RESUMO
Studies have found that cardiomyocytes and cardiac fibroblasts (CFs) can communicate through exosomes, thereby affecting each other's biological functions, but there are few studies on the mechanism. miR-208a/b are specifically expressed in the heart and highly expressed in exosomes derived from various myocardial diseases. Hypoxia induced cardiomyocytes to secrete exosomes (H-Exo) with high expression of miR-208a/b. When H-Exo were added to CFs for co-culture, it was found that CFs took up exosomes, thereby upregulating the expression of miR-208a/b. H-Exo significantly promoted the viability and migration of CFs, enhanced the expression of α-SMA, collagen I and III, and promoted the secretion of collagen I and III. miR-208a or/and miR-208b inhibitors significantly attenuated the effects of H-Exo on CF biological functions. miR-208a/b inhibitors significantly enhanced the levels of apoptosis and caspase-3 activity in CFs, while H-Exo significantly attenuated the pro-apoptotic effects of miR-208a/b inhibitors. Further treatment of CFs with ferroptosis inducer Erastin found that H-Exo further enhanced the accumulation of ROS, MDA and Fe2+, the main indicators of ferroptosis, and inhibited the expression of GPX4, a key regulator of ferroptosis. miR-208a or/and miR-208b inhibitors significantly attenuated the effects of Erastin and H-Exo on ferroptosis. In conclusion, hypoxic cardiomyocyte-derived exosomes can regulate the biological functions of CFs through highly expressed miR-208a/b.
Assuntos
Exossomos , Ferroptose , MicroRNAs , Humanos , Miócitos Cardíacos , Exossomos/metabolismo , MicroRNAs/metabolismo , Apoptose , Hipóxia/metabolismo , Fibroblastos , ColágenoRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Proliferação de Células , Fibroblastos/metabolismo , MicroRNAs/metabolismo , Miocárdio/metabolismo , RNA Circular/metabolismo , Transdução de Sinais , Linhagem Celular , HumanosRESUMO
Finding ways to reduce myocardial ischemia/reperfusion injury in the process of myocardial infarction has been an area of intense study in the field of heart disease. Recent studies have shown that long noncoding RNA (lncRNA) and autophagy play important roles in cardiovascular diseases. In our study, software analysis and dual-luciferase reporter assays have shown that miR-30a has binding sites on both AK088388 and Beclin-1. Continuing experiments found that miR-30a expression is downregulated, while the expressions of AK088388, Beclin-1, and LC3-II are upregulated in hypoxia/reoxygenation (H/R) cardiomyocytes; miR-30a inhibits the expression of AK088388, Beclin-1, and LC3-II in H/R cardiomyocytes, while AK088388 promotes the expression of Beclin-1 and LC3-II and inhibits miR-30a expression. AK088388 small interfering RNA and miR-30a mimics can promote the viability of H/R cardiomyocytes, reduce lactate dehydrogenase release, and reduce apoptosis. Mutations of the miR-30a binding site in AK088388 could not block the effects of miR-30a mentioned above. Therefore, AK088388 can competitively bind to miR-30a, promoting the expression of Beclin-1 and LC3-II, autophagy, and eventually cell damage. This finding provides new evidence for understanding the role of lncRNA in myocardial ischemia/reperfusion injury.
Assuntos
Autofagia/genética , Proteína Beclina-1/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/genética , Animais , Apoptose/genética , Proteína Beclina-1/metabolismo , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular/genética , Células Cultivadas , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/patologia , Oxigênio/metabolismo , Interferência de RNARESUMO
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.
Assuntos
Fibroblastos/citologia , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , MicroRNAs/metabolismo , Miocárdio/citologia , Via de Sinalização Wnt/genética , Adulto , Apoptose , Sequência de Bases , Movimento Celular/genética , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/genéticaRESUMO
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.
Assuntos
Proteínas do Olho/biossíntese , Proteínas de Membrana/biossíntese , MicroRNAs/metabolismo , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Apoptose/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Colágeno Tipo I/biossíntese , Colágeno Tipo III/biossíntese , Fibrose Endomiocárdica/genética , Fibrose Endomiocárdica/metabolismo , Fibrose Endomiocárdica/patologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Miocárdio/citologia , Miocárdio/metabolismo , TransfecçãoRESUMO
The aim of this study is to explore the role of circCELF1/miR-636/DKK2 pathway in myocardial fibrosis (MF). RT-qPCR and western blot were used to detect the expression of circCELF1, miR-636, and DKK2 in activated cardiac fibroblasts (CFs) and the hearts of acute myocardial infarction (AMI) mice. The m6A level of DKK2 was detected by RIP and RT-qPCR. The regulation of circCELF1/miR-636/DKK2 pathway on CF viability, activation, apoptosis, and migration was verified by CCK-8, western blot, flow cytometry, and Transwell. Ang II induced downregulation of circCELF1 expression, while circCELF1 enhanced the expression of DKK2 by adsorbing miR-636. circCELF1 also reduced DKK2 m6A level by upregulating FTO expression, thereby inhibiting the binding of miR-636 to DKK2 and promoting DKK2 expression. Ang II promoted CF viability, activation, and migration through the circCELF1/miR-636/DKK2 pathway. Both miR-636 inhibitors and DKK2 effectively reduced MF and improved cardiac function in AMI mice.