苦荞凝集素靶向抑制结肠癌细胞HCT116增殖的机制

The Mechanism of Tartary Buckwheat Lectin-targeted Inhibition of HCT116 Colon Cancer Cell Proliferation

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-135a&b的反义核酸anti-miR-135a&b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-135a&b的表达,影响Wnt信号通路而抑制结肠癌细胞的增殖。

Tartary buckwheat lectin（ TBL） is a glycoprotein which exhibits ribosome inactivating protein N-glycosidase activity and rutin hydrolase activity,and it has anti-proliferation activity towards CRC cells. Previous works have demonstrated that TBL could down-regulate the expression of some oncomiRs including miR-135ab and inhibit the proliferation of HCT116 colon cancer cells. It was presumed that TBL could suppress the proliferation of HCT116 by oncomiRs regulation. In this study,we cloned 3＇-UTR of APC with potential miR-135ab recognition sequences into dual luciferase reporter. Treating with TBL,the luciferase activity of experiment group was higher than the control group,and the anti-senseoligonucleotides of miR-135ab had the same effect with TBL. Analysis of Western blot showed that after treated with TBL for 24 h,APC and p-β-catenin was up-regulated,whereas total β-catenin was downregulated in a dose-dependent manner and GSK-3β was unaffected. At the same time,the way of TBL enter cell was further investigated,we found that TBL mainly attached to the surface of HCT116,only a part of TBL entered the cells after treating with TBL for 2 hours. In the surface of cells,TBL could compete with galectin-3 to bind to the receptor on cell membrane in a dose-and time-dependent manner.These results suggest that TBL can target the receptor of galectin-3 in the surface of cell membrane and enter the cells by endocytosis. TBL can affect the Wnt signal pathway and inhibit the proliferation of colon cancer cells by regulating the expression of miR-135ab.