| In multicellular organisms, there is a balance between the cell-generating effects of mitosis and cell death which is induced through apoptosis to maintain the number of cells. A disruption of this delicate balance can lead to the development of cancer. It has been put forward that evasion of apoptosis or resisting cell death is a hall mark of cancer. It has been reported that, the inhibition of apoptosis proteins (IAPs), a group of structurally and functionally similar proteins, is the key regulator of cytokinesis, apoptosis and signal transduction. There are eight members of IAPs as follows: Neuronal apoptosis inhibitory protein (BIRC1/NAIP), Cellular-IAPl (BIRC2/c-IAP1), Cellular-IAP2(BIRC3/c-IAP2), X-linked IAP (BIRC4/XIAP), Survivin (BIRC5), Apollon (BIRC6), Livin (BIRC7) and IAP-like Protein-2(BIRC8/ILP-2).Part1:The relation of BIRC6expression with clinicopathologic characteristics and prognosis of HCCThe differences of BIRC6expression in40paired HCC and adjacent non-tumorous tissues were performed by Western blot. Our results showed that the expression of BIRC6increased significantly in the tumor tissues when compared with that in the adjacent non-tumorous tissues, which was testified meaningful by paired t test. In the meanwhile, the result of160paired HCC and adjacent non-tumorous tissues obtained by immunohistochemistry also indicated that the expression of BIRC6in the cancerous tissues was significantly higher than that in the adjacent para-cancerous tissues, which is stastically meaningful.χ2test or Fisher’s exact test showed that the expression of BIRC6in the tumor tissues correlated with vascular invasion and TNM stage. Kaplan-Meier method (log-rank test) utilized for survival analysis indicated that patients with a high BIRC6expression in the tumor tissues have a shorter survival and higer incidence of recurrence. Subsequently, Cox proportional hazards regression analysis aiming at identifing the independent prognostic factors of OS and TTR showed that BIRC6expression in the in the tumor tissues was an independent prognosticator for both of OS and TTR. These data pointed out that BIRC6could be a predictive marker of HCC.Part2:The biological roles of BIRC6in carcinomagenisis and progress of HCCStable BIRC6-knockdown cell lines were established for experiments of cell functions in vitro and tumour-bearing mice model in vivo. The result showed that compared with the control cells, knockdown of BIRC6could significantly inhibit cell proliferation, cell cycle distribution and carcinogenesis in the mice. Furthermore, down-regulation of BIRC6could promote apoptosis. These data demonstrated that BIRC6contributed to proliferation and carcinogenesis of HCC, which coud explained the poor prognosis in HCC patients with elevated BIRC6expression according to the result of the first part.Part3:Mechanisms of BIRC6influence on carcinogenesis of HCCp53is the prototypical tumor suppressor gene and plays a critical role in regulating tumor proliferation and death (apoptosis). Abnormalities in the tumor suppressor p53are among the most common mechanisms of cancer pathogenesis. p53activity, expression, and localization are mainly regulated by posttranslational modifications, such as phosphorylation, acetylation, and ubiquitination. It has been reported that the N-terminal single baculovirus inhibition of apoptosis protein repeat (BIR) domain and a C-terminal ubiquitin-conjugating (UBC) enzyme domain of BIRC6confer the E2/23property of functioning as a chimeric E2/E3ligase, by which SMAC and caspase-9were ubiquitinated and underwent proteolysis. It also been reported that p53is a downstream effector of BIRC6and knockdown of BIRC6could induces apoptosis through incrasing p53stabilisation. However, how BIRC6realized the function is still unknown. Given that BIRC6is itself an E2/E3ubiquitin ligase, it may act similarly to Mdm2, COP1or Pirh2, directly catalysing p53ubiquitination and proteasome degradation. So in this part, we study whether there is a direct interaction between BIRC6and p53and the mechanisms of realizing the interaction. On one hand, we verified the interactivity between BIRC6and p53via co-immunoprecipitation and confocal laser scanning microscopy analysis. On the other hand, we demonstrated that BIRC6promoted ubiquitin-protesome degradation of p53by ubiquitin assay.Taken together, this study provided evidence for the first time that BIRC6could promote cell proliferation and carcionagenesis of HCC and BIRC6could be utilized as a predictive marker of survival and recurrence in HCC patients after curative resection. Moreover, We demonstrated that BIRC6reduced p53expression by ubiquitination and proteasome degradation, thus resisting apoptosis and promoting carcinogenesis. Consequently, BIRC6could be a novel diagnostic marker and therapeutic target for controlling HCC recurrence, providing good significance for prognosis. |
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