| ObjectiveHCC (Hepatocellular Carcinoma, HCC) is one of the most common malignant tumors and the third cause for cancer-related death, due to its high metastasis and postoperative recurrence rate. Because there is still lack of effective treatment, the survival rate of3years for postoperative HCC patients is less than13%.Negative immunoregulatory gene A20induced by tumor necrosis factor alpha, participates in tumor malignant progression by inhibiting cell apoptosis. The similarity between human A20protein and the homologus mice protein is as high as98%. The OUT domain of A20protein can bind to ubiquitin, acting as a deubiquitinating enzyme, while its zinc finger domain has an ubiquitin function. With its two ubiquitin-editing domains, A20can deubiquitinate RAF6and ubiquitinate RIP1for proteasomal degradation, both of the two molecules are key proteins in NF-κB signal pathway. Thus A20can inhibit a variety of inflammatory cytokines. The current researches show that mice defective in the A20is prone to chronic inflammation and multiple organ damage, and die shortly after birth. In addition, A20is closely related to human disease development, such as inflammatory enteritis, allergic asthma, rheumatoid arthritis, systemic, lupus erythematosus (SLE), B cell non-hodgkin’s lymphoma etc. At present, the relationship between A20gene and tumor metastasis is not clear, especially in HCC.This study focuses on the role of A20gene in HCC, espesically its effect on the metastasis of HCC and the corresponding molecular mechanisms. The aim of this study is to explore new targets for the treatment of HCC and provide theoretical basis for clinical treatment.Materials and MethodsⅠ. Expression of A20gene in HCC detected by immunohistochemistry(1) The study includs46liver cancer patients from QILU hospital. The informations of patient’s age, gender, grade differentiation, TNM stage, tumor sizes are from clinical medical records.(2) Expression of A20gene in tumor tissues and tumor-adjacent tissues was detected by immunohistochemistry.Immunohistochemical method was used to detecte the expression of A20gene in tumor tissues and tumor-adjacent tissues. A rabbit anti-human A20antibody was used with the dilution ratio of1:400, while the second antibody was goat anti-rabbit IgG with a dilution ratio of1:1000. Then, incubation was performed at4℃overnight. The results were developed by DAB.Ⅱ. Construction of A20eukaryotic expression vector(1) According to the construction method we constructed A20eukaryotic expression vectorA20gene and pRK5-Basic vector were connected directly after the double enzyme regension digestion. The DH-5a bacteria was transformed by the recombinated pRK5-A20and incubated at37℃with LA plate overnight to select the positive clones. The posive clones were identified by colony PCR and double enzyme digestion after restructuring the vector. At last, the plasmid sample was sequenced.(2) Expression of A20was detected by RT-PCR and Western blot methods in SMMC-7721cell.After A20sequencing, we transfected it with liposome transfection method into SMMC-7721cell lines, then Trizol method was used to extract the total RNA after24h. The expression of A20was detected by RT-PCR at mRNA level; Sample for protein analysis shoud be collected after48h and the expression of A20was detected by Western blot method. The experiment was repeated three times.Ⅲ. Effect of A20on migration in SMMC-7721cell evaluated by cell scratch assay.Using a12-well plate, we drawed a horizontal line in the center of each well as a line marker. SMMC-7721cell was planted with certain concentration after transfeced with pRK5-A20. After6h, we draw a line perpendicular to the horizontal with a10μl pipettor, the cell was washed with PBS three times before culture in serum-free RPIM1640medium. Photoes were taken at three time points (Oh,24h,48h). We used Oh distance to subtract24h and48h scratches distance to get24h and48h migration distance.The experiment set the negative control group, transfected pRK5-Basic plasmid group, transfected pRK5-A20plasmid group. Each group included three wells.Ⅳ Effect of A20on migration of BEL-7402cell line evaluated by Transwell assayBEL-7402cell transfected with A20plasmid was planted in0.4μm Transwell insert in RPMI1640serum-free culture medium with0.1%BSA, with600μl RPM11640containing10%FBS inl2-well plates. After12h the insert was stained by crystal violet. Selected areas under microscope to count the cells which migrated. Statistical analysis was performed. Each experiment set the negative control group, transfected pRK5-basic plasmid group, transfected pRK5-A20plasmid group. The experiment was repeated3times.Ⅴ. Effect of A20on invasion of BEL-7402cell evaluated by cell invasion assayThe procedure is similar to that in metastasis experiment. Matrigel glue and RPMI1640culture was prepared according to1:3proportion. We spread glue by use50μl Matrigel beforehand,after solidified in the room, we planted150μl BEL-7402cell suspension after transfection24h.Ⅵ. Effect of A20on appreciation evaluated by CCK8experimentHCC cells were inoculated in96-well plates, then afer transfected24h we digested and planted the cells with5x105/ml,100μl/hole respectively. we took out to test its absorbance at450nm in accordance to CCK8cell proliferation assay instruction at24h,48h,72h time points and analysised the OD data. The experiment set the negative control group, transfected pRK5-basic plasmid group, transfected pRK5-A20plasmid groups, each group has10holes in96-well plates. The experiment was repeated3times.Ⅶ. Effect of A20on proliferation of HCC cell evaluated by clone formation assayWe digested7402and7721cells lines after transfected24h. Using RPMI1640with10%FBS, we inoculated in6-well plates with1000,5000,2ml/hole respectively.After a week, we watched the formation clones and stained by crystal violet. Selected10different areas under microscope to count the number of forming clones, Statistical analysis was performed. Each experiment set the negative control group, transfected pRK5-basic plasmid group, transfected pRK5-A20plasmid group. The experiment was repeated3times.Ⅷ. Expressions of E-cadherin, and MMP9detected by RT-PCR at mRNA level in SMMC-7721cell lineAfter transfecting PRK5-A20plasmid in7721cell lines, we used Trizol method to extract the total RNA after48h, by using the method of RT-PCR to detect the expressions of E-cadherin and MMP9at mRNA level. The experiment set the negative control group, transfected pRK5-basic plasmid group, transfected pRK5-A20plasmid group. The experiment was repeated3times.ResultⅠ. Immunohistochemical results showed that expression of A20in HCC tissues was lower than that in tumor-adjacent tissues with statistical significance.Ⅱ. A20eukaryotic expression vector was constructed successfully and high expressed in SMMC-7721cell line.Ⅲ. Cell scratch assay showed A20may inhibited migration of HCC cell, precise Transwell assay further showed A20has an inhibitory effect on migration of BEL-7402cell.Ⅳ. CCK8assay showed A20may has a role to promote proliferation of HCC cell, the clone formation assay further showed A20can promote proliferation of HCC cell with statistical significance. Ⅴ. Expression of MMP9was down-regulated by the overexpression of A20gene while the expression of E-cadherin was not.ConclusionⅠ. Compared to tumor-adjacent tissues, the expression in the HCC tissue is lower. A20may have an inhibitory effect on development of HCCⅡ. A20gene can inhibit the invasion and migration of HCC cells.Ⅲ. The inhibitory effect of A20on the invasion and migration of HCC cells may be achieved by lowering the expression of MMP9Innovation and significancesThis is the first study about the relationship between A20gene and HCC occurrence and development.A20gene has an inhibitory effect on the invasion and migration in HCC cells... |
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