Troxerutin attenuates myocardial cell apoptosis following myocardial ischemia-reperfusion injury through inhibition of miR-146a-5p expression

The aim of the current study was to investigate the effects and the underlying mechanisms of troxerutin on myocardial cell apoptosis during ischemia-reperfusion (I/R) injury. Hypoxia/reoxygenation (H/R) model in neonatal rat cardiomyocytes, and I/R model in rats, were established following troxerutin preconditioning. The quantitative real-time polymerase chain reaction analysis was performed to examine the messenger RNA miR-146a-5p expression in cardiomyocytes and myocardial tissues. Hemodynamic parameters and serum creatine kinase, lactate dehydrogenase, tumor necrosis factor-α, and interleukin-10 were evaluated. Infarct size was examined by 2,3,5-triphenyltetrazolium chloride staining. Besides, myocardial apoptosis was detected by terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay. Western blot analysis was performed to determine the protein levels of caspase-3, Bax, and Bcl-2. The results showed that, troxerutin decreased rat cardiomyocyte apoptosis during H/R injury. Furthermore, the antiapoptotic effect of troxerutin against I/R injury was mediated by miR-146a-5p downregulation. In vivo experiments suggested that troxerutin alleviated myocardial I/R injury in rats via inhibition of miR-146a-5p. In conclusion, troxerutin exerted cardioprotective effects during I/R injury by downregulating miR-146a-5p.     

p53 and Mdm2 act synergistically to maintain cardiac homeostasis and mediate cardiomyocyte cell cycle arrest through a network of microRNAs

Defining the roadblocks responsible for cell cycle arrest in adult cardiomyocytes lies at the core of developing cardiac regenerative therapies. p53 and Mdm2 are crucial mediators of cell cycle arrest in proliferative cell types, however, little is known about their function in regulating homeostasis and proliferation in terminally differentiated cell types, like cardiomyocytes. To explore this, we generated a cardiac-specific conditional deletion of p53 and Mdm2 (DKO) in adult mice. Herein we describe the development of a dilated cardiomyopathy, in the absence of cardiac hypertrophy. In addition, DKO hearts exhibited a significant increase in cardiomyocyte proliferation. Further evaluation showed that proliferation was mediated by a significant increase in Cdk2 and cyclin E with downregulation of p21^Cip1 and p27^Kip1. Comparison of miRNA expression profiles from DKO mouse hearts and controls revealed 11 miRNAs that were downregulated in the DKO hearts and enriched for mRNA targets involved in cell cycle regulation. Knockdown of these miRNAs in neonatal rat cardiomyocytes significantly increased cytokinesis with an upregulation in the expression of crucial cell cycle regulators. These results illustrate the importance of the cooperative activities of p53 and Mdm2 in a network of miRNAs that function to impose a barrier against aberrant cardiomyocyte cell cycle re-entry to maintain cardiac homeostasis.     

miR-885 mediated cardioprotection against hypoxia/reoxygenation-induced apoptosis in human cardiomyocytes via inhibition of PTEN and BCL2L11 and modulation of AKT/mTOR signaling

Ischemia/reperfusion (I/R) injury could cause the enhanced cell apoptosis of cardiomyocytes, which is one of key contributors for the development of ischemic heart disease. Recent studies emphasized the role of microRNAs (miRNAs) in regulating cardiomyocyte apoptosis. The study planned to elucidate the molecular actions of miR-885 on mediating human cardiomyocytes (HCMs) apoptosis induced by hypoxia/reoxygenation (H/R) and to explore the potential molecular mechanisms. The present data revealed that H/R stimulation inhibited HCM viability and potentiated HCM apoptosis, and more importantly, the expression of miR-885 in HCMs was markedly repressed after H/R stimulation. Further experimental examinations demonstrated that overexpression of miR-885 attenuated H/R-induced increased in HCM apoptotic rates, while miR-885 knockdown impaired HCM viability and increased HCM apoptotic rates. Moreover, the mechanistic studies showed that miR-885 inversely regulated the expression of phosphatase and tensin homolog (PTEN) and BCL2 like 11 (BCL2L11) in HCMs, and enforced expression of PTEN and BCL2L11 partially antagonized the protective actions of miR-885 overexpression on H/R-induced HCM injury. Moreover, H/R suppressed AKT/mTOR signaling, which was attenuated by miR-885 overexpression in HCMs. In conclusion, the present study for the first time showed the downregulation of miR-885 induced by H/R in HCMs, and provided the evidence that miR-885 attenuated H/R-induced cell apoptosis via inhibiting PTEN and BLC2L11 and modulation of AKT/mTOR signaling in HCMs.     

