| BackgroundCongenital heart disease (CHD) is one of the most common congenital malformations in infants. Most of the complex CHDs are cyanotic, caused by right-to-left shunt in any level of the heart. It is well-known that chronic hypoxia is the basic pathophysiological process. Long-term clinical studies found that patients with cyanotic CHD are still able to survive in a long period of time and rarely progress to heart failure, although they have been in the condition of low-oxygen blood perfusion, suggesting that chronic hypoxia could probably generate compensatory adaptation and exert a protective effect on cardiomyocytes. Therefore, it is urgently required to study the pathophysiological changes of cyanotic CHD and the underlying mechanisms of myocardial adaptation to chronic hypoxia, which will help to improve strategies for cardioprotection and enhance efficiency of clinical therapies.MicroRNAs (miRNAs) are a kind of highly conserved noncoding RNA, which may cause degradation or translational inhibition of the target mRNA, thus playing a functional regulation of gene expression. In cardiomyocytes, several miRNAs were reported to be involved in pathophysiological changes of heart, and were closely related to chronic hypoxia. MiRNA-138 (miR-138) is one of the most popular miRNAs in recent time. In the process of heart development, miR-138 plays a critical role. Once miR-138 is deleted in heart, morphological malformation of cardiomyocytes and cardiac dysfunction would occur. Meanwhile, miR-138 was also found to be related to hypoxia. Hypoxia could induce upregulation of miR-138, and promote proliferation of hypoxic cells, implying that miR-138 may have a compensatory protective effect under hypoxic conditions. However, its specific molecular mechanisms have yet to be further studied.C-jun N terminal kinase (JNK) signaling is one of the most important pathways in the mitogen activated protein kinase (MAPK) family, which has an essential effect on the myocardial apoptosis. Mixed lineage kinase 3 (MLK3) is the upstream kinase of JNK, and it is capable of inducing the phosphorylation of JNK. Activated JNK could specifically bind to the amino terminal region of c-jun, thus enhancing the activity of transcription factors and promoting the expression of some apoptosis-related proteins. The role of MLK3/JNK/c-jun signaling pathway in myocardial adaptation to chronic hypoxia is still not yet studied clearly. Therefore, studying the molecular regulatory mechanisms will provide a new target for clinical myocardial protection.ObjectiveThe present project intends to detect the expression of miR-138 and MLK3 as well as the activity of some protein kinases under chronically hypoxic conditions in myocardial cell lines and clinical samples. Silence and overexpression of miR-138 is achieved by using miRNA agomir or antagomir techniques, in order to evaluate the influence of miR-138 on myocardial apoptosis in hypoxic status. Through establishment of a chronically hypoxic model in cardiomyocytes, the regulatory role of miR-138/MLK3/JNK/c-jun signaling pathway is verified in cellular level. MLK3 is confirmed to be a target protein of miR-138 via dual luciferase reporter gene experiment. By using transfection of shRNA plasmid, expression of MLK3 could be efficiently inhibited. We intend to verify MLK3 involves in the myocardial adaptation to chronic hypoxia via inhibiting the activation of JNK/c-jun signaling.Method1. Establishment of chronical hypoxia model, and collection of human myocardial samples:1.1 H9C2 rat myocardial cell lines are cultured in vitro. After serum-starved overnight, H9C2 cells are placed in an Invivo2000 cultivator containing a gaseous mixture of5% CO2, 94% N2 and 1% O2 at 37℃ for durations of 12,24,48,72 hours respectively, therefore the model of chronic hypoxia could be successfully established.1.2 Hypoxic myocardial samples are taken from the ventricular myocardium of patients with cyanotic CHD (such as Tetralogy of Fallot), while the controlled samples are from patients with acyanotic CHD (such as ventricular septal defect combined with right ventricular outflow tract obstruction). After obtained during operations, all the specimen are quickly stored in liquid nitrogen in prepare for the next experiments.2. Detection of expression of miR-138 and MLK3:2.1 Expression of miR-138 and MLK3 in chronically hypoxic cardiomyocytes or human myocardial samples is detected by using qRT-PCR and Western Blot techniques.2.2 Immunohistochemical analysis is used to localize the expression of MLK3 protein.3. Research on the protective role of miR-138 in myocardiocytes subjected to chronic hypoxia.3.1 H9C2 myocardiocytes are transfected with agomir or antagomir in order to achieve silence or overexpression of miR-138. RT-PCR analysis is used to verify the efficiency of transfection.3.2 Apoptosis caused by chronic hypoxia in H9C2 cells interfered with miR-138 chemical agents is studied by using flow cytometry, TUNEL as well as detection of the activity of some apoptosis-related proteins (such as caspase or bcl-2 family). A microscope is used to observe the cell morphology. Expression of MLK3 protein is detected by Western Blot technique.3.3 Dual luciferase reporter gene is established to confirm that MLK3 is direct target of miR-138. Western Blot method is used to detect expression and phosphorylation of MLK3, JNK, c-jun and some key proteins of MAPK signaling pathway in H9C2 cells interfered with miR-138 chemical agents.4. Research on the role of MLK3 in myocardiomytes subjected to chronic hypoxia.4.1 The shRNA plasmid of MLK3 is constructed to be transfected into H9C2 cells to achieve the silence of MLK3. Western Blot and qRT-PCR are used to