NF-κB反向调控IκB逆转肺癌恶性表型及机制研究

The initiation and development of lung carcinoma is closely related to the uncontrolled inflammatory response that exists in microenvironment, but the mechanism is still unclear.The NF-κB signaling pathway, center of inflammatory response, may play an important role in the regulation of cancer. In previous studies we found that: inhibition of NF-κB reduced the invasion and metastasis capacities of lung cancer cells , with increase of free IκB in cytoplasm,implying that NF-κB may change the malignant phenotype of lung cancer cells through regulating IκB in cytoplasm reversely. This study are planning to observe the influence of NF-κB and IκB on the growth, invasion and metastasis of lung cancer cells, as well as its relationship with clinical prognosis from both cellular and overall level, by changing the expression of NF-κB and IκB from different sources.This is to verify that inhibition of IκB in the cytoplasm is an important mechanism for NF-κB in promoting lung cancer invasion and metastasis. In addition, proteomics technology are used to identify the proteins that interacted with IκB, and explore the molecular mechanism of NF-κB in regulating IκB negatively and reversing lung cancer malignant phenotype. The implement of this project will deepen our understanding of the relationship among key proteins and their functions in the NF-κB signaling pathways, enrich the theory of the relationship between lung cancer and inflammation, and therefore provide evidence for the use of NF-κB signaling pathway in the biological treatment of lung cancer.

肺癌的发生发展与微环境中炎症反应密切相关，但机制并不清楚；NF-κB信号通路是炎症反应中心，可能在肺癌发生发展中发挥重要作用。我们前期研究发现: 抑制NF-κB表达可降低肺癌细胞生长浸润的能力、增加细胞浆内游离IκB，提示IκB具有抗肿瘤特性，NF-κB可能对IκB起反向调控作用。本课题拟通过改变不同类型肺癌细胞内NF-κB和IκB表达，体内外观察NF-κB能否通过调控IκB的功能来逆转肺癌细胞的成瘤性、侵袭和转移能力，验证在细胞浆内抑制IκB是NF-κB促进肺癌浸润转移的重要机制；同时运用蛋白质组学技术鉴定出与IκB相互作用蛋白，观察以此关键蛋白为下游靶点的NF-κB信号通路与肺癌分期及预后的关系，探讨NF-κB反向调控IκB逆转肺癌恶性表型的分子机制。本课题将加深对NF-κB通路中关键蛋白功能及相互关系的认识，丰富炎症与肺癌关系理论，为利用NF-κB信号通路进行肺癌生物治疗提供依据。

此课题按照研究计划基本完成，达到预期目标。我们成功的构建了无NLS（细胞核定位信号）的NF-κBp65质粒系统（即NF-κBp65NLSm质粒），并对NF-κBp65NLSm质粒进行了鉴定；建立了稳定阴性表达NF-κB的肺癌细胞系（即A549/NF-κBp65shRNA细胞系）。将NF-κBp65NLSm质粒转染到A549/NF-κBp65shRNA细胞，免疫荧光结果显示，与未转染组相比，NF-κBp65表达增多，且位于细胞浆内。在TNFα对细胞进行刺激后发现，转染野生型NF-κBp65质粒（即NF-κBp65WT质粒）的A549/NF-κBp65shRNA细胞，细胞核内NF-κBp65表达增多，而转染NF-κBp65NLSm的A549/NF-κBp65shRNA细胞，细胞核内NF-κBp65表达并无明显增多。Real-time PCR结果发现，与转染NF-κBp65WT质粒相比，转染NF-κBp65NLSm质粒的细胞NF-κBp65 mRNA表达无明显差异（P>0.05），但IκBα mRNA的表达显著减少，差异有统计学意义（P<0.01），该项研究结果表明，NF-κB p65NLSm质粒转染A549/NF-κBp65shRNA细胞后，与未转染组相比，NF-κBp65仅在细胞浆内高表达，而不能进入细胞核反馈性促进IκBα的表达。应用MTT和Transwell等实验方法分别对肺癌细胞增殖及迁移等恶性表型进行检测，实验结果显示，转染NF-κBp65NLSm质粒的A549/NF-κBp65shRNA细胞与未转染细胞相比，细胞增殖和迁移能力明显增强，差异有统计学意义（P<0.05）。经上实验结果，我们推测：细胞浆内过表达的无活性NF-κBp65可能与IκBα结合，改变了IκBα在抗肿瘤过程中的作用，使肺癌细胞的恶性表型发生了改变。上述结果不仅加深了对NF-κB信号通路上关键蛋白IκB功能及相互作用关系的认识，而且揭示了抑制 IκBα 可能是 NF-κB促进肺癌增殖、转移的重要机制。本研究为NF-κB信号通路参与的非可控性炎症与肺癌之间的关系提供了切入点，同时也为临床筛选遏制恶性肺癌生长浸润和转移的有效的药物靶点提供理论依据。