利用质粒介导的miRNA-1-2抑制H9C2中Hand2蛋白的表达

Plasmid-mediated miRNA-1-2 specifically inhibits Hand2 protein expression in H9C2 cells

目的构建人miRNA-1-2的真核表达载体,并检测其对大鼠成肌细胞H9C2中Hand2蛋白表达的抑制作用。方法用PCR法体外合成miRNA-1-2前体序列的DNA模板,并定向插入到发夹RNA(hairpin RNA)表达载体pSilencer-4.1-neo上。用脂质体法将构建正确的重组质粒pSilencer-4.1/miRNA-1-2瞬时转染H9C2细胞,检测miRNA-1-2前体转录本(pre-miRNA-1-2)和miRNA-1靶基因Hand2(heart and neural crest derivatives expressed transcript 2)的表达。结果DNA测序表明重组质粒pSilencer-4.1/miRNA-1-2构建正确。pSilencer-4.1/miRNA-1-2转染的H9C2细胞中,pre-miRNA-1-2被有效表达,Hand2 mRNA表达无变化,Hand2蛋白表达被抑制。结论成功构建了miRNA-1-2的真核表达载体,由表达载体介导的miRNA-1能有效抑制Hand2蛋白的表达。

Objective To construct an eukaryotic expression vector for miRNA-I-2 that can be expressed in rat H9C2 cardiomyocytes. Methods The precursor miRNA （pre-miRNA） DNA template for miRNA-1-2 was designed and generated by PCR amplification. The DNA template was inserted into the hairpin RNA expression vector pSilence-4, l-neo and identified by DNA sequencing analysis. The recombinant plasmid DNA was then transfected into H9C2 cells via Lipofectamine, and the green fluorescence protein expression vector pEGFP-N3 served as the transfection marker. Twenty-four hours after transfection, the total cellular RNA was extracted using TRIzol reagent, and thermoscript reverse transcriptase （RT）-PCR was performed to determine miRNA-I-2 precursor expression. Results and Conclusion DNA sequencing indicated that the miR-l-2 expression plasmid was correctly constructed. The precursor miRNA-I-2 was successfully expressed in the H9C2 cells, and the expression of Hand2 protein could be efficiently inhibited by miRNA-1.