microRNA-129 overexpression in endothelial cell-derived extracellular vesicle influences inflammatory response caused by myocardial ischemia/reperfusion injury

Extracellular vesicles (EVs) have the potency to function as modulators in the process of myocardial ischemia/reperfusion (I/R) injury. This investigation was performed to decipher the mechanism of human umbilical vascular endothelial cells (HUVECs)-derived EVs in myocardial I/R injury with the involvement of microRNA-129 (miR-129). HUVECs-secreted EVs were collected and identified. An I/R mouse model was developed, and cardiomyocytes were used for vitro oxygen-glucose deprivation/reperfusion model establishment. Differentially expressed miRNAs in myocardial tissues after EV treatment were assessed using microarray analysis. The target relationship between miR-129 and toll-like receptor 4 (TLR4) was identified using a dual-luciferase assay. Gain- and loss-function studies regarding miR-129 were implemented to figure out its roles in myocardial I/R injury. Meanwhile, the activation of the nuclear factor-kappa-binding (NF-κB) p65 signaling and NOD-like receptor 3 (NLRP3) inflammasome was evaluated. EVs diminished the apoptosis of cardiomyocytes and the secretion of inflammatory factors, and all these trends were reversed by miR-129 reduction. miR-129 bound to the 3′-untranslated region of TLR4 directly. The NF-κB p65 signaling and NLRP3 inflammasome were abnormally activated after I/R injury, whose impairment after EVs was partially restored by miR-129 downregulation. This study illustrated that EVs could carry miR-129 to mitigate myocardial I/R injury via downregulating TLR4 and disrupting the NF-κB signaling and NLRP3 inflammasome.     

miRNA-145-5p induces apoptosis after ischemia-reperfusion by targeting dual specificity phosphatase 6

Disorders mainly caused by ischemia-reperfusion (I/R), including stroke and myocardial infarction, is linked to debilitating health conditions and death. Recent research indicates that microRNAs (miRNAs) mediate the process of ischemic pathology. This study investigated the effects of miR-145-5p in regulating myocardial ischemic injury. The I/R models were established in rat cardiomyocytes H9C2 and rats. Western blot analysis and quantitative polymerase chain reaction was performed to analyze protein expression. Annexin V-FITC/PI staining was conducted to evaluate cell apoptosis. The application of miR-145-5p mimics and inhibitor revealed that miR-145-5p promoted apoptosis in cardiomyocytes. Furthermore, we found that miR-145-5p directly inhibited dual specificity phosphatase 6 (DUSP6) by luciferase reporter assay. The results indicated that DUSP6 was beneficial against I/R injury through inhibiting c-Jun N-terminal kinase pathways. In conclusion, the essential roles of miR-145-5p and DUSP6 in I/R provide a novel therapeutic target to develop future intervention strategies.     

MicroRNA-25 aggravates Aβ1-42-induced hippocampal neuron injury in Alzheimer's disease by downregulating KLF2 via the Nrf2 signaling pathway in a mouse model

Recently, numerous microRNAs (miRNAs) have been considered as key players in the regulation of neuronal processes. The purpose of the present study is to explore the effect of miR-25 on hippocampal neuron injury in Alzheimer's disease (AD) induced by amyloid β (Aβ) peptide fragment 1 to 42 (Aβ1-42) via Kruppel-like factor 2 (KLF2) through the nuclear factor-E2-related factor 2 (Nrf2) signaling pathway. A mouse model of AD was established through Aβ1-42 induction. The underlying regulatory mechanisms of miR-25 were analyzed through treatment of miR-25 mimics, miR-25 inhibitors, or small interfering RNA (siRNA) against KLF2 in hippocampal tissues and cells isolated from AD mice. The targeting relationship between miR-25 and KLF2 was predicted using a target prediction program and verified by luciferase activity determination. MTT assay was used to evaluate the proliferative ability and flow cytometry to detect cell cycle distribution and apoptosis. KLF2 was confirmed as a target gene of miR-25. When the mice were induced by Aβ1-42, proliferation was suppressed while apoptosis was promoted in hippocampal neurons as evidenced by lower levels of KLF2, Nrf2, haem oxygenase, glutathione S transferase α1, glutathione, thioredoxin, and B-cell lymphoma-2 along with higher bax level. However, such alternations could be reversed by treatment of miR-25 inhibitors. These findings indicate that miR-25 may inhibit hippocampal neuron proliferation while promoting apoptosis, thereby aggravating hippocampal neuron injury through downregulation of KLF2 via the Nrf2 signaling pathway.     

