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Study on mechanism of lncRNA NEAT1 in promoting hypoxia and hypoglycemia induced pulmonary fibrosis by targetedly regulating miR-29a
张喜;段效军;李林瑞;陈艳萍;
摘要(Abstract):
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。
关键词(KeyWords): lncRNA NEAT1;miR-29a;缺氧;肺纤维化;凋亡
基金项目(Foundation): 湖南省卫生健康委2020年度科研立项课题(20200641)
作者(Author): 张喜;段效军;李林瑞;陈艳萍;
Email:
DOI: 10.14053/j.cnki.ppcr.202103004
参考文献(References):
- [1] Rangarajan S,Bone NB,Zmijewska AA,et al.Metformin reverses established lung fibrosis in a bleomycin model[J].Nat Med,2018,24(8):1121-1127.
- [2] Kulkarni T,de Andrade J,Zhou Y,et al.Alveolar epithelial disintegrity in pulmonary fibrosis[J].Am J Physiol Lung Cell Mol Physiol,2016,311(2):L185-191.
- [3] de Vega Sánchez B,Disdier Vicente C,Roig Figueroa V,et al.Association between idiopathic pulmonary fibrosis and lung cancer[J].Arch Bronconeumol,2020,56(1):47-49.
- [4] Park J,Kim DS,Shim TS,et al.Lung cancer in patients with idiopathic pulmonary fibrosis[J].Eur Respir J,2001,17(6):1216-1219.
- [5] Zhang Z,Zhu Z,Watabe K,et al.Negative regulation of lncRNA GAS5 by miR-21[J].Cell Death Differ,2013,20(11):1558-1568.
- [6] Yara S,Kawakami K,Kudeken N,et al.FTS reduces bleomycin-induced cytokine and chemokine production and inhibits pulmonary fibrosis in mice[J].Clin Exp Immunol,2001,124(1):77-85.
- [7] Loewen G,Jayawickramarajah J,Zhuo Y,et al.Functions of lncRNA HOTAIR in lung cancer[J].J Hematol Oncol,2014,7:90.
- [8] Yu F,Jiang Z,Chen B,et al.NEAT1 accelerates the progression of liver fibrosis via regulation of microRNA-122 and Kruppel-like factor 6[J].J Mol Med (Berl),2017,95(11):1191-1202.
- [9] Huang S,Xu Y,Ge X,et al.Long noncoding RNA NEAT1 accelerates the proliferation and fibrosis in diabetic nephropathy through activating Akt/mTOR signaling pathway[J].J Cell Physiol,2019,234(7):11200-11207.
- [10] Friedman RC,Farh KK,Burge CB,et al.Most mammalian mRNAs are conserved targets of microRNAs[J].Genome Res,2009,19(1):92-105.
- [11] Lewis BP,Burge CB,Bartel DP.Conserved seed pairing,often flanked by adenosines,indicates that thousands of human genes are microRNA targets[J].Cell,2005,120(1):15-20.
- [12] Reinhart BJ,Slack FJ,Basson M,et al.The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J].Nature,2000,403(6772):901-906.
- [13] Kim VN.MicroRNA biogenesis:coordinated cropping and dicing[J].Nat Rev Mol Cell Biol,2005,6(5):376-385.
- [14] Chen CZ,Li L,Lodish HF,et al.MicroRNAs modulate hematopoietic lineage differentiation[J].Science,2004,303(5654):83-86.
- [15] Yekta S,Shih IH,Bartel DP.MicroRNA-directed cleavage of HOXB8 mRNA[J].Science,2004,304(5670):594-596.
- [16] Roncarati R,Viviani Anselmi C,Losi MA,et al.Circulating miR-29a,among other up-regulated microRNAs,is the only biomarker for both hypertrophy and fibrosis in patients with hypertrophic cardiomyopathy[J].J Am Coll Cardiol,2014,63(9):920-927.
- [17] Xuan J,Guo SL,Huang A,et al.MiR-29a and miR-652 attenuate liver fibrosis by inhibiting the differentiation of CD4+ T cells[J].Cell Struct Funct,2017,42(2):95-103.
- [18] Morlando M,Fatica A.Alteration of epigenetic regulation by long noncoding RNAs in cancer[J].Int J Mol Sci,2018,19(2):570-586.
- [19] Lu Q,Guo Z,Xie W,et al.The lncRNA H19 mediates pulmonary fibrosis by regulating the miR-196a/COL1A1 axis[J].Inflammation,2018,41(3):896-903.
- [20] Jiang H,Chen Y,Yu T,et al.Inhibition of lncRNA PFRL prevents pulmonary fibrosis by disrupting the miR-26a/smad2 loop[J].Am J Physiol Lung Cell Mol Physiol,2018,315(4):L563-L575.
- [21] Chai Y,Liu J,Zhang Z,et al.HuR-regulated lncRNA NEAT1 stability in tumorigenesis and progression of ovarian cancer[J].Cancer Med,2016,5(7):1588-1598.
- [22] Liu G,Friggeri A,Yang Y,et al.miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis[J].J Exp Med,2010,207(8):1589-1597.
- [23] Yang T,Liang Y,Lin Q,et al.miR-29 mediates TGFβ1-induced extracellular matrix synthesis through activation of PI3K-AKT pathway in human lung fibroblasts[J].J Cell Biochem,2013,114(6):1336-1342.
- [24] Wang C,Song X,Li Y,et al.Low-dose paclitaxel ameliorates pulmonary fibrosis by suppressing TGF-β1/Smad3 pathway via miR-140 upregulation[J].PLoS One,2013,8(8):e70725.
- [25] Yang S,Banerjee S,de Freitas A,et al.Participation of miR-200 in pulmonary fibrosis[J].Am J Pathol,2012,180(2):484-493.
- [26] Cui H,Ge J,Xie N,et al.MiR-34a promotes fibrosis in aged lungs by inducing alveolar epithelial dysfunctions[J].Am J Physiol Lung Cell Mol Physiol,2017,312(3):L415-L424.
- [27] Kriegel AJ,Liu Y,Fang Y,et al.The miR-29 family:genomics,cell biology,and relevance to renal and cardiovascular injury[J].Physiol Genomics,2012,44(4):237-244.
- [28] Qin RH,Tao H,Ni SH,et al.MicroRNA-29a inhibits cardiac fibrosis in Sprague-Dawley rats by downregulating the expression of DNMT3A[J].Anatol J Cardiol,2018,20(4):198-205.
- [29] Bibaki E,Tsitoura E,Vasarmidi E,et al.MiR-185 and miR-29a are similarly expressed in the bronchoalveolar lavage cells in IPF and lung cancer but common targets DNMT1 and COL1A1 show disease specific patterns[J].Mol Med Rep,2018,17(5):7105-7112.