Lnc CPS1-IT1靶向调控内皮-间质转化抑制OSA所致肺动脉高压的机制  

LncRNA CPS1-IT1 inhibits obstructive sleep apnea-induced pulmonary hypertension by targeting endothelial-mesenchymal transition

作　　者：魏丽 陈昊 王秋波 张泽明 Wei Li;Chen Hao;Wang Qiubo;Zhang Zeming(Department of Respiratory Medicine,Zhoupu Hospital,Shanghai University of Medicine&Health Sciences,Shanghai 200003,China)

机构地区：[1]上海健康医学院附属周浦医院呼吸内科,上海200003

出　　处：《国际呼吸杂志》2024年第9期1012-1022,共11页International Journal of Respiration

基　　金：上海市卫生健康委员会卫生行业临床研究专项(202140352)。

摘　　要：目的探讨在低氧环境下,长链非编码RNA CPS1内含子转录本1(Lnc CPS1-IT1)通过影响内皮-间质转化(EndoMT)治疗阻塞性睡眠呼吸暂停(OSA)所致肺动脉高压(PH)的分子调控机制。方法本研究为实验研究。以人脐静脉内皮细胞构建OSA的细胞模型,并使用该模型分别构建空白对照组,空过表达载体组(阴性对照过表达载体),长链非编码RNA CPS1内含子转录本1(Lnc CPS1-IT1)过表达组(Lnc CPS1-IT1过表达载体),空沉默载体组(阴性对照沉默载体),沉默Lnc CPS1-IT1组(沉默Lnc CPS1-IT1的短发夹RNA);使用吡咯烷二硫代甲酸铵(PDTC)阻断细胞核因子κB(NF-κB)信号通路构建Lnc CPS1-IT1过表达+PDTC组。比较空过表达载体组与Lnc CPS1-IT1过表达组,空沉默载体组与沉默Lnc CPS1-IT1组EndoMT相关分子标志物[血小板-内皮细胞黏附分子(CD31)、α-平滑肌肌动蛋白(α-SMA)、锌指转录因子(SNAIL)]和胶原蛋白(Ⅰ型胶原、Ⅲ型胶原)mRNA、蛋白表达,以及促炎细胞因子[肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-10、IL-13]的水平。通过Transwell测定法检测Lnc CPS1-IT1对于细胞迁移能力的影响。RNA免疫沉淀和荧光素酶报告基因法检测Lnc CPS1-IT1和缺氧诱导因子1α(HIF-1α)的结合情况。比较PDTC处理前后EndoMT相关分子标志物和胶原蛋白mRNA、蛋白表达,以及促炎细胞因子的水平。结果Lnc CPS1-IT1过表达组CD31 mRNA和蛋白表达均较空过表达载体组升高,α-SMA、SNAIL、Ⅰ型胶原和Ⅲ型胶原的mRNA和蛋白表达均较空过表达载体组降低,差异均有统计学意义(均P<0.001)。沉默Lnc CPS1-IT1组中CD31 mRNA和蛋白表达均较空沉默载体组降低,α-SMA、SNAIL、Ⅰ型胶原和Ⅲ型胶原的mRNA和蛋白表达均较空沉默载体组升高,差异均有统计学意义(均P<0.001)。Lnc CPS1-IT1过表达组TNF-α、IL-10、IL-13的水平均较空白过表达载体组降低,沉默Lnc CPS1-IT1组TNF-α、IL-10、IL-13的水平均较空白沉默载体组升高,�Objective To investigate the molecular regulatory mechanism of long non-coding RNA carbamoyl-phosphate synthase 1(CPS1)intron transcript 1(LncRNA CPS1-IT1)in the treatment of obstructive sleep apnea(OSA)-induced pulmonary hypertension(PH)by influencing endothelial-mesenchymal transition(EndoMT)in a hypoxic environment.Methods This was an experimental study.Human umbilical vein endothelial cells(HUVECs)were used to construct an in vitro OSA model.HUVECs were transfected with negative overexpression plasmid,LncRNA CPS1-IT1 overexpression plasmid(oe-CPS1-IT1),shRNA negative control,and LncRNA CPS1-IT1 shRNA.In HUVECs transfected with LncRNA CPS1-IT1 shRNA,they were additionally induced with pyrrolidine dithiocarbamate(PDTC)to inactivate the nuclear factor kappa B(NF-κB)signaling pathway.Protein and mRNA levels of EndoMT-associated markers(platelet/endothelial cell adhesion molecule-1[CD31],alpha smooth muscle actin[α-SMA]and the zinc-finger transcription factor[SNAIL])and collagen(typeⅠcollagen,typeⅢcollagen)were detected.Relative levels of pro-inflammatory factors,including tumor necrosis factor-α(TNF-α),interleukin-10(IL-10)and IL-13 were examined.Cell migration influenced by LncRNA CPS1-IT1 was detected by Transwell assay.The binding of LncRNA CPS1-IT1 with hypoxia-inducible factor-1α(HIF-1α)was assessed by RNA immunoprecipitation and dual luciferase reporter assay.Protein and mRNA levels of EndoMT-associated markers,collagens and pro-inflammatory cytokines before and after PDTC induction were compared.Results Overexpression of LncRNA CPS1-IT1 significantly upregulated the mRNA and protein expression levels of CD31,but downregulated the mRNA and protein levels ofα-SMA,SNAIL,typeⅠcollagen and typeⅢcollagen(all P<0.001).Knockdown of LncRNA CPS1-IT1 significantly downregulated the mRNA and protein expression levels of CD31,but upregulated the mRNA and protein expression levels ofα-SMA,SNAIL,typeⅠcollagen and typeⅢcollagen(all P<0.001).Overexpression of LncRNA CPS1-IT1 significantly reduced the le

关 键 词：睡眠呼吸暂停 阻塞性 高血压 肺性 RNA 长链非编码 NF-κB 缺氧诱导因子1 Α亚基 CPS内含子转录本1 内皮-间质转化 

分 类 号：R544.1[医药卫生—心血管疾病] R766[医药卫生—内科学]