Dissertation
Dissertation > Medicine, health > Oncology > Oncology experimental study

ZNF300Gene Enhances Growth of Cancer Cells through Activating TRAF2Mediated NF-κB Dependent Pathway

Author WangXianGuo
Tutor LiWenZuo
School Wuhan University
Course Microbiology
Keywords ZNF300 proliferation of Hela cells TRAF2 NF-κB tumor growth andmetastasis
CLC R73-3
Type PhD thesis
Year 2010
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Zinc finger gene family is the largest one of human gene families, which participates in cell differentiation, development of embryo and the disease generation. And about1/3of zinc finger genes belong to C2H2-type family, many C2H2-type ZFPs have been proved to function as transcription factors and involved in pathophysiology progresses. But the functions of many C2H2-type ZFPs are still needed to be unraveled.ZNF300, which harbors a classic KRAB domain in its N-terminus and twelve C2H2zinc finger motifs in its C-terminus, was originally cloned from an early human embryo cDNA library by our lab. It was reported that ZNF300is located to5q33.1, and previously studies had revealed that ZNF300were colocalized in the plasma and nuclear, all of these results suggested that ZNF300might act as a transcription factor. It is reported that ZNF300can bind to the DNA which harbor sequence C(t/a)GGGGG(g/c)G in vitro, and the promoters of some genes have this sequence, such as IL-2, IL2RB, CD44, p53, TNF-a, TRAF2, et al, most of which play important roles in cell proliferation, apoptosis and differentiation. The immunohistochemical staining analysis showed that the expression of ZNF300was higher in cancer cells and paracancerous tissues cells than that in normal tissues cells. Furthermore, the study of the effect of ZNF300on cell found that overexpression of ZNF300enhanced the proliferation of Hela cells, while inbibition of ZNF300by antisense cDNA slowed down the proliferation of Hela cells.Base on the results above, this research will unravel the function of ZNF300as a transcription factor to regulate the proliferation of cell, and will further examine the effect of ZNF300on cell in vivo.As we investigated The Eukaryotic Promoter Database (EPD), the upstream of TRAF2was found to have a potential ZNF300binding site (TTGGGGG,-19to-27). Many evidences had suggested that TRAF2was required for activating NF-κB depended pathway, which is thought to have tight relation with the proliferation of cell. So we concluded that ZNF300may regulate the proliferation of cell by modulating the NF-κB depended pathway mediated by TRAF2. Then Hela cell was taken as a model, transient transfection was applied to enhance or inhibit the expression of ZNF300, then RT-PCR and Western-blot showed that overexpression of ZNF300upregulated the expression of TRAF2, and inhibition of ZNF300by antisense cDNA downregulated the expression of TRAF2. Then we used luciferase analysis to assess the activation of NF-κB depended pathway, and found that overexpression of ZNF300can enhance the activation of NF-κB depended pathway.To further investigate the mechanism that ZNF300regulate TRAF2gene, firstly, the wild type promoter of TRAF2, promoter with mutation on the potential binding site of ZNF300and the truncate form of the promoters were cloned by PCR, then luciferase analysis was applied. The result showed that overexpression of ZNF300can enhance the activation of the wild type TRAF2promoter compared to other forms, and mutation or absence of the the potential binding site of ZNF300disrupted the activation. At last, EMSA and CHIP assay further identified the direct interaction between ZNF300and the promoter of TRAF2. All these results suggested that ZNF300promoted the expression of TRAF2directly, then the NF-κB depended pathway was also activated subsequently, and finally the proliferation of Hela cells was promoted.To assess the effect of ZNF300on proliferation of tumor cell in vivo, nude mouse was selected as the xenograft model in this study. Firstly, three stable cell lines were selected by G418, which ectopic overexpression of GFP (GFP), ZNF300(ZNF300) and inhibition of ZNF300by antisense cDNA (As-ZNF300group), respectively. Then three groups of nude mice were injected subcutaneously with these three stable cell lines respectively, and also a group of nude mice being injected subcutaneously with saline as negative control. Then the time of tumor formation, the tumor growth rate were recorded, the results showed that the tumor of the ZNF300group was firstly found at the seventh day, the tumor of the GFP group was firstly found at the tenth day and the tumor of the As-ZNF300group could not be detected until the thirteenth day. On the day20, the rate of tumor formation for ZNF300group was100%,70%for the GFP group and50%for the AS-ZNF300group. Then we concluded that overexpression of ZNF300promote the formation of tumor formation, while inhibition of ZNF300delayed the formation of tumor formation. The results also noted that the mean volume of tumor of the ZNF300group is3times bigger than that of the GFP group and the AS-ZNF300group. AS the IL-6and IL-8were inflammation factors which had tight relation with tumorgenies and tumor metastasis, so we examined the concentration of IL-6and IL-8in the blood serum by ELISA. The expression level of IL-6and IL-8in blood serum from ZNF300group was2-3times more than that from GFP group, but the expression level of IL-6and IL-8in blood serum from AS-ZNF300was about half of that from GFP group. The expression levels of ZNF300of tumor tissues in different groups were checked by immunohistochemical analysis and tumor cells metastasis were examined by HE staining at last, noted that overexpression of ZNF300promoted both the prolifertation of tumor and tumor metastasis.Here we found ZNF300could enhance the expression of TRAF2and activate the NF-κB dependent pathway, which then enhance growth of human tumor cells. Furthermore, the results also showed that overexpression of ZNF300promoted both the prolifertation of tumor and tumor metastasis in vivo, So we conclud that ZNF300activate the NF-κB dependent pathway by transactivating the expression of TRAF2, and finally promote the proliferation of cancer cells in vitro, tumorgenies and tumor metastasis in vivo. Previously study had reported that the expression of ZNF300can be induced by proinflammation factors, such as TNF-a and LPS. So it also seems that ZNF300could be a candidate oncongen, and a potential gene which links inflammation and tumorgenies.

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