RNA聚合酶Ⅱ转录的ALU序列对HEK293细胞凋亡的影响

Effect of RNA PolⅡ Driven ALU Transcripts on Apoptosis of HEK293 Cells

目的探讨RNA聚合酶Ⅱ转录的ALU序列对人胚肾293(HEK293)细胞凋亡的影响以及干扰素(IFN)在此机制中的作用。方法取对数生长期的HEK293细胞,分为6组,ALU-293组(瞬时转染重组质粒pcDNA3.1-ALU)、pcDNA3.1-293组[瞬时转染空质粒pcDNA3.1(-),作为阴性对照]、Poly I∶C-293组[瞬时转染dsRNA的多聚肌苷胞苷酸(Poly I∶C),作为阳性对照]、IFNβ-293组(加入1.65×104 U IFNβ,作为阳性对照)、空白对照组(未经处理的HEK293细胞)和HBs-293组(瞬时转染重组质粒pcDNA3.1-HBs),转染后48 h,采用MTT法检测细胞的增殖活性;Cellular DNA Fragmentation ELISA和DNA Ladder法检测细胞的凋亡情况;Real-time PCR检测细胞中IFNβ基因mRNA的水平。结果瞬时转染重组质粒pcDNA3.1-ALU能够抑制HEK293细胞增殖,并促使其凋亡,且细胞中IFNβmRNA的水平显著上调。结论 RNA聚合酶Ⅱ转录的ALU序列能够通过激活干扰素系统来诱导细胞凋亡。

Objective To investigate the effect of RNA PolⅡ driven ALU transcripts on the apoptosis of human embryonic kidney 293（HEK293） cells as well as the role of IFN in this mechanism.Methods The HEK293 cells at logarithmic growth phase were divided into six groups.The cells in ALU-293,pcDNA3.1-293,Poly I ∶ C-293 and HBs-293 groups were transiently transfected with recombinant plasmid pcDNA3.1-ALU,empty vector pcDNA3.1（-）（negative control）,Poly I ∶ C（positive control）,recombinant plasmid pcDNA3.1-HBs respectively,while those in IFNβ-293 group was added with 1.65 × 104 U IFNβ（positive control）.However,the cells in blank control group were untreated.The cells in various groups 48 h after transfection were determined for proliferative activity by MTT method,for apoptosis by Cellular DNA Fragmentation ELISA and DNA Ladder,and for IFNβ mRNA level by real-time PCR.Results Transient transfection with recombinant plasmid pcDNA3.1-ALU inhibited the proliferation and promoted the apoptosis of HEK293 cells,and up-regulated the mRNA level in the cells significantly.Conclusion PolⅡ driven ALU transcripts induced the apoptosis of HEK293 cells by activating interferon system.