TBL Inhibit The Proliferation Of Colon Cancer Cells By Regulation On MiR-135a&b

MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that play important role in development and growth of cancer cells. miRNAs can serve as novel therapeutic targets for cancer. Anti-cancer activity of exogenous substances and their regulation in miRNAs can provide a new therapeutic approach for cancer, viral infections and other diseases. In this paper, we studied the anti-cancer mechanism of a novel protein——tatary buckwheat lectin (TBL).The proliferation of 11 kinds of cell lines, which treated with different concentrations of TBL, was determined MTT assay. The morphology of HCT116 cell was observed by an inverted microscope. Cell apoptosis was observed by DAPI dyeing and flow cytometry. The rabbit reticulocyte lysate system and luciferase detection system were used to detect the N-glyectcosidase activity and the inhibition in protein synthesis of TBL.19 miRNAs regulated by TBL validated by qRT-PCR analysis. Double-luciferase reporter assay were used to examine the expression of APC (adenomatous polyposis coli, APC), the target gene of miR-135a&b, treated with TBL. Several key protein levels in Wnt signaling pathway in HCT116 cell, which was treated with TBL, were determined by Western blot.Experiments showed that, TBL significantly and specially inhibited the growth of DLD-1, HCT116 and SW480 cells in a dose-dependent manner. Furthermore, TBL had no proliferative inhibition on human normal liver cells HL7702, human normal colon cells FHC and other types of cancer cells. After treating with TBL, the number of HCT116 reduced, the volume increased significantly, and the nucleus became larger, but we did not find the typical apoptotic bodies around nucleus. These appearances indicated that TBL did not induce apoptosis but other modes of cell death in HCT116 cell. TBL is an atypical type II ribosome inactivating protein, exhibited N-glyectcosidase activity and the inhibition effect in protein synthesis. TBL decreased the expression of six kinds of oncogenic miRNAs, which are miR-21, miR-135a, miR-135b, miR-17-5p, miR-331 and miR-183. The expression of APC, who is the target gene of miR-135a&b, was up-regulated in manner of the inhibition miR-135a&b level by TBL, and then inhibited the level of Wnt pathway.In conclusion, TBL is a type II ribosome-inactivating proteins (ribosome inactivating protein, II-RIP), with higher specificity and lower toxicity. TBL inhibited the growth of colon cancer cell, did not induce apoptosis. The expression of APC, who is the target gene of miR-135a&b, was up-regulated in manner of the inhibition miR-135a&b level by TBL. Furthermore, TBL decreased the level of Wnt pathway. These findings provide new evidences and ideas for anti-cancer activity of natural extracts.