The Mechanism Of Human Cytomegalovirus IE86Protein Promoting Survival Of Glioma Cells Through Acetylating Regulation

Malignant gliomas, the most common subtype of primary brain tumors, are aggressive, highly invasive, and neurologically destructive tumors considered being among the deadliest of human cancers. Interestingly, several reports have confirmed that human cytomegalovirus (HCMV) sequences and viral gene expression exist in most malignant gliomas, while not in surrounding normal brain, and HCMV could modulate the malignant phenotype in glioblastomas by interacting with key signaling pathways, indicating a potential association between gliomas and HCMV. HCMV immediate-Early2(ie2) gene product IE86form complexes with p300and the CREB-binding protein (CBP), which can serve as adaptor proteins to enhance transcriptional activation in host cells. This process plays an important role during HCMV infection. Our previous results indicated that IE86can interact with activating transcription factor5(ATF5), a marker recently shown to be highly expressed by neural tumors. Egr-1, Mcl-1and Bcl-2are downstream targets of ATF5. In addition, ATF5can also interact with p300/CBP. This research will firstly confirm the relationship among HCMV infection, WHO classification and ATF5expression in paraffin sections of surgically excised gliomas. The interaction between ATF5and IE86will be studied by CoIP and Confocal in vitro. Then this research will detect the acetylation level of ATF5alter IE86overexpression. A nude mice model bearing human glioma (HCMV IE86+) will be established to investigate the influence on cellular biological behaviors and ATF5acetylation by IE86overexpression. Taking epigenetic regulation as the breakthrough point, the mechanism that how the IE86regulates the trans criptional activation in host cells and involves in transformation of neural cells will be studied over HCMV persistent infection in central nervous system.Objective:1. To confirm the existence of HCMV and ATF5in gliomas and determine if there is a relationship among ATF5expression, HCMV infection and glioma classification.2. To study the interaction between ATF5and IE86and investigate the influence on cellular biological behaviors and ATF5acetylation by IE86overexpression in glioma cell line.3. To confirm the correlation and mechanism of IE expression on the growth of human glioma cells in a nude mice model bearing human glioma. Methods:1. The expressions of ATF5and IE86in glioma sections specimens were detected by immunohistochemisty. Then the relationship among ATF5expression, HCMV infection and glioma classification was analyzed by SPSS.2. U87cells were prepared using MEM culture medium. Then the cells were divided into serum deprivation group, HCMV infection group and IE86overexpression group. The expression of ATF5and Egr-1was detected by qRT-PCR and western-bolt. Cell proliferation was measured by CCK-8in addition to cell morphology observation. Flow cytometry along with AnnexinV/7-AAD double staining was used to detect cells apoptosis after serum deprivation and IE86overexpression. Meanwhile, RNAi-ATF5U87cell line was established to study the mechanism of ATF5during this process.3. The colocalization of ATF5and IE86in U87cells was detected by confocal microscope. And the interaction between IE86and ATF5was further confirmed by immunoblotting of the immunoprecipitation. The acetylation level of ATF5was analyzed by immunob lotting of the immunoprecipitation in order to further determine the mechanism underlying ATF5-dependent IE86apoptosis rescue.4. U87cells were overexpressed IE8648h before inoculating and maintained in serum free MEM medium. And a RNAi-ATF5U87cell line was established to confirm the mechanism of ATF5. Tumor cells in100μl of PBS were inoculated s.c. into the armpits of the athymic BALB/c nude mice were weighed and tumors were measured with external calipers every3d and each tumor volume in cubic millimeters was calculated by the following formula:V=0.5×D×d2(V, volume; D, longitudinal diameter; d, latitudinal diameter). After2weeks, mice were sacrificed by cervical dislocation, xenografts were resected and weighted. The acetylation level of ATF5was also analyzed by immunob lotting of the immunoprecipitation.Results:1. The expressions of ATF5and IE86in GBM and Anaplastic is higher than in LGG and Hyperplasia. And there is a positive correlation between the expressions of ATF5and IE86(r=0.810).2. Serum deprivation (SD) treatment evokes morphological changes, proliferation inhibition and apoptosis in U87cells, whereas HCMV infection or IE86-expressing cells displayed significant resistance to cell death. Compared with HCMV infection, IE86overexpression suppressed apoptosis more significant. ATF5is highly expressed in U87cell, so a shRNA delivering by lentivirus vector was used to inhibit the expression of ATF5in U87cells. In this condition, HCMV infection or IE86-expressing could not rescue the cells from apoptosis induced by SD.3. Serum deprivation led to depletion of the endogenous ATF5in U87cells whereas HCMV infection and overexpression of IE86increased ATF5expression.4. Immunofluorescence staining showed that the staining patterns of IE86and ATF5partially overlapped in the nucleus. Immunoblotting of the immunoprecipitated IE86from U87cells with an anti-ATF5antibody showed that endogenous ATF5was coprecipitated with IE86. Additional immunoprecipitation immunob lotting analysis showed that C-terminal regions of ATF5interacted with IE86.5. The association between ATF5and p300was interrupted48h after serum deprivation and ATF5acetylation level was descended. Whereas the expression of IE86enhanced the association and acetylation of ATF5interrupted by SD and rescued cells from apoptosis evoked by SD. But the depletion of p300by siRNA abrogated the ATF5acetylation induced by IE86overexpression after serum deprivation.6. In nude mice model, tumors of IE overexpression group grew much larger and faster than vector transfected group. At the endpoint, the tumor volume and weight of vector group was significantly smaller than that of IE group. While in ATF5interference groups, subcutaneous xenograft tumors did not grew much larger and faster with the HCMV IE overexpression. The IE86enhanced the acetylation of ATF5in vivo.Conclusions:1. The expressions of ATF5and IE86in GBM is higher than in LGG and Hyperplasia. And there is a positive correlation between the HCMV infection and expressions of ATF5, glioma classification.2. Overexpresstion of IE86increases cell proliferation and display significant resistance to cell death induced by serum deprivation in vitro. And IE86overexpressions can enhance the tumor growing through ATF5acetylation in vivo.3. IE86and ATF5partially colocalize in the nucleus, and interacted with each other. This interaction can enhance ATF5acetylation level then display significant resistance to proliferation inhibition and apoptosis evoked by serum deprivation.