Preliminary Study On Differential Expression Of MicroRNA In Opening MitoKATP To Anti-myocardial Ischemia Reperfusion Injury

Objective:The model of rat ischemia-reperfusion was made by Langendorff isolated perfusion device.Analysing the expression pattern of miRNA and gene in myocytes during normol state,ischemia state,mitoKATP-opening state,mitoKATP-closing state,screening and authenticating the characteristic miRNA in the four states mentioned above.Using bioinformatics to analyse the regulating network and the key node of the characteristic miRNA and obtaining the regulating network on anti-myocardial ischemia-reperfusion injury and micro RNA mediated by mitoKATP,to provide new theoretical basis and strategy on clinical myocardial ischemia-reperfusin injury preventions and treatments.Methods:Standard healthy SD male ratsweighed between 250-300 gram were intraperitoneal anesthetized by injecting pentobarbital sodium,then fixed stably on a slab and took out the heart and built the Langendoff isolated heart reperfusion model.48 rats were randomly divided into four groups:normal group（N group）,ischemia-reperfusion group（I/R group）,DZ group,5-HD group,each group included 12 rats.N group:perfused K-H fluid continuously for 120min;I/R group: perfused K-H fluid for 20 min,and reperfused for 70 min after 30 min paused-perfusion;DZ group: perfused K-H fluid for20 min,then perfused DZ for 5min immediately after 30 min paused-perfusion;5-HD group:perfused K-H fluid for 20 min,then perfused 5-HD for 5min after 25 min paused-perfusion,and then perfused DZ for 5min.Every group except group N balanced for 20 min and adjusted the perfusion flow to mean perfusion pressure between 60 to 70 mm Hg,and then continued to perfused K-H fluid for 70 min as before after heart ischemia for 30 min at 32 ℃ with stopping prefuse K-H fluid.The detection indexes are as follows:1)Recorded heart hemodynamics indexes at the end of balance and reperfusion,respectively;2)Detected the infarct size at the end of reperfusion by using TTC method;3)Extracted the left ventricular heart tissue at the end of reperfusion and stored in liquid nitrogen rapidly,every four hearts consisted a sample.Using TRIzol method to extracted the total RNA.Built a sequencing library after the 12 samples receiving Fragmentation process.Using Illumina Sequencing platform to sequence.4)Selected the differential expression miRNA and verified the reliability of sequencing results by using q RT-PCR method;5)Analysed the biological regulation pathway and network of the difference genes by using Gene Ontology,KEGG pathway databases.Results:1)Indexes of LVDP、LVEDP、CF and +dp/dtmax except HR had significant difference among groups comparison;2)Compared with N group,I/R group had larger infarct size（P<0.05）;the infarct size of group DZ decreased obviously（P<0.05）;the infarct size of group 5-HD was larger than group DZ（P<0.05）;3)The Sequencing results:compared with group N,I/R group had 192 up-regulation and 77 down-regulation miRNA;compared with group I/R,DZ group had 118 up-regulation and 48down-regulation miRNA;compared with group DZ,5-HD group had 37 up-regulation and123 down-regulation miRNA;4)Selected 17 miRNA had highly expression and significant difference among groups to verify the reliability by using q RT-PCR method,we found 8miRNA also had significant difference,they were rno-mi R-328a-3p,rno-mi R-676,rno-mi R-128-3p,rno-mi R-30c-5p,rno-mi R-30d-5p,rno-mi R-148a-3p,rno-mi R-novel-chr5₅9812,rno-mi R-novel-chr1-36162;5)Analysed the 8 miRNA mentioned above and the possible regulatory pathways and networks of m RNA.Conclusion:The possible mechanism of DZ to alleviate myocardial ischemia-reperfusion injury is opening mitoKATP channel.