MicroRNA-1 and microRNA-499 directly reprogram cardiac myofibroblasts into cardiomyocyte-like cells

doi:10.3969/j.issn.2095-4344.2013.45.016

Abstract

Abstract:

BACKGROUND: MicroRNAs are a class of small non-coding RNA molecules. MicroRNA is involved in the regulation of gene expression, and plays an important role in promoting the proliferation and differentiation of precursor cells. Whether microRNAs can promote progenitor cells to differentiate into myocardial cells has been rarely reported.
OBJECTIVE: To investigate the feasibility of direct reprogramming of neonatal mouse cardiac myofibroblasts to cardiomyocyte-like cells by specific microRNAs (microRNA-1 and microRNA-499).
METHODS: Cardiac fibroblasts isolated from neonatal mice were cultured in vitro at 37 ℃ under normal oxygen, cells were used at the second passage for the following experiments, and then cultured at 37 ℃ in 3% O2 for 48 hours. Immuno?uorescent staining was used to detect expression of vimentin which is a specific marker for Cardiac fibroblasts. Immuno?uorescent staining and flow cytometry assay were employed to detect expression of α-smooth muscle actin which is a specific marker for cardiac myofibroblasts. There were four groups in the experiment: microRNA-1 group, microRNA-499 group, microRNA-1/microRNA-499 co-tranfection group, and blank control group. MicroRNA profile between cardiac myofibroblasts and myocardial cells was determined by microarray analysis via the miRCURYTM Array microarray kit. MicroRNA microarray results were validated by real-time quantitative PCR.
RESULTS AND CONCLUSION: After overexpression of microRNA-1 and microRNA-499 in cardiac myofibroblasts, specific factors of heart development at different stages had different expressions; compared with the blank control group, the expression of GATA4, Tbx5, Mef-2c, α-MHC increased significantly in the remaining three groups, but Mesp1, Isl1 expression was not significantly changed). The results provide strong evidence for the capacity of microRNAs to induce expression of cardiomyocyte markers in cardiac myofibroblasts and demonstrate the potential of specific microRNAs that can direct reprogram cardiac myofibroblasts to cardiomyocytes in vitro.

Key words: stem cells, myocytes, cardiac, microRNAs, fibroblasts

CLC Number:

R394.2

Zhang Cheng1, Zhang Shao-zhong, Zhang Ya-zhou, Zhang Guo-heng, Wang Yun-ru, Dong Hong-yan, Zhang Zhong-ming. MicroRNA-1 and microRNA-499 directly reprogram cardiac myofibroblasts into cardiomyocyte-like cells[J]. Chinese Journal of Tissue Engineering Research, doi: 10.3969/j.issn.2095-4344.2013.45.016.

Figures/Tables 5

心脏成纤维细胞常氧传代至P2代，37 ℃低氧(体积分数3%O2)培养48 h，免疫荧光双染色观察波形蛋白和α-平滑肌肌动蛋白，流式细胞技术检测α-平滑肌肌动蛋白表达量。结果显示，α-平滑肌肌动蛋白特异性表达于心脏肌成纤维细胞，心脏成纤维细胞转化为心脏肌成纤维细胞效率为96.8%，见图2。琼脂糖电泳和A260/A280的比值测定结果显示，所得RNA样本浓度和纯度达到了miRNA芯片和反转录为cDNA和real-time qPCR的实验要求。miRNA芯片分析了心肌细胞与心脏肌成纤维细胞中miRNA的表达差异，结果表明，miR-1和miR-499在心肌细胞中高表达。real-time qPCR对芯片验证结果也显示，与心脏肌成纤维细胞相比，心肌细胞中miR-1和miR-499表达量显著增加(P < 0.05)，见图3，表2。 2.2 miR-1和miR-499转染心脏肌成纤维细胞的效率携带特定miRNA的慢病毒(MOI=40)转染心脏肌成纤维细胞，3 d后，荧光显微镜下可见细胞表达绿色荧光，细胞计数检测病毒转染成功率约为80%，见图4。 2.3 心脏肌成纤维细胞中过表达miR-1和miR-499对心肌细胞发育不同阶段特异性因子的影响 Real-time qPCR检测结果显示，心脏肌成纤维细胞中过表达miR-1和miR-499后，与对照组相比，单纯转染miR-1、miR-499和联合转染miR-1/miR-499后第7天，GATA4、Tbx5、Mef-2c表达明显增高，与转染第7天比较，转染第14天时，GATA4、Tbx5、Mef-2c表达进一步增高(P < 0.05)。与对照组相比，转染第7及14天，单纯转染miR-1、miR-499和联合转染miR-1/miR-499组Mesp1、Isl1表达均无显著变化(P > 0.05)。转染第7天，与对照组相比，单纯转染miR-1、miR-499和联合转染miR-1/miR-499组α-MHC表达显著增高，在第14天时，α-MHC表达进一步增高(P < 0.05)，cTnT在转染第14天开始增高。"

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