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目的:研究RNA结合基序单链相互作用蛋白3(RNA-binding motif,single-stranded-interacting protein 3,RBMS3)对胃癌细胞侵袭的影响,并探讨其可能的分子作用机制。方法:首先采用实时荧光定量PCR和蛋白质印迹法检测人正常胃黏膜上皮细胞GES-1以及胃癌细胞MKN-28、MKN-45、NCI-N87和SGC-7901中RBMS3的表达水平。然后采用脂质体转染法将RBMS3过表达载体[RBMS3-pcDNA3.1(+)]转染至胃癌MKN-45和SGC-7901细胞中,同时设置空白对照组和空载体转染的阴性对照组。采用实时荧光定量PCR和蛋白质印迹法验证RBMS3过表达效果后,Transwell小室法检测细胞侵袭能力的变化,实时荧光定量PCR和蛋白质印迹法检测细胞中上皮-间质转化相关蛋白以及Wnt信号通路中β-连环蛋白(β-catenin)的表达水平变化。最后,用Wnt通路激动剂氯化锂(lithium chloride,LiCl)处理RBMS3过表达的胃癌MKN-45和SGC-7901细胞后,再次采用Transwell小室法检测细胞侵袭能力的变化。结果:与正常胃黏膜细胞相比,4种胃癌细胞中RBMS3 mRNA和蛋白的表达水平均明显降低(P值均<0.05)。RBMS3过表达质粒转染后,胃癌MKN-45和SGC-7901细胞中RBMS 3基因过表达,2种细胞的侵袭能力随之明显降低(P值均<0.05)。在RBMS3过表达的胃癌MKN-45和SGC-7901细胞中,N-钙黏蛋白(N-cadherin)和β-catenin表达水平均明显降低(P值均<0.05),而E-钙黏蛋白(E-cadherin)表达水平明显升高(P值均<0.05)。用LiCl处理RBMS3过表达的MKN-45和SGC-7901细胞后,RBMS3对2种细胞侵袭的抑制作用均被部分抵消(P值均<0.05)。结论:RBMS3可能通过阻滞Wnt/β-catenin信号通路,抑制人胃癌细胞的侵袭和上皮-间质转化。
Objective: To investigate the effect of single-stranded-interacting protein 3 (RNA-binding motif) RBMS3 on the invasion of gastric cancer cells and to explore its possible molecular mechanism. Methods: The expression of RBMS3 in human normal gastric epithelial cells GES-1 and gastric cancer cells MKN-28, MKN-45, NCI-N87 and SGC-7901 were detected by real-time fluorescence quantitative PCR and Western blotting. Then RBMS3 overexpression vector [RBMS3-pcDNA3.1 (+)] was transfected into gastric cancer MKN-45 and SGC-7901 cells by lipofection method. At the same time, blank control group and empty vector transfected negative control group. After the RBMS3 overexpression was verified by real-time fluorescence quantitative PCR and Western blotting, the invasion ability of RBMS3 cells was detected by Transwell chamber assay. The expressions of epithelial-mesenchymal transition associated protein (TGF-β1) and Wnt signaling pathway were detected by real-time fluorescence quantitative PCR and Western blotting - Changes in the expression of β-catenin. Finally, the invasiveness of MKN-45 and SGC-7901 cells expressing RBMS3 over-expressed by Wnt pathway activator lithium chloride (LiCl) was detected by Transwell chamber assay. Results: Compared with normal gastric mucosal cells, the expressions of RBMS3 mRNA and protein in four kinds of gastric cancer cells were significantly decreased (all P <0.05). RBMS3 gene overexpression in gastric cancer MKN-45 and SGC-7901 cells after transfection with RBMS3 overexpression plasmid significantly decreased the invasion ability of both cell lines (all P <0.05). The expression of N-cadherin and β-catenin in gastric cancer MKN-45 and SGC-7901 overexpressed in RBMS3 were significantly decreased (all P <0.05), whereas E-cadherin E-cadherin expression was significantly increased (P value <0.05). The inhibitory effect of RBMS3 on invasion of both cell lines was partially offset by LiCl treatment of MKN-45 and SGC-7901 cells overexpressing RBMS3 (P <0.05). Conclusion: RBMS3 may inhibit invasion and epithelial-mesenchymal transition of human gastric cancer cells through blocking Wnt / β-catenin signaling pathway.