Screening Target Genes Of MiR-210 In Rats' Liver Under Cold Stress And Its Effects On Proliferation And Apoptosis

The aim of this study was to investigate the target genes of miR-210 under cold stress.In this study,rat liver and liver cell line were used to be subject investigated,the expression of mi R-210 was analyzed by qRT-PCR in rats' liver,then using bioinformatics analysis to analyze the target genes of miR-210 by TargetScan,miRanda,PicTar and mirDB four kinds of prediction software.Screening the best transfection conditions of up-regulation and down-regulation of mi R-210 in rat liver cell line,then 8 target genes of miR-210(ISCU,PSMB6,RAD52,GPD1,GPD2,ATP1b1,E2F3 and Rap1b)were screened by qRT-PCR and Western Blot.The target genes were screened according to the cell experiments,and then verified these target genes in rats' liver.Finally,apoptosis and proliferation experiments were carried out on rat hepatocytes.The results showed that the expression of miR-210 in rat liver was significantly(P < 0.01)up-regulated.In the subsequent down-regulation and up-regulation experiment,we found that the optimal up-regulation condition was cultured for 24 h and transfected into mimics 100 pmoL.the optimal down-regulation condition was cultured for 24 h and transfected into inhibitor 150 pmol.QRT-PCR results in cell showed that GPD2,ISCU,PSMB6,RA52 and E2F3 were significant(P< 0.05)down-regulated when miR-210 up-regulated,and these target genes were up-regulated when miR-210 down-regulated,and the expression of target protein ISCU and PSMB6 have the same trend.The results showed that the increase of miR-210 could inhibit cell proliferation and induce cell death,the decrease of mi R-210 did not affect cell death but promoted cell proliferation.The following tests in rats' liver of the 5 target genes GPD2,ISCU,PSMB6,RA52 and E2F3 were verified,we found that ISCU,PSMB6,RA52 and E2F3 were significantly down-regulation(P < 0.05),GPD2 did not change significantly,The target proteins ISCU and PSMB6 in the livers of cold-stressed rats were significantly different(P < 0.05)or extremly significantly different(P < 0.01).Conclusion: the increase of miR-210 may cause some damage to rat liver under cold stress,especially the effect of proliferation and apoptosis of rat liver cells;reduce the immunity of rats;and inhibit the respiratory chain electron transfer in rats' liver;but also reduce the oxidative damage of rat liver resulting in excessive mobilization.