Glucose induces apoptosis of cardiomyocytes via microRNA-1 and IGF-1

Glucose toxicity is an important initiator of cardiovascular disease, contributing to the development of cardiomyocyte death and diabetic complications. The present study investigated whether high glucose state could induce apoptosis of rat cardiomyocyte cell line H9C2 through microRNA regulated insulin-like growth factor (IGF-1) signaling pathway. Our data showed that H9C2 cells exposed to high glucose have increased miR-1 expression level, decreased mitochondrial membrane potential, increased cytochrome-c release, and increased apoptosis. Glucose induced mitochondrial dysfunction, cytochrome-c release and apoptosis was blocked by IGF-1. Using prediction algorithms, we identified 3′-untranslated regions of IGF-1 gene are the target of miR-1. miR-1 mimics, but not mutant miR-1, blocked the capacity of IGF-1 to prevent glucose-induced mitochondrial dysfunction, cytochrome-c release and apoptosis. In conclusion, our data demonstrate that IGF-1 inhibits glucose-induced mitochondrial dysfunction, cytochrome-c release and apoptosis and IGF-1’s effect is regulated by miR-1.     

Decreased circulating miR-375: A potential biomarker for patients with non-small-cell lung cancer

MicroRNAs (miRNAs) are directly involved in cancer initiation, progression and metastasis. Alterations of miRNAs expression in cancer tissue may be reflected in circulation. We attempted to investigate the expression and clinical significance of plasma miR-20a, miR-31 and miR-375 in patients with non-small cell lung cancer (NSCLC). The plasma levels of miR-20a, miR-31 and miR-375 in 164 NSCLC patients and 164 healthy controls (discovery cohort) were evaluated and compared among various clinicopathological characteristics. The relationship between miRNA expression and clinical outcome of NSCLC patients was examined in an independent cohort (53 cases and 53 controls). The expression level of miR-375 in tissue was also examined. Plasma miR-375 levels in NSCLC patients were significantly decreased in both patient cohorts (P < 0.05). In addition, patients with metastatic NSCLC had lower plasma miR-375 expression than those with non-metastatic NSCLC (P < 0.05). Survival analysis showed that patients with low miR-375 expression had worse overall survival rates than those with high miR-375 expression (hazard ratios (HR) = 1.537 (1.046–2.258), P = 0.029). This association was independently validated in a separate cohort of 53 NSCLC patients (HR = 2.406, 95% CI 1.170–4.945, P = 0.017). The expression level of miR-375 was also found to be significantly down-regulated in NSCLC tissues compared with paracancerous tissues (P < 0.001). These findings indicate that miR-375 has an important role in NSCLC initiation and progression, and may be an independent poor prognostic factor in NSCLC patients.     

miR-223 contributes to the AGE-promoted apoptosis via down-regulating insulin-like growth factor 1 receptor in osteoblasts

Advanced glycation end products (AGEs) have been confirmed to induce bone quality deterioration in diabetes mellitus (DM), and to associate with abnormal expression of miRNAs in DM patients or in vitro. Recently, miRNAs have been recognized to mediate the onset or progression of DM. In the present study, we investigated the regulation on miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells, with real-time quantitative PCR assay. And then we examined the inhibition of insulin-like growth factor 1 receptor (IGF-1R) expression by miR-223, via targeting of the 3′ UTR of IGF-1R with real-time quantitative PCR, western blotting and luciferase reporter assay. Then we explored the regulation of miR-223 and IGF-1R levels, via the lentivirus-mediated miR-223 inhibition and IGF-1R overexpression in the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It was demonstrated that AGE-BSA treatment with more than 100 μg/ml significantly up-regulated miR-223 level, whereas down-regulated IGF-1R level in MC3T3-E1 cells. And the up-regulated miR-223 down-regulated IGF-1R expression in both mRNA and protein levels, via targeting the 3′ UTR of IGF-1R. Moreover, though the AGE-BSA treatment promoted apoptosis in MC3T3-E1 cells, the IGF-1R overexpression or the miR-223 inhibition significantly attenuated the AGE-BSA-promoted apoptosis in MC3T3-E1 cells. In summary, our study recognized the promotion of miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells. The promoted miR-223 targeted IGF-1R and mediated the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It implies that miR-223 might be an effective therapeutic target to antagonize the AGE-induced damage to osteoblasts in DM.     