Repression of Cyclin D1 Expression Is Necessary for the Maintenance of Cell Cycle Exit in Adult Mammalian Cardiomyocytes

The hearts of neonatal mice and adult zebrafish can regenerate after injury through proliferation of preexisting cardiomyocytes. However, adult mammals are not capable of cardiac regeneration because almost all cardiomyocytes exit their cell cycle. Exactly how the cell cycle exit is maintained and how many adult cardiomyocytes have the potential to reenter the cell cycle are unknown. The expression and activation levels of main cyclin-cyclin-dependent kinase (CDK) complexes are extremely low or undetectable at adult stages. The nuclear DNA content of almost all cardiomyocytes is 2C, indicating the cell cycle exit from G₁-phase. Here, we induced expression of cyclin D1, which regulates the progression of G1-phase, only in differentiated cardiomyocytes of adult mice. In these cardiomyocytes, S-phase marker-positive cardiomyocytes and the expression of main cyclins and CDKs increased remarkably, although cyclin B1-CDK1 activation was inhibited in an ATM/ATR-independent manner. The phosphorylation pattern of CDK1 and expression pattern of Cdc25 subtypes suggested that a deficiency in the increase in Cdc25 (a and -b), which is required for M-phase entry, inhibited the cyclin B1-CDK1 activation. Finally, analysis of cell cycle distribution patterns showed that >40% of adult mouse cardiomyocytes reentered the cell cycle by the induction of cyclin D1. The cell cycle of these binucleated cardiomyocytes was arrested before M-phase, and many mononucleated cardiomyocytes entered endoreplication. These data indicate that silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit and suggest a mechanism that involves inhibition of M-phase entry.     

MicroRNA-15b enhances hypoxia/reoxygenation-induced apoptosis of cardiomyocytes via a mitochondrial apoptotic pathway

Myocardial ischemia reperfusion (I/R) can induce altered expression of microRNAs (miRNAs). The miRNAs—miR-15a, miR-15b and miR-16 have been shown to play a role in apoptosis, although not in cardiac-related models. We investigated the roles of miR-15b in hypoxia/reoxygenation (H/R)-induced apoptosis of cardiomyocytes. Quantitative real time polymerase chain reaction results showed that the expression of miR-15a and miR-15b were up-regulated in Sprague–Dawley rat hearts subjected to I/R. Expression levels of miR-15b increased more than four fold above basal levels. Similar results were obtained for cardiomyocytes exposed to H/R. Recombinant adenoviral vectors were generated to explore the functional role of miR-15b in cultured cardiomyocytes exposed to H/R. Overexpression of miR-15b enhanced cell apoptosis and the loss of mitochondrial membrane potential, as determined by flow cytometric analysis. Conversely, down-regulated expression was cytoprotective. The effects of miR-15b can by mimicked by Bcl-2 short-interfering RNAs. The inhibition of miR-15b increased expression levels of the Bcl-2 protein without affecting Bcl-2 mRNA levels, suppressed the release of mitochondrial cytochrome c to the cytosol and decreased the activities of caspase-3 and 9. It is possible that miR-15b is the upstream regulator of a mitochondrial signaling pathway for H/R induced apoptosis.     

Overexpression of microRNA-378 attenuates ischemia-induced apoptosis by inhibiting caspase-3 expression in cardiac myocytes

MicroRNAs (miRNAs) are a novel class of powerful, endogenous regulators of gene expression. In an intact rat model of myocardial ischemia caused by coronary artery ligation, this study identified 17 miRNAs that changed more than 1.5-fold in the myocardium subjected to 4-h ischemia. Using miRNA microarray analysis, most of these aberrantly expressed miRNAs were confirmed by quantitative RT-PCR. MiR-378, a significantly down-regulated miRNA, was selected for further function study. In serum deprived rat H9c2 cardiomyocytes exposed to hypoxia (1% O₂), miR-378 expression was down-regulated as well. The overexpression of miR-378 resulting from miR-378 mimic transfection significantly enhanced cell viability, reduced lactate dehydrogenase release, and inhibited apoptosis and necrosis. By contrast, miR-378 deficiency resulting from miR-378 inhibitor transfection aggravated the hypoxia-induced apoptosis and cell injury. In accordance, miR-378 inhibitor caused significant apoptosis and cell injury to cardiomyocytes cultured under normoxia. Using bioinformatic algorithms, caspase-3, a key apoptosis executioner, was predicted as a putative target of miR-378. The quantitative RT-PCR showed no effects of miR-378 mimic or inhibitor on caspase-3 mRNA level. However, the amount of caspase-3 proteins was reduced by miR-378 mimic, whereas increased by miR-378 inhibitor. Furthermore, the luciferase reporter assay confirmed caspase-3 to be a target of miR-378, and the apoptosis and cell injury caused by miR-378 inhibitor in both normoxic and hypoxic cells were abolished by a caspase-3 inhibitor. This study first showed that miR-378 inhibited caspase-3 expression and attenuated ischemic injury in cardiomyocytes. It may represent a potential novel treatment for apoptosis and ischemic heart disease.     

