LncRNA n341773作为ceRNA在肺纤维化中的作用及机制

LncRNAs are associated with pulmonary fibrosis, our first stage study and documents proved lncRNA n341773 significantly down-regulated in myofibroblast and in lung fibrosis tissue, reduction of the lncRNA n341773 level in fibroblasts increase the expression of α-SMA, but the mechanism is unknown. Online bioinformatics analysis indicated that lncRNA n341773 have binding sites for miR-199a-3p, lncRNA n341773 and miR-199a-3p participate in the regulation of PTEN gene. Our previous studies show that lncRNA n341773 increase the expression level of PTEN while miR-199a-3p decrease the expression level of PTEN. Therefore, we propose hypothesis that lncRNA n341773 function as competitive endogenous RNAs (ceRNAs) by sponging miR-199a-3p play an important role in lung fibrosis. This project is proposed to induce the lung fibroblasts phenotype transformation with TGF-β1 and establish pulmonary fibrosis in mices, construct lentiviral vector targeting lncRNA n341773 and miR-199a-3p and transfect into the lung fibroblast cells. PTEN level was detected by RT-PCR and western blot methods, the interaction between lncRNA n341773 and miR-199a-3p were detected by Real-time Quantitative PCR and Luciferase reporter gene technique. PTEN as target gene for lncRNA n341773 and miR-199a-3p was detected by RNA pulldown and RNA immunoprecipitation . The cell activity was determined by CCK8 assay,cell migration ability was detected by Would healing assay and Transwell assay. Flow cytometry was used to monitor the changes of cell cycle distribution. This study will explore the new target on intervention of lung fibrosis.

LncRNAs参与调控肺纤维化，前期实验及文献证实：lncRNA n341773在肺肌成纤维细胞及肺纤维化组织中表达下降，lncRNA n341773与α-SMA表达水平负相关，生物信息学分析提示：lncRNA n341773与miR-199a-3p可能存在交互作用，均靶向调控PTEN。我们的研究发现：lncRNA n341773与PTEN表达呈正相关，而miR-199a-3p与PTEN呈负相关。据此提出假设：LncRNA n341773作为miR-199a-3p的ceRNA，在肺纤维化中起重要作用。本课题拟lncRNA n341773、miR-199a-3p、TGF-β1等处理肺成纤维细胞，研究lncRNA n341773、miR-199a-3p的交互作用，验证两者对靶基因PTEN的调控作用；并研究lncRNA n341773对小鼠肺纤维化的作用。该研究将进一步揭示肺纤维化的发病机制。

LncRNA在肺纤维化的发生发展过程中发挥重要作用。本课题用TGF-β1 诱导肺成纤维细胞表型转化，并建立博来霉素肺纤维化小鼠模型，利用基因芯片技术筛选出肺纤维化小鼠肺组织差异表达的lncRNAs。在TGF-β1诱导的HELF细胞表型转化模型中，lncRNA n341773低表达，同时PI3K/Akt/mTOR通路被激活。用慢病毒载体上调HELF细胞中的lncRNA n341773，上调的lncRNA n341773能够使TGF-β1诱导PI3K/Akt/mTOR通路失活，同时抑制成纤维细胞表型转化。随后用慢病毒载体下调HELF细胞中的lncRNA n341773，下调的lncRNA n341773能够放大TGF-β1诱导的PI3K/Akt/mTOR信号通路,同时促进肺成纤维细胞表型转化。本课题通过对细胞功能实验的研究、以及相关蛋白的检测，提示lncRNA n341773、 miR-199a可以调控PI3K/Akt/mTOR信号通路。发现lncRNA n341773通过调控成纤维细胞增殖以及成纤维细胞表型转化参与肺纤维化的发生发展。验证了lncRNA n341773、miR-199a的交互作用，两者对PTEN的调控作用；并研究lncRNA n341773对小鼠肺纤维化的作用。本课题证明lncRNA n341773通过与miR-199a内源性竞争性调控PI3K/Akt/mTOR信号通路参与调节成纤维细胞增殖以及成纤维细胞表型转化，从而在肺纤维化的发生发展过程中发挥重要作用。