细胞核FAK对胃癌细胞增殖、迁移及侵袭的调控作用机制

Gastric cancer is one of the most common malignant tumors in China. The dysfunction of multiple signaling pathways contribute to the occurrence and progression of gastric cancer. Focal adhesion kinase (FAK)is a non-receptor and non-membrane protein tyrosine kinase (PTK),which always over-expressed in various types of cancer and its over-expression is highly correlated with tumor differentiation, invasion and lymph node metastasis. FAK is shuttled between cytoplasm and nucleus with its nuclear localization sequence and nuclear export signal. The cytosolic FAK is activated by various extracellular signals to promote cancer cell proliferation, migration, invasion and inflammatory cytokines production, While the regulation mechanisms of nuclear FAK remain unknown. Therefore, we propose that nuclear FAK regulate expression of certain genes by interacting with transcription factors of certain genes and these genes expression products further to promote the gastric cancer cell proliferation, migration, invasion and inflammatory factors secretion.In this study, we plan to use the gastric cancer cell MKN45 and FAK gene knock-out cells as study materials. Based on the results of differentially expressed genes and relevant transcription factors with transcriptome sequencing, We plan to adopt the MTT assay, migration assay, invasion assay, RCR-based site-directed mutagenesis, cell transfection, qPCR, ELISA, Co-IP, ChIP-seq, mass spectral analysis and Western blot techniques to explore the roles and regulation mechanisms of nuclear FAK in regulation of certain genes expression. This study will promote to further elucidate the pathogenesis of gastric cancer and propose the effective cancer therapies.

胃癌是常见的恶性肿瘤，严重威胁国人的生命健康，其发病机制涉及多个信号通路的异常。FAK是酪氨酸激酶，在胃癌和其它多种癌组织中都过表达。FAK分子通过核定位和核输出序列穿梭于细胞质和细胞核中发挥作用。细胞质FAK接受细胞外信号促进细胞增殖、迁移、侵袭及肿瘤炎性微环境的形成，细胞核FAK的作用机制尚不清楚。我们提出胃癌细胞中细胞核FAK通过和转录因子相互作用而调控特定基因的表达最终促进细胞增殖、迁移、侵袭及炎性微环境的形成。基于胃癌细胞和FAK基因敲除细胞转录组测序差异表达基因检测和转录因子预测的结果，本课题拟采用胃癌细胞、FAK基因敲除细胞为材料，细胞增殖、迁移及侵袭实验、Co-IP、质谱、ChIP-seq、PCR定点突变、qPCR、ELISA、Western blot等手段，阐明胃癌细胞中细胞核FAK调控特定基因表达的作用及机制。本研究将为阐明胃癌发病机制及有效治疗提供理论基础和技术手段。

胃癌是常见的恶性肿瘤，严重威胁国人的生命健康，其发病机制涉及多个信号通路的异常。FAK是酪氨酸激酶，在胃癌和其它多种癌组织中都过表达。FAK分子通过核定位（NLS）和核输出序列（NES）穿梭于细胞质和细胞核中发挥作用。本研究聚焦于细胞核FAK对胃癌细胞增殖和迁移的调控作用及机制，研究内容：（1）临床胃癌组织中FAK的表达。（2）FAK在胃癌细胞中的核定位；（3）分别构建FAK和FAK核定位序列突变慢病毒表达载体及功能验证。（4）细胞核FAK对胃癌细胞增殖、克隆形成、迁移及炎症因子表达的调控作用。（5）细胞核FAK对胃癌腹膜转移的影响。（6）FAK重新表达细胞和FAK核定位突变表达细胞转录组测序分析及验证。（7）细胞核 FAK相互作用的蛋白质谱检测。（8）转录组和蛋白质谱结果关联分析及验证。（9）细胞核FAK和转录因子YY1的相互作用。主要结果：（1）FAK在胃癌组织细胞中过度表达，其表达水平对预后有不利的影响。FAK在MKN45，NUGC3和NCIN87三种胃癌细胞的细胞质和细胞核中均有表达。（2）成功构建FAK慢病毒表达载体和FAK核定位突变慢病毒表达载体。（3）细胞核FAK入核受阻会抑制胃癌细胞的增殖、克隆形成、迁移及炎症因子IL-6与IL-8的表达。（4）细胞核FAK入核受阻会抑制小鼠腹膜转移模型中肿瘤的体积、数量和重量。（5）转录组测序结果表明，细胞核FAK入核受限制的细胞组中共有160个基因表达发生显著性变化。（6）细胞核FAK调控FGF10和IL-8的表达。（7）FGF10和IL-8促进胃癌细胞的增殖和迁移。（8）细胞核FAK质谱分析与预测的FGF10和IL-8的转录因子取交集得到转录因子YY1，分子对接软件也预测到FAK与YY1分子的相互作用。综上所述，FAK在三种胃癌细胞的细胞质和细胞核中均有表达；FAK核定位突变显著降低了胃癌细胞的增殖、迁移、克隆形成以及炎性因子的表达；FAK核定位突变也降低了胃癌腹膜转移模型中肿瘤的腹膜播散程度；细胞核FAK调控胃癌细胞FGF10和IL-8的表达；重组FGF10和IL-8促进胃癌细胞的增殖和迁移。细胞核FAK可能通过和转录因子YY1相互作用进而调控FGF10和IL-8的表达最终促进胃癌细胞的增殖和迁移。细胞核FAK在胃癌细胞中的调控作用及机制研究将为阐明胃癌的发病机制及提出有效治疗方法提供理论基础和技术手段。