Molecular Cloning Of Human Fas/FasL Gene And The Effect Of Gene Transfection Combined With Cisplatin On Rectal Carcinoma Cells In Vitro

Colorectal cancer is a major public health problem.Conventional therapies for advanced colorectal cancer are uneffective.There is a pressing need for effective treatment of colorectal cancer and colorectal liver metastases.Gene therapy has made greatly progress for the treatment of cancer in the last ten years. Fas/ FasL gene, one of the apoptosis genes, is becoming a hotspot for the cancer trerapy.Objectives This research is to clone Fas/ FasL gene and construct its eukaryotic expression vector. We also want to study the effect of Fas/FasL gene transfection combined with cisplatin on rectal carcinoma cells in vitro.Methods By using the RT-PCR technique,a full length Fas/FasL was cloned from actived peripheral mononuclear cells of healthy donors.The fragment was ligated with the pGEM-T Easy and sequenced.The construction of pcDNA3.1-Fas/FasL,the constructed vector was transfected into 8348 cells with lipofectin,the change in expression of Fas/ FasL gene was determined by RT-PCR. The apoptosis and proliferation of rectal carcinoma cells pre and posttransfection induced by cisplatin were analysed by MTT methods.Results RT-PCR products were digested with the restriction enzyme and the size of the produced fragement was in accord with the expected size.The sequenceing result showed that we had cloned full reading frame Fas/FasL cDNA which is correct compared with the sequence deposited in GeneBank.The recombinant eukaryotic expression vector for Fas/FasL was digested with XhoI and EcoRI,and the eletrophoresis of the digested products showed objective gene fragment and eukaryotic expression vector fragment.The Fas/FasL gene transfected 8348 cells showed prominently elevated mRNA expression of Fas/FasL. Transfection of Fas gene can significantly upregulate the expression of Fas in human rectal carcinoma 8348 cells.Bying using different concentration of cisplatin,the suppression rates of Fas transfection group and control group were 47.2%,51.8%,57.2%,65.4%,71.0% and 29.6%,33.0%,37.8%,41.4%,47.0% respectly,whose difference were all significant(t=15.33,p<0.01). Transfection of FasL gene can significantly upregulate theexpression of FasL in human rectal carcinoma 8348 cells.Apoptosis and decrease of proliferation were difficultly induced by cisplatin in the transfected 8348 cells. Bying using different concentration of cisplatin,the suppression rates of FasL transfection group and comparison were 11.0%,25.4%,31.2%,37.8%,42.4% and 26.1%,34.4%,37.6%,42.9%,53.2% respectly,whose difference were all significant(t=4.43 ,p<0.05).Conclusions This study had cloned full reading frame Fas/FasL gene of the normal human being, and constructed its eukaryotic expression vector, providing a basis for further study on the function of Fas/FasL. Transfection of Fas gene by lipofectin can significantly upregulate the expression of Fas in target cells.A strong suppression of rectal carcinoma cells was observed by combination of Fas gene transfection and cisplatin . Transfection of FasL gene by lipofectin can significantly upregulate the expression of FasL in target cells.The synergistic cytotoxic effect obtainted in 8348 cells suggested that combined use of FasL gene transfection and cisplatin may help in the treatment of cisplatin resistant rectal carcinoma...