The Transcription Factor Otc4A Stimulates the Proliferation, Invasion, and Stemness of Colorectal Cancer Cells by Inhibiting the Regulation of miR-7-5p on TLR4.

Abstract

Background To investigate the effects and mechanism of octamer-binding transcription factor 4 (Otc4A) on proliferation, invasion, and stemness of colorectal cancer (CRC) cells. Methods Firstly, normal fetal human cells (FHC, colon epithelial cells) and HT29 cells (CRC cells) were cultured. The expression levels of Otc4A, miR-7-5p, and TLR4 in cells were then detected by qRT-PCR. CCK-8 was adopted to measure cell proliferation rate after Otc4A, miR-7-5p, and TLR4, respectively, were either knocked out or overexpressed in HT29 cells. Later, the cell viability was detected by cell cloning assay; cell invasion by transwell; cell sphere-forming ability by sphere-formation assay; protein expression level of Otc4A, p65, p-p65, and TLR4 by western blot; and the targeting relationships between miR-7-5p and Otc4A as well as miR-7-5p and TLR4 by dual-luciferase reporter assay. Finally, chromatin immunoprecipitation was applied to verify the interaction between Otc4A and miR-7-5p. Results In HT29 cells, Otc4A expression was significantly increased. Additionally, the knockdown of Otc4A prevented HT29 cells from proliferating, migrating, forming spheres, and activating NF–B signaling. Otc4A could negatively regulate miR-7-5p, and miR-7-5p could target TLR4 expression. Besides, a negative correlation was found between Otc4A and miR-7-5p. Finally, the knockdown of miR-7-5p or overexpression of TLR4 could significantly reverse the effect of the knockdown of Otc4A on HT29 cells. Conclusion The transcription factor Otc4A can regulate the level of TLR4 by inhibiting the expression of miR-7-5p and then promote the proliferation and invasion of CRC cell HT29 as well as enhance cell stemness.