miR-196b-5p promotes myoblast proliferation and differentiation.

MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules about 22 nucleotides in length and are encoded by endogenous genes, and are involved in the regulation of post-transcriptional gene expression in animals and plants. Many studies have shown that microRNAs regulate the development of skeletal muscle, mainly manifested in the activation of muscle satellite cells and biological processes such as proliferation, differentiation, and formation of muscle tubes. In this study, miRNA sequencing screening of longissimus dorsi (LD, mainly fast-twitch fibers) and soleus muscle (Sol, dominated by slow-twitch fibers) identified the miR-196b-5p as a differentially expressed and highly conserved sequence in different skeletal muscles. Studies of miR-196b-5p in skeletal muscle have not been reported. In this study, miR-196b-5p mimics and inhibitor were used in miR-196b-5p overexpression and interference experiments in C2C12 cells. The effect of miR-196b-5p on myoblast proliferation and differentiation was analyzed by western blotting, real-time quantitative RT-PCR, flow cytometry, immunofluorescence staining, and the target gene of miR-196b-5p was identified by bioinformatics prediction and analyzed by dual luciferase reporter assays. The results showed that overexpression of miR-196b-5p could significantly increase the mRNA and protein expression of Cyclin B, Cyclin D and Cyclin E (P<0.05); Cell cycle analysis showed that overexpression of miR-196b-5p significantly increased the proportion of cells in the S phase (P<0.05), indicating that miR-196b-5p could accelerate cell cycle progress. Results of EdU staining showed that overexpression of miR-196b-5p significantly promoted cell proliferation. Conversely, inhibition of miR-196b-5p expression could significantly reduce the proliferation capacity of myoblasts. Further, overexpression of miR-196b-5p could significantly increase the expression levels of myogenic marker genes MyoD, MoyG and MyHC (P<0.05), thereby promoting myoblast fusion and accelerating C2C12 cell differentiation. Bioinformatics predictions and dual luciferase experiments demonstrated that miR-196b-5p could target and inhibit the expression of the Sirt1 gene. Altering the Sirt1 expression could not rescue the effects of miR-196b-5p on the cell cycle, but could weaken the promoting effects of miR-196b-5p on myoblast differentiation, suggesting that miR-196b-5p promoted myoblast differentiation by targeting Sirt1.MicroRNA (miRNA)是一类由内源基因编码的长度约为22个核苷酸的非编码单链RNA分子，在动植物中参与转录后基因表达调控。大量研究表明，miRNA调控骨骼肌的发育，主要表现在肌卫星细胞的激活及增殖、分化、肌管的形成等生物学过程。本实验室前期对大白猪背最长肌(longissimus dorsi，LD)和比目鱼肌(soleus muscle，Sol)进行miRNA测序，筛选鉴定到一个在不同骨骼肌中差异表达并且序列高度保守的miR-196b-5p，目前miR-196b-5p在骨骼肌方面的研究尚未见报道。本研究进一步设计合成miR-196b-5p mimics和inhibitor对C2C12细胞进行miR-196b-5p过表达及干扰表达，利用蛋白免疫印迹、实时荧光定量PCR检测、流式细胞术、免疫荧光染色等方法探究miR-196b-5p对成肌细胞增殖分化的影响，并利用生物信息学预测和双荧光素酶报告系统鉴定了miR-196b-5p的靶基因。结果显示，过表达miR-196b-5p显著增加细胞周期基因Cyclin B、Cyclin D和Cyclin E的mRNA和蛋白表达水平(P<0.05)；流式细胞周期检测结果显示，过表达miR-196b-5p显著增加S期细胞比例(P<0.05)，表明miR-196b-5p能够加快细胞周期进程；EdU染色结果显示，过表达miR-196b-5p显著促进细胞增殖能力；相反，抑制miR-196b-5p的表达可以显著减弱成肌细胞增殖能力。进一步的功能研究发现，过表达miR-196b-5p能够显著增加成肌标志基因MyoD、MyoG和MyHC的表达水平(P<0.05)；MyHC免疫荧光染色结果显示miR-196b-5p可以促进成肌细胞肌管形成，表明miR-196b-5p可以促进成肌细胞分化。生物信息学预测和双荧光素酶实验证明miR-196b-5p可以靶向Sirt1基因，抑制该基因的表达。改变Sirt1表达，不能够挽救miR-196b-5p影响细胞周期的表型，可以减弱miR-196b-5p对成肌细胞分化的促进作用，表明miR-196b-5p通过靶向Sirt1促进成肌细胞分化。.