| BackgroundDiabetes is a disease of the endocrine system.The diagnosis of abnormal blood glucose level is one of the most common diseases in the world and the fastest growing number of patients.Diabetic retinopathy(DR)is one of the most common microvascular complications of diabetes,and current therapies especially drugs are limited in overcoming DR.Therefore,it is urgent to study new therapeutic strategies for DR and seek new therapeutic targets for DR.The presence of inflammatory cells was found in the early stages of DR.With the development of DR,the development of DR is accelerated by excessive persistent inflammatory mediators induced by a persistent hyperglycemic and hypoxic microenvironment.H19 is a conservative lncRNA that is involved in neuroinflammation and brain injury.The inflammatory mediating mechanism of H19 in atherosclerosis and neuroinflammation is clear,but the effect of H19 on inflammation during the progression of DR is unclear.miR-19 b is essential for normal development and disease pathology.miR-19 b may be a disease biomarker in asymptomatic diabetes patients.However,the regulatory function and mechanism of miR-19 b in DR has not been reported.SIRT1(a family of histone deacetylases that catalyze the deacetylation of histone and non-histone lysine residues)is a homologue of SIR2,which is related to metabolic syndrome,tumor and neurodegenerative diseases.It is also considered as a potential target for the treatment of human diseases such as DR.SIRTIt has been reported that a variety of miRnas are involved in regulating SIRT1.Therefore,to explore the role and mechanism of miRNA and SIRT1 in the occurrence and development of DR is conducive to the development of prevention and treatment strategies for DR.ObjectiveThis study intends to explore the regulatory mechanism of H19,miR-19 b and SIRT1 on inflammatory response under high glucose(HG)conditions by constructing a human retinal epithelial cell ARPE-19 model treated with HG,whether LncRNA H19 can inhibit inflammatory factors under HG conditions through H19/ Mir-19b/SIRT1 signaling pathway.This work provides new insights into the role and mechanism of H19,miR-19 b and SIRT1 in the formation of DR,and provides a new idea and a good theoretical basis for clinical treatment of DR and its related diseases.Methods1.After HG treatment,mRNA and protein expression levels of TNF-α,IL-6 and IL-1β in ARPE-19 cells were detected by qPCR and ELISA.The expression levels of H19,miR-19 b and SIRT1 were detected by qPCR and WB.2.The transfection efficiency of miR-19 b mimics and miR-19 b inhibitor was detected by qPCR.The mRNA and protein expression levels of TNF-α,IL-6 and IL-1β were detected by qPCR and ELISA after miR-19 b intervention.3.Bioinformatics predicted the binding of miR-19 b and SIRT1,and the interaction was verified by dual luciferase reporter genes.qPCR and WB were used to detect the changes of SIRT1 expression after transfection with miR-19 b inhibited and overexpressed mimics.The mRNA and protein expression levels of TNF-α,IL-6 and IL-1β were detected by qPCR and ELISA after the inhibition of miR-19 b expression and silencing of SIRT1.4.Bioinformatics predicted the binding of miR-19 b and H19,and the targeting relationship was verified by the double luciferase reporter gene.Changes in miR-19 b expression were detected by qPCR after overexpression of H19.The mRNA and protein expression levels of TNF-α,IL-6 and IL-1β in H19 and miR-19 b were detected by QRT-PCR and ELISA.5.Overexpression of H19 and inhibition of SIRT1 expression were detected by qPCR and WB.The mRNA and protein expression levels of TNF-α,IL-6 and IL-1βin H19 overexpression and SIRT1 inhibition were detected by q RT-PCR and ELISA.6.The DR animal model was constructed and H19 overexpressed,and the changes in the expression levels of inflammatory factors,miR-19 b and SIRT1 in mice were detected.7.The expression of H19,miR-19 b and SIRT1 in serum of DR patients and normal volunteers was detected by qPCR;Spearman correlation analysis of the correlation between H19 and miR-19 b,miR-19 b and SIRT1.Results1.qPCR and ELISA showed that the mRNA and protein expression levels of TNF-α,IL-6 and IL-1β in ARPE-19 cells were significantly increased in HG treatment group.The mRNA and protein expression levels of H19 and SIRT1 in cells in HG treatment group were significantly decreased,while the mRNA and protein expression levels of miR-19 b in cells were significantly increased.2.After transfection with miR-19 b mimics,the expression of miR-19 b was significantly increased;After transfection with miR-19 b inhibitor,miR-19 b expression was significantly inhibited.After transfection with miR-19 b inhibitor,the mRNA and protein expressions of TNF-α,IL-6 and IL-1β were significantly increased,while after transfection with miR-19 b inhibitor,the mRNA and protein expressions of TNF-α,IL-6 and IL-1β were significantly decreased.3.miR-19 b and SIRT1 bind to each other.After transfection with miR-19 b mimics,the luciferase activity in the wild-type SIRT1 group was significantly decreased,while it had no effect on the luciferase activity in the mutant group.After transfection with miR-19 b mimics,the mRNA and protein expressions of SIRT1 were significantly decreased,while after transfection with miR-19 b inhibitor,the mRNA and protein expressions of SIRT1 were increased.The mRNA and protein expression levels of TNF-α,IL-6 and IL-1β were significantly decreased when only miR-19 b expression was inhibited,while the mRNA and protein levels of inflammatory factors were completely reversed after simultaneous transfection with sh-SIRT1.4.miR-19 b is a potential downstream miRNA target of H19.After transfection with miR-19 b mimics,the luciferase activity in the wild-type H19 group was significantly decreased,but it had no effect on the luciferase activity in the mutant group.After overexpression of H19,the mRNA expression level of H19 was significantly increased,while the mRNA expression level of miR-19 b was significantly decreased.The mRNA and protein expression levels of TNF-α,IL-6 and IL-1β were significantly decreased when only H19 was overexpressed,while the mRNA and protein expression levels of inflammatory factors were completely reversed when miR-19 b was overexpressed.5.After overexpression of H19,the mRNA and protein expressions of SIRT1 in cells were significantly increased,while the increased SIRT1 mRNA and protein expressions were significantly reversed after simultaneous inhibition of SIRT1 expression.The mRNA and protein expression levels of TNF-α,IL-6 and IL-1β were significantly decreased when only H19 was overexpressed,while the mRNA and protein expression levels of inflammatory factors were completely reversed after simultaneous transfection of sh-SIRT1.6.In the DR model,the expression levels of inflammatory factors and miR-19 b were significantly decreased in the H19-overexpressing mice group,while the expression level of SIRT1 was significantly increased.7.Compared with normal volunteers,the mRNA expression levels of H19 and SIRT1 in serum of DR patients were significantly decreased,while the mRNA expression level of miR-19 b was significantly increased;The results of Spearman correlation analysis showed that serum H19 and miR-19 b and miR-19 b and SIRT1 were negatively correlated in serum of DR patients.ConclusionsLncRNA H19,SIRT1 and miR-19 b were differentially expressed in ARPE-19 cells under HG condition;miR-19 b targets SIRT1 and aggravates the inflammatory response of ARPE-19 cells;H19 targets miR-19 b,up-regulates the expression of SIRT1 through the H19/ Mir-19b/SIRT1 signaling axis,thus alleviating the inflammatory response of ARPE-19 induced by high glucose.In DR animal models,H19 can regulate the expression of miR-19 b and SIRT1 and affect the inflammatory response of DR.In DR patients,H19 was negatively correlated with miR-19 b,miR-19 b was negatively correlated with SIRT1. |
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