Expression Of α1, 3 Gal Epitopes At The Cell Surface Inhibits The Tumorigenic Properties Of Hepatocellular Carcinoma Cell Line Huh7

Objective: To elucidate whether expression of ? Gal epitope at the cell surface could impact the tumorgenic behaviors of tumor cells and explore the feasibility of gene therapy utilizing the murineα1,3 galactosyltransferase (α1,3 GT) in Hepatocellular carcinoma (HCC).Methods: 1. Preparation of the plasmid construct that containing the full lengthα1,3 GT gene cDNA : The full lengthα1,3 GT gene cDNA fragment was amplified from the murine heart tissue by using RT-PCR and then cloned into the pcDNA 3.1 plasimd to generate an euarokaytic plasmid construct , referred to as pCDNA-α1,3GT, which was further validated by enzymatic digestion and the DNA sequencing; 2. Preparation of the Huh7 cell line with stable expression ofα1,3GT:①The pCDNA-α1,3GT was transfected into the Huh7 cell line using Lipofectamine? 2000.After selected with G418, stable expression cell clone , designated as Huh7-GT, was obtained; Meanwhile, Huh7 cells were also transfected with the pcDNA 3.1(+) empty vector , the screened cell clone was referred to as Huh7- pcDNA and used as control;②RT-PCR analysis was carried out to verify the expression of ?1,3GT mRNA in the Huh7-GT, Huh7- pcDNA and Huh7 cells;③The expression of ? Gal epitope at the cell surface of Huh7-GT, Huh7- pcDNA and Huh7 cells were observed and analyzed by fluorescence microscopy and cell Flow cytometry using the fluorescein isothiocyanate (FITC)-labeled IB4 that specifically recognizes the terminal galactose of Gal ?1,3 Gal-R structure.④To further confirm the expression of ?1, 3 Gal epitope, human serum induced cytotoxicity assays was performed on the Huh7-? GT cells and control cells; 3. Observation of the changes in the tumorgenicity of Huh7-? GT cells in vitro:①The proliferative capacity of Huh7-? GT cells was determined by MTT method ;②The ability of colony formation of the Huh7-? GT cells was evaluated by Colony formation assay;③The apoptosis of Huh7-? GT cells was also measured by Cell Flow cytometry. 4. Finally, the tumorgenicity of Huh7-? GT cells was assessed by in vivo xenograft transplantation in nude mice. 12 nude mice was subdivided into 3 arms on average and inoculated with the Huh7-GT, Huh7- pcDNA and Huh7 cells, respectively; The Endpoints, such as time interval for tumor formation, tumor volume and tumor weight were analysized among these arms.Results: 1. A cDNA fragment with the length of about 1100bp was amplified from the murine heart tissue and then successfully cloned into the the pcDNA 3.1 plasimd which confirmed by enzymatic digestion , DNA sequencing suggested that the sequence of this cDNA fragment was identical to that of the murineα1,3 GT and the full length was 1080bp.2. The Huh7 cell line underwent gene transfection with the pCDNA-α1,3GT and pCDNA3.1 , respectively which generated two tansfected cell lines, Huh7-GT and Huh7-pcDNA ; RT-PCR analysis indicated that a ?1,3GT transcript was detected in Huh7-?GT cells whereas no amplification of such transcript was obtained in Huh7-pcDNA and Huh7 control cells; A strong expression of ? Gal epitope was observed at the cell surface of Huh7-? GT by fluorescence microscopy and Cell Flow cytometry; An elevated susceptibility of these cells to human serum was observed using a complement-dependent cytotoxicity test. 3. MTT method demonstrated that the growth speed of Huh7-? GT was obviously slower than that of the Huh7-pcDNAand Huh7 cells; A colony formation assay showed a significant decrease of colony formation in the Huh7 cells as compared to the controls, In addition, a significant increase of the percentage of apoptotic cells in Huh7-?GT cells was detected by cell Flow cytometry ; 4. A prolonged time interval for tumor formation in the nude mice inoculated with Huh7-?GT cells was observed as compared the control nude mice; the tumor weights and tumor volume were significantly reduced in the experimental group injected with Huh7-?GT cells compared to the control group of mice injected with Huh7-pcDNA and Huh7 cells.Conclusions: This study suggested that intracellular gene Transfer of the murineα1, 3GT could obviously inhibit the malignancy of cells which was possibly applicable to the cancer gene therapy of HCC.