Targeting Gene Therapy Of HSV-tk/hIL12 Fused Gene For Hepatocarcinoma Cells In Vitro

Hepatocellular carcinoma(HCC) is one of the most frequent cancers worldwide.The morbidity is ascensus year by year following the increase of etiological factor for liver cancer.For about 90% of patients, no effective treatment can be proposed although surgical resection,conventional chemotherapy or radiotherapy and embolization can improve the natural history and the survival of certain cases.Liver transplantion is now considererd as the most effective therapeutic strategy,however,it is not applicable universally because of the shortage of organ donations. Therefor there are an urgent need for effective treatment of HCC.The use of gene therapy has been researched for decades,extensive experience and clinical indications has been gained and reported.Different gene therapy strategies for gene therapy has been used for HCC.Many trials have shown that the use of single gene can not work efficiently for liver cancer on account of the complexity of tumor development molecule mechanism.Multi- gene conjoination are used to improve the efficiency of cancer therapy.Both hepes simplex virus thymidine kinase(hsv-tk)and IL-12 are strong and effective gene in immunotherapy as have been shown in many studies and trials. HSV-tk/GCV and human IL-12 can induce cell apootosis and immunological reaction so as to accentuate the gene therapy effect..The targeting gene therapy for liver cancer is aimed to transfer gene to liver cancer cells or to regulate aim gene expression in HCC cells so as to specificly kill liver cancer cells without any injury to nomal cells. Alpha-fetoprotein expresses only in foetal period,no expression will be detected in adult .AFP promoter can regulate gene expression in cancer cells specificly.The study is aimed to construct the combination of interleukin12(IL-12) and simplex virus-thymidine kinase (HSV-tk) gene eukaryotic expression vector under the control of alpha-fetoprotein (AFP) promoter. Targeting gene therapy of liver cancer may be obtained by AFP promoter and the effect of gene therapy may be improved by the combination of IL-12 and HSV-tk gene.1 The clone of AFP promoter and enhancerThe specifici primers were designed according to ribonucleotide sequence of fetoprotein promoter and enhancer as documented. AFP promoter and enhancer were obtained by polymerase chain reaction(PCR) from the genome of human peripheral blood mononuclear cell .The gene fragment was cloned into T vector , The nucleotide sequence analysis of AFP promoter and enhancer was finished using the T vector. The nucleotide sequences of human AFP promoter and enhancer cloned are conformity with that were recoded in Genbank.The AFP promoter nucleotide number is about 300bp, and located at -252bp--+29bp of 5'flank transcriptional control region of AFP gene; The AFP enhancer nucleotide number is about 1100bp, and located at -4404bp—-3295 bp of 5'flank transcriptional control region of AFP.2 To construct vector for gene expression under the control of AFP promoter and enhancerWe replaced the cytomegaoviyns(cmv) promoter and enhancer of pcDNA3.1(+)vector with the AFP promoter and enhancer to construct the expression vector so that the gene expression can be controlled by AFP promoter.The two vectors are pcDNA3.1(+)-eCMV-pAFP and pcDNA3.1(+)-eAFP-pAFP. The restriction digestion results showed the expression vector with AFP promoter and enhancer were constructed successfuly.3 To evaluate the gene specific expression in liver cancer under the control of AFP promoter and enhancerTheβ-galactosidase (β-GAL) reporter gene was cloned respectively into pcDNA3.1(+)-eCMV-pAFP and pcDNA3.1(+)-eAFP-pAFP vector,then pcDNA3.1(+)-eCMV-pAFP-β-gal and pcDNA3.1(+)-eAFP-pAFP-β-gal vecor were constructed.We transfected pcDNA3.1(+)-β-gal and pcDNA3.1(+)-eCMV-pAFP-β-gal and pcDNA3.1(+)-eAFP-pAFP-β-gal vector respectively into HepG2 cells and HeLa cells using liposome.Theβ-gal gene expression were detected byβ-gal cell staining in situ.We founded that the cloned AFP promoter and enhancer successfuly controlled theβ-GAL gene expreesion