Emerin Knockdown Induces Migration And Invasion Of HCC Cells By Up-regulating Cytoplasmic P21

Background and purpose:Hepatocellular carcinoma（HCC）,the most common histological type of primary liver cancer,is the sixth most common cancer in the world,and the third leading cause of cancerrelated deaths.Extrahepatic metastasis of the HCC cells is one of the main causes for the low cure rate and high recurrence of HCC,therefore,it is of great significance to further understand the molecular mechanism of HCC metastasis and to explore the biological targets to inhibit HCC metastasis.Human Emerin（EMD）gene is located in Xq28.EMD protein is a wellconserved,ubiquitously expressed protein in the inner nuclear membrane,and plays a key role in maintaining nuclear structure and function.It has also been shown that EMD can bind to the pointed end of growing actin filaments to regulate the assembly of actin filaments.Accumulating evidence indicates that EMD interacts with a number of transcription regulators and plays diverse functions,including the regulation of gene expression,cell signaling,nuclear structure and chromatin architecture.EMD interacts with some proteins to regulate the expression of genes involved in tumor cell migration and invasion,however,the biological role of EMD relevant to the occurrence and development of HCC has not yet been illustrated.Therefore,the present study will focus on the role of EMD in HCC cell migration and invasion and the underlying mechanism.The findings of this study might provide a novel target for the prevention and treatment of HCC.Methods:1.Immunohistochemical staining was performed on tissue microarray（TMA）containing 103 cases of HCC tissues and their adjacent tissues to analyze the differential expression of EMD between HCC tissues and their matched adjacent tissues.2.The lentiviral vectors encoding short hairpin RNA（shRNA）targeting EMD were constructed by molecular cloning approaches,and the lentiviral particles were packaged and harvested.HCC cell lines SMMC-7721 with low metastatic potential and HCC-LM3 with high metastatic potential were transduced with the lentiviruses and selected with puromycin.Stably EMD knocking down HCC cell strains were validated by reverse transcriptionquantitative polymerase chain reaction（RT-qPCR）and Western Blot.3.CCK-8 cell viability assay,EdU cell proliferation assay and cell cycle analysis were performed to study the effect of EMD knockdown on the proliferation of SMMC-7721 and HCC-LM3 cells.4.Transwell cell migration and invasion assays and the rhodamine-conjugated phalloidin for F-actin staining were performed to evaluate the effect of EMD knockdown on the migration,invasion and the assembly of actin cytoskeleton filaments in SMMC-7721 and HCC-LM3 cells.5.In vivo mouse model for experimental metastasis was established by tail vein injection with EMD stably knockdown HCC-LM3 cells or control to evaluate the effect of EMD knockdown on the HCC cell metastasis in vivo.6.RNA sequencing（RNA-seq）was performed to detect the changes of transcriptome of HCC cells caused by EMD knockdown,and the expression of some genes was verified by RTqPCR and Western Blot.7.Western Blot analysis and immunofluorescence staining were used to explore the expression and distribution of p21 protein in EMD knockdown HCC cells.8.Transwell cell migration and invasion assays and the rhodamine-conjugated phalloidin for F-actin immunofluorescence staining were performed to evaluate the effect of silencing of p21 in stably EMD-knocked down HCC cells on the migration,invasion,and the assembly of actin cytoskeleton filaments in HCC cells.Western Blot analysis was used to verify the effect of silencing of p21 in stably EMD-knocked down HCC cells on the expression of epithelial to mesenchymal transition（EMT）related genes.Results:1.Results from immunohistochemical staining of TMA showed that EMD expression in HCC tissues was higher than that in corresponding paracancerous tissues,and the positive EMD staining was mainly observed in the nuclear region in the HCC tissues and in the cytoplasm in the paracancerous tissues.2.Two constructed lentiviral vectors encoding shRNA targeting EMD were successfully constructed.RT-qPCR and Western Blot analysis showed that EMD expression was significantly downregulated in both SMMC-7721 and HCC-LM3 cells after infection with the lentiviruses encoding shRNA targeting EMD,suggesting that stably EMD knockdown HCC cell models were successfully established.3.The results from CCK-8 assay,EdU cell proliferation assay and cell cycle analysis demonstrated that EMD knockdown had no significant effect on the proliferation of both SMMC-7721 and HCC-LM3 cells.4.Data from Transwell cell migration and invasion assays showed that EMD knockdown promoted the migration and invasion of SMMC-7721 and HCC-LM3 cells when compared with those of control cells.The results from the rhodamine-conjugated phalloidin for F-actin immunofluorescence staining displayed more lamellipodia at the leading edge in EMD knocking down HCC cells when compared with the control.5.The results from in vivo mouse model for experimental metastasis showed that,compared with the control,the tumor metastatic potential of HCC-LM3 cells in the liver and lung of mice was facilitated by the silencing of EMD.6.The results from RNA-seq combined with RT-qPCR and Western Blot demonstrated that,compared with the control,the expression of EMT-related genes such as E-cadherin（ECAD）was down-regulated,whereas the expression of Vimentin（VIM）,N-cadherin（N-CAD）,and Snail were up-regulated in EMD knocking down HCC cells.In addition,the upregulation of p21 was also shown in EMD-knocked down HCC cells.7.Data from Western Blot analysis and immunofluorescence staining showed that EMD knockdown increased the distribution of p21 protein in the cytoplasm of HCC cells when compared with those of control cells.8.Results from Trans well cell migration and invasion assays and the rhodamineconjugated phalloidin for F-actin immunofluorescence staining showed that silencing of p21 in stably EMD-knocked down HCC cells significantly decreased the migratory ability and invasiveness of HCC cells,and reduced the formation of lamellipodia,when compared with those of control cells.In addition,the Western Blot analysis further demonstrated that silencing of p21 partially reversed EMD knockdown-mediated induction of EMT-related gene expression including E-CAD,VIM,N-CAD and Snail,in HCC cells.Conclusion:1.EMD knockdown promoted the migration,invasion,and in vivo metastasis of HCC cells.2.EMD regulated the expression of EMT-related genes presumably by up-regulating p21 in HCC cells and promoting the enrichment of p21 protein in cytoplasm,thereby promoting the migration,invasion and in vivo metastasis of HCC cells.