miR-301a-3p对大鼠星形胶质细胞Cx43转录后的靶向调控作用

miR-301a-3p regulates connexin 43 expression in rat astrocytes

目的:研究大鼠星形胶质细胞中微小RNA-301a-3p(miR-301a-3p)对缝隙连接蛋白43(Cx43)表达的靶向调控作用及其作用位点。方法:合成miR-301a-3p agomir和miR-301a-3p antagomir,转染至星形胶质细胞,Western blot检测各组细胞中Cx43蛋白的表达情况;构建重组载体wt-pEZX-MT05-Cx43和mut-pEZX-MT05-Cx43,采用双萤光素酶报告基因实验验证miR-301a-3p的靶基因;构建表达载体pcDNA3.1-Cx43,通过回复实验分析miR-301a-3p对细胞凋亡的影响。结果:将miR-301a-3p agomir转染到星形胶质细胞后,Western blot检测显示,与对照组相比,Cx43蛋白表达显著降低(P<0.05)。双萤光素酶报告基因实验结果表明,miR-301a-3p能够与Cx43的3′-UTR结合,对其表达产生负调控;将不含Cx43 3′-UTR的重组载体pcDNA3.1-Cx43转染星形胶质细胞后,能够回复miR-301a-3p对Cx43蛋白表达的负调控作用,引起细胞凋亡。结论:Cx43是miR-301a-3p的一个靶基因,miR-301a-3p通过作用于Cx43 mRNA的3′-UTR而抑制其在大鼠星形胶质细胞中的表达。

AIM: To explore the mechanism of microRNA-301 a-3 p(miR-301 a-3 p) regulating connexin 43(Cx43) expression in the rat astrocytes. METHODS: miR-301 a-3 p agomir, miR-301 a-3 p antagomir and microRNA negative control(miR-NC) were synthesized and transfected into the astrocytes. The protein expression of Cx43 was determined by Western blot. The recombinant vectors wt-pEZX-MT05-Cx43 and mut-pEZX-MT05-Cx43 were constructed. The target gene of miR-301 a-3 p was validated by dual-luciferase reporter assay. The expressional vector pcDNA3.1-Cx43 was constructed and the biological activity regulation of miR-301 a-3 p on astrocytes was analyzed by restore experiment. RESULTS: The results of Western blot showed that the Cx43 expression was inhibited by miR-301 a-3 p agomir transfection(P<0.05). The results of dual-luciferase reporter assay showed that miR-301 a-3 p bound to 3′-UTR of Cx43, thus regulating expression of Cx43 negatively. Transfection with recombinant vector pcDNA3.1-Cx43 without 3′-UTR of Cx43 into astrocytes resulted in the restoration of the negative function of miR-301 a-3 p on Cx43 expression, also induced the apoptosis of astrocytes. CONCLUSION: miR-301 a-3 p binds to 3′-UTR of Cx43 mRNA in the rat astrocytes, thus regulating expression of Cx43 negatively.