Construction Of Multi-fluorescent EMT-indicated Model In Hepatocarcinoma And Preliminary Study On Its Function

Background: Malignant tumor is a serious threat to human health. Among all cancercases, hepatocellular carcinoma (HCC) accounts for more than5%and is the fifthleading cause of cancer mortality worldwide. Like other malignant tumors, recurrenceand metastasis are the main causes responsible for the failure of surgery inhepatocellular carcinoma.In the process of metastasis of cancer, the cytology mechanism that loss ofcell-cell contacts, acquisition of migratory designated as EMT(Epithelial-mesenchymal transition) has been more and more popular amongresearchers. However, the most important characteristic of EMT is that the loss ofepithelial marker expression such as E-cadherin and upregulation of mesenchymalmolecular markers. Now, accumulating evidences have suggested that EMT is a keyevent in the metastasis of HCC, which plays a pivotal role in the invasion andmetastasis of HCC. So, how to dynamicly monitor EMT event in HCC is mostimportant for the further analysis on it and the development of specific therapystrategies targeted it.Fluorescence protein reporter gene has become the first choice in theestablishment of reporter system for its advantages such as stable expression inheterologous cells, unnecessary any additional factors, easy to detect, no cytotoxicity,multiple marking on cells by different fluorescent protein combinations, etc. As thelentiviral vector holds characteristics of transfection to dividing and non-dividing cells,low rate of immunological response and no cytotoxicity, effectively integrating intohost genome for stable and long-term expression of therapeutic gene, it has been themost effective ideal gene transfer vectors in gene therapy and scientific research, etc.Therefore, construction of fluorescent EMT indicated model based on lentiviral vector will have a bright clinical significance.Purpose: To construct the biomarker relevant to EMT such as E-cadherin, slugpromoter driven fluorescence reporter system based on lentiviral vector, to lay afoundation for the further study on tracing hepatoma EMT cells, dynamicly displayingEMT process of hepatoma cells as well as establishingt in EMT induction model ofhepatoma cells in vitro.Methods: E-cadherin and Slug promoters were amplified from Hela cell genome, andligased to red、yellow、blue fluorescence proteins respectively, then subcloned into alentiviral vector plasmid to construct the lentiviral vectors of E-cadherin/Slugpromoter with multi-fluorescences: Lv-pE-cad-RFP/YFP/BFP andLv-pSlug-RFP/YFP/BFP. Lentiviruses were produced by calcium phosphatetransfection method following the co-transfection of expressing plasmids andpackaging plasmids in293FT cells. The harvested virus of Lv-pE-cad-YFP thentransfected PLC/PRF/5cells and these cells sorted by FCM were used to verify thevalidity of the virus by qRT-PCR, Western blotting, etc.Results: Fluorescennt reporter plasmids driven by E-cadherin/slug promoter weresuccessfully constructed and confirmed by restriction analysis and DNA sequencing.Lentiviruses were obtained by calcium phosphate transfection method. The virusLv-pE-cad-YFP can effectively infected PLC/PRF/5cells and E-cadherin stablyexpressed in target cells after transfection, moreover, the virus was verified to be validwith a series of function tests.Conclusion: The multi-fluorescent reporter system driven by E-cadherin/Slugpromoter was successfully constructed and the virus Lv-pE-cad-YFP was verified tobe valid, which will lay a good foundation for further study on the relevant function ofHCC with EMT.