒为鳞状细胞癌的基因治疗提供了一种新思路。
To investigate the inhibitory effect of small interfering RNA (siRNA) on the gene expression of survivin in human skin squamous cell carcinoma A431 cell line and its effect on apoptosis, proliferation and cell cycle. The constructed Survivin targeting siRNA expression plasmid was transfected into A431 cell line by liposome. The expression of survivin mRNA of A431 cells was detected by RT-PCR. The expression of survivin protein was detected by Western blot. The apoptotic cells were detected by flow cytometry and the cell cycle was analyzed. Cell proliferation was analyzed by MTT assay. The results showed that compared with the untreated A431 cell line and A431 cell line transfected with empty vector, transfection of siRNA expression plasmid significantly inhibited the expression of survivin gene in transcription and translation; the apoptosis rate of siRNA expression plasmid group was significantly increased Reached 21.3%; 24,48,72 and 96h cell proliferation inhibition rate also increased significantly, and over time inhibition efficiency becomes higher; cell cycle analysis found that inhibition of survivin expression in A431 cell line showed G2 / M phase arrest phenomenon. Thus, survivin-targeted siRNA expression plasmids specifically inhibit survivin expression, significantly inhibit tumor cell proliferation and induce apoptosis. Survivin-targeting siRNA expression plasmids provide a new way of gene therapy for squamous cell carcinoma.