The Effect And Mechanism Of Linc00261 On Suppressing Epithelial-Mesenchymal Transition In Hepatocellular Carcinoma

Metastatic recurrence is a major factor influencing the long-term prognosis of patients witih hepatocellular carcinoma(HCC)after radical resection.Epithelialderived tumor cells undergo epithelial-mesenchymal transition(EMT)and acquire the characteristics of mesenchymal cells,which are the basis for invasion and metastasis.Therefore,elucidating the molecular mechanism of EMT is of great value.In this study,the next generation sequencing was used to screen out some long non-coding RNAs(LncRNAs)that were differentially expressed between HCC and normal liver tissues.LncRNAs that might participate in EMT were selected and researched by in vitro cell lines functional experiments and clinical tissue samples detection to elucidate the role of this lncRNA in EMT,which laid the foundation for future elucidation of the mechanism of metastasis and recurrence of HCC.1.LncRNA sequencing,validation in clinical HCC samples and functional prediction of target gene.Three HCC clinical samples and two normal liver samples were obtained from HCC patients and hemangioma patients,and 248 differentially expressed LncRNAs were discovered by lncRNA sequencing.Gene Ontology analysis shows that linc00261 participates in the regulation of multiple genes of the components,biological pathways of cell-cell adhesion process,which means linc00261 might control the process of EMT of HCC.By bioinformatics analysis,we find that linc00261 have the highest expression in normal liver tissues,while in hepatocellular carcinoma,low linc00261 expression correlate with a shorter overall survival period.2.Linc00261 expression in hepatocellular carcinoma and its clinical significance.Linc00261 expression level is lower in tumor tissues than adjacent normal tissues in 32%(32/100)of HCC patients.Meanwhile,the relative linc00261 expression correlates with preoperative serum AFP level,tumor size,the existence of microvascular invasion,TNM staging and recurrence status.Patients with lower expression of linc00261 in HCC tissues had a shorter relapse-free survival(RFS)than that in higher linc00261 expression group.And COX regression analysis showed that the low expression of linc00261 in HCC tissues was an independent prognostic factor of RFS in HCC patients.Fluorescence in situ hybridization showed that the expression level of linc00261 in micro-vascular tumor cells was significantly lower than that in normal liver cells,suggesting that cancer cells with low expression of linc00261 may have more invasive and metastatic capacity.3.Effect of Linc00261 on the proliferation,migration,invasion and EMT process in hepatocellular carcinoma cells.To investigate the effect of linc00261 on the biological characteristics of hepatoma cells,we knocked-down and overexpressed linc00261 with siRNA and lentivirus,respectively.CCK-8 assay,transwell assays,western blotting and immunofluorescence staining showed that linc00261 had no significant effect on proliferation.After linc00261 expression knockdown,the migration and invasion ability were significantly enhanced,the process of EMT was promoted,whereas after overexpression of linc00261,the migration,invasion ability and EMT process were inhibited.4.The role of linc00261-FOXA2 axis in hepatoma cell migration,invasion and EMT.The expression of linc00261 and FOXA2 were firstly proved positively correlated in cell lines and in tissue samples.Meanwhile,real-time quantitative PCR,western blotting,immunofluorescence staining showed that linc00261 had the effect of upregulating the expression of FOXA2 mRNA and protein;Subsequently,the transwell assays and western blotting showed that FOXA2 had the effect of inhibiting migration,invasion and EMT process on liver cancer cells.Finally,knockdown of FOXA2 after linc00261 overexpression restored cell migration,invasion ability and EMT process,which predicts that linc00261-FOXA2 axis inhibits cell migration,invasion and EMT process.In conclusion,we found that the low expression of linc00261 in HCC tissue is an independent risk factor for patient-free survival.Linc00261 can inhibit migration,invasion and the EMT process by up-regulating the expression of its adjacent gene FOXA2.The linc00261-FOXA2 axis is expected to be a therapeutic target for HCC metastasis and recurrence.