lncRNA NEAT1通过靶向抑制miR-29a促进低氧低糖诱导的肺纤维化进程的机制研究

目的探究lncRNA NEAT1通过负调控miR-29a促进低氧低糖诱导的肺纤维化进程的机制。方法收集健康者和缺血再灌注引起急性肺损伤患者的血清样本,通过qRT-PCR检测血清样本中NEAT1与miR-29a的表达。构建肺纤维化细胞模型和动物模型,进行离体和在体的RNA干扰,通过显微镜观察细胞的形态学改变,Hoechst染色检测细胞凋亡,HE染色和Masson染色评估肺组织病理改变,肺组织羟脯氨酸(HYP)含量判定肺组织纤维化程度,Western blot分析细胞中纤维化标志蛋白col1、col3a1、α-SMA、TGF-β的表达,qRT-PCR检测NEAT1、miR-29a的表达;再通过RIP和双荧光素酶报告基因验证NEAT1与miR-29a之间的相互作用及靶向关系。结果在缺血再灌注引起急性肺损伤患者的血清样本中,NEAT1表达上调,miR-29a表达下调。在细胞模型和动物模型中,同样发现NEAT1上调和miR-29a下调。在低氧低糖诱导的肺纤维化细胞模型中,发现敲除NEAT1可以抑制因低氧低糖造成的肺泡上皮细胞形态由鹅卵石型向长梭型变化,同时抑制肺泡上皮细胞的凋亡,降低纤维化标志蛋白col1、col3a1、α-SMA、TGF-β的表达;RIP实验揭示了NEAT1与miR-29a直接结合;双荧光素酶报告基因检测发现,NEAT1靶向调控miR-29a;抑制miR-29a可以逆转敲除NEAT1对低氧低糖诱导肺泡上皮细胞凋亡和降低纤维化标志蛋白col1、col3a1、α-SMA、TGF-β表达的抑制作用,进而促进肺纤维化进程。结论lncRNA NEAT1通过靶向抑制miR-29a促进低氧诱导的肺纤维化进程,为低氧诱导肺纤维化的机制和新药的研发提供新思路。

Study on mechanism of lncRNA NEAT1 in promoting hypoxia and hypoglycemia induced pulmonary fibrosis by targetedly regulating miR-29a

Objective To investigate the mechanism by which lncRNA NEAT1 negatively regulates miR-29 a to promote the progression of pulmonary fibrosis induced by hypoxia and low glucose.Methods Serum samples from healthy subjects and patients with acute lung injury induced by ischemia-reperfusion were collected and the expression of NEAT1 and miR-29 a in serum samples was detected by qRT-PCR.The cell model and the animal model of pulmonary fibrosis cells were built;the in vitro and in vivo RNA was interfered;the morphologic change of cells was observed through microscope;the apoptosis was detected by Hoechst staining;the pathologic change of lung tissue was evaluated by HE staining and Masson staining;the degree of lung tissue fibrosis was determined by lung tissue hydroxyproline(HYP)content;the expression of fibrosis marker proteins in cells,such as col1,col3 a1,α-SMA and TGF-β,was detected by Western blot;the expression of NEAT1 and miR-29 a was detected by qRT-PCR;the interaction and targeting relationship between NEAT1 and miR-29 a were verified by RIP and dual luciferase reporter genes.Results In serum samples from patients with acute lung injury induced by ischemia-reperfusion,NEAT1 expression was up-regulated and mir-29 a expression was down-regulated.Up-regulation of NEAT1 and down-regulation of miR-29 a were also found in cell and animal models.In the cell model of pulmonary fibrosis induced by hypoxia and hypoglycemia,it was found that NEAT1 knockout could inhibit the change of alveolar epithelial cell morphology caused by hypoxia and hypoglycemia from cobblestone type to long-spindle type,inhibit the apoptosis of alveolar epithelial cells,and reduce the expression of fibrotic marker proteins col1,col3 a1,α-SMA and TGF-β.RIP experiment revealed the direct binding of NEAT1 to miR-29 a.Dual luciferase reporter gene detection revealed that NEAT1 targetedly regulated miR-29 a.Inhibition of miR-29 a could reverse the inhibitory effect of NEAT1 knockout on hypoxia and hypoglycemia-induced apoptosis of alveolar epithelial cells and reducing the expression of fibrotic marker proteins col1,col3 a1,α-SMA and TGF-β,which then promoted the progression of pulmonary fibrosis.Conclusion lncRNA NEAT1 promotes hypoxia-induced pulmonary fibrosis by targeted inhibition of miR-29 a,providing new ideas for studying the mechanism of hypoxia-induced pulmonary fibrosis and developing new drugs.