lncRNA Tug1调控notch1通路参与增生性瘢痕形成

Regulation of Notch1 Pathway and Participation in the Formation of Hypertrophic Scar by LncRNA Tugl

目的 探讨长链非编码RNA(lncRNA)牛磺酸上调基因1(Tug1)调控notch1信号途径参与增生性瘢痕形成的作用机制。方法 取增生性瘢痕组织及正常皮肤组织,RT-qPCR法检测lncRNA-Tug1、miR-299-3p表达,Western blot法检测notch1蛋白表达;取人增生性瘢痕成纤维细胞系(HSF),探究敲低lncRNA-Tug1或激活notch信号后,HSF增殖活性及凋亡变化;敲低lncRNA-Tug1的同时激活Notch信号测HSF细胞notch1/TGF-β/Smad纤维化通路变化。双荧光素酶报告试验验证miR-299-3p与notch1靶向关系。建立小鼠增生性瘢痕模型,共同注射Tug1低表达载体(si-Lv-Tug1)及miR-299-3p抑制物(miR-299-3p inhibitor),探究干预miR-299-3p表达对lncRNA-Tug1及notch信号的调节作用,及对增生性瘢痕小鼠瘢痕增生指数及成纤维细胞密度形成的影响。结果 与正常皮肤组织及上皮细胞系相比,增生性瘢痕组织及HSF细胞系中lncRNA-Tug1及notch1表达升高,miR-299-3p表达降低(P<0.05)。敲低HSF细胞中lncRNA-Tug1,可上调miR-299-3p表达、促进凋亡、降低notch1-TGF-β/Smad纤维化活性激活(P<0.05)。notch激活剂可削弱lncRNA-Tug1敲低发挥的抑制纤维化、促进凋亡等作用(P<0.05)。miR-299-3p与notch1之间有靶向负调控作用(P<0.05)。抑制miR-299-3p表达,可加重耳瘢痕模型小鼠结缔组织纤维化增生等病理损伤,并削弱lncRNA-Tug1敲低发挥的抑制notch1/TGF-β/Smad活化、改善耳瘢痕模型小鼠耳部组织纤维增生症状(P<0.05)。结论 敲低lncRNA-Tug1,可通过上调miR-299-3p,使notch1途径失活,缓解增生性瘢痕纤维化发展。

Objective To explore the mechanism of long non-coding RNA(lncRNA) taurine up-regulated gene 1(Tug1) in the control of the Notch1 signaling pathway and the formation of hypertrophic scars.Methods The expression of lncRNA-Tug1,miR-299-3 p and the Notch1 protein in hypertrophic scar tissue and normal skin tissue was done by RT-qPCR and Western blot, respectively.The proliferation activity and apoptosis of the human hypertrophic scar fibroblast cell line(HSF) were assessed after knocking down lncRNA-Tug1 or activating the notch signal. Moreover, knocking down lncRNA-Tug1 and activating notch signal simultaneously was used to measure the changes in Notch1/TGF-β/Smad fibrosis pathway in HSF cells.The dual-luciferase reporter test verified the relationship between miR-299-3 p and notch1.The mouse hypertrophic scar model was established, and Tug1 low expression vector(si-Lv-Tug1) and miR-299-3 p inhibitor(miR-299-3 p inhibitor) were combined to explore the effect of the miR-299-3 p expression on lncRNA-Tug1 and notch signals and scar proliferation index and fibroblast density in hypertrophic scar mice.Results Compared with normal skin tissues and epithelial cell lines, the expression of lncRNA-Tug1 and notch1 in hypertrophic scar tissue and HSF cell lines increased, and the expression of miR-299-3 p decreased(P<0.05).Knockdown of lncRNA-Tug1 in HSF cells up-regulated the expression of miR-299-3 p, promoted apoptosis, and reduced the activation of notch1-TGF-β/Smad fibrosis activity(P<0.05).Notch activator weakened the effects of lncRNA-Tug1 knockdown in inhibiting fibrosis and promoting apoptosis(P<0.05).There was a specific negative regulation effect between miR-299-3 p and notch1(P<0.05).Inhibiting the expression of miR-299-3 p aggravated pathological damage such as connective tissue fibrosis and hyperplasia in ear scar model mice and weakened the effect of lncRNA-Tug1 knockdown(P<0.05).Conclusion Knockdown of lncRNA-Tug1 can up-regulate miR-299-3 p, inactivate the notch1 pathway, and alleviate the development of hypertrophic scar fibrosis.