Hong-Ying Diao

Down-regulation of microRNA-320 suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1

Insulin-like growth factor-1 (IGF-1) is an important regulator of cardiomyocyte homeostasis and cardiac structure, and the prosurvival and antiapoptotic effects of IGF-1 have been investigated. However, the effect of microRNA-320 (miR-320) in ischemia and reperfusion (I/R) by targeting IGF-1 is rarely discussed. We investigated the role of miR-320 in I/R injury. A total of 192 healthy female Wistar rats were divided into eight groups (n = 24). Rat heart I/R model was established. Hemodynamics, infarct size weight (ISW), heart function, and rat cardiomyocyte apoptosis were measured. Hypoxia-reoxygenation (H/R) in rat cardiomyocyte was used to simulate the I/R process. The mRNA levels of miR-320 and IGF-1, and proteins levels of IGF-1, IGF-1R, p-IGF-1R, p-ASK1, p-JNK, p-p38, Bcl-2, Bax and Caspase-3 were measured. In vivo inhibition of miR-320 expression significantly increased IGF-1 and IGF-1R mRNA levels, elevated the absolute values of SBP, DBP, MAP, ± dp/dtmax, LVEF and LVFS, decreased ISW, LVESD and LVEDd and the number of TUNEL positive cells, lowered the levels of p-ASK1, p-JNK, p-p38, Bax and Caspase-3 and increased expression of Bcl-2 compared to the I/R + NC group. Compared to H/R + NC group in vitro, miR-320 inhibition increased IGF-1 mRNA levels, inhibited cardiomyocyte apoptosis, down-regulated p-ASK, p-JNK, p-p38, Bax and Caspase-3 levels, and up-regulated Bcl-2 level. MiR-320 inhibition target elevated IGF-1 mRNA and protein levels, suppress early cardiomyocyte apoptosis of I/R, and inhibited ASK1-JNK/p38 pathway, which provides a new target for clinical study of I/R injury.

The Protective Effect of MicroRNA-320 on Left Ventricular Remodeling after Myocardial Ischemia-Reperfusion Injury in the Rat Model

The primary objective of this study investigated the role of microRNA-320 (miR-320) on left ventricular remodeling in the rat model of myocardial ischemia-reperfusion (I/R) injury, and we intended to explore the myocardial mechanism of miR-320-mediated myocardium protection. We collected 120 male Wistar rats (240–280 g) in this study and then randomly divided them into three groups: (1) sham surgery group (sham group: n = 40); (2) ischemia-reperfusion model group (I/R group: n = 40); and (3) I/R model with antagomir-320 group (I/R + antagomir-320 group: n = 40). Value changes of heart function in transesophageal echocardiography were recorded at various time points (day 1, day 3, day 7, day 15 and day 30) after surgery in each group. Myocardial sections were stained with hematoxylin and eosin (H&E) and examined with optical microscope. The degree of myocardial fibrosis was assessed by Sirius Red staining. Terminal dUTP nick end-labeling (TUNEL) and qRT-PCR methods were used to measure the apoptosis rate and to determine the miR-320 expression levels in myocardial tissues. Transesophageal echocardiography showed that the values of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular systolic pressure (LVSP) and ±dp/dtmax in the I/R group were obviously lower than those in the sham group, while the left ventricular end-diastolic pressure (LVEDP) value was higher than that in the sham group. The values of LVEF, LVFS, LVSP and ±dp/dtmax showed a gradual decrease in the I/R group, while the LVEDP value showed an up tendency along with the extension of reperfusion time. The H&E staining revealed that rat myocardial tissue in the I/R group presented extensive myocardial damage; for the I/R + antagomir-320 group, however, the degree of damage in myocardial cells was obviously better than that of the I/R group. The Sirius Red staining results showed that the degree of myocardial fibrosis in the I/R group was more severe along with the extension of the time of reperfusion. For the I/R + antagomir-320 group, the degree of myocardial fibrosis was less severe than that in the I/R group. Tissues samples in both the sham and I/R + antagomir-320 groups showed a lower apoptosis rate compared to I/R group. The qRT-PCR results indicated that miR-320 expression in the I/R group was significantly higher than that in both the sham and I/R + antagomir-320 groups. The expression level of miR-320 is significantly up-regulated in the rat model of myocardial I/R injury, and it may be implicated in the prevention of myocardial I/R injury-triggered left ventricular remodeling.

Anti‐Apoptotic Effect of MicroRNA‐30b in Early Phase of Rat Myocardial Ischemia‐Reperfusion Injury Model

This study aimed to investigate the effect of microRNA‐30b (miR‐30b) in rat myocardial ischemic‐reperfusion (I/R) injury model. We randomly divided Sprague–Dawley (SD) rats (n = 80) into five groups: 1) control group; 2) miR‐30b group; 3) sham‐operated group; 4) I/R group, and 5) I/R+miR‐30b group. Real‐time quantitative polymerase chain reaction, immunohistochemical staining and Western blot analysis were conducted. TUNEL assay was employed for testing cardiomyocyte apoptosis. Our results showed that miR‐30b levels were down‐regulated in I/R group and I/R + miR‐30b group compared with sham‐operated group (both P < 0.05). However, miR‐30b level in I/R + miR‐30b group was higher than I/R group (P < 0.05). Markedly, the apoptotic rate in I/R group showed highest in I/R group (P < 0.05). Additionally, the results illustrated that protein levels of Bcl‐2, Bax, and caspase‐3 were at higher levels in ischemic regions in I/R group, comparing to sham‐operated group (all P < 0.05), while Bcl‐2/Bax was reduced (P < 0.05). Bcl‐2 level and Bcl‐2/Bax were obviously increased in I/R + miR‐30b group by comparison with I/R group, and expression levels of Bax and caspase‐3 were down‐regulated (all P < 0.05). We also found that in I/R + miR‐30b group, KRAS level was apparently lower and p‐AKT level was higher by comparing with I/R group (both P < 0.05). Our study indicated that miR‐30b overexpression had anti‐apoptotic effect on early phase of rat myocardial ischemia injury model through targeting KRAS and activating the Ras/Akt pathway. J. Cell. Biochem. 116: 2610–2619, 2015.

