心肌细胞中微小核糖核酸let-7c对肌营养素基因表达的调控作用

发布时间：2018-02-03 01:21

本文关键词： 微小核糖核酸肌营养素核转录因子-κB　出处：《中国循环杂志》2016年12期 　论文类型：期刊论文

【摘要】：目的:探讨心肌细胞中微小核糖核酸(mi R)let-7c(mi R-let-7c)对肌营养素基因表达是否具有调控作用以及可能的作用机制。方法:首先构建携带肌营养素基因3'非编码区(3'-UTR)片段的荧光素酶报告基因载体,与mi R-let-7c前体共转染Hela细胞,双荧光素酶报告基因系统检测荧光素酶活性以验证mi R-let-7c与肌营养素基因的靶向调控关系。进而培养大鼠心肌细胞H9c2,Taqman实时定量聚合酶链式反应(PCR)法检测mi R转染效果,蛋白免疫印迹(Western blot)法检测mi R-let-7c及mi R-let-7c抑制物转染心肌细胞后肌营养素蛋白表达,以及心肌肥厚相关信号通路分子核转录因子-κB(NF-κB)的活性变化。结果:荧光素酶活性实验结果表明,与重组荧光素酶报告基因表达载体(p MIR-MTPN)+mi R前体阴性对照组相比,p MIR-MTPN+mi R-let-7c前体组荧光素酶活性显著降低[(59.30±9.90)%vs(98.10±15.10)%]。Western blot结果表明,mi R-let-7c前体组与mi R阴性对照组相比,肌营养素蛋白表达水平显著降低([0.28±0.05)vs(0.90±0.09)],此外,NF-κB蛋白水平显著降低([0.25±0.06)vs(0.75±0.07)];相反,mi R-let-7c抑制物组与抑制物阴性对照组相比,肌营养素蛋白表达水平显著升高[(1.14±0.09)vs(0.44±0.09)],同时,NF-κB蛋白水平也显著升高[(1.09±0.05)vs(0.71±0.06)],差异均有统计学意义(P0.05)。结论:miR-let-7c能够通过作用于3'UTR区域,抑制肌营养素基因表达,并影响心肌肥厚关键信号通路分子NF-κB的活性。
[Abstract]:Objective: to study the microribonucleic acid (RRL) -7 ct mi R-let-7c (R let-7c) in cardiomyocytes. Methods: firstly, the luciferase reporter gene vector carrying the 3'non-coding region of mytrophin gene was constructed. Hela cells were co-transfected with mi R-let-7c precursor. Double luciferase reporter gene system was used to detect luciferase activity in order to verify the targeted regulatory relationship between mi R-let-7c and myotrophin gene, and then cultured rat cardiomyocytes H9c2. Taqman real-time quantitative polymerase chain reaction (PCR) was used to detect the transfection effect of miR. Western blotting was used to detect the expression of myotensin protein after mi R-let-7c and mi R-let-7c inhibitor were transfected into cardiomyocytes. Results: the activity of luciferase was determined by the changes of nuclear transcription factor-魏 B of NF- 魏 B in cardiac hypertrophy related signal pathway. Compared with the recombinant luciferase reporter gene expression vector (pMIR-MTPNNN) mi R precursor negative control group. Luciferase activity of p MIR-MTPN mi R-let-7c precursor group decreased significantly. [59.30 卤9.90% vs 98.10 卤15.10%. Western blot results showed that. Compared with the negative control group, the expression of myotrophin protein was significantly decreased in the precursor group of miR-let-7c (P < 0.05). [In addition, the level of NF- 魏 B protein decreased significantly (P < 0.05). [On the contrary, compared with the negative control group, the expression of mycotrophin protein in the R-let-7c inhibitor group was significantly higher than that in the negative control group (0.25 卤0.06 vs 0.75 卤0.07). [The protein level of NF- 魏 B also increased significantly. [1. 09 卤0. 05 卤0. 05 vs 0.71 卤0. 06). Conclusion: 1 miR-let-7c can inhibit the expression of myotrophin gene by acting on 3 UTR region. It also affects the activity of NF- 魏 B, a key signal pathway in myocardial hypertrophy.
【作者单位】：山西医科大学基础医学院生物化学与分子生物学教研室;
【基金】：国家自然科学基金(81500364) 山西省基础研究项目(2015021187) 山西省高等学校科技创新项目(2015149)
【分类号】：R54
【正文快照】： 微小核糖核酸(miR)通过降解靶mR NA或抑制靶mR NA的翻译来调控基因表达[1]。最近研究发现miR-let-7家族在心脏组织中高表达,参与许多重要的心脏功能调控[2],miR-let-7c可能在心肌肥厚中发挥重要作用,但其具体作用靶标和机制仍不清楚[3]。肌营养素(myotrophin,MTPN)是从原发性

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