Role And Mechanism Of LncRNA-TUG1 In Hypoxic Cardiomyocytes By Affecting Mitochondrial Dysfunction And Inflammatory Response

Objective:1.To explore the function of lncRNA-TUG1 in hypoxic cardiomyocytes;2.To investigate the regulatory mechanisms of targeting lncRNA-TUG1 to improve mitochondrial dysfunction and inflammatory response in hypoxic cardiomyocytes.Methods:The si-HIF-1αand the control plasmid si-NC were transfected into the HL-1 cells,respectively.Groups:normoxia group,hypoxia group,hypoxia+si NC group and hypoxia+si HIF-1αgroup.The normoxia group was placed in normoxia（O₂=21%）and the rest were incubated in a hypoxic state（O₂<3%）for 24 h.The expression of HIF-1αin each group was measured by Western blotting;The expression of lncRNA-TUG1 in each group was determined by q PCR;A luciferase reporter was used to verify the effect of HIF-1αon the transcriptional activity of lncRNA-TUG1.Then,si-TUG1 and si-NC were constructed and transfected into HL-1 cells.HL-1 cells were divided into normoxia group,hypoxia group,hypoxia+si NC group and hypoxia+si TUG1 group.Cell proliferation in each group was analyzed by CCK-8.The medium supernatant before and after moulding was collected.ELISA was used to detect the release of myocardial necrosis markers（c Tn I,CK-MB,MYO）.The expression of mitochondrial biosynthesis-related proteins（PGC-1,Tfam,NRF1）was furtherly measured by WB;The expression of PGC-1,Tfam,NRF1 related to mitochondrial biosynthesis and inflammatory factors IL-1β,IL-6,TNF-αwas determined by quantitative PCR;The release of the inflammatory factors IL-1β,IL-6 and TNF-αwas determined by ELISA.Normally distributed measurement data were expressed as mean±standard deviation（mean±SD）.Comparisons between the two groups were performed using a t-test.One-way ANOVA was used for more than two groups.They were considered statistically significant at P<0.05.Results:Cells were transfected with si-HIF-1αand control plasmid si-NC,and after hypoxia modeling,the expression of HIF-1αwas measured by WB.Compared with the normoxia group,HIF-1αprotein expression was significantly up-regulated in the hypoxia group and hypoxia+si NC group（Figure 1,***P<0.001）,while the protein expression of HIF-1αwas decreased in the hypoxia+si HIF-1αgroup.The expression of lncRNA-TUG1was measured by RT-PCR.It was found that the expression of lncRNA-TUG1 was significantly increased in the hypoxia group and hypoxia+si NC group compared with the normoxia group（Figure 2,***P<0.001）,while the expression of lncRNA-TUG1 was decreased in the silencing HIF-1αgroup.Luciferase reporter showed that HIF-1αbinds the lncRNA-TUG1 promoter region（Figure 3,**P<0.01）.si TUG1 HL-1 cells and si NC HL-1 cells were constructed,and they were molded by hypoxia.After hypoxia modeling,the proliferation of cells was measured by using CCK-8.The results showed that cardiomyocyte proliferative capacity was significantly reduced during hypoxia compared with the normoxia group（Figure 4,***P<0.001）,while the reduction in HL-1 cells could be reversed by the downregulation of lncRNA-TUG1 expression.The changes of myocardial necrosis markers:troponin I（c Tn I）,creatine kinase isoenzyme（CK-MB）and myoglobin（MYO）were detected by ELISA.The results found that troponin I（c Tn I）,creatine kinase isoase（CK-MB）and myoglobin（MYO）in the hypoxia group and hypoxia+si NC group were all significantly increased compared with the normoxia group（Figure 5,***P<0.001）.However,they were decreased in the hypoxia+si TUG1 group.Then,the expression of mitochondrial biosynthesis-related proteins PGC-1,Tfam and NRF1 in each group was detected by WB.The results showed that the expression of PGC-1,Tfam and NRF1 were significantly reduced in the hypoxia and hypoxia+si NC group compared with the normoxia group（Figure6,***P<0.001）,while the expression levels of PGC-1,Tfam and NRF1 in the silenced lncRNA-TUG1 group were recovered;The m RNA expression levels of their mitochondrial biosynthesis-related PGC-1,Tfam and NRF1 were measured by RT-PCR,which was consistent with the results of Western blotting（Figure 7,**P<0.01,***P<0.001）.This suggested that the inhibition of lncRNA-TUG1 would improve mitochondrial dysfunction caused by hypoxia and restore the function of mitochondria.The expression of the inflammatory factors IL-1β,IL-6 and TNF-αof the cells in each group was determined by q PCR.It was found that inflammatory factors IL-1β,IL-6 and TNF-αwere significantly increased in the hypoxia group and hypoxia+si NC group compared with the normoxia group（Figure 8,**P<0.01,***P<0.001）.However,the expression of inflammatory factors IL-1β,IL-6 and TNF-αwas decreased after the inhibition of lncRNA-TUG1;The release of inflammatory factors IL-1β,IL-6 and TNF-αwas determined by ELISA,which was consistent with q PCR（Figure 9,*P<0.05,***P<0.001）.It was suggested that downregulation of lncRNA-TUG1 could improve the inflammatory response in cardiomyocytes induced by hypoxia.Conclusions:HIF-1αtranscriptionally regulates the expression of lncRNA-TUG1 in hypoxic cardiomyocytes.TUG1 is involved in the regulation of mitochondrial function and inflammatory response in hypoxic cardiomyocytes.The downregulation of lncRNA-TUG1 can improve the mitochondrial dysfunction and inflammatory response to reduce the damage of hypoxic cardiomyocytes,which is expected to provide new targets and ideas for the treatment of myocardial infarction.