Genetic Element-based Gene Circuits Selectively Recognize And Kill Hepatoma Carcinoma Cells

Research background and purposeThe incidence of liver cancer is on the rise worldwide.Liver cancer is expected to affect more than 1 million people annually by 2025.Hepatitis B virus and hepatitis C virus infection are the main predisposing factors of liver cancer in China whereas in western countries,metabolic syndrome and diabetes related-non-alcoholic steatohepatitis are more common causations.Liver cancers can be briefly divided into two groups:hepatocellular carcinoma(HCC)and non-HCC.The former accounts for 90% of total liver cancer cases.Chemotherapy and radiotherapy are main traditional treatment for HCC,however,their efficacy is hampered by poor accuracy and side effects.Therefore,it is urgent to develop an effective targeted therapy strategy.Selective killing of cancer cells has been a focus of research on targeted therapy of HCC in recent years.To selectively recognize and kill the cancer cells has become a promising strategy for HCC treatment in recent decades.Synthetic biology aims to use standardized molecular components and genetic modules to build biological devices with new functions that can sense input signals and regulate gene expression and cell behavior.A variety of synthetic component switches and circuits have been developed to control gene expression at the transcriptional,translational and post-translational levels.In this study,we used synthetic biological techniques to integrate specific promotors,sensing elements(micro RNA sponge)and switch(L7Ae:K-turn),to create a genetic switching plasmid that can respond distinctly to the different miRNA-enriched environment in the cytoplasm of HCC and normal cells,resulting in specifically switch on the pro-apoptosis element in the HCC cells but not nonHCC cells.Our study provides a new approach for gene therapy of HCC.Research methods and results1.Screening and verification of specific miRNAsmiRNA sequencing was performed in a variety of cell lines from different tissue by using high-throughput sequencing,to screen HCC-specific miRNAs.An HCC-specific miRNA,miRNA21,was selected,as well as miRNA122 was selected as non-HCC miRNA.Previously reported miRNA-196 a was used as positive control.A series of plasmids were constructed harboring different miRNA sponge repeated sequences corresponding to specific miRNA.Plasmids were co-transfected with miRNAs into 293 T cells and the results showed that the sponge repeated sequences in the plasmid could interact with corresponding miRNA by which inhibit the expression of effector gene that located upstream of the sponges.Additionally,transfection with the plasmids alone into two HCC cells and three non-HCC cells also exhibited inhibition of the effector,indicating that miRNA sponges are able to bind endogenous miRNA.Thus,we proved that plasmids with miRNA sponges could response to corresponding miRNAs in the cell cytoplasm,and the binding efficiency is about 60%.2.Construction and functional verification of genetic switchesTwo effector element-containing plasmids were constructed,containing fluorescent protein element and apoptosis-inducing element as effector,respectively.Then the verified miRNA sponges and L7Ae:K-Turn switch were cloned into the effector elementcontaining plasmids,to generate genetic switch plasmids that were expected to switch on effector expression in HCC cell lines but switch off or inhibit the expression of effector in non-HCC cells.After co-transfection into 293 T cells with miRNAs that mimicked HCC and non-HCC miRNA-enriched environment,HCC-mimicking 293 T cells showed enhanced effector expression and cell apoptosis whereas non-HCC-mimicking 293 T showed reduced effector expression and less apoptosis.These data demonstrated that genetic switches could exert killing effect specifically in HCC miRNA-enriched environment.3.Screening of liver-specific promotersTo increase the specificity of the genetic switch,CMV promotor in genetic switch plasmid can be replaced with a specific promoter that has similar activity to CMV.One tumor-specific promoter and three liver-specific promoters,namely the survivin gene(SUR)promoter,serum albumin gene(ALB)promoter,human thyroxin-binding globulin(TBG)promoter and human A1pha-1 antitrypsin(AAT)promoter,were selected for screening.Four liver-specific promoter plasmids were constructed,with CMV promoter as control and GFP protein as effector element.Plasmids were transfected into non-HCC cell lines,293 T,SH-SY5 Y,RD,HCT116,Caco-2and A549 cells,and HCC cell lines,PLC,Huh-7 and Hep G2 cells,respectively.The expression of GFP were determined.The results showed that the positive rate of GFP protein of AAT promoter in HCC cells was about 60% of that of CMV promoter in HCC cells,and the positive rate of GFP protein in non-HCC cells was about 20% of that of CMV promoter.The activity of AAT promoter was significantly different in non-HCC cells and HCC cells,suggesting AAT as a good replacement to CMV in genetic switches to promote effector expression.4.Construction and functional verification of liver-specific promoter-based genetic switchesAAT promoter was cloned into the genetic switch plasmids harboring apoptosisinducing element to replace CMV promoter.AAT-genetic switch plasmids were transfected into a variety of non-HCC cells and HCC cells and cell growth and apoptosis were measured.The results showed that the AAT-genetic switches recognized HCC cells with good accuracy and induced cell apoptosis in HCC cells without affecting other nonHCC cells.ConclusionUsing the liver-specific promoter,HCC-high miRNA sponge sequences and the L7Ae:K-turn switch,we successfully constructed a genetic switch that could specifically recognize and induce apoptosis in HCC cell.Our work provides a new approach for gene therapy of HCC.