The Mechanism Of Human Cytomegalovirus Affecting The Biological Behaviors Of Glioma Cells Through Up-regulating Astrocytes Secretion Of IGF2

Human cytomegalovirus(HCMV) infection is closely related to malignant glioma. In glioma microenvironment, HCMV latency infection subtly influence the development of glioma cells. The influence pathway include: astrocytes infected with HCMV increase the expression of IGF2, and then IGF2 protein secrete increasingly, which lead to the activation of MAPK-ATF5 signal pathway through binding and activation of IGF-IR widely expressed on U87 cell surface, and in the end affect the biological behaviors of glioma cells and promote glioma cells proliferation, differentiation, and inhibiting apoptosis. First, astrocytes before and after HCMV infection IGF2 expression level are detected by PCR technique, and IGF2 is measured by ELISA technique. Immunofluorescence staining demonstrates that the number of receptors underwent phosphorylation in membrane surface increases. In addition, we can evaluate HCMV infection via IGF-IR mediating MAPK-ATF5 signal pathway exercise a great influence on gliomas metabolism and molecule changes in cells at molecular level. At the cellular level, we need to investigate that HCMV infection have an effect on gliomas cells proliferation and differentiation in vitro and in vivo. We are trying to clarify the molecular relationship between HCMV latent infection and brain tumor cells proliferation and differentiation. Our study will provide new drug targets and preventions of diseases for HCMV latent infection.Objective: 1. The detection of the change of IGF2 gene expression and protein secretion in astrocytes before and after infection, the secretion of astrocytes have an effect on the levels of ontogeny of gliomas related cytokines in HCMV latent infection in the microenvironment of brain glioma. 2. To establish an in vitro model with co-cultured astrocytes and gliomas cells, simulate the glioma microenvironment. By comparing and researching membrane surface receptor in gliomas cells co-cultured with between HCMV-infected astrocytes and mock-infected astrocytes, we observe and analyze signal transduction pathway and gliomas cell biological behavior alterations.Methods:1. Measuring the optimum moi stimulated HCMV latency infection states, we infect astrocytes with HCMV(m.o.i. = 2). q RT-PCR is performed on RNA to detect IGF2 gene expression in astrocytes. Detect IGF2-secretion of astrocytes with HCMV infection by ELISA. 2. q RT-PCR and Western blot verify that MAPK-ATF5 signal pathway downstream of IGF-IR is stimulated, which will influence all aspects of gliomas cells development. 3. When co-culture system is established, methods on the base of immunoflourescens stain, use the confocal laser scanning microscope to observe the phosphorylation of IGF-IR in gliomas cells co-cultured with between HCMV-infected astrocytes and mock-infected astrocytes. The activation level of MAPK signal pathway is detected by q RT-PCR and Western blot. Glioma cells co-cultured with astrocytes proliferation capability is detected by CCK-8, and apoptosis is detected using flow cytometry.Results: 1. When astrocytes are infected by HCMV AD169 at a concentration of m.o.i. = 2, astrocytes persisting and secreting IGF2, have a better cellular growth behavior and keep their proliferation ability with regular cell shape, which lays the basis for the further research in the bioactivity of the gliomas cells. 2. As time goes on, the expression level of IGF2 in the HCMV-infected astrocytes shows an increasing trend. With the increasing of the expression level of IGF2, IGF2 secretion is also increased. 3. After establishing co-cultured system, we observe that the ratio of the phosphorylation of IGF-IR in gliomas cells co-cultured with HCMV-infected astrocytes will rapid rise by confocal lasermicroscopy. It was also found that the ratio of the phosphorylation of IGF-IR shows an increasing trend with the increase of co-cultured time. The ratio of the phosphorylation of IGF-IR in gliomas cells co-cultured with mock-infected astrocytes remains constant. The nonphosphorylationlevel of IGF2 is superior in numbers 4. The activation of MAPK signal pathway is detected by q RT-PCR and Western blot, which affects glioma cells development. 5. CCK-8 assays show that gliomas cells co-cultured with HCMV-infected astrocytes and gliomas cultured with exogenous IGF2 could promote tumor cell proliferation, and the latter one’s function is greater. But, the proliferation of gliomas cells co-cultured with mock-infected astrocytes is evidently behind of proliferation of the two groups. the apoptosis rate of gliomas cells co-cultured with HCMV-infectedastrocytes and gliomas cultured with exogenous IGF2 has the trend of decrease,but flow cytometry analysis indicated that the apoptosis rate of gliomas cells co-cultured with mock-infected astrocytes remains about the same.Conclusions: 1. HCMV-infected astrocytes up-regulate the expression of IGF2 while promoting the release of IGF2. 2. IGF-IR in glioma cells co-cultured with HCMV-infected astrocytes is activated, and the phosphorylation level of IGF-IR increases. Then MAPK signal pathway is activated, which leads to the overexpression of ATF5. 5. High expression of ATF5 will influence glioma cells growth and survival, enhancing the proliferation ability of glioma cells, inhibiting the apoptosis of glioma cells, and accelerates the deterioration of the patient’s conditions or even death.