Construction And Functional Identification Of Adenoviral ShRNA Suppressing Human CFLIP Expression

Objectives:To construct adenoviral vector for specially inhibiting the cFLIP expression, detect the transfected efficiency of adenovirus vector in HepG2cells, determinate the protein expression of cFLIP shRNA in the transfected cells and the effect on cell proliferation of cFLIP shRNA of Adenovirus.Methods:1.According the sequence of cFLIP mRNA, we designed and synthesized four pairs of cFLIP gene shRNA. We connected the cFLIP sh RNA to the interference vector and transfected HepG2, analysis the gene expression by RT-PCR and Western blotting.2. Using of the above mRNA single-stranded target fragment, we made ligation with two complementary single-stranded oligonucleotide and builded adenovirus expression vector with the Best interference fragment.3. We transfected the recombinant plasmids into HepG2cells and double digested the best cFLIP shRNA fragments. After ligation, transformation, restriction endonuclease digestion we detected the cFLIP shRNA fragments. The positive clones were transfected into the293cells by calcium phosphate method and the recombinant adenovirus Ad5.shcFLIP were obtained after packaging, amplification and purification. Setting the control group, we determined the virus titer. The inhibition rate of HepG2cells was detected by MTT.Results:1. cFLIP expression in tumor cells:Real time PCR was ued to detect the expression of cFLIP in various tumor cells; the results showed that cFLIP in liver cancer cells present a high level of expression.2. Results of recombinant plasmid restriction enzyme digestion and sequencing identification:After linearized by HindⅢ and BamH Ⅰ, plasmid pGenesil-1was ligated with fragments synthesis of shRNA. The product was transformed and cultured in LB medium. After plasmid extraction, new plasmid was got. The new plasmid was identified by restriction enzyme SalⅠ restriction enzyme digestion and detected by electrophoresis in1%agarose gel. Positive plasmids were obtained which were named pGeneSil-cFLIP-1, pGeneSil-cFLIP-2, pGeneSil-cFLIP-3, pGeneSil-cFLIP-4, pGeneSil-control. Positive plasmids were sequenced to the sequencing of the company and the results fully meet the design requirements.3. Detection of cell transfection efficiency:Gene expression of green fluorescent protein reporter of HepG2cells was surveyed with inverted fluorescence microscope after transfection by the LipofectamineTM2000for48h to detect the transfection efficiency. Fluorescence showed strong fluorescence expressing at48h, the transfection rate was about70%.4. Detection of cFLIP protein expression of the cells after shRNA transfected: HepG2cells (including shRNAl, shRNA2, shRNA3, shRNA4, shRNAC) were transfected with the same quality of shRNA vector applicating the liposome2000TM Western blotting analysis the protein expression48h later. The results showed that four pairs of shRNA could inhibit the expression of cFLIP on varying degrees, that the strongest inhibition is pGeneSil-cFLIP-1.5. Purification and interference effects detection of adenovirus Ad5.shcFLIP:Though vector construction, transfection, packaging, purification we obtained the adenovirus Ad5.shcFLIP and Ad5.shcontrol which could express cFLIP shRNA and shcontrol. The CPE picture was below. The interference effect of cFLIP was detected after the virus infecting HepG2for48h.6. Effection on cell proliferation by Adenovirus cFLIP shRNA:Adding Ad5.shcontrol and Ad5.shcFLIP into96-well plates, MTT method was adopted to test the inhibitory effect on HepG2cells at12h,24h and48h. A blank cell was cultured as a negative control. The results showed that Ad5.shcFLIP HepG2cause a time-dependent inhibition of growth in hepatocellular carcinoma cells. Experimental group and control group were significantly different (P<0.05).Conlusion:1. Apoptosis inhibitory protein cFLIP expresses in tumor cells which was high expression in hepatoma cells.2. Constructing the adenovirus for especially down-regulated the cFLIP expression in HCC using linearized plasmid and synthetic shRNA fragment by ligation, transformation, lipofection.3. Adenovirus of cFLIP of shRNA could specifically interfere with the expression cFLIP protein.4. Adenovirus cFLIP shRNA can specifically interfere with the expression of cFLIP protein, which lay the foundation for studying cFLIP in the proliferation of human hepatocellular carcinoma and clinical application.