specificly in liver cancer cells.4 To link the two subunites gene p35 and p40 of IL-12The two bovine elastin motifs (ten amino acid) genes were used as linkage peptide for linking two subunits gene so that the p40 peptide and p35 peptide can form a fusion peptide to formate IL-12. The p40 gene products with 1000bp nucleotide numbers and the p35 gene products with 600bp nucleotide numbers were obtained by PCR. The partly linkage peptide gene sequences and Kpn I digestion site are arranged separately to 3'end of p40 gene and 5'end of p35 gene. The PCR products of p35 and p40 gene were digested separately with Kpn I restriction endonuclease.The two digestion products were ligated by T4DNA ligase after purificatin of the two digestion products. The ligated products was explanded by PCR with sense primer of p40 gene and antisense primer of p35 gene. The ligated products of two subunits of IL-12 with 1600bp nucleotide numbers were obtained. The PCR products was cloned into T clone vector,and we picked out positive clones , to evaluate the T clone vector by restriction endonuclease digestion. The nucleotide sequences of ligated products was judged by sequencing analysis. The sequencing analysis results of ligated products were the same as the gene nucleotide sequences of p35 and p40 gene and linkage peptide. The two subunit of IL-12 was successful linked with linkage peptide with this method.5 To construct the vector for the expression of HSV-tk and IL-12The IL-12 and HSV-tk were cloned orderly and separately into pcDNA3.1(+)and pcDNA3.1(+)-eAFP-pAFP vector .In each vector,the HSV-tk and IL-12 gene were linked by internal ribosome entry site(IRES) so that the two gene drived from the same promoter can be expressed isochronously . The restriction digestion results showed the two expression vectors were constructed successfully.6 The specific expresison of hsv-tk gene and its efficiencypcDNA3.1 ( + ) -HSV-tk-IRES-IL-1 and pcDNA3.1 ( + ) -eAFP-pAFP -HSV-tk-IRES- IL-12 vector were transfected separately into HepG-2 cells and HeLa cells. The mRNA levels of HSV-tk gene were examed by reverse transcriptase PCR (RT-PCR), The antitumor effects and the bystander effects of HSV-tk/GCV system were evaluated by MTT. HSV-tk mRNA expression can be detcted in HepG2 cells and HeLa cells transfected with pcDNA3.1(+)-HSV-tk-IRES-IL-12, HSV-tk mRNA expression can be detcted only in HepG2 cells transfected with pcDNA3.1(+)-eAFP-pAFP -HSV-tk-IRES- IL-12. Both HepG2 cells and HeLa cells transfected with pcDNA3.1(+)-HSV-tk-IRES-IL-12 were sensitive to ganciclovir(GCV) ,while in pcDNA3.1(+)-eAFP-pAFP -HSV-tk-IRES- IL-12 group ,only HepG2 cells is sensitive to ganciclovir(GCV). The HepG2 cells with HSV-tk showed a"bystander effects".7 Specific expresison of the IL-12 gene and their biological activitypcDNA3.1 ( + ) -HSV-tk-IRES-IL-1 and pcDNA3.1 ( + ) -eAFP-pAFP -HSV-tk-IRES- IL-12 vector were transfected separately into HepG-2 cells and HeLa cells. The mRNA levels of HSV-tk gene were detected by reverse transcriptase PCR (RT-PCR), and ELISA was used to mensurate IL-12 protein levels. The biology activity of IL-12 were evaluated by both the proliferation of lympholeukocyte and the secretion of IFN-γproducted . IL-12 expression were detected in both HepG2 cells and HeLa cells transfected with pcDNA3.1(+)-HSV-tk-IRES-IL-12 .We verified that the expression of IL-12 can stimulate the proliferation of lympholeukocyte and IFN-γproduction. Whereas in pcDNA3.1(+)-eAFP-pAFP -HSV-tk-IRES- IL-12 group ,only the HepG2 cells has IL-12 . We verified that IL-12 expressed in HepG-2 cells can stimulus lympholeukocyte proliferation and IFN-γproduction..The combination of human IL-12 and HSV-tk gene eukaryotic expression vectors were constructed in our study under the control of AFP promoter and enhancer. The human IL-12 and HSV-tk gene can be expressed specificly in liver cancer cells,and showed antitumer biological activity .In summary ,our research achieved the aim of expressing targeting gene combination in liver cancer cells simultaneously and improved the safety and validity of gene therapy for liver cancer.