| Background:As development of science and technology,diagnosis and treatment level of congenital heart disease has a great improvement.However,complex congenital heart disease remains a tough problem,with high mortality.The most common problem is cyanosis congenital heart disease.Furthermore,chronic hypoxia is the common pathophysiological basis of the cyanosis congenital heart disease.The specific mechanism is still unknown,including how cardiomyocytes can complete the transformation of cell signal transduction,protein synthesis and metabolism to adapt the chronic hypoxia condition.Therefore,the study on adaptive mechanism of cardiomyocytes under chronic hypoxia may contribute to the treatment and prognosis of cyanosis congenital heart disease.miR-17-92 gene cluster is a typical highly conserved cluster containing multicistronic promoter.This cluster is located on human chromosome 13q31.3,the third intron of C13orf25 gene and its transcriptional precursor contain six serial stem loop hairpin structures,which can finally produce six mature miRNA including miR-17,miR-18,miR-19a,miR-20,miR-19b and miR-92.miR-17-92 gene cluster plays a critical role in tumorigenesis and normal development of lung,heart,and immune organ.miR-17-5p that belong to miR-17-92 gene cluster and located on human chromosome 13q31 is multicistronic miRNA which code six miRNAs.Some research found that miR-17-5p can regulate the growth of Myeloid-derived suppressor cells(MDSC)via STAT3 signal pathway.Recent study suggested that,in the mice model of myocardial ischemic and reperfusion injury and mice cardiomyocyte model induced by H202,the expression of miR-17-5p increased,and reduced the expression of STAT3 through combining the 3'UTR of STAT3 to promote cardiomyocyte apoptosisMany studies show that IL-6-JAK-STAT3 signal pathway possesses protective effect on pathology and physiology of heart.Our previous studies have proved that,during myocardial chronic hypoxia,as the self-induced regulatory factor of STAT3,the SOCS3-involved regulatory crosslinks between IL-6/JAK2/STAT3 signal pathway and NF-?B signal pathway has great significance for the survival and function regulation of cardiomyocytes.Thus,this experiment mainly focuses on the expression change of miR-17-5p and STAT3 and its effect on hypoxic cardiomyocyte apoptosis under myocardial chronic hypoxia.Objective:In this study,we intend to detect the expression change of miR-17-5p in clinical myocardial sample and in H9c2 cells.The expression of miR-17-5p is changed by inhibitor and enhancer,and its effect on STAT3 and downstream target gene c-myc is detected in the cardiomyocyte chronic hypoxia model in vitro.In addition,we also detect its effect on the H9c2 cardiomyocytes.Methods:1.Study of the expression change of miR-17-5p in clinical myocardial sample and in H9c2 cells.1.1 We collect human myocardial sample.The cyanosis group includes patients with tetralogy of Fallot,while the acyanosis group is the patients with ventricular septal defect and right ventricular outflow tract stenosis.The clinical sample was obtained from the right ventricular outflow tract,and the tissue RNA was extracted.We detect the expression of miR-17-5p via PCR in the clinical sample of both groups.1.2 The same batch of H9c2(rat ventricular myocyte line)cells were placed in hypoxia incubator(1.0%02?5.0%CO2)as the hypoxia group and cultured Oh?12h?24h?48h and 72h respectively,while the cells in the control group were placed and cultured in common incubator(21.0%02).The expression of miR-17-5p in cardiomyocyte line of both groups was detected via PCR.2.Study of the function of miR-17-5p in the chronic hypoxic H9c2 cells and its regulation mechanism on STAT3 signal pathway.2.1 TheH9c2 cell line was transfected with the negative control with green stain in normoxia and the transfection efficiency was detected by fluorescence microscope.2.2 H9c2 cells were transfected with agomir,antagomir and NC,as the agomir group,antagomir group and control group respectively.The black group was the cell without transfection.2.3 The transfected cardiomyocytes and cells in black group were cultured 24h in normoxia,and then were cultured 48h in hypoxia.The apoptosis condition of the entire group was detected by TUNEL method.2.4 CCK8 kit detects the proliferation of cells in every group cultured in hypoxia for 0 h,12 h,24 h,48 h.2.5 TUNEL experiments detect the apoptosis of cells in every group cultured in hypoxia for 48 h.Western Blot detects the expression of STAT3,p-Tyr-705-STAT3 and its downstream protein c-myc in cells of each group after hypoxia 48h.Results:1.Compared to acyanosis group(0.482±0.163),the expression of miR-17-5p is higher in myocardial tissue of cyanosis group(1.45±0.705,P<0.01).2.The RT-PCR result suggested that the expression of miR-17-5p gradually increased in H9c2 cells under chronic hypoxia0h(0.933±0.115)?12h(1.802±0.130)?24h(1.973±0.140)?48h(2.030±0.134)and 72h(2.113±0.220).3.After H9c2 cells were transfected with antagomir fluorescence negative control,the transfection efficiency was more than 75%via fluorescence microscope.4.CCK8 experiments showed that after hypoxia Oh,12h,24h,48h,the proliferation of H9c2 cells was significantly lower in up-regulated group than control group(P<0.01),while there was no statistically significant difference between down-regulated group and black group(P>0.05).5.TUNEL experiments suggested that the apoptosis of H9c2 cells was significantly higher in up-regulated group(0.1599±0.02016)than control group(0.1063±0.010969)(P<0.01),while there was no statistically significant difference between down-regulated group(0.0941±0.01913)and black group(0.0948± 0.013286)(P>0.05).6.The expression of STAT3,p-Tyr-705-STAT3 and downstream protein c-myc was decreased significantly(P<0.01),after miR-17-5p was overexpression in H9c2 cells under hypoxia 48h;after inhibition of miR-17-5p in H9c2 cells under hypoxia 48h,there was no significant change in the expression of STAT3,p-Tyr-705-STAT3 and downstream protein c-myc.Conclusion:1.Compared with the non-cyanotic patients,the expression of miR-17-5p increased about 3 times in myocardial samples of cyanotic patients.2.Under chronic hypoxia,miR-17-5p expressing cells gradually increased.in H9c2 cells.3.Under chronic hypoxia,overexpression of miR-17-5p in H9c2 cells has a significant inhibitory effect on the STAT3 signaling pathway in cardiac muscle,and promotes the cardiomyocyte apoptosis and decreases its proliferation.On the other side,there is no significantly change on the STAT3 signaling pathway by inhibition of miR-17-5p in H9c2 cells,and no effect on the cardiomyocyte apoptosis or proliferation. |
PDF Full Text Request