Relationship Between Expression Of BubR1 And Cenp-E Protein Of Spindle Checkpoint And Chromosmal Numerical Abnormality In Human Hepatoma Cells

Objective:To study the differential expression of BubR1 and Cenp-E in human hepatoma tissue and human hepatic tissue, and then, to further study their localization and role in hepatoma cells by indirect immunofluorescence and RNAi.It imply important information for exploring the mechanism of chromosome numerical abnormality in human hepatoma cells.Method:1. We detected expression of BubR1 and Cenp-E in human hepatoma tissue and human hepatic tissue by FQ-PCR and Western-blot, indirect immunofluorescence.2. We analyzed the chromosome numerical abnormality of human hepatoma cell line (HepG-2) and normal human hepatic cell line (LO2) by chromosome analysis.3. We treated the HepG-2 cells and LO2 cells with Nocodazole at the same time to synchronize them at mitosis, and then, determined their synchronization at mitosis with flow cytometry before and after treatment. 4. We detected the mRNA and protein expression of BubR1 and Cenp-E in the two kinds of cells before and after treating with Nocodazole by FQ-PCR and Western-blot.5. We observed the localization and expression of BubR1 and Cenp-E protein in the two kinds of cells by indirect immunofluorescence.6. After Cenp-E was interfered in LO2 cells by RNAi, we detected the mRNA and protein expression of BubR1 and Cenp-E before and after interference by FQ-PCR and Western-blot, and then, further evaluated the cells, function after interference by MTT and indirect immunofluorescence.Results:1. By FQ-PCR,Western-blot and indirect immunofluorescence, we detected the expression of BubR1 and Cenp-E in the human hepatic tissue was significantly higher than that of human hepatoma tissue。2. By chromosome analysis, we discovered the chromosome numerical abnormality of HepG-2 cells was significantly higher than that of LO2 cells.3. Using flow cytometry to determine the HepG-2 cells and LO2 cells before and after treating with Nocodazole, we found the ratio of the two kinds of cells in G2-M phase before treatment was no significant difference, and the ratio of the two kinds of cells in G2-M phase after treatment both increased significantly, but the ratio of the two groups of cells in metaphase was no significant difference.4. The results of FQ-PCR and Western-blot showed that the expression of BubR1 and Cenp-E in the two kinds of cells before treating with Nocodazole was no significant difference, the up-regulated expression level of BubR1 and Cenp-E in hepatic cell line L02 was higher than that in human hepatoma carcinoma cell line HepG2 during cell division phase5. Indirect immunofluorescence showed that Cenp-E protein mainly located in the nucleus, and that the expression of Cenp-E protein in the nucleus with abnormality mitosis was significantly lower than that in the nucleus with normal mitosis.6. After Cenp-E was interfered, MTT showed the growth of LO2 cells slowed down significantly. Indirect immunofluorescence showed the ratio of dyskaryosis in the cells with interfered Cenp-E was significantly higher than that in the normal cells.Conclusion:The expression of BubR1 and Cenp-E in human hepatic tissue was significantly higher than that in human hepatoma tissue. the up-regulated expression level of BubR1and Cenp-E in human hepatic cell line L02 was higher than that in human hepatoma carcinoma cell line HepG2 during cell division phase .We discovered the cell proliferation was inhibited in the LO2 cells with interfered Cenp-E. The low expression of BubR1 and Cenp-E may be an important reason of chromosome numerical abnormality in human hepatoma cells.