Mechanism Of Liver Metastasis In Colorectal Cancer By Mir-17/92 Cluster

Background/AimColorectal cancer(CRC)is one of the most common cancers with the highest mortality in the world.The high metastatic and recurrence is the main reason for high mortality of colorectal cancer.MicroRNAs(miRNAs)in serum or cancer tissues have been widely reported as biomarkers of CRC.In this study,we found that some of the miRNAs in miR-17/92-cluster were significantly elevated in primary colorectal cancer tissues compared with adjacent tissues.Further analysis revealed that miR-17-5p?miR-19a-3p?miR-92a-3p directly suppressed PTEN,thereby inhibiting the inhibitory effect of PTEN on the phosphoinositide 3-kinase(PI3K)/AKT pathway.When the PI3K/AKT pathway is activated,the downstream canonical Wnt/?-catenin pathway is facilitated.Thus,nuclear translocation of ?-catenin is promoted,which led to C-myc,SNAIL and other transcription factors are up-regulated.This series of processes eventually induced epithelial-mesenchymal transition(EMT)and enhanced metastasis of CRC cell in vitro and in vivo.The results of this study suggested a critical role of miR-17/92-cluster in promoting CRC liver metastasis.Methods1 Bioinformatics analysis of RNA-seq and miRNA-seq in colorectal cancer patients from the TCGA database to identify dysregulated miRNAs and mRNAs in cancer and adjacent tissues.The miRNA and target genes were predicted by using the UALCAN and Starbase 3.0 platforms to analyze the correlation between selected miRNAs and mRNAs.2 Wound-healing assay,immunoblotting and Real-time Quantitive PCR were used respectively to detect the migration ability,protein expression and miRNA expression of colorectal cancer cell lines such as sw480,sw620,Hct116 and DLD-1.3 Transwell migration assay was used to observe the in vitro migration ability of sw480 cell line after transfection of miRNA mimic.4 The Real-time Quantitive and dual luciferase reporter experiments were used to verify the effect of miR-17/92-cluster on the target gene.Immunoblotting was used to detect the expression of EMT-related proteins in the sw480 cell line after transfection of miR-17/92-cluster.5 Immunofluorescence assay was used to observe the localization of E-cadherin and ?-catenin after transfection of miRNA mimic.Co-immunoprecipitation was used to verify the binding of E-cadherin to ?-catenin in the sw480 line.Top-Flash assay was used to verify the activity of TCF/LEF-1.6 The mouse liver metastasis model was constructed by injecting sw480 cells into the spleen.After the tumor formation,the experimental group was injected with the agomiR-19a-3p by tail vein injection.The liver metastasis condition was observed after the fith time injection.Results1 MiR-17/92-cluster is highly expressed in colorectal cancer tissues and high metastatic colorectal cancer cell lines.2 MiR-17-5p,miR-19a-3p,and miR-92a-3p can directly act on the 3'UTR of PTEN to inhibit the expression of PTEN protein,which in turn affects the PTEN/PI3K/AKT pathway.3 Activation of the AKT pathway affects the expression of transcription factors such as C-myc and SNAIL associated with cell transfer by affecting the classical Wnt/?-catenin pathway,thereby affecting the stability of the epithelial cell marker E-cadherin.Conclusion1 MiR-17/92-cluster showed high expression in colorectal cancer patients.The expression of PTEN in all stages of colorectal cancer patients was lower than that in normal subjects,and the two were negatively correlated.2 MiR-17-5p,miR-19a-3p,miR-92a-3p can activate the AKT/SNAIL pathway and then regulate WNT/p-catenin by inhibiting the expression of PTEN protein to promote the EMT process of colorectal cancer cell lines,which will enhance its metastasis ability.