The Effects Of Silencing Of BubR1 On Human HBV-related Hepatoma Cell Proliferation And The Related Mechanism

Objective:To detect the expression of BubR1 in hepatocellular carcinoma(HCC) cells, explore the effects of small interfering RNA(siRNA)-targeting BubR1 via RNA interference on the proliferation, apoptosis and cell cycle of HBV-related HCC cells, examine the interaction between BubR1 and HBV X protein(HBx) and uncover the underlying mechanism. Methods:First, the cell line with higher expression of BubR1 from HCC cells(HepG2, SMMC7721, Huh7 and HepG2.2.15) was screened using Real-time PCR and Western Blot analysis. Three specific siRNAs of the targeting BubR1 mRNA were synthesized and five groups including Normal group, Control group, BubR1-1 group, BubR1-2 group and BubR1-3 group were designed. HCC cells were transfected with three siRNAs targeting BubR1 and scrambled control siRNA using lipofectamine 2000. The siRNA with the highest efficiency was used for subsequent analyses. Then,the effects of BubR1 down-regulation on the cell proliferation was assessed using MTT assay, the influence on apoptosis and cell cycle progression were assessed using flow cytometry methods. Finally, the expression changes of MAPKs(ERK1/2, JNK, p38MAPK)and NF-?B signaling pathways and the two pro-apoptotic regulators, Bax and Caspase-3 after silencing BubR1 were observed by Western Blot. And Co-immunoprecipitation and immunofluoresence assays were performed to examine the interaction between BubR1 and HBx proteins. Results:RT-PCR and Western Blot revealed that the expression of BubR1 in HBV-related HCC cells(HepG2.2.15 cells) was higher than in HBV-unrelated HCC cells. RT-PCR and Western Blot consistently showed three siRNAs(BubR1-1, BubR1-2 and BubR1-3) reduced the BubR1 expression levels with different efficacy in HepG2.2.15 cells and BubR1-3 showed the highest efficiency. The MTT assay and colony formation assay showed that the proliferation rate of BubR1-3 transfected cells was significantly lower than control cells. Flow cytometric analysis indicated that cell apoptosis of BubR1-3 transfected cells was remarkably higher than control cells. Cell cycle analysis indicated that BubR1 silencing caused an inhibition of cell cycle progression and an S phase arrest. At the protein level, Western Blot revealed that the levels of phosphorylation of ERK1/2 and NF-?B were remarkably decreased after interfering BubR1, and the protein levels of the two pro-apoptotic regulators, Bax and Caspase-3, were markedly up-regulated in BubR1-3 transfected cells compared to the control cells. Co-Immunoprecipitation assay and immunofluorescence assay together indicated that BubR1 binds to HBx and co-localizes with HBx at the nucleus in HepG2.2.15 cells. Conclusions:BubR1 is overexpressed in HBV-related HepG2.2.15 cells. Down-regulation of BubR1 inhibits proliferation and colony formation, causes an S phase arrest of cell cycle and induces apoptosis in HepG2.2.15 cells. BubR1 plays a tumor-promoting role in HBV-related HCC cells, which is probably associated with the interaction with HBx and regulation of NF-?B and ERK1/2 activation.