Retinoic Acid-induced RAI2 Gene Expression Inhibits Wnt/β-catenin Signaling Pathway And Promotes Chemotherapy Sensitivity In Colorectal Cancer

Objective:To investigate the role of retinoic acid induced protein 2 gene(RAI2)as a novel antagonist of Wnt/β-catenin signaling pathway in colorectal cancer(CRC)and its mechanism.Methods: There were 298 eligible CRC patients in our study.Kaplan-meier curves were used to analyze the relationship between 5-year overall survival time and relapse-free survival and RAI2 expression,while cox analyses were performed to evaluate the prognostic significance of RAI2 in CRC.Cell spheroidization test and flow cytometry were employed to evaluate the influences of RAI2 expression on stem cell-like properties of CRC cells,while MTT assay was adopted to estimate the effects of RAI2 on chemosensitivity of CRC cells.Protein immunoprecipitation,immunofluorescence and dual luciferase reporter system were applied to investigate the interaction between RAI2 and CTBP2,and the regulation of RAI2 on CTBP2 expression was detected by western blot.Based on immunohistochemistry,cell transfection and western blot analyses,the influences of RAI2 on activation of Wnt/β-catenin signaling pathway was evaluated.Results: Low RAI2 expression was significantly associated with dismal 5-year overall survival(OS)(P = 0.0102)and 5-year recurrence free survival(RFS)(P = 0.0029).According to Cox proportional risk model,low expression of RAI2 was an independent prognostic biomarker for poor 5-year OS(P = 0.027)and RFS(P = 0.022).The re-expression of RAI2 significantly suppressed the cell sphere formed by CD133 + cells,inhibited the expression of stem cell markers such as CD133,SOX2 and Oct4.The re-expression of RAI2 enhanced the chemosensitivity of CRC cells to oxaliplatin(L-OHP)and fluorouracil(5-FU),while knockout of RAI2 significantly reduced the chemosensitivity of CRC cells.RAI2 could interact with CtBP2 through ALDLS sequences,consequently regulating CtBP2 expression in CRC.The analysis of TCGA data set showed the negative association between RAI2 and CtBP2 expression.The expression of RAI2,p-β-catenin,ASCL2 and LGR5 in 298 CRC samples detected by IHC showed that the expression of RAI2 was positively correlated withβ-catenin phosphorylation(r=0.8866,P < 0.0001).It was negatively correlated with ASCL2expression(r=-0.1674,P=0.0037)and LGR5 expression(r=-0.1580,P=0.0063).Immunofluorescence assay and Western blot analysis showed that RAI2 expression suppressed the fluorescence activity of Wnt signal,increased the phosphorylation of β-catenin,with down-regulated target genes c-Myc,cyclin D1,ASCL2 and LGR5.In contrast,the mutated RAI2,which can’t interact with CtBP2,has no above effects.Conclusions: As an independent poor prognostic marker,RAI2 inhibits Wnt signaling by interacting with or down-regulating CtBP2,resulting in repression of stem cell-like properties and increased chemosensitivity of CRC cells,Thus,RAI2-based detection may provide a novel therapeutic target for the treatment of patients with CRC.