背景:体细胞命运转变在组织再生中具有重要意义。目前,研究的重点是通过将多种细胞重编程为心肌细胞样细胞来实现心脏组织再生。这里,我们研究了miRNAs对成纤维细胞转分化为心肌样细胞的可能影响.
方法:使用生物信息学技术通过比较心脏组织与其他身体组织的基因表达谱来鉴定第一个心脏特异性miRNA。在鉴定心脏特异性miRNA后,使用miRWalk和miRBase数据库研究了它们的细胞和分子功能。然后将候选miRNA克隆到慢病毒载体中。Follows,培养人真皮成纤维细胞并用化合物毛喉素处理,丙戊酸,和CHIR99021。24小时后,将携带miRNA基因的lentivector转染到细胞中以启动转分化过程。最后,经过两周的治疗,转分化的效率是通过检查细胞的外观和使用RT-qPCR和免疫细胞化学技术测量心脏基因和蛋白质的表达水平来检查的。
结果:鉴定出9个miRNA在心脏中有较高的表达。miR-2392由于其在心脏中的功能和特异性表达而被提名为候选miRNA。该miRNA与参与细胞生长和分化的基因有直接联系;例如,MAPK和Wnt信号通路。根据体外结果,心脏基因和蛋白质在同时接受三种化学物质和miR-2392的成纤维细胞中的表达增加。
结论:考虑到miR-2392在成纤维细胞中诱导心脏基因和蛋白质表达的能力,它可以诱导成纤维细胞分化为心肌样细胞。因此,miR-2392可以进一步优化用于心肌细胞再生,组织修复,和药物设计研究。
BACKGROUND: Somatic cell fate transition is now gained great importance in tissue regeneration. Currently, research is focused on heart tissue regeneration by reprogramming diverse cells into
cardiomyocyte-like cells. Here, we examined the possible effect of miRNAs on the transdifferentiation of fibroblasts into
cardiomyocyte-like cells.
METHODS: First heart-specific miRNAs were identified by comparing the gene expression profiles of heart tissue to other body tissues using bioinformatic techniques. After identifying heart-specific miRNAs, their cellular and molecular functions were studied using the miRWalk and miRBase databases. Then the candidate miRNA was cloned into a lentiviral vector. Following, human dermal fibroblasts were cultured and treated with compounds forskolin, valproic acid, and CHIR99021. After 24 h, the lentivector harboring miRNA gene was transfected into the cells to initiate the transdifferentiation process. Finally, after a two-week treatment period, the efficiency of transdifferentiation was examined by inspecting the appearance of the cells and measuring the expression levels of cardiac genes and proteins using RT-qPCR and immunocytochemistry techniques.
RESULTS: Nine miRNAs were identified with higher expression in the heart. The miR-2392 was nominated as the candidate miRNA due to its function and specific expression in the heart. This miRNA has a direct connection with genes involved in cell growth and differentiation; e.g., MAPK and Wnt signaling pathways. According to in vitro results cardiac genes and proteins demonstrated an increase in expression in the fibroblasts that simultaneously received the three chemicals and miR-2392.
CONCLUSIONS: Considering the ability of miR-2392 to induce the expression of cardiac genes and proteins in fibroblast cells, it can induce fibroblasts to differentiate into
cardiomyocyte-like cells. Therefore, miR-2392 could be further optimized for
cardiomyocyte regeneration, tissue repair, and drug design studies.