Retracted: LncRNA-LET relieves hypoxia-induced injury in H9c2 cells through regulation of miR-138

Ischemic heart disease (IHD) is a common cardiovascular disease, occurs when coronary artery blood circularity cannot match with the heart's need. The present work attempted to study the effects of long noncoding RNA (lncRNA) low expression in tumor (LET) on the progression of IHD. H9c2 cells were injured by hypoxia to mimic a cell model of IHD. The effects of lncRNA-LET on hypoxia-injured H9c2 cells were tested by using cell counting kit-8 assay, flow cytometry, and Western blot analysis. MicroRNA-138 (miR-138) expression was tested by a quantitative real-time polymerase chain reaction, and the expression of c-Jun N-terminal kinase (JNK) and p38MAPK (p38–mitogen-activated protein kinase) proteins was measured by Western blot analysis. We found that hypoxia exposure significantly repressed the viability of H9c2 cells, and induced apoptosis. Meanwhile, phosphorylation of JNK and p38MAPK was enhanced by hypoxia. The expression of lncRNA-LET was repressed by hypoxia. Overexpression of lncRNA-LET attenuated hypoxia-induced injury in H9c2 cells. Moreover, miR-138 was a downstream effector of lncRNA-LET, that miR-138 was highly expressed in lncRNA-LET-overexpressed cell. The cardioprotective effects of lncRNA-LET were abolished when miR-138 was silenced. In conclusion, this study revealed the cardioprotective function of lncRNA-LET. lncRNA-LET conferred its cardioprotective effects possibly via upregulation of miR-138 and thus repressing the JNK and p38MAPK pathways.     

ERK1／2信号通路对黄芪苷Ⅳ抗H2O2诱导H9c2细胞氧化损伤的作用

目的：研究黄芪苷Ⅳ（AST）是否通过细胞外信号调节激酶1／2（ERK1／2）通路发挥对H2O2诱导的H9c2细胞氧化损伤的保护作用。方法：用200μmoL／L的H2O2处理细胞6h,采用MTT法检测细胞存活率,建立H2O2诱导的H9c2细胞氧化损伤模型;比色法测定细胞培养液中乳酸脱氢酶（LDH）活性、总超氧化物歧化酶（T—SOD）和锰超氧化物歧化酶（Mn—SOD）活力以及丙二醛（MDA）含量;Western blot检测H9c2细胞ERK1／2蛋白的磷酸化水平。结果：在H2O2浓度为200μmol／L作用6h条件下,细胞存活率降低程度适中,实验结果重复性好,确定后续实验采用200μmol／L H2O2作用6h建立模型。与H2O2组比较,10mg／L及20mg／L AST均显著提高细胞存活率（P〈0．01）,使细胞培养液中LDH活性显著降低（P〈0．01）,T—SOD及Mn—SOD活力显著提高（P〈0．01）,MDA含量显著降低（P〈0．01）。10mg/L及20mg/L AST均显著增加H2O2损伤的H9c2细胞p—ERK1／2蛋白的表达（P〈0．01）,当用PD98059（ERK1／2的抑制剂）预处理后,AST的作用则被取消。结论：黄芪苷Ⅳ可以通过ERK1／2通路发挥对H2O2诱导的H9c2细胞氧化损伤的保护作用。     

Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Abstract

Background. Insulin protects cardiomyocytes from reactive oxygen species (ROS)-induced apoptosis after ischemic/reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from ROS injury. Methods. Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H₂O₂) or insulin at various concentrations for various periods of time, or with insulin and H₂O₂ for various periods of time. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results. The percentage of viable cells was significantly and inversely associated with H₂O₂ concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H₂O₂ or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H₂O₂-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion. Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H₂O₂/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.     

microRNA-193b protects against myocardial ischemia-reperfusion injury in mouse by targeting mastermind-like 1

