MiR-223-3p increases resistance of colorectal cancer cells to 5-fluorouracil via targeting SORBS1.

OBJECTIVES:5-Fluorouracil (5-FU) is the first-line drug for treating colorectal cancer (CRC), and the resistance of tumor cells to 5-FU is the main cause of chemotherapeutic failure. However, the resistant mechanism is still unclear. This study aims to explore the tumor suppressor genes involved in 5-FU resistance in CRC, and to find the microRNA (miRNA) that regulates these genes. METHODS:CRC data sets GSE28702 and GSE69657 were downloaded from Gene Expression Omnibus (GEO) database, and gene expression profiles of patients in the FOLFOX chemotherapeutic response group and the non-response group were analyzed, and differential expression genes were identified between the 2 groups. Target gene was then selected. Online bioinformatics databases TargetScan, miRwalk, and miRDB were used to predict miRNA targeting the interested gene sorbin and SH3 domain containing 1 (SORBS1). siSORBS1, HA-SORBS1, miR-223-3p mimic, anti-miR-223-3p, and their corresponding negative controls (siNC, HA, miR-NC, and anti-miR-NC) were transfected into CRC cell lines of HCT116 and SW620 by transient transfection technique, respectively. Co-transfection was done with miRNA and plasmid (miR-NC+HA, miR-223-3p mimic+HA, or miR-223-3p mimic+HA-SORBS1) or anti-miRNA and siRNA (anti-miR-NC+siNC, anti-miR-223-3p+siNC, or anti-miR-223-3p+siSORBS1) in HCT116 cells. Real-time reverse transcription PCR (real-time RT-PCR) and/or Western blotting were used to detect the expression levels of SORBS1 and miR-223-3p in cells. After transfection, the cells were treated with different concentrations of 5-FU, and the cell viability was detected by methyl thiazolyl tetrazolium (MTT) method. The targeting relationship between miR-223-3p and SORBS1 was comfirmed by dual luciferase reporter gene assay. RESULTS:There were 409 and 528 highly expressed genes in the FOLFOX chemotherapeutic response group of GSE69657 and GSE28702, respectively. There were 22 overlapping genes in the response group, among which exist 3 tumor suppressor genes might be involved in chemosensitivity in CRC, and SORBS1 was selected as the target gene for further study. Three online bioinformatics databases predicted miRNAs targeting SORBS1 and obtained an intersection molecule miR-223-3p. After treatment with 5-FU (25 µmol/L) for 12-36 h, the levels of miR-223-3p in HCT116 and SW620 cells were significantly down-regulated (all P<0.05). After transfection with siSORBS1 or miR-223-3p mimic, the expression levels of SORBS1 in HCT116 and SW620 cells were down-regulated, and the cell viability was increased (all P<0.05). After transfection with HA-SORBS1 or anti-miR-223-3p, the expression levels of SORBS1 in HCT116 and SW620 cells were up-regulated, and the cell viability was decreased (all P<0.05). The result of dual luciferase reporter gene assay showed that the luciferase activity of cells co-transfected with SORBS1 3'-UTR wild plasmid and miR-223-3p mimic was significantly lower than that of the 3'-UTR wild plasmid and miR-NC cells (P<0.05). Compared with co-transfection with miR-223-3p mimic and HA, the cell viability of cells co-transfected with miR-223-3p mimic and HA-SORBS1 was decreased significantly (P<0.01). Compared with the co-transfected anti-miR-223-3p and siNC, the cell viability of the co-transfected anti-miR-223-3p and siSORBS1 was significantly increased (P<0.05). CONCLUSIONS:MiR-223-3p increases 5-FU resistance in CRC cells by targeting SORBS1,and miR-223-3p is expected to become a new target for clinical treatment of CRC.