MicroRNA-135a Regulates Apoptosis Induced by Hydrogen Peroxide in Rat Cardiomyoblast Cells.

Oxidative stress and apoptosis are the most important pathologic features of ischemic heart disease. Recent research has indicated that microRNAs (miRs) play an essential role in apoptosis. However, whether miRs might regulate B cell lymphoma-2 (Bcl-2) protein in apoptosis during ischemic heart disease is still unclear. The aim of this study, therefore, was to confirm the regulation of microRNA-135a (miR-135a) in oxidative stress injuries induced by hydrogen peroxide (H2O2) in rat cardiomyoblast cells H9c2. To this end, we analyzed the effects of H2O2 treatment on miR-135a expression in rat cardiomyocytes. Furthermore, we upregulated and inhibited miR-135a using mimics and inhibitors, respectively, and examined the effects on cell viability and apoptosis-related proteins. We observed that miR-135a was markedly up-regulated under H2O2 treatment in rat cardiomyoblast cells. Overexpression of miR-135a blocked the Bcl-2 protein and enhanced the apoptosis induced by H2O2, and miR-135a inhibition restored Bcl-2 protein expression. Interestingly, miR-135a inhibition did not attenuate H2O2-induced apoptosis with Bcl-2 knockdown. The results of the present study indicate that miR-135a regulates H2O2-induced apoptosis in H9c2 cells via targeting Bcl-2, and that miR-135a may be a novel therapeutic target for ischemic heart disease.

Inhibition of long noncoding RNA IGF2AS promotes angiogenesis in type 2 diabetes

BACKGROUND In this study, the angiogenic effect of long noncoding RNA (lncRNA), insulin growth factor 2 antisense (IGF2AS) in type 2 diabetes was evaluated. METHODS Between Wistar rat and Goto-Kakizaki (GK) rat, a genetic model of type 2 diabetes, mRNA expressions of IGF2AS and IGF2 in myocardial microvascular endothelial (mMVE) cells were compared by qRT-PCR. In GK mMVE cells, IGF2AS was inhibited by siRNA. Its effects on cell proliferation and invasion were evaluated by MTT and wound-healing assays. Also, changes of IGF2, VEGF and IGF1 in siRNA-transfected GK mMVE cells were evaluated by qRT-PCR and western blot. In IGF2AS-inhibited GK mMVE cells, IGF2 was further downregulated to evaluate its role in IGF2AS-associated angiogenic regulation, using MTT, wound-healing qRT-PCR and western blot assays, respectively. RESULTS IGF2AS was upregulated, whereas IGF2 was downregulated, in diabetic GK mMVE cells. IGF2AS inhibition augmented proliferation and invasion in GK mMVE cells. It also upregulated IGF2 and VEGF (not IGF1) at both molecular and protein levels. Conversely, IGF2 downregulation upregulated IGF2AS and reversely inhibited angiogenic effect of IGF2AS inhibition in GK mMVE cells. It also downregulated VEGF but had no effect on IGF1. CONCLUSION IGF2AS inhibition has angiogenic effect in diabetic GK mMVE cells. The functions of IGF2AS in type 2 diabetes are very likely through the inverse regulation of IGF2, but independent of IGF1.

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

BackgroundOxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2).ResultsUsing geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H2O2. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.ConclusionTaken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

MicroRNA-210 alleviates oxidative stress-associated cardiomyocyte apoptosis by regulating BNIP3

Oxidative stress-induced myocardial apoptosis and necrosis are involved in ischemia/reperfusion (I/R) injury. This study was performed to investigate microRNA (miR)-210’s role in oxidative stress-related myocardial damage. The expression of miR-210 was upregulated in myocardial tissues of I/R rats, while that of Bcl-2 adenovirus E1B 19kDa-interacting protein 3 (BNIP3) was downregulated. To simulate in vivo oxidative stress, H9c2 cells were treated with H2O2 for 48 h. MiR-210 level was increased upon H2O2 stimulation, peaked at 8 h, and then decreased. An opposite expression pattern of BNIP3 was observed. BNIP3 was demonstrated as a direct target of miR-210 via luciferase reporter assay. H2O2-induced cell apoptosis was attenuated by miR-210 mimics, whereas aggravated by miR-210 inhibitor. MiR-210 knockdown-induced cell apoptosis in presence of H2O2 was attenuated by BNIP3 siRNA. Our work demonstrates that miR-210 plays a protective role in H2O2-induced cardiomyocyte apoptosis at least by regulating the pro-apoptotic BNIP3. MiR-210 targeted 3’-UTR and CDS of BNIP3 mRNA in H9c2 cells (A). The miR-210 knockdown-induced apoptosis was attenuated by BNIP3 siRNA (B).

Diagnostic Value of Serum YKL-40 Level for Coronary Artery Disease: A Meta-Analysis.

This meta‐analysis aimed to identify the value of serum YKL‐40 level for the diagnosis of coronary artery disease (CAD).