Rab20在结直肠癌中高表达并调控结直肠肿瘤细胞的增殖与干性

Overexpression of Rab20 in Colorectal Cancer Mediates the Proliferation and Stemness of Colorectal Cancer Cells

目的探究Ras蛋白家族成员Rab20对结直肠肿瘤细胞增殖及干性的影响。方法应用TIMER2和GEPIA2数据库分析Rab20在人结直肠肿瘤组织及正常组织中的表达;采用Western blot方法检测人正常肠上皮细胞与结直肠癌细胞系XhCRC、SW620中Rab20的表达水平;应用TCGA和STRING数据库对Rab20相关基因进行富集分析;构建敲减Rab20的XhCRC、SW620细胞;转染ts045-VSVG-GFP质粒,采用免疫荧光方法检测Rab20对结直肠癌细胞囊泡运输的影响;CCK-8和细胞球体形成实验检测Rab20敲减对结直肠癌细胞增殖及干性的影响;Western blot法检测β-catenin、Cyclin D1的表达水平。结果人结直肠癌组织及细胞系中Rab20的表达水平明显高于正常组织与细胞。Rab20相关基因富集分析表明Rab20与胞吞胞吐、囊泡运输等过程相关。敲减Rab20可明显抑制结直肠癌细胞的囊泡运输能力。敲减Rab20后,XhCRC细胞增殖及干性显著抑制,且抑制作用在加入Wnt3a蛋白后被抵消。同样在敲减Rab20组中,β-catenin、Cyclin D1蛋白表达水平显著降低,而在加入Wnt3a蛋白后其表达被逆转。结论Rab20在结直肠癌中高表达,调控结直肠癌细胞囊泡运输,并通过Wnt/β-catenin信号通路增强结直肠癌细胞的增殖与干性。

Objective To investigate the effect of Rab20 on the proliferation and stemness of colorectal cancer cells.Methods The expression of Rab20 in human colorectal tumor and normal tissues were analyzed through TIMER2 and GEPIA2 databases.The expression of Rab20 in human normal intestinal epithelial cells and colorectal cancer cell lines(XhCRC and SW620)were detected by Western blotting.Gene Set Enrichment Analysis(GSEA)of Rab20-related genes was performed by using TCGA and STRING database.The Rab20-knockdown XhCRC and SW620 cell lines were obtained and transfected with ts045-VSVG-GFP plasmid to detect the effect of Rab20 on vesicle traffic by immunoflurescence method.The effect of Rab20 knockdown on the proliferation and stemness of colorectal cancer cells were detected by CCK-8 and sphere formation assay.The expression levels ofβ-catenin and Cyclin D1 were detected by Western blotting.Results The expression of Rab20 in human colorectal cancer tissues and cell lines were significantly higher than that in normal tissues and cells.GSEA result indicated that Rab20 was related to phagocytosis,exocytosis and vesicle traffic.Knockdown of Rab20 significantly reduced the vesicular traffic capacity of XhCRC cells.Knockdown of Rab20 impaired the proliferation and stemness of XhCRC cells and the effects were counteracted after adding Wnt3 a protein.Similarly,in shRab20 group,the expression levels ofβ-catenin and Cyclin D1 were significantly reduced,whereas their expression was reversed after the addition of Wnt3 a.Conclusion Rab20 is highly expressed in colorectal cancer,which regulates the vesicle traffic of colorectal cancer cells and promotes the proliferation and stemness of colorectal cancer cells via Wnt/β-catenin signaling pathway.