BC047440 Silencing In HepG2 Cells Mediated By Lentiviral Vector

The purpose and significance:Primary hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and its incidence rate and mortality are high in cancer. Resection and liver transplantation are considered to be the most effective treatments; however, most of the liver cancer are found late and progress rapidly, so just a few patients can be successfully implemented resection or liver transplantation.There are so many studies show that the carcinogenesis of liver cancer is a multi-step process which involves multiple genes. Abnormal activation or inactivation gene such as oncogene, tumor suppressor genes, cell cycle regulating genes, apoptosis genes is the molecular basis of liver cancer. In recent years, gene therapy on HCC has been a hot point, restraining tumor cell-specific gene expression by RNA interference, observe the effects on tumor cells and then speculated the gene's role in tumor development, lead for gene therapy.A function gene unknown called BC047440 which was highly expressed in liver tissue and positively correlated with the degree of malignancy of liver cancer was discovered in the previous study in our laboratory. In our experiment, for the purpose of silencing human gene BC047440 gene, by RNAi technology, we designed BC047440 gene specific small interfering RNA (small interference RNA, siRNA) to build its short hairpin RNA (short hairpin RNA, shRNA) ,and constructed recombinant lentiviral vector and confirmed by PCR and DNA sequencing. Using the packaging cells called 293FT cells to package the restructuring vector, and then collect, concentrate virus supernatant. After infecting 293FT cells with recombinant lentiviral, we detect the expression of green fluorescent protein in 293FT cells, and figure out the viral titer in order to detect its optimal multiplicity of infection (MOI) in HepG2 cells; Separate HepG2 cells into BC047440-shRNA group, control-shRNA group and HepG2 group, infect HepG2 cells with the optimal MOI, detect BC047440 mRNA and BC047440 protein by RT-PCR and Western blot, and verify the silencing effect of the BC047440 gene in HepG2 cells, the changes in HepG2 cell cycle among groups were determinated by MTT and influence on biological characteristics of HepG2 cell was determinated after watching silence BC047440 gene. So as to understand BC047440 on the biological behavior of HepG2 cells more and to explore its mechanism, so we can find new ways to lay the foundation for liver cancer treatment.Methods:1. Firstly, design and synthesize human BC047440 gene specific DNA oligo nucleotides and connect to the pFU-GW-iRNA vector plasmid which has been double digestion linearized by Hpa I and Xho I. Secondly, transform it into Escherichia coli DH5αcompetent cells, select and amplify the positive colonies plasmid, and last to be confirmed by PCR and DNA sequencing.2. On one hand, mix the recombinant lentiviral vector pFU-shBC047440, pHelper 1.0 vector, pHelper 2.0 vector with Opti-MEM; on the other hand, mix the Lipofectamine 2000 reagent and Opti-MEM. Mix the vectors with Lipofectamine 2000 dilution, incubated at room temperature for 20 minutes, add 293FT cell culture and mix them, and put into cell incubator at 37℃, 5% CO2. Collect 293FT cells and its supernatant after 48 hours, centrifugate and concentrate the virus, repackage and store at -70℃refrigerator.3. Use recombinant virus particles to infect 293FT cells with slow dilution titer,and calculate the titer of recombinant lentiviral particles.4. Select the appropriate titer of lentiviral particles to infect HepG2 cells, view the expression of green fluorescent protein by the ordinary light microscope and the corresponding fluorescence microscopy 72 ,96 hours later. Chose the Multiplicity Of Infection (MOI) in better condition whose expression of green fluorescence intensity is higher as the optimal MOI value. Infect the HepG2 cells with the optimal MOI values, and then use flow cytometry (FCM) to detect the expressing of green fluorescent protein-positive rate and intensity of HepG2 cells, eventually determine the optimal MOI value.5. Firstly, separate HepG2 cells into BC047440-shRNA group, control-shRNA group and HepG2 group, secondly, add the corresponding recombinant lentivirus and control lentivirus, and extract the total RNA and total protein of each group 96 hours later as well as detect them by RT-PCR and Western blot.6. Changes in HepG2 cell cycle among groups were determinated by MTT. Results:1. According to human BC047440 gene mRNA sequence, referring to previous experimental results, select a target sequence, designing and synthesizing one pair of complementary oligo nucleotide template, recombined it into the pFU-GW-iRNA lentiviral vector. Recognize the positive clones successfully.2. Choose several positive clones from the recombinant lentiviral vector, confirmed them by PCR and DNA sequencing.3. Restructuring plasmid constructed by the 293FT packaging cells produces slow virus particles and concentrates. Using recombinant virus particles infect 293FT cells with slow dilution titer, collect and analyze the images under the microscope and fluorescence microscope, calculate the titer of recombinant lentiviral particles is 5×108 TU / ml.4. Infect HepG2 cells with lentiviral particles at an appropriate titer, we see the cell's condition better, the fluorescence expression higher, the fluorescence intensity stronger when the MOI is 20; and with infection is longer, fluorescence intensity becomes stronger. So select the 20 as the optimal MOI. Flow Cytometry confirms that HepG2 cells expression green fluorescent percentage is 88.15%, the average fluorescence intensity is 33.0%, finally select the 20 as the optimal MOI value.5. After detected by RT-PCR, Western blot, the expression of BC047440 mRNA and protein in BC047440-shRNA group is obviously lower than the control-shRNA group and HepG2 group (Inhibition ratio 58.96%), but there is no visible differences between the control-shRNA group and HepG2 group.6. After analyzing the changes in HepG2 cell cycle among groups, it was determinated that HepG2 cell in BC047440-shRNA group proliferated more slowly than HepG2 and control-shRNA groups, while there were no obvious differences between control-shRNA and HepG2 groups.Conclusions:1. The lentivirus RNAi vector of BC047440 has been constructed successfully.2. Pack the lentiviral particles use of recombinant lentiviral vector constructed. And successfully measure the titer of recombinant lentiviral particles. So that it is confirmed that the recombinant virus particles can efficiently infect HepG2 cells.3. Infect GepG2 cells at optimum MOI value. By means of RT-PCR and Western blot, the purpose of specific silencing BC047440 gene expression in human hepatoma cell line HepG2 is achieved, HepG2 cell in the interference groups can be proliferated slowly. So it is laid the foundation of further research of the BC047440 gene effection on biological behavior of HepG2 cells, its mechanism and new ways to treat liver cancer.