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真核翻译起始因子4γ2调控N-乙酰氨基半乳糖转移酶1 mRNA对胃癌细胞恶性表型的影响

Effect of eukaryotic translation initiation factor 4γ2 on the malignant phenotype of gastric cancer cells regulated by N-acetylgalactosaminotransferase 1 mRNA

    摘要
  • 摘要:
    目的 探讨真核翻译起始因子4γ2(EIF4G2)通过调控N-乙酰氨基半乳糖转移酶1(GALNT1) mRNA对胃癌进展的影响。
    方法 通过生物信息数据库分析GALNT1、EIF4G2在胃癌细胞和胃癌组织中的表达及与胃癌患者预后的关系, 并分析胃癌组织中EIF4G2、GALNT1表达的相关性。采用实时荧光定量聚合酶链反应(RT-qPCR)检测GALNT1 mRNA、EIF4G2 mRNA在人胃黏膜上皮细胞GES-1和人胃癌细胞(AGS细胞等)中的表达水平，并采用蛋白质印迹法(Western blot)检测GALNT1、EIF4G2蛋白表达水平。建立干扰GALNT1和过表达EIF4G2的AGS细胞株，采用CCK-8法和克隆形成实验检测细胞增殖能力，采用TUNEL实验检测细胞凋亡能力，采用划痕实验和Transwell实验分别检测细胞迁移和侵袭能力，采用Western blot检测凋亡和转移相关蛋白表达水平，采用RIP实验和RNA下拉实验检测GALNT1 mRNA与EIF4G2的结合关系。
    结果 胃癌组织和胃癌细胞中的GALNT1、EIF4G2水平分别高于正常组织和人胃黏膜上皮细胞，差异有统计学意义(P < 0.001), 且与胃癌患者预后差相关; 胃癌组织中, EIF4G2表达与GALNT1表达呈正相关。敲低GALNT1后, AGS细胞活力降低，克隆形成数、迁移和侵袭细胞数减少，凋亡细胞数增加, Bcl-2、MMP2、MMP9蛋白表达降低, Bax、cleaved-caspase3、cleaved-caspase9蛋白表达增加，差异有统计学意义(P < 0.001)。EIF4G2可与GALNT1 mRNA结合，促进GALNT1 mRNA、GALNT1蛋白表达及GALNT1 mRNA稳定性，差异有统计学意义(P < 0.001)。共转染sh-GALNT1-1与oe-EIF4G2可逆转sh-GALNT1-1对AGS细胞增殖、凋亡、迁移和侵袭的作用，差异有统计学意义(P < 0.05或P < 0.01或P < 0.001)。
    结论 EIF4G2可通过稳定GALNT1 mRNA促进胃癌细胞增殖、迁移、侵袭并抑制细胞凋亡，其或可成为胃癌临床诊疗的新靶点。

     

    Abstract:
    Objective To explore the impacts of eukaryotic translation initiation factor 4γ2 (EIF4G2) on gastric cancer progression via stabilizing N-acetylgalactosaminyltransferase 1 (GALNT1) mRNA.
    Methods Bioinformatics database detected GALNT1 and EIF4G2 expression in gastric cancer cells and tissues, the association between GALNT1, EIF4G2 expressions and the prognosis of gastric cancer patients and the correlation between GALNT1 and EIF4G2 expression in gastric cancer tissues. Reverse-transcription quantitative polymerase chain reaction(RT-qPCR) examined levels of GALNT1 and EIF4G2 mRNA expression and western blot examined GALNT1 and EIF4G2 protein levels in human gastric mucosal epithelial GES-1 cell line and gastric cancer cells. AGS cell lines with GALNT1 interference or EIF4G2 overexpression plasmids were established. Cell proliferation was estimated by CCK-8 and colony formation assays. Cell apoptosis was evaluated by TUNEL assay. Cell migration and invasion were respectively measured through wound healing and transwell assays. Western blot was used to test the expression levels of apoptosis-and metastasis-associated proteins. RIP and RNA pull down assays was used to detect the binding relationship between GALNT1 mRNA and EIF4G2.
    Results The expression levels of GALNT1 and EIF4G2 were significantly higher in gastric cancer tissues and cells than those in the normal tissues and human gastric mucosal epithelial cell line (P < 0.001), and associated with poor prognosis of gastric cancer patients. EIF4G2 expression was positively correlated with GALNT1 expression in gastric cancer. After GALNT1 was knocked down, cell viability was significantly reduced, the number of colony formation, migrated and invasive cells was significantly decreased and the number of apoptotic cells was significantly increased (P < 0.001). Bcl-2, MMP2 and MMP9 protein expressions were significantly decreased, while Bax, cleaved-caspase3, cleaved-caspase9 protein expressions were significantly increased (P < 0.001). EIF4G2 bonded to GALNT1 mRNA, and significantly enhanced GALNT1 mRNA expression, GALNT1 protein expression and GALNT1 mRNA stability (P < 0.001). Further more, co-transfection of sh-GALNT1-1 and oe-EIF4G2 significantly reversed the impacts of sh-GALNT1-1 on the proliferation, apoptosis, migration and invasion of AGS cells (P < 0.05, P < 0.01 or P < 0.001).
    Conclusion EIF4G2 might accelerate the proliferation, migration, invasion and suppress the apoptosis of gastric cancer cells via stabilizing GALNT1 mRNA, which is expected to become a novel target for clinical diagnosis and treatment of gastric cancer.

     

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