长链与环状非编码RNA共同调控MUC3A/5AC/13在肺癌发生发展中的作用研究

Long and circular non-coding RNA (LncRNA and CircRNA) both play important roles in cancer development with a possibly synergistic effect. By using the RNA-seq and circRNA chip, we found that the MUC gene family and their chromosome lncRNA and circRNA neighbors intensively up expressed. We then tested the expression of those molecules in more samples and found three significant correlations between those molecules including RP11-395B7.2 and MUC3A, lnc-MUC5B-4 and MUC5AC, and circ-MUC5AC-ol.3 and MUC5AC. Meanwhile, those molecules were up regulated in cancer tissues. The weighted correlation network analysis (WGCNA) further indicated that the above molecules and another family member MUC13 worked in a common function model. Because MUC proteins involve tumor immunity, we hypothesized that the above LncRNAs and CircRNA can influence the development of lung cancer via their combinational effects on MUC3A/MUC5AC/MUC13. Here, this project intends to investigate the role of those ncRNA molecules on lung cancer diagnosis and prognosis through clinical studies; To reveal the biological phenotypes as well as immune regulation function of and to analysis the biological mechanism underlying the correlation between those ncRNAs and their targeted MUC proteins with a series of functional assays conducted on stable transfected lung cell lines and humanized mice model, which were built by the tandem expression vectors and CRISP-Disp technology, respectively. Results from this project will provide effectively scientific evidences for molecular diagnosis and targeted therapy of lung cancer.

长链与环状非编码RNA（LncRNA与CircRNA）在肿瘤发生发展中起着重要作用，可能存在协同效应。预实验检测肺癌与癌旁组织转录组，发现黏蛋白（MUC）家族及其染色体区域内LncRNA与CircRNA密集表达上调，扩大样本分析显示RP11-395B7.2与MUC3A，lnc-MUC5B-4、circ-MUC5AC-ol.3皆与MUC5AC表达显著相关，且均表达上调。加权相关网络分析提示上述分子及另一个家族成员MUC13共处一个功能模块。MUC参与调节肿瘤免疫，故推测上述非编码RNA可共同调控MUC3A/MUC5AC/MUC13而影响肺癌的发生发展。本项目拟通过临床样本研究，探讨上述非编码RNA在肺癌诊断和预后评估中的作用；应用串联共表达载体和CRISP-Disp技术构建稳转细胞系和人源化模式小鼠，探索其生物学表型及免疫调控作用，揭示其表达调控机制，为肺癌的分子诊断和靶向治疗提供科学依据。

基于粘蛋白家族（MUCs）在肿瘤发生发展过程中的重要意义，本项目旨在探索由二代测序提示的、与MUCs表达潜在相关的长链和环状非编码RNA（nc-MUCs），分析它们在肺癌发生发展中的作用及其调控MUCs分子表达的生物学机制。项目在广州地区收集并建立了276对肺癌组织与癌旁正常组织数据库，成功随访并获取了163例肺癌患者的生存信息。在上述样本中检测三个MUCs分子（MUC3A、MUC5AC、和MUC5B）和4个nc-MUCs（Circ_0001166和Circ_0064019、linc01125和RP11-395B7.2-2）的表达，发现MUC5AC、MUC5B和Circ_0001166在肺癌组织中的表达显著高于癌旁正常组织，Circ_0064019和linc01125在前者中的表达显著低于后者，而MUC3A和RP11-395B7.2-2在两组间的表达差异不显著。其中，MUC5AC高表达能显著增加肺癌致死率（HR = 1.43；95%CI = 1.00-2.05），Circ_0064019高表达能显著降低肺癌致死率（HR = 0.55；95%CI = 0.37-0.83），而其它分子与肺癌患者预后无显著关联。系列功能实验显示，Circ_0064019可能与RNA结合蛋白相互作用而下调MUC5AC的表达，其过表达可显著抑制肺癌细胞的增殖、克隆形成、裸鼠成瘤、转移和侵袭能力，促进细胞凋亡并影响细胞周期，而其去表达效应则相反；linc01125可竞争性结合miR-19b-3p而上调TNFAIP3的表达，进而间接抑制MUC5AC的表达，其过表达可显著抑制肺癌细胞的增殖、克隆形成、裸鼠成瘤、转移和侵袭能力，促进细胞凋亡。然而，Circ_0001166对肺癌细胞的恶性表型无显著效应。此外，血浆中环状nc-MUCs的表达对肺癌诊断无显著价值。以上结果说明，Circ_0064019和linc01125可抑制MUC5AC表达而发挥抑癌效应，有望成为肺癌治疗的新分子靶标。