Synergistic induction of miR-126 by hypoxia and HDAC inhibitors in cardiac myocytes

HDAC inhibitors are under clinical development for the treatment of hypertrophic cardiomyopathy and heart failure although the mechanisms of protection are incompletely understood. Micro-RNA 126, an endothelium-specific miR has been assigned essential developmental roles in the heart by activating survival kinases ERK1/2 and Akt and increasing pro-angiogenic signaling. Here we provide the first evidence that hypoxia and HDAC inhibitors selectively and synergistically stimulate expression of miR-126 in cardiac myocytes. MiR-126 expression was increased 1.7-fold (p < 0.05) after 1 h of hypoxic exposure and this was further enhanced to 3.0-fold (p < 0.01) by simultaneously blocking HDAC with the pan-HDAC inhibitor Tricostatin A (TSA). TSA alone did not increase miR-126. In parallel, hypoxia and TSA synergistically increased p-ERK and p-Akt without effecting VEGF-A level. Knockdown of miR-126 with si-RNA eliminated inductions of p-ERK and p-Akt by hypoxia, whereas miR-126 overexpression mimicked hypoxia and amplified p-ERK and p-Akt in parallel with miR-126. The results suggest that miR-126 is a hypoxia-inducible target of HAT/HDAC and its activation in cardiac myocytes may contribute to cardioprotection by activating cell survival and pro-angiogenic pathways selectively during ischemia.     

Evidences showing association of interleukin-1B polymorphisms with increased risk of gastric cancer in an Indian population

Helicobacter pylori infection is strongly associated with gastric cancer. In the present study, the relationship between interleukin-1B (IL-1B) polymorphism, H. pylori infection, and prevalence of gastric cancer (GC) in patients of North India was evaluated using genomic DNA directly extracted from biopsy tissues for performing PCR-RFLP. A total of 136 GC cases and 110 healthy controls were included for studying polymorphisms in the genotypes of IL-1B−511, −31, +3954 and IL-1RN both in the presence and absence of H. pylori active infection. Results showed that the frequency of IL-1RN 2/2 was significantly higher in GC cases (21.32%) than the controls (9.09%) with an odds ratio (OR) of 4.391 (95% CI 1.093-10.131). The risk of GC was also found higher in other genotypes of IL-1B namely, −511 TT (χ² = 18.975, p < 0.001), −31CC (χ² = 21.219, p < 0.001), +3954 CT (χ² = 21.082, p < 0.001) and IL-1RN 1/2 (χ² = 30.543, p < 0.001) with active infection of H. pylori. Our findings indicate that the IL-1B and IL-1RN polymorphisms are associated with the development of GC and H. pylori infection markedly increases the risk of GC in North Indian population. Additionally, IL-1B−511 C/C and IL-RN 2/2 polymorphisms seem to be involved in the development of GC in H. pylori uninfected patients.     

Downregulation of microRNA-214 and overexpression of FGFR-1 contribute to hepatocellular carcinoma metastasis

miR-214 is one of the most significantly downregulated microRNAs (miRNAs) in hepatocellular carcinoma (HCC). Fibroblast growth factor receptor 1 (FGFR-1) is a miR-214 target gene implicated in the progression of HCC. However, the roles of miR-214 and FGFR-1 in HCC are not fully understood. Here, we analyzed the expression of miR-214 and FGFR-1 in 65 cases of HCC and paired non-neoplastic tissue specimens using real-time PCR and Western blot (WB), respectively. Our data indicated that miR-214 was downregulated and FGFR-1 was overexpressed in HCC compared to the paired non-neoplastic tissues. The low miR-214 expression was correlated with portal vein invasion (p = 0.016) and early recurrence (p = 0.045) in HCC patients. Moreover, the low miR-214 expression was correlated with high positive rate of FGFR-1 in HCC cases (p = 0.020). Our data further demonstrated that miR-214 overexpression in SK-HEP1 and HepG2 cells downregulated FGFR-1 expression and inhibited liver cancer cell invasion. The Luciferase assay results further demonstrated the targeted regulation of FGFR-1 by miR-214. In conclusion, our data indicate that the downregulation of miR-214 in HCC and the upregulation of its target gene FGFR-1 is associated with HCC progression. Therefore, miR-214 and FGFR-1 are potential prognostic markers and therapeutic targets in HCC.     