The current study aimed to explore the functions and roles of microRNA-193b (miR-193b) in the myocardium with ischemia-reperfusion (I/R) injury and a potential therapeutic method for myocardial I/R injury. The mice were subjected to myocardial I/R with or without miR-193b pretreatment. The infarct size and myocardial enzymes were detected. The terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was conducted to investigate the effect of miR-193b on cardiomyocyte apoptosis. The expression levels of miR-193b and mastermind-like 1 (MAML1) were validated by quantitative real-time polymerase chain reaction and Western blot analysis. The results suggested that the miR-193b expression level was significantly downregulated in the myocardium with I/R injury compared with control group. miR-193b overexpression is able to reduce infarct size and myocardial enzymes after myocardial I/R injury. Furthermore, overexpression of miR-193b could alleviate the apoptosis level after myocardial I/R injury. Taken together, the present study demonstrated that upregulated miRNA-193b alleviated myocardial I/R injury via targeting MAML1.     

MicroRNA‐21 protects against cardiac hypoxia/reoxygenation injury by inhibiting excessive autophagy in H9c2 cells via the Akt/mTOR pathway

MicroRNAs and autophagy play critical roles in cardiac hypoxia/reoxygenation (H/R)‐induced injury. Here, we investigated the function of miR‐21 in regulating autophagy and identified the potential molecular mechanisms involved. To determine the role of miR‐21 in regulating autophagy, H9c2 cells were divided into the following six groups: control group, H/R group, (miR‐21+ H/R) group, (miR‐21‐negative control + H/R) group, (BEZ235+ H/R) group and (miR‐21+ BEZ235+ H/R) group. The cells underwent hypoxia for 1 hr and reoxygenation for 3 hrs. Cell count kit‐8 was used to evaluate cell function and apoptosis was analysed by Western blotting. Western blotting and transmission electron microscopy were used to investigate autophagy. We found that miR‐21 expression was down‐regulated, and autophagy was remarkably increased in H9c2 cells during H/R injury. Overexpression of miR‐21 with a miR‐21 precursor significantly inhibited autophagic activity and decreased apoptosis, accompanied by the activation of the AKT/mTOR pathway. In addition, treatment with BEZ235, a novel dual Akt/mTOR inhibitor, resulted in a significant increase in autophagy and apoptosis. However, we found that miR‐21‐mediated inhibition of apoptosis and autophagy was partly independent of Akt/mTOR activation, as demonstrated in cells treated with both miR‐21 and BEZ235. We showed that miR‐21 could inhibit H/R‐induced autophagy and apoptosis, which may be at least partially mediated by the Akt/mTOR signalling pathway.     

Gastrodin relieves inflammation injury induced by lipopolysaccharides in MRC‐5 cells by up‐regulation of miR‐103

The beneficial function of gastrodin towards many inflammatory diseases has been identified. This study designed to see the influence of gastrodin in a cell model of chronic obstructive pulmonary disease (COPD). MRC‐5 cells were treated by LPS, before which gastrodin was administrated. The effects of gastrodin were evaluated by conducting CCK‐8, FITC‐PI double staining, Western blot, qRT‐PCR and ELISA. Besides this, the downstream effector and signalling were studied to decode how gastrodin exerted its function. And dual‐luciferase assay was used to detect the targeting link between miR‐103 and lipoprotein receptor‐related protein 1 (LRP1). LPS induced apoptosis and the release of MCP‐1, IL‐6 and TNF‐α in MRC‐5 cells. Pre‐treating MRC‐5 cells with gastrodin attenuated LPS‐induced cell damage. Meanwhile, p38/JNK and NF‐κB pathways induced by LPS were repressed by gastrodin. miR‐103 expression was elevated by gastrodin. Further, the protective functions of gastrodin were attenuated by miR‐103 silencing. And LRP1 was a target of miR‐103 and negatively regulated by miR‐103. The in vitro data illustrated the protective function of gastrodin in LPS‐injured MRC‐5 cells. Gastrodin exerted its function possibly by up‐regulating miR‐103 and modulating p38/JNK and NF‐κB pathways.     