detect the efficiency of transfection.4.2 In different time points of chronic hypoxia, cell survival curve is drafted by using MTT analysis in H9C2 cells with stable silence of MLK3. Apoptosis and activity of H9C2 cells are detected by using LDH, TUNEL and Typan Blue staining techniques. Western Blot is used to detect expression of some apoptosis-related proteins such as caspase or Bcl-2 family.4.3 The expression of phosphorylation of JNK, c-jun and some key proteins of MAPK signaling pathway is detected by using Western Blot technique in H9C2 cells transfected with MLK3 shRNA plasmid.Results1. Myocardial samples were collected in the process of operations. By using Trizol, total RNA in cells or tissues was extracted. The specific stem-loop primers of miR-138 were used for qRT-PCR. Results suggested that expression of miR-138 was significantly increased in patients with cyanotic CHDs than in acyanotic CHDs. H9C2 rat myocardial cells were placed in chronically hypoxic incubator for 12h,24h,48h and 72h. Total RNA was extracted by using Trizol for qRT-PCR. Results found that expression of miR-138 is gradually increased with the prolonged hypoxic time.2. Agomir, agomir-NC, antagomir or antagomir-NC was transfected into H9C2 cells with Lipofectamine 2000 respectively. After 48h, total RNA was extracted to detect the efficiency of transfection. Compared to corresponding negative control group, the expression of miR-138 was increased significantly in agomir group while the value was reduced in antagomir group. Cells interfered with chemical agents were seeded in flaks or plates, and cultured in hypoxic incubator for 72h. Cell survival curve in different time point was drafted by using MTT analysis, and results suggested that agomir group had the faster survival rate of H9C2 cells while antagomir group had the slowest. Cell death rate was detected by LDH release. After 72h in hypoxic conditions, overexpression of miR-138 significantly reduced cell death, while nhibition of miR-138 resulted in higher cell death. Flow cytometry, TUNEL and Hoechst staining was used to analyze cell apoptosis after 72h exposure to hypoxia. Hypoxia could greatly induce myocardial apoptosis. However, once exogenous miR-138 was added, the pro-apoptosis action of hypoxia was attenuated almost close to the normoxic conditions. The antagomir group had the highest amount of apoptotic cells in six groups. Expression of some apoptosis-related proteins was detected by using Western Blot method. Hypoxia increased the expression of cleaved-caspase 3 and its substrated cleaved-PARP, which could be eased up by agomir and promoted by antagomir. In addition, overexpression of miR-138 increased the level of Bcl-2, together with reduced expression of Bad. Although Bax had no obvious changes in various groups, the ratio of Bcl-2/Bax significantly decreased in agomir group. Opposite trends were also observed in antagomir group.3. Wild or mutant type plasmid of MLK3 3’UTR was construced, and co-transfected into 293T cells with miR-138 agomir. By using dual luciferase reporter gene analysis, we observed that overexpression of miR-138 could significantly reduce the relative fluorescence intensity, implying that MLK3 was a direct target of miR-138. Expression of MLK3 also had obvious changes in H9C2 cells transfected with agomir or antagomir. Meanwhile, miR-138 could significantly influence the phosphorylation of JNK and c-jun.4. Western Blot and immunohistochemical techniques were used to detect and localize the expression of MLK3 in human myocardial samples. Results suggested that expression of MLK3 in patients with cyanotic CHD was significantly lower than in the patients with acyanotic CHD. In hypoxic H9C2 cells, the amount of MLK.3 protein was gradually reduced with prolonged time of hypoxia. However, by using qRT-PCR analysis, the expression of MLK3 mRNA showed no significant difference no matter in human myocardial samples or in hypoxic H9C2 cells.5. The shRNA plasmid of MLK3 and Lipofectamine 2000 were co-transfected into H9C2 cells, and the effeiciency of transfection was detected by using Western Blot and qRT-PCR techniques after 48h. Cells were cultured in hypoxic incubator for more 72h. After silence of MLK3, the growth of hypoxic H9C2 cells could be significantly promoted according to the results of MTT, LDH as well as Trypan Blue analysis. By using TUNEL and Hoechst staining, the amount of apoptotic cells was obviously reduced in the MLK3 silence group. Apoptosis-related proteins were also detected by Western Blot technique. The results demonstrated that silence of MLK3 could significantly upregulate the expression of cleaved-caspase 3, cleaved-PARP, Bad and Bax, together with the downregulation of Bcl-2. Meanwhile, MLK3 also had an important role in the phosphorylation of its downstream proteins, JNK. and c-jun.Conclusion1. The expression of miR-138 is significantly up-regulated in cyanotic group, and is gradually increased in H9C2 cells with prolonged time of hypoxia.2. Up-regulation of miR-138 could attenuate myocardial apoptosis induced by hypoxia, and promote cardiomyocyte survival in hypoxic conditions.3. MLK3 is the target protein of miR-138. MLK3/JNK/c-jun signaling pathway could be obviously affetecd by the interference of miR-138.4. The expression of MLK3 protein is significantly reduced in hypoxic cardiomyocytes, but the level of mRNA does not exhibit obvious changes, suggesting that hypoxia may play a post-transcriptional regulatory role in MLK3 expression.5. Silence of MLK3 could protect cardiomyocytes from hypoxia-induced apoptosis, and the activation of its downstream proteins, JNK and c-jun, is also inhibited. |
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