Molecular Mechanism Of MiR-760 Targeting EphB3 In The Regulation Of Colorectal Cancer Progression

Colorectal cancer(CRC)is a common gastrointestinal malignancy in the world.Its global incidence rate ranks third behind lung cancer,breast cancer,and the mortality rate ranks fourth behind lung cancer,liver cancer,and gastric cancer.Postoperative recurrence and metastasis are the main cause of death.Therefore,the study about invasion and metastasis mechanism of colorectal cancer is an effective way to reduce the incidence and the mortality of colorectal cancer.MicroRNAs are a class of endogenous small molecule single-stranded non-coding RNAs,regulating the target genes by degrading mRNA or inhibiting its translation,playing a role as a tumor suppressor or promoter.It is important to study the regulation of miRNAs and their target genes in the diagnosis of colorectal cancer.miR-760 is one of the differentially expressed miRNAs screened from the methylation microarray of CRC in our previous research.At present,the role of miR-760 in CRC progression is still not fully understood.EphB3 is a candidate target gene of miR-760 predicted by bioinformatic methods.The aim of this study is to reveal the roles underlying mechanisms of miR-760 and its target gene in colorectal cancer progression,which may provide a new molecular target for the diagnosis and treatment of colorectal cancer.Methods and results1 Expression of miR-760 in colorectal cancer cells in vivo and in vitro and its clinical significance(1)Bioinformatics predicts low expression of miR-760 in CRC cancers,QRT-PCR detects that miR-760 is down-regulated in 40 cases of matched colorectal cancer tissues,and its expression is negatively correlated with lymph node metastasis.(2)QRT-PCR shows that miR-760 is highly expressed in SW480 and HT29,and lowly expressed in SW620 and HCT116.2 The function of miR-760 in colorectal cancer cell lines in vitroMTT assay,Transwell invasion assay,plate colony formation assay and wound healing assay indicate that upregulation of miR-760 inhibits the proliferation,migration and invasion ability of CRC cell lines,while downregulaiton of miR-760 enhances the proliferation,migration and invasion ability of CRC cell lines.3 Identification of miR-760-target gene EphB3 and exploration of their relationship(1)Bioinformatic prediction of miR-760 target genes and verification Bioinformatic assay predictes that EphB3 is one of the target genes of miR-760.Double luciferase reporter system verifies that miR-760 can bind to 3'UTR of EphB3.(2)Analysis of expression of EphB3 and its correlation with miR-760Western blot sresauls show that EphB3 is highly expressed in SW620 and HCT116,and lowly expressed in SW480 and HT29,indicating a negative correlation between the expression of EphB3 and miR-760 in colorectal cancer cells.The results of Western blot and real-time quantitative PCR show that the expression of EphB3 is down-regulated after CRC cells transfected with miR-760 mimics,while the expression of EphB3 reverses after transfected with EphB3 mRNA su.bsequently.Bioinformatics predicts high expression of EphB3 in CRC cancers,and the result of IHC shows that EphB3 is overexpressed in 84 cases of colorectal cancer tissues,and it is positively related with lymph node metastasis and associated with the poor prognosis of CRC patients.miR-760 is negatively correlated with EphB3 expression in 40 cases of matched colorectal cancer tissues,and both are associated with lymph node metastasis.4 Biological function of EphB3 in colorectal cancer cells in vitro and in vivoMTT assay,Transwell invasion assay,plate colony formation assay and vivo tumourigenesis assay in nude mouse indicate that downregulation of EPHB3 inhibit the proliferation and migration ability of CRC cells in vitro and in vivo..5 Explore the molecular mechanism of miR-760/EphB3 in promoting CRC progression(1)The results of flow cytometry assay show that downregulation of EphB3 inhibits the progress of CRC cells from G1 phase to S phase.Up-regulation of P21 protein and downregulation of E2F1,CDK6 and CDK4 proteins were detected in EphB3-silencing CRC cell lines by both qRT-PCR and Western blot assays.(2)The results of flow cytometry assay show that the apoptotic rate of CRC cells is significantly increased in EphB3-silencing CRC cell lines as compared with control cell lines.The expression levels of Caspase-3,Caspase-6,Caspase-8,Bax were increased in EphB3-silencing cell lines as detected by qRT-PCR and WB assay.(3)The results of immunofluorescence assay suggest that down-regulating of EphB3 inhibits EMT and CRC cell sternness by down-regulating beta-catenin,Vimentin,Nanog and up-regulating E-cadherin.(4)The results of WB and Co-IP show that there is an interaction relationship between EphB3 and Tiaml,suggesting a potential role of EphB3 in regulation of Ras-Rac signaling pathway.Conclusion1 miR-760 is down-regulated in CRC,negatively associated with lymphatic metastasis of CRC patients.miR-760 inhibits the proliferation and migration ability of CRC cell lines in vitro.2 EphB3 is one of the target genes of miR-760.EphB3 is upregulated in CRC and promotes CRC cell proliferation and migration.Overexpression of EPHB3 is associated with lymphatic metastasis and poor prognosis of CRC patients.3 miR-760/EphB3 promotes CRC progression by accelerating cell cycle progress,inhibiting cellular apoptosis,and promoting EMT and celluar stemness.The underlying mechanism may be due to activation of Ras-Rac signaling pathway by interaction between EphB3 and Tiaml proteins.