血小板通过TGF-β1/SH2B3轴介导上皮间质转化促进肺腺癌侵袭转移

Platelets interact with cancer cells and play an important role in growth and metastasis. Our previous studies have found that inhibition of the SH2B3 expression to respond to platelets induces epithelial-mesenchymal transition (EMT) phenotype and promotes invasion and metastasis of lung adenocarcinoma. However, the detailed molecular mechanisms are still unknown. Platelets contain a lot of transforming growth factor -β1 (TGF-β1), which is one of the main EMT-inducing factor. We hypothesize that the activation of platelets has been suggested to promote the release of TGF-β1 and be involved in EMT via down-regulating the expression of SH2B3. In this study, gene transfection, yeast two-hybrid, protein phosphorylation chip, laser co-focus microscopy and western blot were used to investigate the key EMT-related proteins of SH2B3-associated signal transducing network and the difference in phosphorylation level in lung adenocarcinoma cells before and after SH2B3 gene knockout and platelets co-culture. Analysis was made of the expression of key protein in lung adenocarcinoma tissues as well as its associations with recurrence and metastasis. Besides, relevant functional experiment, both in vivo and in vitro, were conducted. The objectives of this study are to clarify the mechanisms of SH2B3 in EMT and invasion and metastasis of lung adenocarcinoma induced by platelet activation, in the meanwhile to provide clues for new treatment targets for lung adenocarcinoma.

肿瘤相关血小板在癌症生长和转移中起重要作用。前期我们研究发现血小板可下调肺腺癌细胞接头蛋白SH2B3的表达，诱导上皮间质转化（EMT）表型，促进肿瘤侵袭转移，但机制不清。血小板富含大量EMT相关细胞因子转化生长因子β1（TGF-β1），因此推测血小板活化释放的TGF-β1,导致肺腺癌细胞SH2B3表达下调，活化系列下游信号通路，参与了肿瘤侵袭转移。本课题将在前期研究基础上，拟采用基因转染、核外酵母双杂交及免疫印迹等技术筛选SH2B3互作蛋白及调控EMT发生的关键蛋白；激光共聚焦、蛋白磷酸化芯片等研究SH2B3敲除前后、血小板共培养前后肺腺癌细胞EMT相关功能蛋白表达、易位及磷酸化水平；同时分析关键蛋白在肺腺癌组织中的表达及其与转移复发的关系，并进行系列体内、体外功能分析。以期阐明SH2B3在血小板活化诱导肺腺癌EMT和侵袭转移中的作用机制，为寻找肺腺癌治疗新靶点提供实验依据。

肺癌是全世界最常见的恶性肿瘤，每年约有180万例新发病例与160万例因肺癌所致的死亡病例，其中，约40%新发及死亡病例发生在中国。目前国内外治疗肺癌最有效的方式仍是手术治疗，然而，肺癌患者单纯手术远期疗效仍不能令人满意，癌症全基因组关联分析（GWAS）提示携带易感基因的患者暴露于致癌物时更易发展为肺癌。为了从分子水平深入了解肺癌的发生发展机制，我们系统研究了SH2B3在肺癌中的作用及其可能的机制。SH2B3除参与肥胖、糖尿病等内分泌疾病外，近期研究聚焦于SH2B3与血液系统及实体肿瘤的发生发展方面，可作为候选促癌基因参与多种癌症。. 采用定量逆转录聚合酶链反应(QRT-PCR)、Western blotting、免疫组化、免疫组化等方法检测转化生长因子β1（TGF-β1）、SH2B3、JAK2/STAT3、SHP2/GrB2/PI3K/AKT、EMT相关分子的表达水平。采用流式细胞仪、CCK-8、软琼脂集落形成实验、划痕实验和Transwell实验检测癌细胞凋亡、增殖、迁移和侵袭能力。CO-IP实验验证SH2B3/JAK2和SH2B3/SHP2的相互作用。采用裸鼠移植瘤模型检测SH2B3在体内肿瘤生长中的作用。. 我们研究结果证实：肺癌组织和细胞中SH2B3表达降低，TGF-β1表达升高。SH2B3降低、TGF -β1高表达与预后不良相关。SH2B3过表达可抑制失巢凋亡抵抗、内皮细胞转化和癌细胞增殖、迁移和侵袭，而TGF -β1通过减少SH2B3促进上述过程。SH2B3直接与JAK2和SHP2结合，分别抑制JAK2/STAT3和SHP2/Grb2/PI3K/AKT信号通路，从而降低失巢凋亡抵抗、EMT和癌细胞的增殖、迁移和侵袭。SH2B3过表达可抑制肺癌细胞体内生长。. 我们实验阐明了SH2B3在肺癌中的重要作用，揭示了TGF-β1/SH2B3轴通过JAK2/STAT3和SHP2/Grb2/PI3K/AKT信号通路调控肺癌细胞的失巢凋亡抵抗和转导过程。这些发现为未来治疗的发展提供了潜在的靶点。