GKN2在胃癌细胞增殖、转移中的作用及机制

目的探讨胃动蛋白2(gastrokine 2, GKN2)对胃癌细胞的生长增殖、侵袭、转移的影响及分子机制。方法利用qRT-pCR和Western blot法检测GKN2在胃癌组织中的表达;构建对照细胞株AGS-CON、SGC-7901-CON与GKN2过表达细胞株AGS-GKN2、SGC-7901-GKN2,应用qRT-pCR和Western blot法检测GKN2在各细胞株中的表达变化,RTCA系统和克隆形成实验检测细胞增殖功能,Transwell及划痕实验检测细胞迁移和侵袭功能,并通过Western blot观察GKN2表达变化对PI3K/AKT/PTEN/mTOR信号通路相关蛋白的作用。结果 GKN2在胃癌组织及多株胃癌细胞株中的表达明显低于癌旁组织和正常胃上皮细胞;RTCA系统显示GKN2过表达组的增殖活性明显低于对照组;克隆形成实验结果显示,GKN2过表达组的细胞克隆数和大小明显低于对照组;Transwell及划痕实验结果表明,GKN2过表达组纵向迁移/侵袭数和横向迁移数明显低于对照组。Western blot结果显示,GKN2过表达下调增殖相关蛋白PCNA、Survivin,抗凋亡蛋白BCL-2及转移相关蛋白MMP-7、MMP-9和MMP-2表达水平,而上调Timp2表达水平。Western blot结果亦显示,GKN2影响PI3K/AKT/PTEN/mTOR通路的激活水平,特别是明显上调PTEN的表达。结论 GKN2在胃癌组织及多株胃癌细胞株中呈低表达或表达丢失。GKN2影响胃癌细胞的增殖和转移,可能通过PI3K/AKT/PTEN/mTOR通路促进胃癌的发生、发展。

Role and mechanism of GKN2 in gastric cancer cell proliferation and metastasis

Purpose To explore the effect of gastrokine 2(GKN2)on the growth,proliferation,invasion and metastasis of gastric cancer cells and its molecular mechanism.Methods qRT-pCR and Western blot were used to dectect the expression of GKN2 in gastric cancer tissues.Lentivirus vector was used to build control cell lines AGS-CON,SGC-7901-CON and GKN2 over-expression cell lines AGS-GKN2,SGC-7901-GKN2.While qRT-pCR and Western blot detect GKN2 of expression changes in various cell lines.The proliferation of the gastric cancer cell was quantified by RTCA and clone formation experiment,while the migration and invasion was quantified by Transwell and wound healing proliferation test.The effect of GKN2 expression on the proteins of the PI3K/AKT/PTEN/mTOR signaling pathway was observed by Western blot.Results The expression of GKN2 in gastric cancer tissues and multiple gastric cancer cell lines was significantly lower than that in paracancerous tissues and normal gastric epithelial cells.RTCA showed that the proliferative activity of GKN2 overexpression group was significantly lower than that of control group.The results of clone formation experiments showed that the number and size of cells in the GKN2 overexpression group were significantly lower than those in the control group.Transwell and wound healing experiments results showed that the number of longitudinal migration/invasion and transverse migration in the GKN2 overexpression group was significantly lower than that in the control group.Western blot results showed that GKN2 down-regulated proliferation-related protein PCNA,Survivin,anti-apoptotic proteins BCL-2 and transfer-related proteins MMP-7,MMP-9 and MMP-2,while up-regulated the expression of Timp2.Western blot results also showed that the GKN2 affected the activation level of PI3K/AKT/PTEN/mTOR signaling pathway,especially the significantly up-regulated level of PTEN.Conclusion The expression of GKN2 is under-expressed or lost in gastric cancer tissues and multiple gastric cancer cell lines.Overexpression of GKN2 affects the proliferation and metastasis of gastric cancer cells and may promote the occurrence and development of gastric cancer through PI3K/AKT/PTEN/mTOR.