| Cardiovascular diseases (CVDs) account for more than 17 million deaths globally each year, which is estimated to grow to 23.6 million by 2030.Ischaemic heart disease was alone led to 7 million deaths worldwide in 2010, which was increased to 35% compared to that of 1990. Cardiomyocyte apoptosis and cardiac fibrosis resulted from the proliferation and differentiation of cardiac fibroblasts lead to ventricular remodeling that is pathological basis of heart failure, which contributes to the main cause of death in patients with ischaemic heart disease. Based on serious consequences induced by cardiac apoptosis and fibrosis, it is very urgent to explore the molecular mechanisms, investigate new targets and effective drugs, which has drew wide attentions of the cardiovascular experts at home and abroad.MicroRNAs (miRNAs) are conserved endogenous, non-coding small RNAs, measuring about-22 nucleotides of extension that play crucial roles in the regulation of physiological and pathological events by inhibiting the translation or by promoting the degradation of target messenger RNAs (mRNAs).The miR-208 family is composed of miR-208a, miR-208b, and miR-499 encoded by a-cardiac muscle myosin heavy chain gene (?-MHC/Myh6), (?-MHC/Myh7, and Myh7b, respectively, which plays a vital role in regulating muscle myosin content, myofiber identity, and muscle performance. Strikingly, miR-208a is cardiac-specific expression in the adult heart, whereas miR-208b and miR-499 are expressed in embryonic heart and skeletal muscles. In animal experiments, studies have found that upregulated miR-208a can induce ventricular hypertrophy, fibrosis and cardiac disfunction, while silencing of miR-208a can reverse ventricular hypertrophy, myocardial fibrosis and improve cardiac function under stress condition induced by thoracic aorta ligation, high salt diet and myocardial ischemia. Clinical studies also found that an abnormal increase of circulating miR-208a is one of the diagnostic markers for patients with acute myocardial infarction (AMI).However, so far, the underlying mechanism of miR-208a in regulating cardiomyocyte apoptosis and cardiac fibrosis in response to stress is still not clear. Nemo-like kinase (NLK) was conserved endogenous extracellular signal regulated kinases-2 (ERK-2)/mitogen activated protein kinase (MAPK) kinase, which is universally expressed in the body. NLK could regulate cell proliferation, migration and apoptosis via phosphorylating or ubiquitinating many key molecules of signal pathway. Using a publicly available database (http://www.mirbase.org), we found that miR-208a-3p could bind to the 3'-untranslated region(3'-UTR) of NLK, and NLK may be a direct target of miR-208a-3p.However, the molecular mechanism that miR-208 modulates cardiac apoptosis and the proliferation and differentiation of cardiac fibroblasts remains to be elucidated. In this project, we explored the following contexts:Firstly, rats were underwent permanent coronary artery ligation to establish AMI model. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the expression of miR-208a and NLK mRNA after AMI. Secondly, in cultured H9C2 cells, we explored the functional role of miR-208a in the progression of angiotensin ? (Ang ?)-induced cell apoptosis. Western blot and luciferase reporter assay were performed to indentify NLK as a target of miR-208a, and further evaluated the role of NLK in cardiomyocyte apoptosis. Finally, we evaluated the functional role of miR-208a and NLK in the proliferation and differentiation of cardiac fibroblasts.1, miR-208a was upregulated and NLK was downregulated after AMIFour weeks after AMI in rats, obvious cardiomyocyte apoptosis and cardiac fibrosis were observed. HE staining showed apparent cardiomyocyte necrosis and infiltration of inflammatory cells in the infract size, and cardiomyocyte hypertrophy and the proliferation of cardiac fibroblasts were detected in the border zone, while cardiomyocytes were normal in sham group. TUNEL staining found that the ratio of apoptotic cardiaomyocyte was increased in the non-infarcted zone compared to sham group. Meanwhile, to further evaluate cell apoptosis in vivo, the anti-apoptosis protein Bcl-2 measured by western blot that was significantly decreased but the pro-apoptosis protein Bax and caspase-3 were increased in MI group. Meanwhile, masson staining demonstrated an increase of collagen volume fraction in model group. Western blot showed that the expression of a-SMA and collagen ? proteins was increased in model rats.Upregulated miR-208a in MI rats was verified by qRT-PCR. Strong miR-208a staining was found via using in situ hybridization (ISH) in the cytoplasm of