Inhibition of C6 rat glioma proliferation by [Ru2Cl(Ibp)4] depends on changes in p21, p27, Bax/Bcl2 ratio and mitochondrial membrane potential

The ruthenium compound [Ru₂Cl(Ibp)₄] (or RuIbp) has been reported to cause significantly greater inhibition of C6 glioma cell proliferation than the parent HIbp. The present study determined the effects of 0-72 h exposure to RuIbp upon C6 cell cycle distribution, mitochondrial membrane potential, reactive species generation and mRNA and protein expression of E2F1, cyclin D1, c-myc, pRb, p21, p27, p53, Ku70, Ku80, Bax, Bcl2, cyclooxygenase 1 and 2 (COX1 and COX2). The most significant changes in mRNA and protein expression were seen for the cyclin-dependent kinase inhibitors p21 and p27 which were both increased (p < 0.05). The marked decrease in mitochondrial membrane potential (p < 0.01) and modest increase in apoptosis was accompanied by a decrease in anti-apoptotic Bcl2 expression and an increase in pro-apoptotic Bax expression (p < 0.05). Interestingly, COX1 expression was increased in response to a significant loss of prostaglandin E₂ production (p < 0.001), most likely due to the intracellular action of Ibp. Future studies will investigate the efficacy of this novel ruthenium-ibuprofen complex in human glioma cell lines in vitro and both rat and human glioma cells growing under orthotopic conditions in vivo.     

Overexpression of apolipoprotein-B improves cardiac function and increases survival in mice with myocardial infarction

Background

The heart produces apolipoprotein-B containing lipoproteins (apoB) whose function is not well understood. The aim of this study was to evaluate importance of myocardial apoB for cardiac function, structure and survival in myocardial infarction (MI) and heart failure (HF).

Methods and results

MI was induced in mice (n = 137) and myocardial apoB content was measured at 30 min, 3, 6, 24, 48, 120 h and 8 weeks post-MI. Transgenic mice overexpressing apoB (n = 27) and genetically matched controls (n = 27) were used to study the effects of myocardial apoB on cardiac function, remodeling, arrhythmias and survival after MI. Echocardiography was performed at rest and stress conditions at baseline, 2, 4 and 6 week post-MI and cumulative survival rate was registered. The myocardial apoB content increased both in the injured and the remote myocardium (p < 0.05) in response to ischemic injury. ApoB mice had 2-fold higher survival rate (p < 0.05) and better systolic function (p < 0.05) post-MI.

Conclusion

Overexpression of apoB in the heart increases survival and improves cardiac function after acute MI. Myocardial apoB may be an important cardioprotective system in settings such as myocardial ischemia and HF.     

Blocking exosomal miRNA-153-3p derived from bone marrow mesenchymal stem cells ameliorates hypoxia-induced myocardial and microvascular damage by targeting the ANGPT1-mediated VEGF/PI3k/Akt/eNOS pathway

It has been widely reported that exosomes derived from mesenchymal stem cells (MSCs) have a protective effect on myocardial infarction (MI). However, the specific molecules which play a damaging role in MSCs shuttled miRNAs are much less explored. MiRNA-153-3p (miR-153-3p) is a vital miRNA which has been proved to modulate cell proliferation, apoptosis, angiogenesis, peritoneal fibrosis and aortic calcification. Here, we aim to study the effect and mechanism of miR-153-3p in MSC-derived exosomes on hypoxia-induced myocardial and microvascular damage. The exosomes of MSCs were isolated and identified, and the MSCs-exosomes with low expression of miR-153-3p (exo-miR-153-3p⁻) were constructed to interfere with the endothelial cells and cardiomyocytes in the oxygen-glucose deprivation (OGD) model. The viability, apoptosis, angiogenesis of endothelial cells and cardiomyocytes were determined. Additionally, ANGPT1/VEGF/VEGFR2/PI3K/Akt/eNOS pathway was detected by ELISA and/or western blot. The results illustrated that exo-miR-153-3p⁻ significantly reduced the apoptosis of endothelial cells and cardiomyocytes and promoted their viability. Meanwhile, exo-miR-153-3p⁻ can promote the angiogenesis of endothelial cells. Mechanistically, miR-153-3p regulates the VEGF/VEGFR2/PI3K/Akt/eNOS pathways by targeting ANGPT1. Intervention with VEGFR2 inhibitor (SU1498, 1 μM) remarkably reversed the protective effect of exo-miR-153-3p⁻ in vascular endothelial cells and cardiomyocytes treated by OGD. Collectively, MSCs-derived exosomes with low-expressed miR-153-3p notably promotes the activation of ANGPT1 and the VEGF/VEGFR2 /PI3K/Akt/eNOS pathways, thereby preventing the damages endothelial cells and cardiomyocytes against hypoxia.     

