The Role Of MiR-206/Cx43 Axis In Melatonin Against Oxidative Stress-induced Cardiomyocytes Injury And Its Underlying Mechanism

Objective:Melatonin,secreted by the pineal gland,is an indole hormone.It was reported that melatonin conferred protection against free radical-mediated cardiovascular diseases,and the mechanisms has not been systematically illustrated.Our study aims to illustrate the mechanism of melatonin against oxidative stress injury in cardiomyocytes,and to provide novel ideas about the molecular mechanisms associated with cardiovascular diseases.Methods:1.To explore the effect of melatonin on H₂O₂-induced oxidative stress injury in H9c2 cells.H9c2 cells were pretreated with 100μM melatonin for 24 h and then treated with 100μM H₂O₂for 30 min to induce oxidative stress injury.The cell viability of cardiomyocytes was detected by CCK-8 kit,the ROS level was detected by DCFH-DA fluorescent probe,the mitochondrial membrane potential was detected by JC-1 fluorescent probe,the protein expressions of Bax and Bcl-2 were detected by Western blot.2.To explore the effect of melatonin on the expression of miR-206 and Cx43in H9c2 cells induced by H₂O₂.The protein expression of Cx43 was detected by Western blot,and the expression of miR-206 was detected by qRT-PCR.3.To explore the effect of overexpression of miR-206 on melatonin and Cx43protein expression.H9c2 cells with miR-206 overexpression were constructed by transfecting 30 nM miR-206 mimic for 24 h.The expression of miR-206 gene and Cx43 protein,cell activity,the ROS level,mitochondrial membrane potential and the expression of apoptosis proteins were detected.4.To explore the effect of knockdown of Cx43 on melatonin.H9c2 cells with Cx43 knockdown were constructed by transfecting 50 nM Cx43 siRNA for 24 h.The expression of Cx43 protein,cell activity,the ROS level,mitochondrial membrane potential and the expression of apoptosis proteins were detected.5.To explore the targeting relationship of miR-206 to Cx43.Double luciferase reporter gene assay verified the targeting relationship between miR-206 and Cx43.The expression of miR-206 gene and Cx43 protein were detected when 30 nM/100nM miR-206 mimic/inhibitor was transfected in H9c2 cells respectively.6.To explore the effect of melatonin receptor on the regulation of miR-206/Cx43 axis by melatonin in alleviating oxidative stress injury in H9c2 cells.10μM luzindole（Melatonin receptor inhibitor）was added at the same time as melatonin.The expression of miR-206 gene and Cx43 protein,cell activity,ROS level,mitochondrial membrane potential and the expression of apoptosis proteins were detected.Results:1.Melatonin protected cardiomyocyte against oxidative stress injury induced by H₂O₂.After melatonin pretreatment,cell viability was significantly increased（p<0.01）,ROS level（p<0.001）and mitochondrial membrane potential（p<0.001）were improved,the expression of Bcl-2 was significantly increased（p<0.01）,but the expression of Bax was significantly decreased（p<0.05）.2.Melatonin reversed the expression of miR-206 and Cx43 in H₂O₂-induced cardiomyocyte oxidative stress injury.After melatonin pretreatment,the expression of miR-206 was significantly decreased（p<0.01）,and the protein expression of Cx43 was significantly increased（p<0.001）.3.Melatonin protected cardiomyocyte against oxidative stress injury induced by H₂O₂through inhibition of miR-206.Compared with mimic NC,after transfection with miR-206 mimic,the expression of miR-206 was significantly increased（p<0.001）,the expression of Cx43 was decreased（p<0.01）;the cell viability（p<0.001）,mitochondrial membrane potential（p<0.01）and the expression of Bcl-2（p<0.01）were significantly decreased,the expression of Bax（p<0.01）and ROS（p<0.001）level were significantly increased.4.Melatonin protected cardiomyocyte against oxidative stress injury induced by H₂O₂via upregulating Cx43.Compared with si-NC,after transfection with Cx43siRNA,the expression of Cx43 protein was significantly decreased（p<0.001）;cell viability（p<0.001）,mitochondrial membrane potential（p<0.01）and the expression of Bcl-2（p<0.001）were significantly decreased,the expression of Bax（p<0.001）and ROS（p<0.001）level were significantly increased.5.Cx43 was a target of miR-206.MiR-206 mimic significantly decreased the luciferase activity of Cx43 wild-type plasmid（p<0.01）.After transfection of miR-206 inhibitor,the expression of Cx43 was significantly increased（p<0.001）,while the expression of Cx43 was significantly decreased by transfection of miR-206 mimic（p<0.001）.6.Melatonin against cardiomyocyte oxidative stress injury induced by H₂O₂was dependent onMelatonin receptor.After co-treatment with luzindole and melatonin,the expression of miR-206 was significantly increased（p<0.01）,the expression of Cx43 protein was significantly decreased（p<0.01）;the cell viability（p<0.01）,mitochondrial membrane potential（p<0.01）and the expression of Bcl-2（p<0.01）were significantly decreased,the expression of Bax（p<0.05）and ROS level（p<0.001）were significantly increased.Conclusion:Melatonin can protect cardiomyocyte against oxidative stress injury via the miR-206/Cx43 axis,and melatonin receptors mediate this protective effect.