cardiomyocytes in border area, whereas the hybridization signal was obviously weak in sham group. Moreover, the level of NLK was reduced both at mRNA and protein level in MI rats compared to the sham group. Immunohistochemistry assay demonstrated that NLK in the cytoplasm of cardiomyocytes from non-infarcted area was decreased compared with sham rats. These data suggested that miR-208a and NLK are associated with cardiomyocyte apoptosis and cardiac fibrosis.2. Ang ? induced cell apoptosis and upregulated the expression of miR-208aThe effect of Ang ? on the miR-208a expression was evaluated by qRT-PCR. Ang ? induced the expression of miR-208a at a dose dependent manner, and miR-208a was transiently increased with a peak at approximately 24 h after treatment with Ang ? (100nM). FCM analysis revealed that the percentage of apoptotic cells was increased compared to vehicle after treatment with Ang ? (100nM) for 24 h. The Bcl-2 expression was decreased, while the level of Bax and caspase-3 were increased evaluated by western blot in H9C2 cells treatment with Ang ?.3. Upregulated miR-208a aggravated Ang ?-induced cell apoptosis via inhibiting NLKTo modulate the expression of miR-208a, transfecting miR-208a mimics, inhibitors and their controloligos for 8h in H9C2 cells, respectively. After treatment with Ang ? for another 24h, miR-208a mimics (200nM) aggravated the ratio of Ang ?-induced cell apoptosis, whereas the percentage of cells apoptosis was reduced after transfection of miR-208a inhibitors (200nM). In addition, overexpression of miR-208a reduced NLK while downregulation of miR-208a increased NLK protein level, and the anti-apoptotic Bcl-2 protein level was also reduced in miR-208a mimic-treated group, conversely, miR-208a inhibitors treatment led to opposite effect. These results suggest that miR-208a can promote Ang ?-induced cell apoptosis via inhibiting NLK and Bcl-2. 4. NLK as an effective target of miR-208a After transfecting miR-208a mimics, inhibitors and their controloligos for 8h, respectively, western blot analysis revealed that upregulation of miR-208a reduced NLK protein expression, whereas the level of NLK protein was significantly increased in miR-208a-downregulated cells. More importantly, overexpression of miR-208a reduced but downregulation of miR-208a increased luciferase activity of NLK in H9C2 cells after cotransfecting mimics, inhibitors with DNA of NLK 3'-UTRwt plasmid, respectively. 5. NLK modulated Ang ?-induced cell apoptosis To verify the biological function of NLK in miR-208a-mediated cell apoptosis, we modulated the level of NLK by transfection of siRNA-NLK and pcDNA3.1(+)-NLK plasmid in H9C2 cells, respectively. Compared with siRNA control, downregulation of NLK by transfection of siNLK inhibited Bcl-2 protein expression but increased cell apoptotic rate induced by Ang ? (100nM). Overexpressed NLK via transfection of pcDNA3.1(+)-NLK effectively upregulated the level of Bcl-2 protein while reduced the percentage of apoptotic cells after treatment with Ang ? (100nM). These results suggested that NLK could suppress Ang ?-induced cardiac myocyte apoptosis by regulation of Bcl-2.6. Upregulated miR-208a promoted the proliferation and differentiation of cardiac fibroblastsIn cultured cardiac fibroblasts, qRT-PCR analysis revealed that Ang ? (100nM) induced the expression of miR-208a at a dose dependent manner in cardiac fibroblasts. The a-SMA expression was also increased after treatment with Ang ? evaluated by western blot. Interestingly, western blot analysis found miR-208a mimics (200nM) increased but miR-208a inhibitors (200nM) decreased the level of a-SMA and collagen ? induced by Ang ?. FCM assay investigated that miR-208a mimics (200nM) promoted but miR-208a inhibitors (200nM) prevented Ang ?-induced cell proliferation. These findings suggested that miR-208a could promote the proliferation and differentiation of cardiac fibroblasts, which may be the main cause of miR-208a-induced cardiac fibrosis.7. Downregulated NLK inhibited the proliferation and differentiation of cardiac fibroblastsTransfecting siRNA-NLK and siRNA control in cardiac fibroblasts for 8h, qRT-PCR and western blot demonstrated that Ang ? upregulated the expression of NLK both at mRNA and protein level, while the level of NLK was downregulated in siRNA-NLK group compared to the control group. FCM analysis revealed that downregulated NLK inhibited the proliferation and differentiation of cardiac fibroblasts induced by Ang ?. Compared with control siRNA, downregulated NLK by transfecting siRNA-NLK reduced the protein level of a-SMA and collagen ? in cardiac fibroblasts. |
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