Microrna-145 Aggravates Hypoxia-Inducedlnjury By Targeting Rac1 In H9c2 Cells

BackgroundMyocardial infarction endanger human life and health,can lead to cardiac death.Myocardial infarction accompanied by a large number of myocardial ischemia and hypoxia,can cause irreversible cell death or apoptosis,eventually resulting in cardiac insufficiency.Early diagnosis of myocardial infarction,timely and accurate diagnosis is one of the important conditions to improve the prognosis of patients,so looking for high sensitivity and specificity of molecular markers,will be used in the prevention,diagnosis,treatment and prognosis of myocardial infarction,Has become the focus and hot point of medical research.MicroRNA(miRNA)is a kind of non-coding single-stranded small RNA that has been deeply studied so far.It is related to a series of physiological and pathological processes.miR-145 is a member of the miRNA family,and its function and mechanism in malignant tumors are studied more deeply.Recent reports show that miR-145 can be used as a new molecular marker in predicting cardiac function,while also assessing the risk of developing heart failure.But so far there is no miR-145 in the occurrence and development of myocardial infarction related research.ObjectiveThe study is order to investigate the role of miR-145 in the pathogenesis and progression of myocardial infarction.We systematically studied the expression and role of miR-145 in cardiomyocytes under hypoxia conditions,and explored its target genes and their downstream factors Provide experimental and theoretical basis for finding new molecular markers for the detection and treatment of myocardial infarction.Method(1)A total of 40 acute myocardial infarction patients were selected with onset of 2 h at Affiliated Hospital of XX University from May 2014 to May 2016.Thirty patients with angina pectoris and 30 elderly volunteers who underwent physical examination in our hospital were enrolled.Peripheral venous blood-was collected from the above subjects,and the serum levels of miR-145 and creatine kinase isoenzyme-MB(CK-MB)were measured.(2)Sprague-Dawley rat model of myocardial infarction was established.The Sprague-Dawley rats were randomly divided into MI group,control antagomir group,MI + control antagomir group and MI + miR-145 antagomir group.Serum CK-MB of each group was measured.Myocardial tissue samples were taken for HE staining to observe pathological changes.Expression of miR-145 in Myocardial Tissue.TUNEL method was used to detect cardiomyocyte apoptosis in each group.(3)Establish hypoxia model of myocardial H9C2 cells.In order to detect the effect of hypoxia on the biological behavior of cardiomyocytes,H9C2 cells were divided into control group and Hypoxia group.The proliferation,apoptosis,invasion and migration of H9C2 cells were detected and the expressions of Bcl-2,Bax,P-The expressions of caspase-3,C-caspase-3,P-caspase-9 and C-caspase-9 were detected by RT-Hypoxia group,mimic control group,miR-145 mimic group,ASO control group and ASO-miR-145 group were divided into control group,Hypoxia group,mimic control group,and Hypoxia group.The proliferation,apoptosis,invasion and migration of the above groups of cells were detected.The expressions of Bcl-2,Bax,P-caspase-3,C-caspase-3,P-caspase-9 and C-caspase-9 were detected.The biological software was used to predict the target gene of miR-145 and validated.In order to understand the effect of miR-145 on Racl expression,H9C2 cells were randomly divided into control group,mimic control group,miR-145 mimic group,ASO control group and ASO-miR-145 group.The expression of Racl mRNA and protein in each group was detected.In order to investigate the role of miR-145 targeting Racl in cardiomyocyte biological behavior,H9C2 cells were randomly divided into Hypoxia group,control group,ASO control group,ASO-miR-145 group,ASO-miR145 + si-Racl group.The proliferation,apoptosis,invasion and migration of the above groups were detected and the expressions of Bcl-2,Bax,P-caspase-3,C-caspase-3,P-caspase-9 and C-caspase-9 were detected..In order to understand the effect of miR-145 targeting Racl on PI3K/AKT and MAPK/ERK signaling pathway,H9C2 cells were randomly divided into control group,ASO control group,ASO-miR-145 group,ASO-miR145 + si-Racl group.The expressions of Racl,p-PI3K,PI3K,p-PI3K,p-AKT,AKT,p-MAPK,MAPK,p-ERK and ERK in the above groups of cells were detected.Results(1)According to the analysis of the general information of the participants,there was no significant difference in gender and age group between the two groups(P>0.05).The expression level of miR-145 in each group was not related to hypertension and diabetes(P>0.05),but not to coronary heart disease risk factors.The results of serum miR-145 showed that there was no significant difference in serum miR-145 expression in patients with angina pectoris compared with healthy controls(P>0.05),while the level of miR-145 in patients with acute myocardial infarction was significantly higher Increased(P<0.01).The expression of miR-145 was positively correlated with CK-MB(r = 0.88,P ?0.001).