Abstract
ABSTRACT This study aims to investigate the role of targeting lncRNA myocardial infarction-associated transcript (MIAT) in protection against hypoxia/reoxygenation (H/R) injury in H9c2 cells in vitro and myocardial ischemia/reperfusion (I/R) injury in vivo by regulating expression of NF-kB and p53 upregulated modulator of apoptosis (PUMA). H9C2 cells were infected with lentivirus expressing the short-hairpin RNA direct against human MIAT gene (Lv-MIAT shRNA) or lentivirus expressing scrambled control (Lv-NC shRNA) or PUMA siRNA or p65 siRNA or their control siRNA respectively. Then the H9c2 cells were infected with Lv-shRNA to 2 hours of hypoxia (H) and 24 hour of reoxygenation (R). 100 ul of Lv-MIAT shRNA (1 × 108 PFU) or Lv-NC shRNA was transfected into mouse hearts, then the hearts were subjected to I/R (1h/72 h). We discovered targeting MIAT remarkably enhanced H9c2 cell viability, decreased H/R-induced cell apoptosis and LDH leakage and significantly decreased I/R-induced myocardial infarct size, reduced myocardial apoptosis and enhanced the heart function. Targeting MIAT downregulated p65 nuclear translocation, NF-κB activity and anti-apoptotic protein cleaved-caspase-3, Bax, and upregulated anti-apoptotic protein Bcl-2 induced by H/R or I/R. Our study suggests that targeting MIAT may protect against H9c2 cardiomyoblasts H/R injury or myocardial I/R injury via inhibition of cell apoptosis, mediated by NF-κB and PUMA signal pathway.
Highlights
Ischemia induced by the interruption of heart blood flow evokes significant cardiac myocytes damages
Lv-myocardial infarction-associated transcript (MIAT) shRNA transfection significantly decreased MIAT expression in the H9C2 cells subjected to h followed by reoxygenation (H/R) compared to the Lv-NC shRNA-transfected H9C2 cells subjected to H/R
lactate dehydrogenase (LDH) activity increased 4.6-fold in the H9C2 cells subjected to H/R, but Lv-MIAT shRNA transfection significantly attenuated the LDH activity in the H9C2 cells subjected to H/R (Figure 1(b))
Summary
Ischemia induced by the interruption of heart blood flow evokes significant cardiac myocytes damages. A more complete understanding of cardiomyocyte apoptosis holds great therapeutic potential to the medical community due to the phenomena’s prominent role in a variety of cardiac diseases, including acute myocardial infarction (AMI) rat, heart failure, and cardiac allograft [3]. MIAT, termed as Gomafu in human or Rncr in mouse [6,7,8], is a promising functional factor among all disease-associated lncRNAs, and exhibits deregulation in multiple diseases, including upregulation in ischemic stroke, myocardial infarction, neuroendocrine prostate cancer, non-small-cell lung cancer, diabetic cardiomyopathy, cataract, chronic chagas disease cardiomyopathy, chronic lymphocytic leukemia and down-regulation in schizophrenia, diabetic nephropathy, bone disease [9]. It has recently found that MIAT is upregulated in hearts of a mouse model of AMI, and knocking it down improves cardiac function by inhibition of activation of NF-κB signaling pathway [10].
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