Role Of Long Non-coding RNA BANCR In Colorectal Cancer

[Background] Long non-coding RNAs (lncRNAs) are newly identified regulators in tumorigenesis and tumor progression. Studies have shown that BRAF-activated long non-coding RNA (BANCR) is overexpressed in melanoma and has a potential functional role in melanoma cell migration. This study tries to detect the expression level of BANCRin colorectal cancer andthe effects of BANCRon colorectal cancer cell function, to reveal the function and molecular mechanisms of BANCR in colorectal cancer preliminary.[Methods]1. Fifty six surgical samples of colorectal cancer were collected, the expression levels of BANCR were detected by qRT-PCR, the relationship between the expression of BANCR and the clinicopathological factors of colorectal cancer was analyzed; the expression of BANCR levels in colorectal cancer cell line HCT116, LoVo, HT-29, SW480, SW620, Caco-2and the normal intestinal epithelial cell line CCD841CoN were detected by qRT-PCR.2. By plasmid and lentivirus construction, stably transfected cell lines of BANCR-downregulated and BANCR-upregulated were established respectively, the changes of cell proliferation, apoptosis and migration were detected by CCK-8assays, flow cytometry, wound healing assays and transwell assays.3.Changes in cell morphology were observed under inverted microscope before and after the BANCR expression was changed; the changes on theprotein level and mRNA level of EMT associated gene (E-Cadherin and Vimentin) in BANCR-downregulated and BANCR-upregulated cells were detected by qRT-PCR and Western blot, respectively.4. After using MEK pathway inhibitor U0126in HCT116cells and BANCR-overexpressed HCT116cells, changes of migration were tested by transwell assays, and the changes of the expression of E-Cadherin and Vimentin were detected by qRT-PCR and Western blot.[Results]1. The relative expression of BANCR in cancer tissues was1.6±0.4, which was significantly higher than0.9±0.7of the adjacent normal tissues (p<0.05). The results of clinicopathological analysis showed that this overexpression was significantly correlated with lymphatic metastasis and the tumor stage (p<0.05), the result of multivariate analysis showed that lymph node metastasis and tumor stage(stage Ⅲ-Ⅳ) were the independent risk factors influencing the high expression of BANCR. Compared with normal intestinal epithelial cells, expression of BANCR in HCT116, LoVo, HT-29, SW480, SW620cells increased significantly (P<0.05), while no significant difference was found in Caco-2cells (P>0.05).2. Stably transfectedcell lines of BANCR-downregulated and BANCR-upregulated were established successfully,the wound healing experimentsand transwellassays demonstrated that BANCR can promote the migration of colorectal cancer cells. But CCK-8assays andflow cytometryshowed that BANCR had no obvious effects on cell proliferation and apoptosis.3. Under an inverted microscope, we saw the morphology of HCT116cells turned round or oval rather than spindle, similar tofibroblastcellsafter the BANCR wasknocked-down. Western blot, qRT-PCR results showed that the downregulation of BANCR levels in HCT116cells was associated with downregulated Vimentin and upregulated E-cadherin both on the mRNA and protein levels, and in BANCR-overexpressed Caco-2cells, E-cadherin was downregulated on mRNA level, while Vimentin expression was upregulated on the mRNA and protein levels.4. After using MEK inhibitor U0126, the transwell assays showed the migrated cells decreased and E-cadherin was upregulated, while Vimentin expression was downregulated.[Conclusion] Theupregulated BANCR expression was related to the TNM stage and LN metastasis, BANCR was upregulated in CRC cells, compared with normal intestinal epithelial cells. Moreover, BANCR promotes the migration of colorectal cancer cells, and indcesepithelial mesenchymal transition through an ERK pathway dependent mechanism, but had no obvious effects on cell proliferation and apoptosis.