MiR-144-3p Inhibited The Invasion And Metastasis Of Colorectal Cancer By Targeting ZEB1/2

Purposes:miRNAs play an important role in the occurrence and development of tumors,and they can regulate various biological processes such as tumor proliferation,apoptosis,metastasis,and angiogenesis.Studies showed that miR-144-3p was low expressed in tumor tissues and acted as a tumor suppressor to inhibit tumor metastasis.However,the clinical significance and biological function of miR-144-3p in colorectal cancer(CRC)have not been elucidated.This study aimed to explore the function and mechanism of miR-144-3p in colorectal cancer,and provide new targets for the prevention and treatment of colorectal cancer.Methods and Contents:1.Expression of miR-144-3p in CRC and its relationship with clinicopathological features and prognosis of patientsThe expression levels of miR-144-3p were detected by RT-PCR in CRC cell lines and tissues from a training cohort including 160 CRC patients,and a validation cohort consisting of 140 matched colorectal cancer tissues and corresponding para-carcinoma nontumor tissues was used for validation analysis.The correlation of miR-144-3p expression level with clinicopathological characteristics and survival prognosis of patients in training cohort and validation cohort was statistically analyzed,and Cox proportional hazards regression model was used to identify independent prognostic factors related to OS and DFS.2.The effect of miR-144-3p on the proliferation and growth of colorectal cancer cellsMethyl thiazolyl tetrazolium(MTT)experiment,cell clone formation experiment,5-ethynyl-2’-deoxyuridine(Ed U)experiment and other in vitro experiments were used,and animal subcutaneous xenograft tumor models were constructed to detect the effect of miR-144-3p on the proliferation of CRC cells.3.The effect of miR-144-3p on the invasion and metastasis of colorectal cancer cellsIn vitro experiments such as cell migration assay,cell invasion assay,homogenous adhesion assay,and heterogeneous adhesion assay were used,and in vivo liver metastasis models were constructed to detect the effect of miR-144-3p on the invasion and metastasis of CRC cells.4.The effect of miR-144-3p on EMT of colorectal cancer cellsThe expression levels of epithelial-mesenchymal transition(EMT)-related molecular markers were detected by RT-qPCR and Western Blot(WB),and the remodeling of cytoskeleton and the change of cell morphology were observed by Immunofluorescence assay(IF)to explore the effect of miR-144-3p on EMT.5.Identification and validation of downstream target genes of miR-144-3pThe target genes of miR-144-3p were preliminarily predicted by bioinformatics analysis of Targetscan,miRDB,miRDIP and miRTar Base,among which EMT-TFs were selected.The targeted regulatory relationship between miR-144-3p and target genes was verified by dual-luciferase gene reporter experiment.Results:1.The low expression of miR-144-3p in CRCCompared with human normal colonic epithelial cells(FHC),the expression level of miR-144-3p was low in CRC cells;the expression level of miR-144-3p was significantly down-regulated in cancer tissues compared with matched paracarcinoma nontumor tissues.2.Low miR-144-3p expression was associated with malignant clinicopathological features and poor prognosis in patients with CRCIn the training cohort,miR-144-3p expression level was correlated with tumor size,pT stage,pN stage,distant metastasis,and lymphatic/microvascular/neural invasion;in the validation cohort,miR-144-3p expression level was correlated with tumor Size,tumor differentiation,pT stage,pN stage,distant metastasis,and lymphatic/microvascular/nerve infiltration.Cox regression analysis showed that low expression of miR-144-3p was an independent risk factor for 5-year disease-free survival(DFS)and 5-year overall survival(OS)in patients with colorectal cancer.3.miR-144-3p inhibited the proliferation and growth of colorectal cancer cellsIn vitro functional experiments showed that miR-144-3p-mimics inhibited the in vitro proliferation ability of LoVo cells,while miR-144-3p-inhibitors promoted the proliferation ability of HCT116 cells.Subcutaneous tumorigenesis experiments in nude mice showed that overexpression of miR-144-3p inhibited the growth of colorectal cancer cells in vivo.4.miR-144-3p inhibited the invasion and metastasis of colorectal cancer cellsIn vitro functional experiments showed that miR-144-3p inhibited the in vitro migration,invasion and cell-ECM adhesion ability of colorectal cancer cells,and promoted cell-cell adhesion ability.In vivo liver metastasis model in nude mice showed that overexpression of miR-144-3p inhibited the metastasis of colorectal cancer cells in vivo.5.miR-144-3p inhibited EMT in colorectal cancer cellsmiR-144-3p-mimics promoted the expression of epithelial markers-E-cadherin and inhibited the expression of mesenchymal markers-Vimentin in LoVo cells.Conversely,miR-144-3p-inhibitors inhibited the expression of E-cadherin in HCT116 cells and enhanced the expression of vimentin.Mi R-144-3p also affected F-actin aggregation and cell morphology in CRC cells.6.miR-144-3p inhibited invasion and metastasis by regulating downstream target genes ZEB1/2Bioinformatics analysis preliminarily predicts that EMT-related transcription factors-Zinc Finger E-box binding homeobox-1(ZEB1)and ZEB2 were potential targets of miR-144-3p,which were verified in dual luciferase gene reporter assay.The experiments confirmed that miR-144-3p could target and inhibit the expression of ZEB1/2.Mir-144-3p-mimics inhibited the expression of ZEB1 and ZEB2 in LoVo cells,while Mir-144-3P-inhibitors promoted the expression of ZEB1 and ZEB2 in HCT116 cells.Conclusions:1.The expression of miR-144-3p was down-regulated in colorectal cancer,and its low expression was significantly correlated with the poor prognosis of colorectal cancer patients.2.miR-144-3p inhibited the proliferation,growth,invasion,metastasis and EMT of CRC cells.3.miR-144-3p inhibited the invasion and metastasis of colorectal cancer by regulating ZEB1 and ZEB2.