Calcium-sensing receptors induce apoptosis in cultured neonatal rat ventricular cardiomyocytes during simulated ischemia/reperfusion

Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which regulate systemic calcium homeostasis and also participate in cell proliferation, differentiation and apoptosis. We have previously shown that CaSR can induce apoptosis in isolated rat adult hearts and in normal rat neonatal cardiomyocytes. However, no knowledge exists concerning the role of CaSR in apoptosis induced by ischemia and reperfusion in neonatal cardiac myocytes. Therefore, in the present study, we incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2h, then re-incubated them in a normal culture medium for 24h to establish a model of simulated ischemia/reperfusion (I/R). We assayed the apoptotic ratio of the cardiomyocytes by flow cytometry; observed morphological alterations by transmission electron microscope; analyzed the expression of caspase-3, Bcl-2, CaSR, extracellular signal-regulated protein kinase (ERK), and Fas/Fas ligand (FasL) by Western blotting; and measured the concentration of intracellular calcium by Laser Confocal Scanning Microscopy. The results showed that simulated I/R increased the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further enhanced CaSR expression, along with increases in intracellular calcium and apoptosis in cardiomyocytes during I/R. Activation of CaSR down-regulated Bcl-2 expression, up-regulated caspase-3 and Fas/FasL expression and stimulated ERK1/2 phosphorylation. In summary, CaSR is involved in I/R injury and apoptosis of neonatal rat ventricular cardiomyocytes by inhibiting Bcl-2, inducing calcium overload and activating the Fas/FasL death receptor pathway.     

Downregulation of tripartite motif protein 11 attenuates cardiomyocyte apoptosis after ischemia/reperfusion injury via DUSP1-JNK1/2

Currently, the prevention of ischemic diseases such as myocardial infarction associated with ischemia/reperfusion (I/R) injury remains to be a challenge. Thus, this study was designed to explore the effects of tripartite motif protein 11 (TRIM11) on cardiomyocytes I/R injury and its underlying mechanism. Cardiomyocytes AC16 were used to establish an I/R injury cell model. After TRIM11 downregulation in I/R cells, cell proliferation (0, 12, 24, and 48 h) and apoptosis at 48 h as well as the related molecular changes in oxidative stress-related pathways was detected. Further, after the treatment of TRIM11 overexpression, SP600125, or DUSP1 overexpression, cell proliferation, apoptosis, and related genes were detected again. As per our findings, it was determined that TRIM11 was highly expressed in the cardiomyocytes AC16 after I/R injury. Downregulation of TRIM11 was determined to have significantly reduced I/R-induced proliferation suppression and apoptosis. Besides, I/R-activated c-Jun N-terminal kinase (JNK) signaling and cleaved caspase 3 and Bax expression were significantly inhibited by TRIM11 downregulation. In addition, the overexpression of TRIM11 significantly promoted apoptosis in AC16 cells, and JNK1/2 inhibition and DUSP1 overexpression potently counteracted the induction of TRIM11 overexpression in AC16 cells. These suggested that the downregulation of TRIM11 attenuates apoptosis in AC16 cells after I/R injury probably through the DUSP1-JNK1/2 pathways.     

