miRNA-326作用于其靶基因BCL-XL的研究

Analysis of miRNA-326’s action on its target gene BCL-XL

目的研究miRNA-326是否作用于其靶基因BCL-XL,探讨miRNA调控血小板凋亡的分子机制。方法利用萤火虫荧光素酶和海肾荧光素酶组成双荧光素酶报告基因实验系统,分别构建含有BCL-XL 3′端非翻译区(3′untranslated region,3′UTR)片段的野生型和变异型的双荧光素酶载体,设置对照组,转染细胞检测相对荧光强度。结果分别构建了PsiCHECK-BCL-XL-3′UTR-WT(野生型)和PsiCHECK-BCL-XL-3′UTR-MT(变异型)的双荧光素酶报告基因质粒载体;miRNA-326序列和PsiCHECK-BCL-XL-3′UTR-WT质粒共转染测定的相对荧光强度显著低于对照组(miRNA-326阴性对照序列和PsiCHECK-BCL-XL-3′UTR-WT质粒共转染组)(P=0.034);同时也显著低于miRNA-326序列和PsiCHECK-BCL-XL-3′UTR-MT质粒共转染的变异组(P=0.022)。结论miRNA-326作用于BCL-XL基因的3′UTR;这为研究miRNA调控血小板凋亡的分子机制奠定了基础。

Objective To analyze the action of miRNA-326 on its target gene BCL-XL and the molecular mechanism of platelet apoptosis regulated by miRNAs.Methods Dual-luciferase vectors containing respectively the wild-type and mutant 3′-untranslated region(3′UTR)fragments of the BCL-XL gene were constructed with firefly and renilla luciferases and transfected into 293T cells.Relative fluorescence intensities of the transfected cells were measured.Results Dual-luciferase reporter gene vectors for PsiCHECK-BCL-XL-3′UTR-WT(wild-type)and PsiCHECK-BCL-XL-3′UTR-MT(variant)were respectively constructed.Relative fluorescence intensities of the 293T cells co-transfected by miRNA-326 and PsiCHECK-BCL-XL-3′UTR-WT plasmid were significantly lower compared with the control group(co-transfected by a miRNA-326 negative sequence and PsiCHECK-BCL-XL-3′UTR-WT plasmid)(P=0.034).The relative fluorescence intensity was also significantly reduced in cells co-transfected by miRNA-326 and PsiCHECK-BCL-XL-3′UTR-WT plasmid compared with the mutant control group co-transfected by miRNA-326 and PsiCHECK-BCL-XL-3′UTR-MT plasmid(P=0.022).Conclusion miRNA-326 may participate in the regulation of platelet apoptosis by acting on the 3'-UTR of the BCL-XL gene.