MIR-214-5P INHIBITS MIGRATION AND INVASION OF COLORECTAL CANCER CELLS BY TARGETING TWIST1

Lan Jing^1,#, JiangLi Shen^2,#,YueDong Liu³, XiaoYan Zhu¹, XingWei Gu^1,*

¹Department of Anorectal Surgery, The People’s Hospital of Danyang; Affiliated Danyang Hospital of Nantong University, Danyang, 212300, Jiangsu Province, China - 2Department of Anus bowel division, Xianyang center hospita, Xianyang, 712000, China-³Department of anal bowel, Liaoning anorectal hospital, Liaoning university of traditional Chinese medicine, Liaoning, 110033, China

Objective: This study aimed to investigate the specific molecular mechanism of miR-214-5p in colorectal cancer (CRC). 

Methods: miR-214-5p overexpressed or low-expressed CRC cells and low-expressed TWIST1 CRC cells were constructed by transfection with miR-214-5p mimics or inhibitors and TWIST1 siRNA. The expression level of miR-214-5p in CRC cells was detected by qRT-PCR. The expression levels of TWIST 1 protein and EMT-related proteins (E-cadherin, vimentin) were detected by Western blot assay. The migration and invasion ability of CRC cells in each group were detected by a cell scratch test and a Transwell invasion test, respectively. The direct target protein of miR-214-5p was predicted using the TargetScan database, and the possible binding sequence was predicted at the same time. Subsequently, luciferase activity in CRC cells co-transfected with miR-214-5p and TWIST1 3 'UTR plasmid was detected by using a double luciferase assay. The expression level of TWIST1 in CRC patients was collected from clinical samples using the UALCAN database. 

Results: miR-214-5p mimics significantly inhibited the invasion and migration of CRC cells, while miR-214-5p inhibitors had the opposite effect. In addition, the TargetScan database predicted that TWIST1 might be a direct target of miR-214-5p. At the same time, the double-luciferase experiment verified that miR-214-5p could directly act on TWIST1, and miR-214-5p negatively regulated TWIST1 levels at both the transcriptional and protein levels. Analysis of the UALCAN database showed that TWIST1 was highly expressed in CRC patients. At the same time, the downregulation of the TWIST1 gene significantly inhibited the migration ability of CRC cells and the number of CRC cells in a Transwell membrane. Cells transfected with miR-214-5p mimics or inhibitors plus TWIST1 siRNA showed no significant difference in migration and invasion ability compared with cells transfected with TWIST1 siRNA alone. 

Conclusion: In summary, we demonstrated that miR-214-5p could inhibit the invasion and migration of CRC cells by targeting TWIST1 and inhibiting EMT.