title: Impact of miR-208 and its Target Gene Nemo-Like Kinase on the Protective Effect of Ginsenoside Rb1 in Hypoxia/Ischemia Injuried Cardiomyocytes.
abstract: OBJECTIVE
  Ginsenoside Rb1 (GS-Rb1) is one of the most important active pharmacological extracts of the Traditional Chinese Medicine ginseng, with extensive evidence of its cardioprotective properties. Mir-208 has been shown to act as a biomarker of acute myocardial infarction in vivo studies including man. However the impact of miR-208 on the protective effect of GS-Rb1 in hypoxia/ischemia injured cardiomyocytes remains unclear. The current study aims to investigate the target gene of miR-208 and the impact on the protective effect of GS-Rb1 in hypoxia/ischemia (H/I) injuried cardiomyocytes.
  
  
  METHODS
  Primary cultures of neonatal rat cardiomyocytes (NRCMs) was subjected to the H/I conditions with or without GS-Rb1. Cell viability was calculated by MTT assay and confirmed by flow cytometry analysis. Mir-208 was then detected by qRT-PCR. Luciferase reporter assay was carried out to detect the target gene of Mir-208. Then the NRCMs were transfected with miR-208 mimics and inhibitors to evaluate the impact on cardioprotective properties of Rb1.
  
  
  RESULTS
  The miR-208 expression level was clearly upregulated in the H/I treated NRCMs accompanied by the percentage of the apoptotic cells which could be reversed by GS-Rb1 pretreatment. The nemo-like kinase (NLK) mRNA and protein expression levels were decreased in H/I group measured by RT-PCR and western blotting. Luciferase activity assay was then carried out to identify that NLK may be a direct target of mir-208. MTT assay showed that miR-208 inhibitor slightly decreased the protective effect of Rb1 on the H/I impaired NRCMs. However, results showed no statistical difference.
  
  
  CONCLUSIONS
  These findings proved that NLK was a direct target of mir-208 and miR-208 act indirectly during Rb1 protecting H/I impaired NRCMs and further researches were needed to explore the relationship that microRNAs and other signal pathways in the protective effect of GS-Rb1 on the hypoxia/ischemia injuries in cardiomyocytes.
authors: Beijing Haidian Hospital, Haidian Section of Peking University Third Hospital, 29 Zhongguancun Dajie, Beijing, China.
authors: Xu
authors: X
authors: Yan
authors: Jianxun
authors: J
authors: Liu
authors: Hongjin
authors: H
authors: Wu
authors: Yuna
authors: Y
authors: Liu
authors: Sidao
authors: S
authors: Zheng
authors: Chengying
authors: C
authors: Zhang
authors: Cui
authors: C
authors: Yang
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002316:Cardiotonic Agents / Q000494:pharmacology*; D015687:Cell Hypoxia; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D036145:Ginsenosides / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
chemical_list: D002316:Cardiotonic Agents; D036145:Ginsenosides; C569395:MIRN208 microRNA, rat; D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; C442759:ginsenoside Rb1; D008156:Luciferases; C586674:NLK protein, rat; D017346:Protein Serine-Threonine Kinases