Effect Of MiR-146b On Hypoxia-induced Cardiomyocyte Apoptosis And Its Mechanism

BackgroundSince the twentieth century, with the development of neonatal congenital malformation screening technology and interventional techniques, the cure rate of CHD has increased. However, the mortality of patients with complex CHD is still high. Cyanotic congenital heart disease(CHD) is one of the most common complex congenital heart disease. In cyanotic CHD, the chronic hypoxia caused by right-to-left shunt of the blood flow is the common pathophysiology of CHD. Although patients with cyanotic CHD suffer long-term chronic hypoxia, most of them can tolerate the hypoxia until the optimal operation time. It has been proved that the changes of cardiomyocytes under chronic hypoxia can make cardiomyocyte more adaptive to a variety of external stimuli, but the underlying mechanisms are not fully understood till now. Therefore, better understanding of possible compensative mechanism of cardiomyocytes under chronic hypoxic conditions can help to improve clinical curative effect and reduce hypoxic heart disease mortality.Micro RNAs(mi RNAs) is the endogenous, short, non-coding RNA. The mi RNA genes exist in the genome as forms of a single copy, multi copies, gene cluster or ones. They pair with the target m RNA, form RNA induced silencing complex(mi RISC), and inhibit translation of m RNA or accelerate the degradation of m RNA, anticipating in posttranscriptional gene regulation. It is demonstrated that mi RNA plays important roles in the physiologic and pathological process of heart development and disease. For example, mi R-1 promotes the differentiation of embryonic stem cells into cardiomyocytes, while mi R-133 inhibited this process. Inhibition of mi RNA-21 induce cardiomyocyte hypertrophy, while overexpression of mi R-21 attenuated such process. Overexpression of mi R-208 a leads to myocardial hypertrophy and cardiac arrhythmia. The expression of mi R-146 b is reported to be higher in heart tissue of the tetralogy of Fallot than that of the healthy ones by mi RNA-array experiment. Taking other tissues under chronic hypoxia as a reference, the expression of mi R-146 b is up-regulated in lung epithelial cells under hypoxic condition. A group of researchers in Beijing An Zhen Hospital found that in cyanotic heart disease model using miniature pigs, the expression of mi R-146 b is higher in lung tissue compared with the control. These results suggest that mi R-146 b may play a certain kind of role under chronic hypoxic conditions, and further study on its mechanism is needed.Ribonuclease L(RNase L) is a member of the 2-5A system, which belongs to the MX family. Research shows that RNase L can induce apoptosis in a caspase-cascade dependent manner. In addition, once apoptosis occurs, RNase L moves from the cytoplasm to the mitochondria, changes the mitochondrial membrane potential(ΔΨM) and induces the activation of reactive oxygen species(ROS). Knockdown of RNase L in cells causes significantly less apoptosis than that in the non-knockout group, and research shows that this is partially mediated by JNK/c-Jun pathway. RNase L can also indirectly influence the NF- κB signaling pathway via the IKK- kappa and IKK- alpha. Tissue-specific Gene Expression and Regulation(Ti GER) database reveals that m RNA expression of RNase L is high in heart tissue, suggesting it may play an important role in hypoxia-induced cardiomyocyte apoptosis.ObjectiveThe present project aims detect expression of mi R-146 b in both vivo(clinicalmyocardial samples) and vitro(H9c2 cells) under chronic hypoxic conditions. Then weup- and down- regulate expression mi R-146 b using mimic and inhibitor transfectiontechnology in chronic cardiomyocyte hypoxic model, and observe the changes of cellsthereafter. We want to demonstrate that RNase L is the direct target gene of mi R-146busing the dual luciferase reporter method. In addition, we want to investigate whether theactivation of NF-κB/ IL6 signaling axis and STAT3 are involved in myocardial adaptationto chronic hypoxia. We down-regulated RNase L to preliminarily observe its effect incardiomyocyte chronic hypoxiaMethod1. Collection of myocardial specimen, and establishment of chronic hypoxic cell model. Detect the expression of mi R-146 b under hypoxia both in vivo and in vitro.1.1. Myocardial samples were taken during the operation, and were divided into cyanotic group from cyanotic congenital heart disease patients(tetralogy of Fallot or pulmonary atresia with ventricular septal defect) and non-cyanotic group from non-cyanotic congenital heart disease patients(right ventricular outflow tract stenosis complicated with ventricular septal defect); RNA was extracted from the samples and RT-PCR was used to measure the changes of mi R-146 b between the two groups;1.2. Cardiomyocyte H9c2 cell lines were cultured under hypoxic condition(1% O2, 5% CO2) for 24 h, 48 h and 72 h, respectively. The control group was cultured in normal condition(21.0% O2). RT-PCR was used to measure the changes of mi R-146 b among the groups;2. Explore the role of mi R-146 b in H9c2 cells under chronic hypoxia and its mechanism(cell line level):2.1. Transfect inhibitor NC-FAM(normal control with green fluorescent) into H9c2 cells under normal oxygen concentration and measure the transfection efficiency by fluorescence microscope.2.2. In order to explore the effect of mi R-146 b on cardiomyocyte apoptosis, chemically synthesized mi RNA-146 b inhibitor were transfected