miRs在力学拉伸促进成肌细胞增殖过程中的作用

The role of miRs in the process of promoting myoblast proliferation by mechanical stretch

目的探讨3种miRs在力学拉伸促进C2C12成肌细胞增殖过程中的作用。方法周期性拉伸的各种力学条件通过BioFlex加载系统实现。采用CCK-8检测不同拉伸频率对成肌细胞增殖的影响。对促增殖拉伸组和对照组进行高通量测序,反转录定量PCR(qRT-PCR)验证高通量基因测序结果,并进行生物信息学分析。结果 0.125 Hz拉伸组明显促进C2C12成肌细胞增殖(P<0.05),而0.25Hz和0.5 Hz相比于对照组的差异无统计学意义;高通量测序分析表明,11种miRs表达于对照组和0.125Hz拉伸组,其中差异有统计学意义的为3种:mir-44,mir-36,mir-20;qRT-PCR证实这3种miRs的表达改变与测序结果一致;这3种miRs参与了多个信号通路的调控。结论高通量基因测序和生物信息学分析表明3种miRs在应力诱导的C2C12成肌细胞增殖过程中有重要作用。

Objective To explore the role of three kinds of miRs in the process of promoting myoblast proliferation by mechanical stretch. Methods Application of cyclic mechanical strain using the computer-controlled Flexcell Strain Unit cultured C2C12 cells. CCK-8 assay was applied to test myoblast proliferation in different conditions of mechanical stretch. The expression profiles of miRs in mechanical stretch groups and control groups were detected by high-throughput sequencing. The results were verified by reverse transcription quantitative PCR（qRT-PCR） and analyzed by bioinformatics. Results C2C12 cells in 0.125Hz stretching group was significantly observed with promoted proliferation of C2C12 myoblasts（P〈0.05）, but no significant difference of cell proliferation were detected inthe 0.25 Hz and 0.5Hz groups compared to that in the control group;Using high-throughput sequencing technology, we found 11 kinds of miRs which were co-expressed and differentially expressed in the two groups （Control group and 0.125 Hz stretching group）;the difference was statistically significant in the expression of three miRs：mir-44 , mir-36 and mir-20. These three miRs expression was verified by qRT-PCR, and the results were consistent with the sequencing. These three miRs were involved in several signaling pathways. Conclusions High-throughput genome sequencing and bioinformatics analysis show that three kinds of miRs play an important role in the proliferation of C2C12 myoblast.