Circ_LAS1L regulates cardiac fibroblast activation, growth, and migration through miR-125b/SFRP5 pathway.

Current studies have shown that circular RNAs (circRNAs) and microRNAs (miRNAs) are closely related in acute myocardial infarction (AMI). Previous studies have shown that miR-125b promotes fibrosis and up-regulates in cardiac fibroblasts (CFs), and further experiments showed that circ_LAS1L had multiple binding sites of miR-125b, and their expression was inversely related in AMI patients and CFs. RNA immunoprecipitation (RIP), pull down, and dual luciferase reporter gene assay all confirmed that miR-125b directly bound to circ_LAS1L. Circ_LAS1L overexpression promoted the expression of downstream target gene secreted frizzled-related protein 5 (SFRP5), inhibited the expression of alpha-SMA, collagen I, and collagen III, inhibited CF proliferation and migration, and promoted apoptosis. When cotransfected with circ_LAS1L overexpression vector and miR-125b mimics, the above gene expression and CF biological behaviours did not change significantly. But when cotransfected with circ_LAS1L overexpression vector and SFRP5 siRNA, SFRP5 expression was still down-regulated, the expression of alpha-SMA, collagen I, and collagen III was up-regulated, and the proliferation and migration of CFs were increased. Therefore, circ_LAS1L inhibits the activity of miR-125b by adsorbing it, thus promoting the expression of SFRP5 and then regulating the biological characteristics of CFs. These findings may provide an important experimental basis for the regulation of myocardial fibrosis after myocardial infarction. SIGNIFICANCE OF THE STUDY: Studies have shown that circular RNAs (circRNAs) play important roles in cardiovascular diseases, but there are few studies on their roles in the process of myocardial fibrosis. In this study, we found that circ_LAS1L was down-regulated in acute myocardial infarction (AMI) patients and cardiac fibroblasts (CFs), and could bind directly to miR-125b, thereby promoting the expression of downstream target gene secreted frizzled-related protein 5 (SFRP5), ultimately inhibiting the activation, proliferation and migration of CF, and promoting apoptosis. This suggests that circ_LAS1L/miR-125b/SFRP5 pathway can regulate the biological function of CF and may play an important role in the process of myocardial fibrosis, thus providing an important theoretical basis for the regulation of myocardial fibrosis after myocardial infarction.

MiR-154 directly suppresses DKK2 to activate Wnt signaling pathway and enhance activation of cardiac fibroblasts.

Excessive proliferation of cardiac fibroblasts (CFs) and their transdifferentiation into myofibroblasts leads to expression of α-smooth muscle actin (α-SMA), as well as excessive synthesis and secretion of collagens. This process represents an important pathological basis for myocardial fibrosis (MF). MicroRNA (miR)-154 and the Wnt signaling pathway play key roles in the above process, although their specific interactions are poorly understood. After transfecting CFs with miR-154 mimics or inhibitors, miR-154 was found to inhibit the expression of Dickkopf-related protein 2 (DKK2), while miR-154 inhibitors upregulated DKK2 expression in a Western blot analysis. In a subsequent dual-luciferase activity assay, direct binding of miR-154 to DKK2 was detected. Further experiments demonstrated that transfection of DKK2 siRNA or miR-154 resulted in increased levels of ß-catenin, α-SMA, and collagens I and III. Moreover, these changes were observed in association with increases in CF proliferation and migration, and reduced apoptosis. Conversely, transfection of miR-154 inhibitors or DKK2 overexpression vector resulted in lower expression levels of ß-catenin, α-SMA, and collagens I and III, suppressed cell proliferation and migration, and enhanced apoptosis. Furthermore, in each assay, when the DKK2 overexpression vector and miR-154 mimics were co-transfected, the functions of each component were counteracted by the other. Therefore, in CFs, targeting of DKK2 by miR-154 leads to upregulation of ß-catenin expression and activation of the classical Wnt signaling pathway and CFs. These results suggest new targets for the clinical treatment of MF and ischemic heart disease.

MiR-125b regulates SFRP5 expression to promote growth and activation of cardiac fibroblasts.

Myocardial fibrosis (MF), which typically occurs after a myocardial infarction (MI), is a major factor involved in the process of ventricular remodeling and subsequent progression to heart failure. Current studies have found that various microRNAs (miRNAs), such as miR-125b, play an important role in this process. However, few studies have investigated the specific mechanism of miR-125b. Transfection of miR-125b mimics into cardiac fibroblasts (CFs) resulted in significantly increased expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) and vinculin by Western blot analysis, while transfection of miR-125b inhibitors resulted in the opposite effect. Analysis of putative CF target genes for miR-125b revealed that miR-125b specifically inhibits expression of secreted frizzled-related protein 5 (SFRP5). SFRP5 inhibited expression of α-SMA and collagen I and III in CFs, while miR-125b promoted the expression of these proteins. Cotransfection of the SFRP5 overexpression vector and miR-125b mimics did not result in significant upregulation of SFRP5 expression or downregulation of α-SMA and collagen I and III. Further analysis revealed that miR-125b promotes the proliferation and migration of CFs and inhibits their apoptosis, while SFRP5 exhibits the opposite effects. These results indicate that miR-125b can regulate SFRP5 expression and thus influence the growth and activation of CFs. Hence, this study provides important insight into possible approaches for the prevention and treatment of MF after an MI.