The Role Of LncRNA TUG1 In The Pathogenesis Of COL1A1 Synthesis In Systemic Sclerosis Dermal Fibroblasts

Systemic sclerosis is a chronic autoimmune disease that is characterized by skin and multiple organ fibrosis.The etiology and pathogenesis are still partly understood.Long noncoding RNAs are refined as non-protein-coding transcripts longer than 200 nucleotides.Taurineupregulated gene 1(TUG1)is closely related to many human diseases.Numerous studies have shown that TUG1 plays important role in cell migration and proliferation,and also takes part in many fibrotic diseases.Objective:The primary purpose of this study is to explore the pathophysiology of TUG1 in systemic sclerosis dermal fibroblasts,and also to investigate the role of TUG1 in bleomycin-induced SSc mice.Ultimately to provide novel therapeutic theories for anti-fibrotic in SSc.Methods:1.Dermal fibroblasts were isolated and cultivated in healthy donor and SSc patients,and the RNA level of TUG1 in healthy and SSc fibroblasts was detected by qPCR.Then CCK8 and wound healing experiments were utilized to detect the proliferation and migration ability in fibroblasts transfected with si-RNA TUG1(si-TUG1).RNA and protein level of COL1A1 was also detected by qPCR and western blot in si-TUG1 fibroblasts.2.The RNA and protein levels of SNAI2 were detected by qPCR and western blot in healthy and SSc dermal fibroblasts.We also saw the RNA and protein level of COL1A1 in fibroblasts transfected with si-SNAI2 or SNAI2 plasmid separately.We predicted the binding site of Snai2 on the promoter region of COL1A1 then verified by ChIP-qPCR.3.We established the SSc murine model by repeated subcutaneous injection of Bleomycin,then treated the SSc murine with si-Tug1.Fibrosis degree was evaluated by analyzing the histological features.Snai2 protein level was detected by immunohistochemistry in different groups.Results:1.TUG1 in SSc dermal fibroblasts was elevated than in healthy fibroblasts(n=12,**P<0.001).CCK8 and wound healing experiment showed that inhibited TUG1 lead to impaired cell proliferation and migration.COL1A1 was also downregulated when TUG1 was inhibited(n=5,*P<0.05,***P<0.001).2.SNAI2 was detected upregulated in SSc dermal fibroblasts and downregulated when TUG1 was inhibited(n=12,**P<0.001).Besides,we found that the expression level of COL1A1 was positively correlated with SNAI2(n=6,*P<0.05,**P<0.01).We predicted the binding sites between Snai2 and COL1A1 on JASPAR and proved it via ChIPqPCR(n=3,*P<0.05,**P<0.01).3.Repeated subcutaneous injection of bleomycin lead to skin fibrosis in mice,alongside elevated Snai2(n=3,**P<0.01).Si-Tug1 treatment relieved skin fibrosis and also decreased the expression level of Snai2(n=3,**P<0.01)Conclusion:Long non-coding RNA TUG1 is upregulated in SSc dermal fibroblasts,and TUG1 may regulate fibrosis by coordinating with transcription factor SNAI2.