Combined effect of oxidative stress-related gene polymorphisms on atherosclerosis

It is well known that oxidative stress plays critical roles in the pathogenesis of atherosclerosis. In this study, we enrolled 1746 type 2 diabetic subjects, determined 4 common genetic variants related to oxidative stress (glutamate-cysteine ligase modifier subunit (GCLM) C-588T, myeloperoxidase G-463A, human paraoxonase 1 Gln192Arg and NAD(P)H oxidase p22phox C242T polymorphisms), and measured carotid intima-media thickness (IMT) as a surrogate marker for atherosclerosis. GCLM C-588T polymorphism was associated with average IMT (AveIMT) (r = 0.090, p = 0.0008), but the association between the other 3 polymorphisms and AveIMT did not reach the statistical significance. However, AveIMT was significantly greater as the total number of 4 concomitant “pro-oxidant alleles” in each subject was increased (r = 0.108, p < 0.0001). Furthermore, the number of “pro-oxidant alleles” was a risk factor for a high AveIMT independently of conventional risk factors (p = 0.0003). In conclusion, accumulation of oxidative stress-associated alleles was associated with carotid atherosclerosis in type 2 diabetic patients.     

Pyrroloquinoline quinone inhibits oxygen/glucose deprivation-induced apoptosis by activating the PI3K/AKT pathway in cardiomyocytes

The purposes of this study were to examine the protective effect of pyrroloquinoline quinone (PQQ) on oxygen/glucose deprivation (OGD)-induced injury to H9C2 rat cardiomyocytes and to investigate the mechanism. Using H9C2 cells cultured in vitro, we examined changes in cell viability with an MTT assay at 12, 24, and 48 h after injury induced by OGD. Various concentrations of PQQ (1, 10, and 100 μM) were added, and the effect of PQQ on cell viability after OGD was assessed using the MTT assay. Thus, the optimal concentration of PQQ for the protection of cardiomyocytes against oxygen and glucose deprivation injury was determined. We also used flow cytometry analysis to examine the effect of PQQ on H9C2 cells with OGD-induced injury. The molecular probe 2′,7′-dichlorofluorescin diacetate was used to label the H9C2 cells, and flow cytometry was used to detect the effect of PQQ on reactive oxygen species (ROS) content. After labeling the H9C2 cells using a mitochondrial green fluorescent probe (Mito-Tracker Green), we measured the change in the mitochondrial content of PQQ-treated H9C2 cells. Western blotting was used to examine the effect of PQQ on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the H9C2 cells. The results of the MTT assay showed that 48 h of OGD significantly injured the H9C2 cells (p < 0.01) and that treatment with 100 μM PQQ effectively decreased the level of OGD-induced injury (p < 0.01). The results of the flow cytometry analysis showed that PQQ significantly reduced apoptosis in H9C2 cells subjected to OGD (p < 0.05). In addition, OGD significantly increased the ROS level in H9C2 cells (p < 0.01), and PQQ significantly inhibited this increase (p < 0.05). The results of the Mito-Tracker Green staining suggested that PQQ effectively inhibited the decrease in mitochondrial content caused by OGD (p < 0.05). Western blot analysis showed that PQQ partially reversed the decrease in Akt phosphorylation that was caused by OGD (p < 0.05). PQQ treatment dose-dependently protects H9C2 cells from OGD-induced injury by reducing apoptosis, decreasing intracellular ROS levels, and rescuing the OGD-induced decrease in mitochondrial content. The protective effect of PQQ may be related to its effects on the PI3K/Akt pathway.     

Pharmacology and selectivity of various natural and synthetic bombesin related peptide agonists for human and rat bombesin receptors differs