Retracted: MicroRNA-132 protects H9c2 cells against oxygen and glucose deprivation-evoked injury by targeting FOXO3A

Myocardial ischemia/reperfusion injury (MIRI) is a clinically familiar disease, which possesses a great negative impact on human health. But, the effective treatment is still absent. MicroRNAs (miRNAs) have been testified to play a momentous role in MIRI. The purpose of the study aimed to probe the functions of miR-132 in oxygen and glucose deprivation (OGD)-evoked injury in H9c2 cells. miR-132 expression in H9c2 cells accompanied by OGD disposition was evaluated via real-time quantitative polymerase chain reaction. After miR-132 mimic and inhibitor transfections, the impacts of miR-132 on OGD-affected H9c2 cell viability, apoptosis, cell cycle, and the interrelated factors were appraised by exploiting cell counting kit-8, flow cytometry, and western blot analysis. FOXO3A expression was estimated in above-transfected cells, meanwhile, the correlation between miR-132 and FOXO3A was probed by dual-luciferase report assay. Ultimately, above mentioned cell processes were reassessed in H9c2 cells after preprocessing OGD administration and transfection with si-FOXO3A and si-NC plasmids. We got that OGD disposition obviously enhanced miR-132 expression in H9c2 cells. Overexpressed miR-132 evidently reversed OGD-evoked cell viability repression and apoptosis induction in H9c2 cells. In addition, overexpressed miR-132 mitigated OGD-evoked G0/G1 cell arrest by mediating p21, p27, and cyclin D1 expression. Repression of FOXO3A was observed in miR-132 mimic-transfected cells, which was also predicated as a direct gene of miR-132. We discovered that silenced FOXO3A alleviated OGD-evoked cell injury in H9c2 cells via facilitating cell viability, hindering apoptosis and restraining cell arrest at G0/G1 phase. In conclusion, these investigations corroborated that miR-132 exhibited the protective impacts on H9c2 cells against OGD-evoked injury via targeting FOXO3A.     

Adiponectin attenuates lipopolysaccharide-induced cell injury of H9c2 cells by regulating AMPK pathway

Adiponectin,an adipokine synthesized and secreted majorly by adipose tissue,is reported to exert cardioprotective properties via anti-inflammation and antiapoptosis.Lipopolysaccharide(LPS)is a common inflammation and apoptosis inducer of cardiomyocytes.However,few studies have reported the roles of adiponectin on LPS-induced inflammation as well as apoptosis of H9c2 cells,and the possible mechanisms of these effects.In the present study,we found that adiponectin significantly relieved LPS-induced cytotoxicity including decreased viability and elevated LDH release,inhibited LPS-triggered inflammation,which is evidenced by increases in release of TNF-α,IL-1β as well as IL-6,and attenuated the enhanced rates of apoptotic cells as well as increased caspase-3 activity caused by LPS in H9c2 cells.In addition,our data demonstrated that adiponectin upregulated AMP-activated protein kinase(AMPK)activation of H9c2 cells with or without LPS administration.Moreover,we found that blocking AMPK pathway by compound c attenuated the protective effects of adiponectin against the cytotoxicity,inflammatory response,and apoptosis of H9c2 cells resulted from LPS.Our observations bring novel insights for understanding the mediatory role of AMPK pathway implicated in the protective effects of adiponectin against LPS-induced cardiotoxicity.     

Retracted: LncRNA-ROR alleviates hypoxia-triggered damages by downregulating miR-145 in rat cardiomyocytes H9c2 cells