MicroRNA-221 regulates osteosarcoma cell proliferation,apoptosis, migration,and invasion by targeting CDKN1B/p27

MicroRNAs (miRNAs, miR) are of critical importance in growth and metastasis of cancer cells; however, the underlying functions of miRNAs in osteosarcoma (OS) remain largely unknown. This study was aimed to elucidate the role of miR-221 in regulating the biological behavior of OS cells. The proliferation ability was examined by cell counting kit-8 (CCK-8) and cell cycle assay. The abilities of cell migration, invasion, and apoptosis were monitored by transwell assay and flow cytometry, respectively. The effect of miR-221 on cyclin-dependent kinase inhibitor 1B (CDKN1B) expression was evaluated by luciferase assays, real-time polymerase chain reaction, and Western blot analysis. We found that miR-221 was elevated in OS cell lines compared with the normal osteoblastic cell line. Transfection of the miR-221 inhibitor into MG63 and U-2OS cell lines obviously suppressed cell proliferation, migration, and invasion, which is accompanied with cell cycle arrest in G0/G1 phase. Furthermore, luciferase reporter assays indicated that CDKN1B is directly targeted by miR-221 in OS cells. Knockdown of CDKN1B inhibited the effects of miR-221 inhibitor, along with decreased Bax and caspase-3 and increased cyclin E, cyclin D1, Bcl-2, Snail, and Twist1 expression. The results suggested that miR-221 might act as a potentially useful target for treatment of OS.     

Involvement of miR-1 in the protective effect of hydrogen sulfide against cardiomyocyte apoptosis induced by ischemia/reperfusion

The protective effect of hydrogen sulfide (H₂S) against myocardial ischemia/reperfusion (IR) injury via anti-apoptotic signaling is well established, but the underlying mechanism remains unclear. Recently, miRNAs have been identified as important mediators of myocardial injury by regulating apoptosis-related genes. It was found in our previous preliminary study that microRNA-1 (miR-1) expression underwent a significant change in IR group compared to H₂S preconditioned group, indicating that miR-1 possessed myocyte-specific properties. In the present study, we intended to see whether miR-1 participated in H₂S protection of cardiomyocytes against IR-induced apoptosis by regulating apoptosis-related genes. Cardiomyocytes of neonatal rats were subjected to hypoxia/reoxygenation (HR) injury with or without H₂S preconditioning, while the myocardium of adult SD rats was subjected to IR with or without H₂S preconditioning. It was found that HR injury increased apoptosis of cardiac myocytes, up-regulated the expression of miR-1, and down-regulated the expression of Bcl-2. H₂S preconditioning attenuated cardiomyocyte apoptosis and LDH release, as well as enhanced cell viability following HR injury. MiR-1 was up-regulated by HR and down-regulated by H₂S preconditioning. In contrast, Bcl-2 was down-regulated by HR and up-regulated by H₂S preconditioning. In addition, Bcl-2 protein was down-regulated by the miR-1 mimic in a dose-dependent manner. H₂S also attenuated IR-induced cardiomyocyte apoptosis in vivo. MiR-1 regulated H₂S protection of cardiomyocytes against IR-induced apoptosis by stimulating Bcl-2. These results implicate miR-1 as an important regulator of H₂S on the IR myocardium.     

Retracted: Downregulated microRNA-135a ameliorates rheumatoid arthritis by inactivation of the phosphatidylinositol 3-kinase/AKT signaling pathway via phosphatidylinositol 3-kinase regulatory subunit 2

Synovial fibroblasts (SFs) of rheumatoid arthritis (RA) are phenotypically aggressive, typically progressing into arthritic cartilage degradation. Throughout our study, we made explorations into the effects of microRNA-135a (miR-135a) on the SFs involved in RA by mediating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway via regulation of phosphatidylinositol 3-kinase regulatory subunit 2 (PIK3R2). The expression of PI3K was higher, the expression of PIK3R2 was lower, and AKT was phosphorylated in the RA synovial tissues, relative to the levels found in the normal synovial tissues. We predicted miR-135a to be a candidate miR targeting PIK3R2 using an online website, microRNA.org, which was verified with a dual-luciferase reporter gene assay. Subsequently, high miR-135a expression was observed in RA synovial tissues. To study the effect of the interaction between miR-135a and PIK3R2 in RA, the SFs isolated from RA samples were cultured and transfected with mimic, inhibitor, and small interfering RNA. The proliferation, invasion, and apoptosis of the SFs were detected after the transfection. The cells transfected with miR-135a inhibitor showed inhibited cell proliferation, migration, and invasion, while also displaying promoted cell apoptosis, G0/G1 cell ratio, and decreased S cell ratio, through upregulation of PIK3R2 and inactivation of the PI3K/AKT signaling pathway. These findings provided evidence that downregulation of miR-135a inhibits proliferation, migration, and invasion and promotes apoptosis of SFs in RA by upregulating the PIK3R2 coupled with inactivating the PI3K/AKT signaling pathway. The downregulation of miR-135a might be a potential target in the treatment of RA.