Study On The Role And Mechanism Of LncRNA FUNDC2P4 In Residual Hepatocellular Carcinoma After Insufficient Radiofrequency Ablation

Background and purposeAt present,radiofrequency ablation(RFA)has become the main local treatment of advanced hepatocellular carcinoma(HCC)which was not able to be removed.Residual tumor progression after insufficient RFA was reported through promoting epithelial-mesenchymal transition(EMT)to increase the potential of HCC cells invasiveness and metastasis.LncRNAs were reported to be widely involved in tumor growth,invasion and metastasis through their interactions with DNA,protein,and RNA.However,the role of LncRNAs in residual tumor progression after incomplete RFA has remained widely elusive.Materials and methods1.Establishment and characterization of Huh7-H cell lineInsufficient RFA was simulated in vitro by heating Huh7 cells in water bath at 47℃,named as Huh7-H.EMT change was detected by microscope and Western blot.Cell proliferation was determined by CCK8 assay.Cell invasion,migration assays were conducted for functional analysis.2.Identification of LncRNAsDifferential expression profile of EMT related LncRNAs between Huh7-H and Huh7 were analyzed by LncPath human EMT array,and validated by qRT-PCR.The expression of LncRNA FUNDC2P4 was detected by qRT-PCR in HCC cell lines with high invasive potential including SMMC-7721 and HCCLM3 and low invasive potential including Huh7 and Hep 3B.The expression of LncRNA FUNDC2P4 was detected in 25 cases of the human HCC and corresponding adjacent normal tissues,15 cases of the residual HCC tissues after RFA.3.Analysis of the function and mechanism of LncRNA FUNDC2P4GV367-FUNDC2P4 and FUNDC2P4-siRNA were transfected to SMMC-7721 and Huh7 respectively.To investigate the functions of FUNDC2P4,we evaluated its abilities in cell proliferation,migration and invasion.We further investigated the potential mechanism by detecting the expression levels of E-cadherin,N-cadherin and Vimentin.Results1.Establishment and characterization of Huh7-H cell lineWe found that Huh7-H cells showed spindle cell-like morphology.To evaluate EMT changes in Huh7-H cells,we tested several EMT markers.By western blot we found a significant down-regulation of E-cadherin expression,and notably up-regulation of N-cadherin and vimentin.Using CCK8 assay,Huh7-H displayed stronger proliferation ability compared with Huh7.Transwell assays showed that cell migration and invasion were increased in Huh7-H cell compared with Huh7 cell,indicating Huh7-H cells possessed greater abilities in cell migration and invasion compared with Huh7 cells.2.LncRNA FUNDC2P4 is down-regulated in Huh7-H cells and residual HCC tissues after RFALncPathTM Human EMT Array was employed to screen the differential LncRNAs between Huh7 and Huh7-H cells.Hierarchical clustering showed that the significantly differentially expressed LncRNAs were as follows:down-regulated LncRNAs including FUNDC2P4,RPL27P7 and up-regulated LncRNA including MTND4LP14.To validate findings from microarray analysis,we analyzed the expression levels of these three LncRNAs using qRT-PCR between Huh7 and Huh7-H cells.We found that the expression of FUNDC2P4,RPL27P7,MTND4LP14 in Huh7-H cell was 0.137,0.869,1.037 times of that in Huh7 cells,respectively,only LncRNA FUNDC2P4 was observed in significant change,then we chose it for further studies in HCC cell lines.qRT-PCR showed that FUNDC2P4 was expressed significantly lower in HCC cell lines with high invasive potential than that in HCC cell lines with low invasive potential.Furthermore,FUNDC2P4 was highly expressed in normal liver tissues,however,significantly decreased in paired HCC tumors.After treatment with insufficient RFA,FUNDC2P4 dramatically down-regulated in residual HCC tissues even compared with HCC tumors,indicating FUNDC2P4 was negatively correlated with HCC progression.3.LncRNA FUNDC2P4 inhibits cell proliferation,migration and invasionWe developed stably over-expressed cell line of FUNDC2P4 by infecting SMMC-7721 cells with LV-FUNDC2P4.We found that FUNDC2P4 overexpression significantly inhibited cell proliferation ability compared with control cell line.By transwell assay,lower cells were observed under microscopy,indicating that cells were losing the ability of invasion and migration after over expression of FUNDC2P4.By wound healing assay,we observed the same outcomes,SMMC-7721 cells with over-expressed FUNDC2P4 possessed lower potential in wound healing.Then we developed Huh7 cells with stably silencing FUNDC2P4 by infecting with FUNDC2P4-siRNA-121.By depletion of FUNDC2P4,we found that cells gained significantly higher abilities in cell proliferation,invasion and migration compared with control cells.Taken together,these results suggested that FUNDC2P4 suppressed HCC cell progression.4.LncRNA FUNDC2P4 inhibits EMT by regulating E-cadherin expressionWestern blots results showed that the expression of E-cadherin was significantly up-regulated in SMMC-7721 cells with over-expressed FUNDC2P4 compared with that in control cells,whereas neither N-cadherin nor Vimentin expressions was changed.In contrast,the depletion of FUNDC2P4 in Huh7 cells displayed lower expression level of E-cadherin,but no change was found in N-cadherin and Vimentin expressions.Conclusions1.Huh7-H cells show EMT-like morphological changes and stronger abilities of proliferation,migration and invasion,indicating in vitro cell model for mimicking residual tumor cells after insufficient RFA was established successfully.2.Cell and histopathological tests showed that the expression of LncRNA FUNDC2P4 was negatively correlated with the degree of malignancy of HCC.3.LncRNA FUNDC2P4 down-regulation promoted EMT leading to tumor proliferation,invasion and migration by reducing E-cadherin expression in residual HCC after insufficient RFA in vitro.