Chronic hypoxic heart disease (CHD) is a common clinical type of congenital heart disease. Long noncoding RNA regulator of reprogramming (lncRNA-ROR) exerts an important regulating effect in cardiovascular diseases. In our study, we explored the effect of lncRNA-ROR and the possible mechanisms against hypoxia-caused apoptosis in H9c2 cells. H9c2 cells were exposed to hypoxia (1% O₂) to construct the in vitro model of CHD. The level of lncRNA-ROR and microRNA (miRNA/miR)-145 was detected. To upregulate the level of lncRNA-ROR and miR-145, transfection was carried out. Western blot assay was performed to quantified protein expression. The interaction of lncRNA-ROR with miR-145 was verified by RIP and Dual-luciferase reporter assays. The expression of p53 and Bax was largely elevated and Bcl-2 was suppressed by hypoxia induction. We found that lncRNA-ROR was elevated by hypoxia. LncRNA-ROR overexpression was able to relieve the damages of H9c2 cells induced by hypoxia through rescuing viability, suppressing apoptosis, and blocking Cytochrome c release. miR-145 was suppressed by overexpressed lncRNA-ROR and the combination of miR-145 mimic was able to abolish the protective effect of lncRNA-ROR. Moreover, we found that lncRNA-ROR activated Ras/Raf/MEK/ERK and PI3K/AKT transduction cascades by suppressing miR-145. Besides, lncRNA-ROR directly targeted miR-145 and negatively modulated the level of miR-145. Our present study revealed that lncRNA-ROR protected H9c2 cells against hypoxia-caused damages by regulation of miR-145 through activating Ras/Raf/MEK/ERK and PI3K/AKT.     

Induction of caspase-independent apoptosis in H9c2 cardiomyocytes by adriamycin treatment

The cardiotoxicity of adriamycin limits its clinical use as a powerful drug for solid tumors and malignant hematological disease. Although the precise mechanism by which it causes cardiac damage is not yet known, it has been suggested that apoptosis is the principal process in adriamycin-induced cardiomyopathy, which involves DNA fragmentation, cytochrome C release, and caspase activation. However, there has been no direct evidence for the critical involvement of caspase-3 in adriamycin-induced apoptosis. To determine the requirements for the activation of caspase-3 in adriamycin-treated cardiac cells, the effect of a caspase inhibitor on the survival of and apoptotic changes in H9c2 cells was examined. Exposure of H9c2 cells to adriamycin resulted in a time- and dose-dependent cell death, and the cleavage of pro-caspase-3 and of the nuclear protein poly (ADP-ribose) polymerase (PARP). However, neither the reduction of cell viability nor the characteristic morphological changes induced by adriamycin were prevented by pretreatment with the general caspase inhibitor z-VAD.FMK. In contrast, caspase inhibition effectively blocked the apoptosis induced by H₂O₂ in H9c2 cells, as determined by an MTT assay or microscopy. We also observed that p53 expression was increased by adriamycin, and this increase was not affected by the inhibition of caspase activity, suggesting a role for p53 in adriamycin-induced caspase-independent apoptosis in cardiac toxicity. (Mol Cell Biochem 270: 13–19, 2005)These authors contributed equally to this work     

MicroRNA‐328 is involved in the effect of selenium on hydrogen peroxide‐induced injury in H9c2 cells

Oxidative stress induces apoptosis in cardiac cells, and antioxidants attenuate the injury. MicroRNAs (miRNAs) are also involved in cell death; therefore, this study aimed to investigate the role of miRNAs in the effect of selenium on oxidative stress‐induced apoptosis. The effects of sodium selenite were analyzed via cell viability, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) concentration. Flow cytometry was used to evaluate cell apoptosis. Fura‐2AM was used to calculate intracellular Ca²⁺ concentration. Sodium selenite could ameliorate hydrogen peroxide (H₂O₂)‐induced cell apoptosis and improve expression levels of glutathione peroxidase and thioredoxin reductase. Pretreatment with sodium selenite improved SOD activity and reduced MDA concentration. Treatments with H₂O₂ or sodium selenite decreased miR‐328 levels. MiR‐328 overexpression enhanced cell apoptosis, reduced ATP2A2 levels, and increased intracellular Ca²⁺ concentration, while inhibition produced opposite effects. MiR‐328 might be involved in the effect of sodium selenite on H₂O₂‐induced cell death in H9c2 cells.     

Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.     

