分泌型粘蛋白Muc5AC促进Kras相关肺腺癌发生的研究及机制

The activation of Oncogene Kras by mutation contributes to the lung carcinogenesis and progression. Mucins are large extracellular proteins that are heavily glycosylated with complex oligosaccharides to establishish a selective molecular barrier at the epithelial surface and engage in morphogenetic signal transduction. Alterations in mucin expression or glycosylation accompany the development of cancer and influence cellular growth, differentiation, transformation, adhesion, invasion and immune surveillance. In human lungs, MUC5AC is one of the predominant airway mucins physiologically expressed especially encounter some stimulation. Based upon the hypothesis that tumors use mucins like Muc5AC for invasion, metastasis and protection, we will study the role of secreted mucin in lung carcinogenesis by targeting the Muc5AC gene. We introduce the Muc5AC knock out and floxed alleles into K-ras mutant lung cancer animal model, CCSPCre/LSL-K-rasG12D（CC-LR）, to generate Muc5AC-/-/CC-LR. Using these mice, we want to identify the role of Muc5AC in lung carcinogenesis in vivo. We inhibit NF-?B by chemical inhibitors in mucin secreted lung adeno-carcinoma cell line Calu-3 and Kras mutant lung adeno-carcinoma cell line A549 and H2009 to study how mutant Kras gene drive Muc5AC expression through the IKKs-IkB- NF-kB pathway to activate EGFR and strengthen the ability of tumor cells for anti-apoptosis, cell proliferation and extend the survival of tumor cells. We want to find out the role of Muc5AC in Kras induced lung carcinogenesis by in vivo and in vitro study, to explore the function of secreted mucin in this field in order to build up the foundation of future lung caner prevention and treatments.

癌基因Kras的激活是肺腺癌发生的最常见的分子机制。粘蛋白是粘膜屏障的主要分子，涉及多种肿瘤特别是腺癌的发病，Muc5AC是应激情况下呼吸道最主要的分泌型粘蛋白。有学者提出肿瘤细胞通过增加粘蛋白分泌保护自身免受免疫攻击的假说。本研究拟用Muc5AC缺失合并Kras突变的肺癌基因动物模型Muc5AC-/-/CC-LR在体研究Muc5AC对Kras突变相关肺癌发生的促进作用；应用肺腺癌分泌细胞株Calu-3，肺腺癌Kras突变细胞株A-549及H2009，并通过化学法干预肿瘤细胞NF-kB表达，明确Kras突变通过IKKs-IkB-NF-kB通路促进Muc5AC的表达，增强肿瘤细胞抗凋亡，促增殖能力进而发生肺癌的作用机制，从而更好的理解粘膜功能在肺癌发生发展中的地位，为今后改善肺癌的防治效果提供基础研究。

Kras的激活是肺腺癌发生及进展的最常见的分子机制。课题组在MUC5AC-/-背景的Kras突变相关肺癌基因动物CC-LR上，体内评估并证明Kras突变通过上调Muc5ac促进肺腺癌的发生。运用CRISPR-Cas9质粒，成功构建了MUC5AC沉默的A549细胞系，MUC5AC敲除后对细胞增殖能力影响较小；敲除MUC5AC显著抑制 A549细胞株的迁移侵袭能力并明显抑制肺腺癌细胞株在裸小鼠肺内的定植能力（P<0.0001），从而明确Kras突变促进Muc5ac的表达，增强肿瘤细胞侵袭转移进而促进肺癌恶性进展的具体调节过程。在整体动物水平上，运用PAFS法观察到粘蛋白Muc5ac的转运主要是依赖于突触体相关小体蛋白SNAP23。课题组的研究表明，临床标本Muc5ac表达水平与癌症预后相关，Muc5ac高表达的肿瘤病人的预后差（HR:1.53,95%CI:1.158-2.028,P=0.003）.特别是在胆管癌（HR:1.83,95%CI:1.269-2.639,P=0.001）和胃肠恶性肿瘤（HR:1.44,95%CI:1.069-1.949,P=0.017）。同时观察到Mucin4表达水平与癌症预后相关，Mucin4高表达肿瘤病人的预后差（HR:1.87,95%CI:1.58-2.22,P<0.001）。特别是在胆管腺癌（HR:2.41,95%CI:1.69-3.42, P<0.001）和胰腺癌（HR:2.01,95%CI:1.42-2.86, P<0.001）。课题组完成83对肺腺癌临床标本Muc5ac表达水平与肺癌临床分期及预后的关系研究的芯片实验。结果发现Muc5ac高表达组的总生存率显著降低。Muc5ac表达高的病人生存期更短，预后更差（P=0.0084,HR=1.932）。病人临床资料单因素多因素分析发现Muc5ac的表达可作为肺腺癌病人的独立预后因素。