【作者】 赵洁；

【导师】 丁毅鹏；

【作者基本信息】 南华大学 ， 临床医学（内科学）（专业学位）， 2020， 硕士

【摘要】 目的:本课题组前期通过慢性阻塞性肺疾病(Chronic obstructive pulmonary disease,COPD)的miRNA转录组分析发现,miRNA-186-5p在COPD中表达存在差异,可能在COPD的发生发展中起重要作用,但其具体作用机制不明。进一步的生物信息学分析发现,miR-186-5p可与lncRNA RP11-20G6.3及HIF-1α靶向结合。因此,本研究拟从细胞水平探究miRNA-186-5p在COPD中的生物学功能,并揭示miRNA-186-5p与lncRNA RP11-20G6.3及HIF-1α的靶向调控作用,初步在细胞水平探究lncRNA RP11-20G6.3对COPD发生的影响,同时在COPD患者血清中验证lncRNA RP11-20G6.3表达水平,为揭示其分子机制提供理论依据。方法:(1)采用脂多糖(LPS)诱导炎症构建人支气管上皮(BEAS-2B)细胞模型。(2)分别合成miR-186-5p、lncRNA RP11-20G6.3低表达序列并转染细胞,用CCK-8法检测细胞增殖活性、流式检测细胞凋亡及细胞周期;RT-qPCR检测TNF-α、HIF-1α、IL-6的mRNA表达,Western blot检测TLR-4/NF-κB信号通路p65、p-p65蛋白表达水平。(3)双荧光素酶报告实验验证miRNA-186-5p与lncRNA RP11-20G6.3及HIF-1α的靶向调控作用。(4)观察COPD患者及健康对照者外周血lncRNA RP11-20G6.3的表达变化。结果:(1)通过LPS诱导BEAS-2B细胞模拟COPD炎症模型,通过RT-qPCR和Western bolt检测显示HIF-1αmRNA及蛋白水平表达增加,ELISA检测细胞上清中炎症因子显示IL-6、TNF-α的表达增加,证实细胞炎症模型构建成功,可用于后续实验。(2)低表达miR-186-5p可明显降低BEAS-2B的增殖能力,抑制miR-186-5p表达能促进BEAS-2B的凋亡,与细胞周期无显著关系;低表达lncRNA RP11-20G6.3能降低BEAS-2B细胞的增殖。(3)miR-186-5p低表达可以促进NF-κB炎症通路相关基因HIF-1α、TNF-α及IL-6基因表达。miR-186-5p可能通过NF-κB等影响COPD支气管上皮细胞的增生。(4)结合生物信息学分析以及荧光素酶报告实验验证发现miR-186-5p可与lncRNA RP11-20G6.3及HIF-1α靶向结合。(5)lncRNA RP11-20G6.3在COPD患者血清中较对照组表达明显上调。结论:(1)低表达miR-186-5p可明显降低支气管上皮细胞的增殖能力、促进其凋亡,低表达lncRNA RP11-20G6.3可降低其细胞的增殖,二者均在COPD中起重要作用。(2)miR-186-5p低表达可促进NF-κB炎症通路HIF-1α、TNF-α及IL-6的表达。(3)lncRNA RP11-20G6.3与miR-186-5p靶向结合、miR-186-5p与HIF-1α靶向结合,故推测lncRNA RP11-20G6.3可能通过与miR-186-5p结合调控HIF-1α参与NF-κB炎症相关信号通路,影响COPD支气管上皮细胞增殖及凋亡进程,从而参与COPD的发生机制。(4)lncRNA RP11-20G6.3在COPD患者外周血mRNA表达水平增高。更多还原

【Abstract】 Objective: Early miRNA transcriptome analysis in our laboratory revealed that the expression of miR-186-5p was changed in COPD,however,the specific regulatory mechanism of miR-186-5p was unknown.Bioinformatics analysis showed that miR-186-5p could bind to lncRNA RP11-20G6.3 and HIF-1α.Therefore,this study aims to explore the biological function of miR-186-5p in COPD from the cell level,and reveal the targeted-regulating of miR-186-5p with lncRNA RP11-20G6.3 and HIF-1α,and preliminarily explore the effect of lncRNA RP11-20G6.3 on the occurrence of COPD in cell,which provides theoretical basis for its molecular mechanism.Methods:(1)LPS induction and inflammatory cell model construction.(2)miR-186-5p and lncRNA RP11-20G6.3 knockdown sequence were synthesized and transfected into cells.LPS induced cell proliferation activity was detected by CCK-8 method;cell apoptosis and cell cycle were detected by flow detection.The mRNA expressions of TNF-α,HIF-1α and IL-6 were detected by qRT-PCR,and the expression levels of TLR-4/NF-κB signaling pathway p65 and p-p65 were detected by Western blot.(3)Targeted regulation of miR-186-5p with lncRNA RP11-20G6.3 and HIF-1α were verified by double luciferase reporting experiment.(4)Observe the expression of lncRNA RP11-20G6.3 in peripheral blood of COPD patients and healthy people.Results:(1)The expression of HIF-1α was increased by LPS-induced BEAS-2B cells to simulate the COPD model,and the expression of HIF-1α was increased.IL-6 and TNF-α in the supernatant were detected by ELISA,which confirmed the successful construction of the cell inflammation model and could be used in subsequent experiments.(2)Low expression of miR-186-5p significantly decreased the proliferation of BEAS-2B.Inhibition of miR-186-5p expression can promote the apoptosis of BEAS-2B.There was no significant correlation in cell cycle.Inhibition of lncRNA RP11-20G6.3 expression can reduce cell proliferation.(3)miR-186-5p knockdown can promote the expression of TNF-α,HIF-1α and IL-6 genes related to the inflammatory pathway of NF-κB.This suggests that miR-186-5p may affect COPD through NF-κB.(4)Combined with bioinformatics analysis and luciferase reporting experiment,it was found that miR-186-5p can bind to lncRNA RP11-20G6.3 and HIF-1α,thus regulating the downstream signaling pathway and causing the occurrence and development of COPD.(5)lncRNA RP11-20G6.3 is up-regulated in serum.Conclusion:(1)Low expression of miR-186-5p can significantly reduce the proliferation ability and promote apoptosis of bronchial epithelial cells,and low expression of lncRNA RP11-20G6.3 can reduce its cell proliferation,both of which play important roles in COPD.(2)The low expression of miR-186-5p can promote the expression of HIF-1α,TNF-α and IL-6 in the NF-κB inflammatory pathway.(3)lncRNA RP11-20G6.3 targets miR-186-5p and miR-186-5p targets HIF-1α.Therefore,it is speculated that lncRNA RP11-20G6.3 may regulate HIF-1α in combination with miR-186-5p to participate in NF-κB inflammation-related signaling pathways,affect the proliferation and apoptosis of bronchial epithelial cells in COPD,and thus participate in the mechanism of COPD.(4)The expression of lncRNA RP11-20G6.3 mRNA in peripheral blood of COPD patients increased.更多还原