The mammalian bombesin (Bn)-receptor family [gastrin-releasing peptide-receptor (GRPR-receptor), neuromedin B-receptor (NMB receptor)], their natural ligands, GRP/NMB, as well as the related orphan receptor, BRS-3, are widely distributed, and frequently overexpressed by tumors. There is increased interest in agonists for this receptor family to explore their roles in physiological/pathophysiological processes, and for receptor-imaging/cytotoxicity in tumors. However, there is minimal data on human pharmacology of Bn receptor agonists and most results are based on nonhuman receptor studies, particular rodent-receptors, which with other receptors frequently differ from human-receptors. To address this issue we compared hNMB-/GRP-receptor affinities and potencies/efficacies of cell activation (assessing phospholipase C activity) for 24 putative Bn-agonists (12 natural, 12 synthetic) in four different cells with these receptors, containing native receptors or receptors expressed at physiological densities, and compared the results to native rat GRP-receptor containing cells (AR42J-cells) or rat NMB receptor cells (C6-glioblastoma cells). There were close correlations (r = 0.92-99, p < 0.0001) between their affinities/potencies for the two hGRP- or hNMB-receptor cells. Twelve analogs had high affinities (≤1 nM) for hGRP receptor with 15 selective for it (greatest = GRP, NMC), eight had high affinity/potencies for hNMB receptors and four were selective for it. Only synthetic Bn analogs containing β-alanine¹¹ had high affinity for hBRS-3, but also had high affinities/potencies for all GRP-/hNMB-receptor cells. There was no correlation between affinities for human GRP receptors and rat GRP receptors (r = 0.131, p = 0.54), but hNMB receptor results correlated with rat NMB receptor (r = 0.71, p < 0.0001). These results elucidate the human and rat GRP-receptor pharmacophore for agonists differs markedly, whereas they do not for NMB receptors, therefore potential GRP-receptor agonists for human studies (such as Bn receptor-imaging/cytotoxicity) must be assessed on human Bn receptors. The current study provides affinities/potencies on a large number of potential agonists that might be useful for human studies.     

A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin-stimulated proliferation of vascular smooth muscle cells

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA₂) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA₂ are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA₂ and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA₂ by 1.5-fold (p < 0.01), which was blocked by the cPLA₂ inhibitor MAFP (10 μM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 μM; 15 min) and ERK1/2 inhibitor PD98059 (20 μM; 15 min) abolished the INS-mediated increase in cPLA₂ phosphorylation by 59% (p < 0.001), and by 75% (p < 0.001), respectively. Further, inhibition of cPLA₂ with cPLA₂ inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% (p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% (p < 0.001). The effect of INS on VSMCs proliferation was significantly (p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA₂ and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA₂ activity.     

Quercetin up-regulates paraoxonase 1 gene expression with concomitant protection against LDL oxidation

Paraoxonase 1 (PON1) protects the oxidative modification of low-density lipoprotein (LDL) and is a major anti-atherosclerotic protein component of high-density lipoprotein (HDL). Quercetin, a ubiquitous plant flavonoid, has been shown to have a number of bioactivities and may offer a variety of potential therapeutic uses. We explored the roles of quercetin in the regulation of PON1 expression, serum and liver activity and protective capacity of HDL against LDL oxidation in rats. Compared to the pair-fed control group, feeding quercetin (10 mg/L) in the liquid diet for 4 weeks increased (a) hepatic expression of PON1 by 35% (p < 0.01), (b) serum and liver PON1 activities by 29% (p < 0.05) and 57% (p < 0.01), respectively, and (c) serum homocysteine thiolactonase (HCTL) activity by 23% (p < 0.05). Correspondingly, the lag time of low-density lipoprotein (LDL) oxidation was increased by >3-fold (p < 0.001) with plasma HDL from quercetin-fed group compared to the HDL from control group. Our data suggest that quercetin has antiatherogenic effect by up regulating PON1 gene expression and its protective capacity against LDL oxidation.     

A common polymorphism in pre-microRNA-146a is associated with lung cancer risk in a Korean population

Introduction

MicroRNAs (miRs) play important roles in the development and progression of human cancers. MiR-146a down-regulates epidermal growth factor receptor and the nuclear factor-κB regulatory kinase interleukin-1 receptor-associated kinase 1 genes that play important roles in lung carcinogenesis. This study was conducted to evaluate the association between rs2910164C>G, a functional polymorphism in the pre-miR-146a, and lung cancer risk.

Material and methods

The rs2910164C>G genotypes were determined in 1094 patients with lung cancer and 1100 healthy controls who were frequency matched for age and gender.

Results

The rs2910164 CG or GG genotype was associated with a significantly decreased risk for lung cancer compared to that of the CC genotype (adjusted odds ratio = 0.80, 95% confidence interval = 0.66–0.96, P = 0.02). When subjects were stratified according to smoking exposure (never, light and heavy smokers), the effect of the rs2910164C>G genotype on lung cancer risk was significant only in never smokers (adjusted odds ratio = 0.66, 95% confidence interval = 0.45–0.96, P = 0.03, under a dominant model for the C allele) and decreased as smoking exposure level increased (P_trend < 0.001). In line with this result, the level of miR-146a expression in the tumor tissues was significantly higher in the GG genotype than in the CC or CG genotype only in never-smokers (P = 0.02).

Conclusions

These findings suggest that the rs2910164C>G in pre-miR-146a may contribute to genetic susceptibility to lung cancer, and that miR-146a might be involved in lung cancer development.