体外神经细胞过表达Mex3C-1对诱导性Fos表达的影响

Overexpression of Mex3C-1 Enhances and Prolongs the Expression of Induced Fos in Neuroblastoma Cells

目的探讨Mex3C-1对卡巴胆碱诱导的Fos表达的影响。方法将具有可持续表达人Mex3C-1序列的质粒pLV-CMV-Mex3C(OE)在HEK293T细胞中包装成慢病毒载体,同时制备非特异性对照(NC)CmiR0001-MR03的慢病毒载体,分别感染人神经母细胞瘤细胞SH-SY5Y,经嘌呤霉素筛选后得到稳定高表达Mex3C-1和阴性对照细胞系,用Real time PCR和Western blot方法检测Mex3C-1的基因和蛋白表达效果。之后用卡巴胆碱诱导Fos表达,在诱导0、30、60、90和120min后分别提取mRNA,采用Real time PCR方法检测Fos mRNA的相对表达量。结果Real time PCR检测结果显示,OE组的Mex3C-1 mRNA的相对表达量(21.11±0.60)高于NC组(1.03±0.13)(t=32.63,P=0.000)。Western blot结果显示,在82kDa处OE组的Mex3C-1蛋白表达量高于NC组(P<0.001)。Real time PCR检测不同干预时间两组Fos mRNA,除0min外各个时间点OE组均高于NC组,表明Mex3C-1的过表达可以明显上调Fos mRNA的表达;NC组于120min时已基本恢复至基础值,而OE组120min时Fos mRNA表达量仍然较高,Mex3C-1过表达可以延长Fos mRNA的半衰期,增强其稳定性,OE组与阴性对照NC组比较,Fos mRNA表达量差异有统计学意义(F=287.069,P=0.000)。结论持续过表达Mex3C-1的人神经母细胞瘤SH-SY5Y细胞系建立成功,且Mex3C-1过表达能够明显增强卡巴胆碱诱导的Fos表达程度并增强其稳定性。

Objective To investigate the regulation of overexpressed Mex3C-1 on the expression of induced Fos.Methods HEK293T cells were cultured for transfection of pLV-CMV-Mex3C(OE)and its nontargeting control CmiR0001-MR03(NC)to produce lentivirus.Neuroblastoma cells SH-SY5Y were cultured for transduction of the lentivirus to build stable cell lines.Real time PCR and Western blot were then performed to detect Mex3C-1 expression.The mRNA was purified after 0,30,60,90,120 minutes of Fos induction by carbachol,and was reverse transcribed to cDNA for further Fos mRNA detection through Real time PCR to investigate the regulation of Mex3C-1 overexpression on induced Fos.Results The expression of Mex3C-1 mRNA in both OE and NC was detected by Real time PCR.The relative expression of Mex3C-1 mRNA in OE(21.11±0.60)was remarkably higher than NC(1.03±0.13)(t=32.63,P=0.000).Western blot found the protein expression of Mex3C-1 in OE cell line was obviously higher than that of NC cell line at 82kDa.The expression of induced Fos mRNA at different time was analyzed by Real time PCR,showing that except 0min,expression at each time in OE was higher than that of NC,and at 120min the expression of Fos mRNA in NC decreased to basic level while expression in NC was still high.The data revealed that overexpressed of Mex3C-1 increased the expression and half-life of induced Fos mRNA(F=287.069,P=0.000).Conclusion In neuroblastoma cell SH-SY5Y,overexpression of Mex3C-1 enhances and prolongs the expression of Fos induced by carbachol in vitro.