靶向survivin利用RNA干扰技术影响云南个旧肺鳞癌细胞株YTMLC凋亡的初步研究

RNA Interference-mediated Significant Enhancement of Apoptosis in Yunnan Gejiu Lung Squamous Carcinoma Cells(YTMLC) by siRNA Expression Vector Targeting Survivin Gene

目的检测云南个旧肺鳞癌细胞株YTMLC细胞内源凋亡抑制因子survivin基因的表达以及促进YTMLC细胞凋亡的情况。方法构建和转染重组质粒pshRNA-survivin-1和pshRNA-survivin-2至YTMLC细胞株,通过免疫荧光和半定量RT-PCR检测survivin蛋白表达及mRNA转录水平的变化,并通过流式细胞仪使用PI-AnnexinV双染法检测YTMLC细胞株的凋亡。结果构建的2种重组质粒pshRNA-survivin-1和pshRNA-survivin-2均能明显抑制survivin基因的表达;应用免疫荧光检测survivin基因的表达,转染重组质粒pshRNA-survivin-1和pshRNA-survivin-2的实验组survivin荧光强度明显低于转染空载体pGE-1和pshRNA-negative非特异对照质粒;通过半定量RT-PCR检测到sur-vivin基因mRNA转录明显减少,抑制率为80.60%和70.09%;通过PI-AnnexinV双染法检测YTMLC细胞的凋亡分别达(33.42±2.42)%(P<0.01)和(20.09±1.32)%(P<0.01)。结论重组质粒pshR-NA-survivin-1和pshRNA-survivin-2均可明显抑制YTMLC细胞内源survivin的表达和mRNA的转录均可并明显促进YTMLC细胞的凋亡,为survivin介导的肿瘤基因沉寂疗法提供良好的实验基础。

Objective To apply the small interfering RNAs targeting survivin to inhibit expression of endogenous survivin gene in Yunnan Gejiu lung squamous carcinoma cells（YTMLC） and increase the apoptosis of Yunnan Gejiu lung squamous carcinoma cells （YTMLC）. Methods The recombinant plasmid pshRNA-survivin-1 and pshRNA-survivin-2 were constructed and transfected into YTMLC cell line. The expression level of survivin was determined by immunofluorescence staining and the transcription of survivin gene was detected by semi-quantitative RT-PCR. The apoptosis in YTMLC cell line were detected by PI-AnnexinV staining. Results The introduction of plasmids pshRNA-survivin-1 and pshRNA-survivin-2 were showed to efficiently and specifically inhibit the expression of survivin according to results of immunofluorescence staining. Semi-quantitative RT-PCR showed that mRNA transcription of survivin gene was reduced by nearly 80. 60% and 70.09%, respectively. On the contrast, the control plasmid did exhibit no inhibitory effect on the protein expression and mRNA transcription of survivin gene. The apop- tosis in YTMLC cell line were detected by PI-Annexin V staining with apoptosis rates at （33.42±2.42）%（P〈0. 01 ）and （20. 09± 1.32）%（P〈0. 01 ）, respectively. Conclusion The results demonstrate that the small interfering RNA targeting survivin gene shows dramastic inhibition on protein expression and RNA transcription and significant enhancement of apoptosis in YTMLC cell line, and should be a new strategy in gene silence therapy targeting survivin.