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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 kind of glycoprotein which has the activity of ribosomal inactivating protein N-glycosidase and rutin hydrolase, and has a remarkable function of inhibiting the proliferation of colon cancer cells. Previous studies have shown that TBL can down-regulate the expression of some oncomiRs in colon cancer cells HCT116 including miR-135a and miR-135b and can inhibit the proliferation of colon cancer cells HCT116. 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 polyposis coli (APC) containing miR-135a and miR-135b binding sites. The dual luciferase reporter system showed that the luciferase activity of the experimental group was higher than that of the control group after TBL treatment, and similar results were obtained with the experimental group after treatment with miR-135a & b antisense oligonucleotide anti-miR-135a & b. Western blotting analysis showed that the expression of APC and p-β-catenin was up-regulated and the expression of total β-catenin was down-regulated in HCT116 cells treated with TBL for 24 h, and there was a dose-dependent effect, but no significant effect on the expression of GSK-3β . Further explore the TBL into the cell, we found that after adding TBL for 2 h, it is mainly present on the surface of HCT116 cells, partly into the cells, and can compete with the galectin-3 on the cell surface for binding to the cell membrane surface Body, and there is dose and time dependent. These results indicate that TBL targets the galectin-3 receptor on the surface of HCT116 cells and enters the cell via endocytosis, thereby regulating the expression of miR-135a & b in HCT116 cells and affecting the proliferation of colon cancer cells through affecting the Wnt signaling pathway.