miR-335/TGF-β1/Smad通路调控EMT影响胃癌腹膜转移的机制研究

Peritoneal metastasis is one of the most important factors of gastric cancer prognosis. The mechanism is not only related to the heterogeneity of tumor cell, but also closely associated with the interaction of cancer cell and peritoneal microenvironment. We confirmed that miR-335 inhibited gastric cancer cell invasion and metastasis through regulating Bcl-w and SP1 negativly. Moreover, we found that comparing with the primary tumor cell, miR-335 expressed lower in gastric cancer peritoneal metastases significantly. Its low expression promoted the fibrosis, senescence and apoptosis of peritoneal mesothelium cell. Epithelial mesenchymal transformation(EMT) is an important way of epithelial tumor cell metastasis. It is reported that Sp1, Smurf2 were the key control factors of TGF-β1/smad, which is a classic pathway of EMT. Also, TGF beta regulates the expression of Snail through activating Smad. Combined with the inhibition of Sp1, Snail and upregulation of Smurf2 by miRNA-335, we speculated that low expression of miRNA-335 may induced EMT in tumor cell and peritoneal mesothelium cell damage by activating Snail through TGF-β1/smad, thereby promoting peritoneal metastasis of gastric cancer. This study will provide new ideas and targets for the prevention and treatment of gastric cancer peritoneal metastasis.

腹膜转移是胃癌预后不良的重要因素，其发生机制不仅与肿瘤细胞的异质性有关，亦与癌细胞与腹膜微环境相互作用关系密切。我们首次证实miR-335通过负调控靶基因Bcl-w及SP1抑制胃癌细胞侵袭转移。并发现miR-335在胃癌腹膜转移灶较原发灶癌细胞中显著低表达，其低表达促进腹膜间皮细胞纤维化、衰老及凋亡。上皮间质转化（EMT）是上皮来源的肿瘤细胞浸润转移的重要途径。文献报道，Sp1、Smurf2是EMT的经典诱导通路TGF-β1/smad的关键调控因子；TGF-β使胞浆的Smads活化入核后可调节Snail活性。结合我们证实的miRNA-335抑制Sp1、Snail表达，上调Smurf2表达的结果，推测miRNA-335低表达可能通过激活TGF-β1/smad通路活化Snail，诱导肿瘤细胞EMT，并引起腹膜间皮细胞损伤，从而促进胃癌腹膜转移。本研究将为有效防治胃癌腹膜转移提供新的思路与靶点。

本项目的总体思路是验证miR-335在胃癌腹膜转移中的作用，并进一步探讨其诱导间皮致损因子表达引起腹膜间皮细胞损伤，从而促进胃癌腹膜转移的分子机制，为有效防治胃癌腹膜转移提供新的思路与靶点。研究内容按计划进行。首先检测74例胃癌原发灶及腹膜转移灶中miR-335表达，发现在44例原发灶中表达miR-335的患者腹膜转移灶中有37例miR-335表达降低或缺失。应用miR-335低表达的原代胃癌腹膜转移细胞与间皮细胞共培养，间皮细胞出现衰老、成纤维化及凋亡等不同形式的细胞损伤，且衰老相关蛋白Endoglin表达显著升高。进一步检测126例胃癌患者腹膜标本中Endoglin及ID1表达，发现Endoglin高表达／ID1低表达与患者预后呈负相关，Endoglin表达与肿瘤大小、TNM分期、腹膜播散呈正相关。建立间皮细胞损伤模型GC3，与其共培养的间皮细胞中Endoglin、pSmad2/3、p16表达增高，ID1表达降低。沉默Endoglin表达后，Smad2/3磷酸化受到抑制，ID1表达增高；与GC3共培养间皮细胞的侵袭能力增强，表明Endoglin通过促进肿瘤诱导的间皮细胞老化发挥其影响胃癌腹膜转移的作用。芯片分析及实时定量PCR验证，间皮致损因子TGF-β1、TGF-β3 、bFGF、CTGF表达显著升高。通过生物信息学分析并结合腹腔冲洗液差异表达筛选结果确定TGF-β1是miR-335低表达胃癌细胞诱导间皮细胞损伤进而导致腹膜转移的主要致损因子。Tbx3是受TGF-β1信号通路调节的下游转录因子，检测98例胃癌原发灶及12例正常胃粘膜组织中Tbx3表达情况，发现其在胃癌组织呈高表达，并与肿瘤侵袭深度、淋巴结转移、TNM分期、肿瘤复发相关。Tbx3高表达的胃癌组织中，64%伴有E-cadherin缺失，65.8%伴有Vimentin高表达。建立Tbx3高/低表达胃癌细胞株，增殖及侵袭实验证实Tbx3促进胃癌细胞增殖及克隆形成，增强胃癌细胞侵袭能力。Tbx3过表达促进细胞周期进展，上调cyclin D1及c-myc表达，降低G1期细胞比例 ，增加S期和G2期细胞比例。Tbx3下调 E-cadherin表达，上调N-cadherin及vimentin表达。表明Tbx3通过促进细胞周期进展、诱导EMT促进胃癌细胞侵袭转移。