苦荞凝集素靶向抑制结肠癌细胞HCT116增殖的机制

发布时间：2018-10-22 20:48

【摘要】：苦荞凝集素(tartary buckwheat lectin,TBL)是一类兼具核糖体失活蛋白N-糖苷酶活性和芦丁水解酶活性的糖蛋白,具有显著的抑制结肠癌细胞增殖的功能。先前研究表明,TBL可以下调结肠癌细胞HCT116中包括miR-135a和miR-135b在内的一些oncomiRs的表达,并且可以抑制结肠癌细胞HCT116的增殖。因此,我们推测TBL可能通过调控oncomiRs从而抑制HCT116细胞的增殖。本研究中,我们构建了包含miR-135a和miR-135b结合位点的腺瘤性结肠息肉病基因(adenomatous polyposis coli,APC)的3'-UTR区。双荧光素酶报告系统表明,经过TBL处理后,实验组的荧光素酶活性较对照组高,而且用miR-135ab的反义核酸anti-miR-135ab处理后和实验组有相似的结果。Western印迹分析表明,TBL处理HCT116细胞24 h后,细胞中APC和p-β-catenin的表达上调,总β-catenin的表达下调,且存在剂量依赖效应,而对GSK-3β的表达无明显影响。进一步探究了TBL进入细胞的方式,我们发现,加入TBL作用2 h后,其主要存在于HCT116细胞的表面,部分进入细胞内部,在细胞表面可以同半乳糖凝集素galectin-3竞争结合细胞膜表面受体,并且存在剂量和时间依赖性。这些结果表明:TBL以HCT116细胞表面的galectin-3受体为靶点,通过内吞作用进入细胞,进而调控HCT116细胞中miR-135ab的表达,影响Wnt信号通路而抑制结肠癌细胞的增殖。
[Abstract]:Tartary buckwheat lectin (tartary buckwheat lectin,TBL) is a kind of glycoprotein with both ribosomal inactivating protein N-glycosidase activity and rutin hydrolase activity. Previous studies have shown that TBL can down-regulate the expression of some oncomiRs including miR-135a and miR-135b in HCT116 of colon cancer cells and inhibit the proliferation of HCT116 cells. Therefore, we speculate that TBL may inhibit the proliferation of HCT116 cells by regulating oncomiRs. In this study, we constructed the 3'-UTR region of adenomatous colonic polyposis gene (adenomatous polyposis coli,APC containing miR-135a and miR-135b binding sites. The double luciferase report system showed that the luciferase activity of the experimental group was higher than that of the control group after TBL treatment, and the results were similar to those of the experimental group treated with miR-135ab antisense nucleic acid anti-miR-135ab. Western blot analysis showed that TBL treated HCT116 cells for 24 h. The expression of APC and p- 尾-catenin was up-regulated and the expression of total 尾-catenin was down-regulated in a dose-dependent manner, but had no effect on the expression of GSK-3 尾. We found that TBL mainly existed on the surface of HCT116 cells after 2 h of TBL treatment, and some of them entered into the cells. On the cell surface, galactose agglutinin (galectin-3) can compete with galactose agglutinin (galectin-3) to bind to cell membrane receptors in a dose-and time-dependent manner. These results suggest that TBL targeting the galectin-3 receptor on the surface of HCT116 cells enters the cells through endocytosis and then regulates the expression of miR-135ab in HCT116 cells and inhibits the proliferation of colon cancer cells by affecting the Wnt signaling pathway.
【作者单位】：化学生物学与分子工程教育部重点实验室山西大学生物技术研究所;
【基金】：国家自然科学基金项目(No.31171659/31300653) 山西省科技平台项目(No.2014091028)资助~~
【分类号】：R735.35

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