title: [Effects of microRNA-21 on the myocardial cell apoptosis induced by ischemia and hypoxia in rat].
abstract: OBJECTIVE
  To explore the effects of microRNA-21 on apoptosis of myocardial cell of rats as induced by ischemia and hypoxia, and to analyze the underlying mechanisms.
  
  
  METHODS
  (1) Rat myocardial cell line H9C2 was cultured in a serum-free and low glucose DMEM medium using a hypoxic incubator which was filled with 1% oxygen, 5% carbon dioxide, and 94% nitrogen to simulate ischemic environment. The expression of microRNA-21 in normal myocardial cells and cells treated with low oxygen exposure for 6 and 24 h were assessed by real-time fluorescent quantitative RT-PCR. (2) Another portion of myocardial cells were divided into 4 groups according to the random number table: normal control group (NC, ordinary culture without any treatment), ischemia/hypoxia group (IH, treated with ischemia and hypoxia for 24 h), negative transfection control+ischemia/hypoxia group (NC+IH, treated with ischemia and hypoxia for 24 h after the transfection of microRNA mimics control for 24 h), microRNA-21+ischemia/hypoxia group (M+IH, treated with ischemia and hypoxia for 24 h after the transfection of microRNA-21 mimics for 24 h). The cells in the latter three groups were examined immediately after treatment, and cells in group NC were collected and examined at the same time point. Apoptosis rate of myocardial cells was determined by flow cytometer. The mRNA and protein expression levels of programmed cell death 4 (PDCD4) in myocardial cells were determined by real-time fluorescent quantitative RT-PCR and Western blotting respectively. The sample numbers in this experiment were 6 or 3. Data were processed with one-way analysis of variance and LSD- t test.
  
  
  RESULTS
  (1) The expression level of microRNA-21 in normal myocardial cells and cells treated with ischemia and hypoxia for 6 and 24 h were respectively 1.09 ± 0.17, 0.75 ± 0.08, and 0.67 ± 0.08 (F = 11.280, P = 0.009). Compared with expression level of microRNA-21 in normal myocardial cells, those of cells treated for 24 h (t = 4.461, P = 0.004) and 6 h (t = 3.642, P = 0.011) were both lower, and the former was more obvious. Therefore all the ischemia and hypoxia treatment time of cells in the following experiment was 24 h. (2) The apoptosis rate of myocardial cells in group NC was (3.5 ± 0.7)%. After being treated with ischemia and hypoxia for 24 h, the apoptosis rates of myocardial cells in groups IH, NC+IH, and M+IH were respectively (17.3 ± 3.2)%, (16.4 ± 3.0)%, and (7.6 ± 2.0)% (F = 15.176, P = 0.001). Compared with that of group NC, the apoptosis rate of myocardial cells of group IH was significantly increased (t = 5.641, P < 0.001), while it was significantly decreased in group M+IH as compared with group NC+IH (t = 3.588, P = 0.007). The mRNA expression level of PDCD4 in group NC was 1.06 ± 0.21. After being treated with ischemia and hypoxia for 24 h, the mRNA expression levels of PDCD4 in groups IH, NC+IH, and M+IH were respectively 3.01 ± 0.34, 3.05 ± 0.25, and 1.48 ± 0.24 (F = 44.952, P < 0.001). Compared with that of group NC, the mRNA expression level of PDCD4 in group IH was higher (t = 8.945, P < 0.001), while it was significantly lower in group M+IH as compared with group NC+IH (t = 7.253, P < 0.001). The protein expression level of PDCD4 in group NC was 0.44 ± 0.08. After being treated with ischemia and hypoxia for 24 h, the protein expression levels of PDCD4 in groups IH, NC+IH, and M+IH were respectively 0.96 ± 0.13, 1.05 ± 0.12, and 0.58 ± 0.12 (F = 18.804, P = 0.008). Compared with that of group NC, the protein expression level of PDCD4 in group IH was higher (t = 5.429, P = 0.006), while it was significantly reduced in group M+IH as compared with group NC+IH (t = 4.903, P = 0.008).
  
  
  CONCLUSIONS
  Ischemia and hypoxia reduce the expression of microRNA-21 in myocardial cells, while increasing the expression of microRNA-21 can alleviate the ischemia/hypoxia-induced apoptosis by lowering the expression of PDCD4.
authors.affiliation: Institute of Burns, Wuhan City Hospital NO.3 & Tongren Hospital of Wuhan University, Wuhan 430060, China.
authors.forename: Qionghui
authors.initials: Q
authors.lastname: Xie
authors.affiliation: Institute of Burns, Wuhan City Hospital NO.3 & Tongren Hospital of Wuhan University, Wuhan 430060, China.
authors.forename: Chaoli
authors.initials: C
authors.lastname: Zhao
authors.affiliation: Institute of Burns, Wuhan City Hospital NO.3 & Tongren Hospital of Wuhan University, Wuhan 430060, China.
authors.forename: Ziqing
authors.initials: Z
authors.lastname: Ye
authors.affiliation: Institute of Burns, Wuhan City Hospital NO.3 & Tongren Hospital of Wuhan University, Wuhan 430060, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Yang
authors.affiliation: Institute of Burns, Wuhan City Hospital NO.3 & Tongren Hospital of Wuhan University, Wuhan 430060, China.
authors.forename: Qiongfang
authors.initials: Q
authors.lastname: Ruan
authors.affiliation: Institute of Burns, Wuhan City Hospital NO.3 & Tongren Hospital of Wuhan University, Wuhan 430060, China. Email: wgxie@hotmail.com.
authors.forename: Weiguo
authors.initials: W
authors.lastname: Xie
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D000860:Hypoxia; D007511:Ischemia; D035683:MicroRNAs / Q000235:genetics*; D009206:Myocardium; D032383:Myocytes, Cardiac; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D051017:Apoptosis Regulatory Proteins; D035683:MicroRNAs; C467042:Pdcd4 protein, rat; D012333:RNA, Messenger