Effects On The Biological Characteristics In H22 Hepatocellular Carcinoma Cells By Lentiviral Vector-mediated SiRNA Interfering Gene TLR4

Object:Construction of siRNA interfering TLR4 gene lentiviral vector and identified by lentivirus vector. Using lentiviral vector transfected H22 cultured hepatocellular carcinoma cells, and detected the expression of target gene in hepatocellular carcinoma by RT-PCR and West-blotting. H22 cell proliferation was detected by MTT assay.Then we will study the changes of biological characteristics of hepatocellular carcinoma cell H22Methods:1. Construction of siRNA interfering TLR4 gene lentiviral vector:By using the siRNA design’s principle,we could design target sequence by siRNA design software.These sequence would connect U6,then the recombinant lentiviral vector would be builded.2. After transforming the lentiviral vector which was connected U6 into the competent cells.We would extract plasmid from lentiviral vector,and digest the plasmid by restriction enzyme,then sequencing after digesting. Then we could package the lentiviral vector particles.3. H22 hepatocellular carcinoma cells were cultured and infected with the lentiviral vector. The siRNA interfering TLR4 gene sequences were screened by RT-PCR to measuring interference efficiency from each sequences.4. West-blotting detection:Total protein were extracted from selected RNAi group, empty vector transfected group and control group cells,then protein were separated by electrophoresis, caused to develop and we would calculate and compare it’s gray value, The expression of TLR4, MyD88 and Nf-kBwould determine and compare the differences between each group.5. According to a certain concentration, the RNAi group and control group cells were planked.Withered holes were setted.Using MTT method to measure the OD value for 3 consecutive days. Finally, the average OD of each group was used to subtract the OD value of the withering hole to draw the production curve, and the difference was compared.Result:1. Using shRNA design software to design siRNA interfering TLR4 gene sequences and insert lentiviral vector, then construct three siRNA interfering TLR4 gene sequences and a containing empty vector sequence control lentiviral vector.Then we would extract plasmid from lentiviral vector,and digest the plasmid by restriction enzyme,then sequencing after digesting.By comparative analysis, target gene sequence and sequences are completely consistent, lentiviral vector successfully constructed.2. The different titer of lentivirus particles were transfected into H22 hepatoma cells, then we would observe the cells growth after transfection.When geting MOI of 10, H22 hepatoma cells growed well shape, observed under the fluorescence microscope, there were a lot of fluorescence in the field of vision, and it’s the highest fluorescence rate.3. H22 cells were infected by shRNA were determined by RT-PCR to determin the expression of mRNA, and the shRNA#1’s interfering efficiency was the highest.4. The West-biotting test showed that the expression of each protein was different, and the difference was statistically significant, and the expression of RNAi group was the least.5. The proliferation of RNAi group and control group were statistically significant, and the proliferation of RNAi group was decreasedConclution:1.The siRNA interfering TLR4 gene lentiviral vector was successfully constructed by connecting the TLR4 gene with the U6 lentiviral virus vector and packaging production2.The most suitable MOI values were determined, and the infecting H22 cells were successful.3.Using real-time fluorescence quantitative PCR determination showed that shRNA#1 can effectively silence TLR4 gene.4.The siRNA interfering TLR4 gene lentiviral vector can interfere with the hepatocellular carcinoma cells,then decrease expression of TLR4、MyD88 and NF-kB.5. After silencing the TLR4 gene of HCC cells, their proliferation were suppressed.