微小RNA分子miR-21和miR-22对C2C12细胞成肌分化影响的研究

Roles of miR-21 and miR-22 in myogenic differentiation of myoblasts C2C12

目的探讨微小RNA(microRNAs)分子miR-21和miR-22对于C2C12细胞体外成肌分化进程的影响。方法以C2C12细胞体外成肌分化为模型,Northern blot检测miR-21和miR-22两个分子在分化过程中的表达变化及组织表达谱,转染DNA/LNA杂合体反义链进行loss-of-fuction功能研究,RT-PCR分析其对分化相关分子的影响。结果成功建立C2C12细胞体外成肌分化模型,2个分子在分化过程中均表达上调,且它们具有各自的组织分布特点;建立了以DNA/LNA杂合体反义链为手段的microRNAs研究的loss-of-fuction平台;与对照组比较,抑制这2个microRNAs对于成肌分化相关的骨骼肌α-肌动蛋白、成肌素和MEF-2D等分子的表达没有影响。结论抑制miR-21和miR-22在C2C12细胞成肌分化过程中的上调不影响其分化,说明这2个分子对于分化的进程影响不大。

Objective To explore the roles of 2 microRNA molecules, miR-21 and miR-22, in the myogenic differentiation of C2C12 myoblasts. Methods C2C12 cells was taken as the model since they have a tendency of myogenic differentiation when cultured in differentiation medium. The expression levels of miR-21 and miR-22 in the process of myogenic differentiation and different tissues （ the brain, small intestine, skeleton muscle, spleen, kidneys, liver, heart and lungs） were detected by Northern blotting. Loss-of-function about these 2 microRNAs were carried out by transfecting the antisense strands of their DNA/locked nucleic acid （LNA） mixtures respectively and the effects on the expressions of 3 myogenic differentiation related genes, skeletal-α-actin, myogenin and myocyte enhancer factor-2D （MEF-2D） were evaluated by RT-PCR. Results These 2 microRNAs were strongly expressed during the differentiation of C2C12 cells and they had obvious expression profiles in different tissues. Comparing to negative control, inhibiting the up-regulation of miR-21 and miR-22 had no effect on the expressions of above mentioned genes. Conclusion miR-21 and miR-22 have little effects on the myogenic differentiation of the murine myoblast cell line C2C12.