into H9c2 cells to down-regulate mi R-146 b. Four goups were conducted: Normal oxygen concentration group, hypoxia group, hypoxia and 146b-inh transfection group and hypoxia and 146b-inh NC transfection group. After 72 h hypoxia, we measured the degree of cytotoxicity and apoptosis by LDH cytotoxicity, flow cytometry Annexin V-FITC/PI, Hoechst staining, TUNEL staining and JC-1 staining methods. The expression of apoptosis related proteins(cleaved-caspse3,Bcl-2 and Bax) was detected by Western-Blot;3. Targetscan database was used to predict target gene of mi R-146 b. The wild type and mutant dual luciferase report genes were constructed and co-transfected with mi R-146 b mimic(NC) in 293 T cells to detect that whether mi R-146 b can bind to RNase L m RNA. The expression of RNase L after down-regulation of mi R-146 b was also measured in H9c2 cells by Western-Bolt at the same time.4. We first transfected mi R-146 b inhibitor into H9c2 cells, then the key protein of NF-κB-IL6 signaling axis such P65 and STAT3 and their phosphorylated forms were detected in H9c2 cells by Western-Bolt after hypoxia.5. Preliminary study of RNase L in cardiomyocyte under chronic hypoxia.5.1. Expression of RNase L was detected in human cardiac tissues by immunohistochemistry.5.2. Detect the expression of RNase L and RLI in H9c2 after hypoxia using RT-PCR and Western-bolt method.5.3. Construct chemically synthetical si RNA of RNase L, and transfected it into H9c2 cells. RT-PCR method was used to detect interference efficiency. Select effective si RNA sequence.5.4. We transfected RNase L si RNA into H9c2 cells and apoptosis was measured after hypoxia by flow cytometry Annexin V-FITC/PI analysis.Results1. RT-PCR results showed that the expression of mi R-146 b was significantly higher in cyanotic tissues than in acyanotic ones(140% vs the latter, P<0.05).2. RT-PCR results showed that the expression of mi R-146 b gradually increased after hypoxia, and reached its peak at 72h(197% vs NC, P<0.05).3. Inhibitor NC-FAM was transfected into the H9c2 cells using Lipofectamine 2000, results showed that the transfection efficiency is more than 75%, meeting our expectations. Cell death was detected by LDH cytotoxicity experiment. The results showed that up-regulation of mi R-146 b can inhibit cell death by 20.3%(P<0.05), while down-regulation of it can augument cell death by 64%(P<0.05). The apoptosis rate of hypoxic cardiomyocytes were then measured using flow cytometry Annexin V-FITC/PI, TUNEL, Hoechst and JC-1 staining analysis. The results showed that compared with the NC group, the mi R-146 b inhibitor could significantly increase the rate of apoptosis(P<0.05). This conclusion was also verified by Western-Blot results, which showed the down-regulation of mi R-146 b could significantly increase expression of cleaved-caspase 3, Bax by 73.0% and 72.8%, respectively(P<0.05), while Bcl-2 expression was down-regulated by 37.2%(P<0.05), the Bcl-2/Bax ration was also decreased significantly by 64.0%(P<0.05).4. The reporter genes results showed co-transfection with wild-type reporter gene and mimic can significantly reduce the fluorescence intensity by 30.9%(P<0.05), while co-transfected with mutant one did not differ compared with the NC group(P>0.05). The results confirmed that mi R-146 b could bind to RNase L m RNA directly. In addition, the down-regulation of mi R-146 b in H9c2 cells lead to increased expression of RNase L protein by 661%(P<0.05), which verified that the RNase L is the direct target of mi R-146 b.5. mi R-146 b did not affect the expression of p65 and STAT3 protein, but increased both of their phosphorylated forms in the H9c2 cardiomyocyte after 72 h hypoxia,6. Results of immunohistochemistry showed that high expression of RNase L protien was detected in cardiomyocyte and fibroblasts, and relatively low expression in stromal cells(e.g. vascular endothelial cell) in human myocardial tissue samples.7. In the H9c2 cardiomyocytes, both m RNA and protein expression of RNase L reduced gradually after hypoxia(RNase L m RNA decreased by 61.0% 72 h compared with 0h, P<0.05), while expression of its natural inhibitor RLI increased.8. Three pairs of RNase L si RNA were designed and transfected into H9c2 cells, respectively. RT-PCR was conducted to detect transfection efficiency. Only one pair of si RNA could inhibit RNase L at m RNA level by 52.3%(P<0.05). After culturing in hypoxic incubator for 72 h, apoptosis rate was detected by flow cytometry Annexin V-FITC/PI. The results showed that the number of apoptotic cells in RNase L si RNA transfection group was significantly lower than that in the control group(4.36% vs NC 12.4%, P<0.05).Conclusion1. The expression of mi R-146 b is significantly elevated in cyanotic tissues samples compared with that in the acyanotic one, and its expression is gradually increased in H9c2 cells after hypoxia.2. Down-regulation of mi R-146 b in H9c2 cells could augument myocardial hypoxiainduced apoptosis.3. RNase L is a direct target of mi R-146 b.4. NF-κB and STAT3 were significantly activated by down-regulation of mi R-146 b.5. The expression of RNase L protien was high in cardiomyocyte and fibroblasts, and relatively low in stromal cells(e.g. vascular endothelial cell) in human myocardial tissue samples. Both m RNA and protein expression of RNase L are gradually reduced after hypoxia in hypoxic H9c2 cardiomyocytes, while protein of RLI increased. Down-regulation of RNase L may protect cardiomyocytes from hypoxia-induced apoptosis.