Src介导的Cx43蛋白酪氨酸磷酸化在胃癌腹腔转移multicellular aggregates抗失巢凋亡中的作用及机制研究

Abdominal exfoliated gastric cancer cells are a prerequisite for the formation of peritoneal metastasis as the seeding cells during the process of peritoneal metastasis, including two existing types, one is singled,isolated gastric cancer cell, second is multicellular aggregates(MCAs) with the acquired ability of anoikis, which is the major seeding cells during the process of peritoneal metastasis. However,the exact mechanism of regulating the anoikis is unknown.In our previous study, a multicellular aggregates culture model was established successfully,and connexin43 expression was observed in MCAs, predominantly with phosphorylation,together with a positive expression of phosphorylating regulating protein Src, based on data review, a hypothesis is presented that C-terminal tyrosine phosphorylation of Cx43 mediated by Src probably plays an important role in anoikis for gastric cancer MCAs. In order to prove it, biomechanical and immunological techniques are employed to investigate the viscoelastic properties, adhesion forces and expression of apoptotic related proteins, C-teminal tyrosine phosphorylation is assessed as well; RNAi, protein immune coprecipitation, site-mutagenesis and in vivo bioluminescence imaging are adopted to investigate the role and exact mechanism of tyrosine phosphorylation of Cx43 mediated by Src against anoikis for gastric cancer MCAs, thus offered novel theoridical and practical basis for the prevention and treatment of peritoneal metastasis of gastric cancer.

腹腔脱落癌细胞是胃癌腹腔转移的"种子"细胞，通常以两种形式存在，一为散在、单个游离癌细胞，常发生失巢凋亡，二为多细胞聚合形成的团簇（multicellular aggregates, MCAs），具有抗失巢凋亡能力，是胃癌腹腔转移的真正"种子"细胞，但调节胃癌MCAs抗失巢凋亡的机制不清。项目组前期成功建立胃癌MCAs培养模型，发现MCAs缝隙连接蛋白Cx43表达，且以磷酸化为主，其C末端酪氨酸磷酸化调节蛋白Src阳性表达，我们推测Src介导的Cx43 C末端酪氨酸磷酸化可能在胃癌MCAs抗失巢凋亡中发挥重要作用。本课题拟应用生物力学技术，检测胃癌MCAs粘弹性、黏附力等生物学行为和凋亡相关分子表达；应用RNA干扰、免疫共沉淀、定点突变和小动物发光技术，深入研究Src介导的Cx43蛋白C末端酪氨酸磷酸化在胃癌MCAs抗失巢凋亡中的作用及机制，从而为胃癌腹腔转移的防治提供新的理论和实验依据。

胃癌是常见的消化系统恶性肿瘤，死亡率居恶性肿瘤首位。而腹腔转移是胃癌术后最常见的转移形式，是导致胃癌患者死亡的最重要原因。根据经典的“种子－土壤”学说，胃癌腹腔转移包括如下三个环节，即胃癌细胞从原发灶脱落入腹腔；游离癌细胞粘附于腹膜间皮细胞并突破腹膜间皮屏障；癌细胞在腹膜下基质定植，形成转移癌结节。因此，腹腔脱落癌细胞作为胃癌腹腔转移的种子细胞，在胃癌腹腔转移中发挥重要作用。.我们前期研究发现，腹腔脱落胃癌细胞通常以两种形式存在，一为散在的、单个游离癌细胞，二为以多个细胞相互粘连形成多细胞团簇或多细胞球（multicellular aggregates/spheroids, MCAs/MCS）。研究发现腹腔散在的、单个游离癌细胞通常发生失巢凋亡（anoikis），而MCAs具有抗失巢凋亡能力，能够在乏氧的腹腔微环境中生长、增殖，可能是胃癌腹腔转移的真正“种子”细胞。MCAs作为独立的结构和功能单元，如何整体协调的发挥抗失巢凋亡功能，目前国内外未见报道。深入揭示胃癌腹腔转移MCAs抗失巢凋亡的分子机制，对于胃癌腹腔转移的防治具有重要意义。本项目从胃癌腹腔转移患者腹水中成功分离胃癌MCAs，Real-time PCR结果表明：腹水中胃癌MCAs中Src的表达水平明显高于散在游离的胃癌细胞，首次证实胃癌腹腔脱落MCAs具有抗失巢凋亡功能；利用BGC823和MKN5成功建立胃癌多细胞团簇的体外三维培养模型， Western blot结果表明：体外三维培养的胃癌MCAs中Src的表达水平明显高于单层贴壁培养的胃癌细胞。成功构建了Src慢病毒载体，抑制胃癌细胞BGC823和MKN45中Src的表达，可以明显降低其形成多细胞团簇的能力，并明显影响MCAs的活力。流式细胞术实验结果表明抑制Src表达可以显著诱导BGC823 MCAs和MKN45 MCAs发生凋亡。Western blot结果表明抑制 Src的表达后，胃癌MCAs 促凋亡蛋白caspase-3表达明显上调，抗凋亡蛋白bcl-2表达明显下调。首次证实首次证实Src 蛋白在胃癌MCAs抗“失巢凋亡”中发挥重要作用。并进一步用Western blot结合免疫共沉淀证实Src通过磷酸化C43在胃癌MCAs抗失巢凋亡中发挥重要作用。本课题首次系统阐释了胃癌腹腔转移MCAs抗失巢凋亡的机制，为胃癌腹腔转移的防治提供理论和实验依据