RNA干扰COPS3表达对肺癌细胞增殖影响的分析

he effects of RNA-interference-mediated downregulation of COPS3 on the proliferation of lung cancer cell line

目的本研究应用RNA干扰(RNA interference,RNAi)方法抑制COPS3基因表达,分析其对肺癌细胞增殖的影响并探讨其相关机制,以期初步阐明COPS3基因在肺癌发展中的作用。方法以肺癌细胞株A549为研究对象,分别将携带有COPS3基因siRNA(Small interfering RNA)序列的慢病毒载体(si-COPS3)和携有无关序列的对照载体感染A549细胞株,应用实时定量PCR(real-time PCR,RT-PCR)和western blot方法分析COPS3mRNA及蛋白表达水平;BrdU法分析细胞增殖情况;流式细胞术检测细胞周期变化与细胞凋亡情况。结果成功构建了COPS3慢病毒RNAi载体,并成功感染A549细胞;与对照组相比,感染有si-COPS3的细胞COPS3mRNA及蛋白表达水平均显著下降,细胞增殖受抑制,细胞凋亡增加;大部分细胞周期滞于G0/G1期。结论下调COPS3基因水平可以抑制肺癌细胞增殖并促进其凋亡。COPS3慢病毒RNAi的建立为进一步研究COPS3的功能奠定了基础。

Objective To analyze the role of COPS3 in the development of lung cancer by inhibiting its expression using RNA interfering methodology.Methods Lentivirus vector containing siRNA sequence of COPS3（si-COPS3） and control sequence（si-CTRL） were constructed and transfected the lung carcinoma cell line A549,respectively.The expression of COPS3 mRNA and protein were analyzed by real-time PCR and western blot.Cell proliferation was detected by BrdU assay.Flow cytometry was used to analyze the cell cycle and apoptosis.Results Si-COPS3 and si-CTRL vector were successfully constructed and transfected into A549 cells.Compared with the control group,the expression of COPS3 mRNA and protein was significantly down-regulated after delivering si-COPS3 into the cells.Growth inhibition and apoptosis was also shown in the cells transfected with si-COPS3,and cell cycle was mostly arrested at the G0/G1 phase.Conclusion COPS3 may play an important role in promoting proliferation and anti-apoptotic of lung cancer cells.Construction of RNA interference vector should facilitate the further research of COPS3.