小鼠缺血性脑卒中后microRNA-128的表达及其对p38α MAPK调控作用的研究

Expression of microRNA-128 in ischemic stroke of mice and its correlation with p38α MAPK

目的:探讨p38α蛋白在脑缺血后2 h降低的原因以及p38α蛋白水平表达是否受调于microRNA-128。方法:首先通过qPCR和Western blotting来检测p38αmRNA和蛋白的表达水平,其次通过生物信息学软件预测出p38α的表达可能受调于microRNA-128,最后进行细胞水平验证,使用携带有microRNA-128序列质粒的慢病毒载体、携带有microRNA-128反义序列质粒的慢病毒载体以及相应空质粒的慢病毒载体转染SH-SY5Y细胞,观察p38α蛋白的表达情况。结果:小鼠脑缺血后2 h p38αmRNA水平无降低,但其蛋白水平却明显下降,microRNA-128的表达水平明显升高;转染实验发现,转染microRNA-128序列质粒的慢病毒载体的细胞p38α蛋白水平明显降低,转染microRNA-128反义序列质粒的慢病毒载体的细胞p38α蛋白水平明显升高。结论:脑缺血后p38α蛋白的表达水平下降并不是因为p38αmRNA水平的降低,microRNA-128可以调控p38α蛋白的表达,可能是通过结合p38α的mRNA从而抑制其蛋白的翻译表达。

Objective:To investigate the reason of down-regulation of p38a protein2h after whether the expression of p38a protein is regulated by mcroRNA-128.M ethods:First,the p38a mRNAand protein were detected qPCRand Western blotting respectively,and than some predictions weredone by using bioinformatics software,and we found that microRNA-128may target p38a.F in a iy,the cellular levelverification was conducted by using lentiviral vector carrying microRNA-128sequence plasmid,microRNA-128antisense sequence plasmid and empty plasmid to transfect SH-SY5Y cells.Results:p38a mRNAreduced2h after ischemia,while its protein level was significantly decreased,and microRNA-128expression levelssignificantly increased$transfection experiments found that p38a protein levels which cells were transfected witlilentiviral vector carrying microRNA-128sequence plasmid were significantly lower,but p38a protein levels weresignificantly increased by transfecting lentiviral vector carrying microRNA-128antisense sequence plasmid^Conclusion:The decline of p38a protein expression after cerebral ischemia is not due to the reduced level of p38a mRNA,microRNA-128can regulate the expression of p38a protein,which may inhibit its protein expression by bindingp38a mRNA^