(2)Compared with MI group,the expression level of miR-145 and serum CK-MB in myocardium of control antagomir group were significantly decreased(P<0.05),but there was no significant difference in MI + control antagomir group(P>0.05)+ control antagomir group,the expression of miR-145 and serum CK-MB in myocardial tissue of MI + miR-145 antagomir group were significantly decreased(P<0.05).Myocardial cells in MI group were disordered and swollen obviously,most of the nucleus were concentrated and irregular in shape,with incomplete structure,obvious apoptosis and necrosis,edema in interstitium,disorganized myocardial fiber,A large number of neutrophil infiltration;myocardium in control antagomir group showed no significant pathological changes,a small part of myocardial cells can be observed slight swelling;MI ?control antagomir group and MI group roughly the same situation;compared with MI + control antagomir group Myocardial tissue injury in MI + miR-145 antagomir group was significantly reduced,the histological morphology was relatively complete,the number of apoptotic necrosis and the infiltration of inflammatory cells in MI + miR-145 antagomir group were significantly decreased compared with MI + control antagomir group.Compared with MI group,the number of apoptotic cardiomyocytes in control antagomir group was significantly decreased(P<0.05),but there was no significant change in MI +control antagomir group(P>0.05).MI + miR The number of apoptotic cardiomyocytes in-145 antagomir group decreased significantly(P<0.05),as to these in the MI + control antagomir group.(3)Effects of hypoxia on the biological behavior of cardiomyocytes The results showed that compared with the control group,the proliferation,invasion and migration of Hypoxia group were significantly decreased and the apoptosis rate was significantly increased(P<0.05).Compared with the control group the expression of Bcl-2 protein in Hypoxia group was significantly decreased(P<0.05),but Bax protein increased(P<0.01).The activated caspase-3 and caspase-9 were not expressed in control group,However,the expression of miR-145 in Hypoxia group was significantly higher than that in Hypoxia group(P<0.05).The effect of miR-145 expression on hypoxic cardiomyocytes The results showed that compared with the mimic control group,the miR-145 mimic group cells miR-145 expression was significantly increased(P<0.01);ASO control group phase The expression of miR-145 in ASO-miR-145 group was significantly decreased(P<0.01).The proliferation,migration and invasiveness of cardiomyocytes in miR-145 mimic group were significantly decreased(P<0.05),as to these in the mimic control group.Compared with the ASO control group,the proliferation of cardiomyocytes in ASO-miR-Invasion and migration ability were significantly increased,the apoptosis rate was significantly reduced(P<0.05).Compared with mimic control group,the expression of Bcl-2 protein in myocardial cells of miR-145 mimic group was significantly decreased(P<0.05),Bax expression was significantly increased(P<0.05),activated caspase-3 protein and caspase(P<0.001).Compared with ASO control group,the expression of Bcl-2 protein in ASO-miR-145 group was significantly increased(P<0.01)5 and the expression of Bax was significantly increased(P<0.01).The expressions of activated caspase-3 and caspase-9 were significantly decreased(P<0.001).Racl was predicted to be a target gene of miR-145.Luciferase reporter gene Rac1-WT and miR-145 mimic could inhibit luciferase expression in H9C2 cells when compared with mimic control group(P<0.05).Compared with the ASO control group,the expression of Racl mRNA and protein in ASO-miR-145 group was significantly increased(P<0.05),while the expression of Racl mRNA and protein in miR-145 mimic group was significantly decreased<0.01).The results of miR-145 targeting Racl on the biological behavior of cardiomyocytes showed that compared with ASO control group,the proliferation,invasion and migration of cardiomyocytes in ASO-miR-145 group were significantly increased(P<0.05).For ASO-miR-145 group,the proliferation,invasion and migration of cardiomyocytes in ASO-miR-145 + si-Rac1 group were significantly decreased(P<0.01).Compared with ASO control group,the expression of Racl and Bcl-2 protein in ASO-miR-145 group was significantly increased(P<0.01)and the expression of Bax protein was significantly decreased(P<0.01)Racl and Bcl-2 protein expression in ASO-miR-145 group and si-Racl group were significantly decreased(P<0.01),and Bax protein expression was significantly increased(P<0.01).Compared with ASO control group,the expressions of activated caspase-3 and caspase-9 in ASO-miR-145 group were significantly decreased(P<0.01).Compared with ASO-miR-145 group,ASO-miR-+ si-Racl group,the expression of activated caspase-3 and caspase-9 were significantly increased(P<0.01).The results of miR-145 targeting Racl on PI3K/AKT and MAPK/ERK signal pathways showed that Racl protein and activated PI3K,AKT,MAPK and ERK proteins in ASO-miR-145 group(P<0.01).Compared with ASO-miR-145 group,the expressions of Racl,activated PI3K,AKT,MAPK and ERK in ASO-miR-145 group and si-Racl group The levels were significantly reduced(P<0.01).Conclusion(1)The expression of circulating miR-145 is abnormally upregulated in patients with myocardial infarction,and positively correlated with myocardial injury marker CK-MB.(2)miR-145 promotes the occurrence and development of myocardial infarction,when its expression is inhibited,it can effectively relieve myocardial injury and apoptosis caused by myocardial infarction.(3)The expression of miR-145 in cardiomyocytes was significantly up-regulated by continuous hypoxia,while hypoxia-induced cardiomyocyte injury was inhibited when the expression of rmiR-145 was inhibited.MiR-145 inhibited the expression of Racl and then inhibited the expression of PI3K/Activation of AKT and MAPK/ERK signaling pathways.