The Function And Mechnism Of RHBDF1 Promoting The Progression Of Colorectal Cancer Via Wnt/β-catenin Signalling Pathway

Background:Colorectal cancer(CRC)is one of the most common gastrointestinal tumors,which threatens the quality of life of patients and causes serious medical and social burden.Currently,it is still difficult to thoroughly inhibit the pathological progression of CRC.Therefore,it was essential to further explore the molecular mechanism of CRC occurrence and development,which has important scientific significance and clinical value for the early monitoring and intervention of CRC.Previous studies found that RHBDF1 is closely related to the occurrence and development of many cancers,but its potential roles and possible mechanism in CRC have not been fully clarified.Objective:This project intends to explore the potential role and molecular mechanism of RHBDF1 in CRC from the cellular,animal and molecular mechanism levels through gain of function and lose of function,so as to provide new molecular targets and experimental basis for the early diagnosis and treatment of CRC,which contributes to provide novel theoretical basis for the subsequent development of new CRC targeted chemotherapy drugs.Methods:The relationship between RHBDF1 expression and the prognosis of 120 patients with CRC was detected.Meanwhile,the expression of RHBDF1 in CRC and adjacent tissues was detected by q RT-PCR and Western blot.We also detected the expression level of RHBDF1 in CRC cell lines using Western blot,and then constructed stable cell lines with interference and overexpression of RHBDF1 using Lentivirus.In these stable cell lines,we detected the effect of RHBDF1 on the proliferation,invasion and migration of CRC cells by CCK8,clone formation,wound healing and transwell assay.Then,the effect of RHBDF1 on EMT-related markers of CRC cells was detected by Western blot and immunofluorescence assay.We also tested the influence of RHBDF1 on Wnt/β-catenin signaling pathway protein and its downstream related proteins.Furthermore,we design rescue experiments to detect whether RHBDF1 regulate Wnt/β-catenin signaling pathway via APC.Finally,we detected the effect of RHBDF1 on the subcutaneous tumorigenicity of CRC using xenograft models.Results:Immunohistochemical results showed that the expression of RHBDF1 in CRC was significantly higher than that in adjacent tissues.Consistently,the results of q RT-PCR and Western blot also showed that RHBDF1 was highly expressed in CRC.Subsequently,we identified that RHBDF1 was significantly correlated with tumor size,lymph node metastasis,T stage and patient survival.Survival analysis showed that CRC patients with high expression of RHBDF1 was associated with lower overall survival.Western blot showed that RHBDF1 was highly expressed in HCT116 and Lo Vo cell lines,but low expressed in SW480 and SW620 cell lines.Accordingly,we used HCT116 and Lo Vo cell lines to construct stable expression cell lines interfering with RHBDF1,while SW480 and SW620 cell lines for overexpressing RHBDF1.We found that silencing of RHBDF1 significantly inhibited CRC cell proliferation,invasion and migration,while overexpression of RHBDF1 significantly promoted CRC cell proliferation,invasion and migration.Meanwhile,silencing of RHBDF1 reduced the expression of vimentin and N-cadherin and increased E-cadherin and overexpression of RHBDF1 had the opposite effect.Also,we examined the effect of RHBDF1 on Wnt/β-catenin signaling pathway protein.The results indicated that silencing RHBDF1 decreased the expression of total β-catenin and nuclei β-catenin,APC,GSK-3β and Axin-2,which overexpression of RHBDF1 showed oppsite effects.Meanwhile,we explored the influence of RHBDF1 on Wnt/β-catenin signaling pathway downstream related proteins.The results demonstrated that silencing RHBDF1 inhibited the expression of its downstream related proteins(twist,c-myc,Cyclin D1,Tcf4 and MMP-14),while overexpression of RHBDF1 had the opposite effect.The results of rescue experiment suggest the expression levels of downstream genes(TCF4,c-myc,Cyclin D1,MMP-14,and Twist)were increased following APC knockdown in the RHBDF1-silenced HCT116 and LOVO cells,which following APC overexpression,which promotes β-catenin degradation in the cytoplasm,led to diminished TCF4,c-myc,Cyclin D1,MMP-14,and Twist expression in the RHBDF1-overexpressing SW620 and SW480 cells.The results of CRC xenograft mouse model showed that the average tumour volume was typically smaller in the groups with reduced RHBDF1 expression than those with increased RHBDF1 expression.Conclusions:The expression of RHBDF1 in CRC was significantly higher than that in adjacent tissues.RHBDF1 promotes CRC cell proliferation,invasion,migration and EMT through Wnt/β-catenin signaling pathway.APC was the target for RHBDF1 regula-ting Wnt/β-catenin signaling pathway.