miRNA-1和miRNA-133过表达对L6细胞增殖与分化的影响  

Effect of overexpression of miRNA-1 and miRNA-133 on proliferation and differentiation of L6 cells

作　　者：弓贺炜 李波[1] 李文斌[2] 李刚[2] 梁炳生[2] 

机构地区：[1]山西医科大学第二临床医学院,太原030001 [2]山西医科大学第二医院

出　　处：《中华细胞与干细胞杂志（电子版）》2014年第2期11-16,共6页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：国家自然科学基金面上项目(81171722);国家自然科学基金青年科学基金项目(81101365)

摘　　要：目的观察大鼠微小RNA-1(miR-1)、微小RNA-133(miR-133)过表达对L6成肌细胞增殖分化的影响。方法分别构建miR-1、miR-133的重组慢病毒载体,并进行测序鉴定,稳定转染L6细胞后,用RT-PCR Taqman探针的方法检测miR-1、miR-133的表达水平;细胞计数实验(CCK-8试剂盒)评价miR-1、miR-133过表达后对L6细胞增殖的影响。诱导稳定转染后L6成肌细胞进行分化,观察miR-1、miR-133过表达后对L6细胞分化的影响。以Western blot法检测miR-1、miR-133过表达后,α肌动蛋白(skeletalα-actin)表达水平的变化。采用Kruskal-Wallis H检验、重复测量和单因素方差分析进行比较。结果miR-1慢病毒载体经酶切和测序鉴定序列准确,转染48 h后L6细胞miR-1组(4.292±0.50)比control组(0.231±0.86)、miR-133组(0.205±0.48)比control组(3.564±0.45)表达均显著上调(P<0.001);细胞计数和细胞分化实验显示,培养120 h后,过表达miR-1的L6细胞α肌动蛋白(skeletalα-actin)比对照组(0.415±0.02)表达显著升(0.676±0.02,F=222.144,P<0.001),分化明显加快,但增殖无明显变化;而过表达miR-133的L6细胞增殖明显加快,α肌动蛋白表达呈下降趋势(0.363±0.02,F=2385.643,P<0.001),分化受到抑制。结论 miR-1、miR-133慢病毒表达载体稳定转染L6成肌细胞后高效表达miR-1和miR-133,miR-1可促进L6细胞分化,miR-133能促进L6细胞增殖但抑制其分化。Objective To observe the effect of overexpression of rat miRNA-1 and miRNA-133 on the proliferation and differentiation of L6 myoblasts. Methods The recombinant lentiviral vectors containing miRNA-1or miRNA-133 gene were constructed, and confirmed by restriction enzyme digestion（MulI/BamH Ⅰ） and sequencing. L6 cell was stably transfected by lentiviral vector using polygrene reagent. The expression levels of miRNA-1 or miRNA-133 was detected by reverse transcription- polymerase chain reaction which contained Taqman probes. The effect of overexpression of miRNA-1 or miRNA-133 on the proliferation of L6 cells was evaluated by cell count test（CCK-8 kit）. The differentiation of transfected L6 myoblasts was evaluated. The expression levels of skeletalα-actin after overexpression of miRNA-1 or miRNA-133 was detected by Western blot. Difference was compared by using the Kruskal-Wallis H test and one- way ANOVA. Results miRNA-1 and miRNA-133 lentiviral vectors were successfully constructed as indicated by restriction enzyme digestion and sequencing. When the miRNA-1 and miRNA-133 lentiviral vectors were transfected into L6 cells for 48 h, the expression levels of miRNA-1（4.292 ± 0.50）was significantly increased compare with control group（0.231 ± 0.86,P〈0.001） and miRNA-133（4.292 ± 0.50）was also significantly increased compare with control group（3.564 ± 0.45,P〈0.001）. Overexpression of miRNA-1 caused increased expression level of skeletalα-actin in the L6 cells compared with the control group（0.676 ± 0.02 vs 0.415 ± 0.02, F = 222.144, P〈0.001）. Cell differentiation the L6 cells was significant with overexpression of miRNA-1 were, but no significant changes was observed for cell proliferation. However, L6 cells with overexpression of miRNA-133 had significantly increased proliferation. And expression level of skeletalα-actin was significantly decreased at 120 h（0.363 ± 0.02, F = 2385.643, P〈0.001）, differentiation of L6 cells with overexpression of miRNA-133 was inhibited.

关 键 词：慢病毒载体 微RNAS 细胞分化 细胞增殖 肌动蛋白类 

分 类 号：R745[医药卫生—神经病学与精神病学]