Resveratrol for protection against statin toxicity in C2C12 and H9c2 cells

Statins are among the most commonly prescribed drugs for the treatment of high blood cholesterol. Myotoxicity of statins in certain individuals is often a severe side effect leading to withdrawal. Using C2C12 and H9c2 cells, both exhibiting characteristics of skeletal muscle cells, we addressed whether resveratrol (RSV) can prevent statin toxicity. Statins decreased cell viability in a dose and time‐dependent manner. Among the five statins tested, atorvastatin, simvastatin, lovastatin, pravastatin, and fluvastatin, simvastatin is the most toxic one. Simvastatin at 10 µM caused about 65% loss of metabolic activity as measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays in C2C12 cells or H9c2 cells. Inhibition of metabolic activity correlates with an increase in caspase activity. RSV was found to protect H9c2 cells from simvastatin‐induced activation of caspase‐3/7. However, such protection was not found in C2C12 cells. This cell type‐dependent effect of RSV adds to the complexity in muscle cell toxicity of statins.     

Effects of resveratrol on H2O2‐induced apoptosis and expression of SIRTs in H9c2 cells

Resveratrol, a polyphenol found in fruits, has been demonstrated to activate Sir2. Though many studies have demonstrated that resveratrol can activate SIRT1, whether it has effect on other sirtuins (SIRT2–7) are unknown. The present study shows that exposure of H9c2 cells to 50 µM H₂O₂ for 6 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry (FCM), but pretreatment of resveratrol (20 µM) eliminated H₂O₂‐induced apoptosis. Resveratrol also prevented H₂O₂‐induced caspase‐3 activation. Exposure of cells to resveratrol caused rapid activation of SIRT1,3,4,7. Sirtuin inhibitor, nicotinamide (20 mM) attenuated resveratrol's inhibitory effect on cell apoptosis and caspase‐3 activity. These results suggest that resveratrol protects cardiomyocytes from H₂O₂‐induced apoptosis by activating SIRT1,3,4,7. J. Cell. Biochem. 107: 741–747, 2009. © 2009 Wiley‐Liss, Inc.     

HSP70 protects H9C2 cells from hypoxia and reoxygenation injury through STIM1/IP3R

Hypoxia/reoxygenation (H/R) is used as an in vivo model of ischemia/reperfusion injury, and myocardial ischemia can lead to heart disease. Calcium overload is an important factor in myocardial ischemia–reperfusion injury and can lead to apoptosis of myocardial cells. Therefore, it is of great clinical importance to find ways to regulate calcium overload and reduce apoptosis of myocardial cells, and thus alleviate myocardial ischemia–reperfusion injury. There is evidence that heat shock protein 70 (HSP70) has a protective effect on the myocardium, but the exact mechanism of this effect is not completely understood. Stromal interaction molecule 1 and inositol 1,4,5-triphosphate receptor (STIM/1IP3R) play an important role in myocardial ischemia–reperfusion injury. Therefore, this study aimed to investigate whether HSP70 plays an anti-apoptotic role in H9C2 cardiomyocytes by regulating the calcium overload pathway through STIM1/IP3R. Rat H9C2 cells were subjected to transient oxygen and glucose deprivation (incubated in glucose-free medium and hypoxia for 6 h) followed by re-exposure to glucose and reoxygenation (incubated in high glucose medium and reoxygenation for 4 h) to simulate myocardial ischemia reperfusion-induced cell injury. H9C2 cell viability was significantly decreased, and lactate dehydrogenase (LDH) release and apoptosis were significantly increased after oxygen and glucose deprivation. Transfection of HSP70 into H9C2 cells could reduce the corresponding effect, increase cell viability and anti-apoptotic signal pathway, and reduce the apoptotic rate and pro-apoptotic signal pathway. After hypoxia and reoxygenation, the expression of STIM1/IP3R and intracellular calcium concentration of HSP70-overexpressed H9C2 cells were significantly lower than those of hypoxia cells. Similarly, direct silencing of STIM1 by siRNA significantly increased cell viability and expression of anti-apoptotic protein Bcl-2 and decreased apoptosis rate and expression of pro-apoptotic protein BAX. These data are consistent with HSP70 overexpression. These results suggest that HSP70 abrogates intracellular calcium overload by inhibiting upregulation of STIM1/IP3R expression, thus reducing apoptosis in H9C2 cells and playing a protective role in cardiomyocytes.