HCMV Infection For Malignant Glioma Cell Proliferation, Apoptosis And Its Mechanisms Of Influence

Objective:1、To investigate the effects of human cytomegalovirus (HCMV) infection on cell proliferation in U87gliomas cells, and detect the expression of ATF5, Bcl-2and BAX in U87cells after HCMV infection.2、To explore whether HCMV infection increases the malignancy of glioma by regulating ATF5pathway and provide a new treatment idea for glioma.Methods:1、After HCMV infected U87cells at different time, cell proliferation was detect by MTT assay.2、Real-time PCR and Western-blot analyzed the expression level of ATF5, Bcl-2and BAX in U87cells after HCMV infection at0,12,24,48h.3、U87cells were infected by a lentiviral vector for RNA interference(RNAi) of ATF5gene. The infection efficiency was observed by fluorescence microscope. Western-blot was used to detect the expression level of ATF5.We choose the minium ATF5for the next experiment.4、siATF5U87cells were infected with HCMV at different hours(0、12、24、48h) respectively. Viabilities were determined by MTT assay and the apoptotic ratio was detected by TUNEL.5、Western-blot was used to detect the expression levels of Bcl-2and BAX after interference with ATF5in U87cells treated with HCMV infection. And we explored the molecular mechanism of HCMV infection increasing malignancy in U87cells.Results:1. The MTT assay indicated proliferation of U87cells can be promoted by HCMV infection contrast with control.2. Real-time PCR and Western-blot showed that ATF5expression levels were increased in U87cells after HCMV infection and the ratio of Bcl-2/BAX was also increased, which indicated that HCMV infection enhanced the resistance to apoptosis ability in U87cells.3. Control plasmid and other three plasmid containing ATF5lentivirus shRNAs LV-ATF5-RNA(8842), LV-ATF5-RNAi(8843) and LV-ATF5-RNAi(8844) targeting the ATF5coding sequence were separately transduced into U87cells. We observed that the efficiency of infection was more than95%under a fluorescence microscope. The percentage of protein ATF5/β-actin was0.67±0.03,0.38±0.09,0.61±0.06,0.68±0.06respectively. Expression level of ATF5in8842group was lowest. Then8842group was chosen for coming research.4. Loss of ATF5function in U87cells, it occurs to growth and anti-apoptotic ability slightly compared with control.5. We detected the expression of anti-apoptotic Bcl-2and apoptotic BAX protein in HCMV-infected siATF5U87cells by western-blot assay. Compared with control group, there was no distinct alteration in Bcl-2and BAX protein level from0h to48h in HCMV-infected siATF5U87cells.Conclusion:1. HCMV infection increases the malignancy of U87cells by regulating ATF5pathway.2. Prevention of HCMV infection may be a potential and promising approach for the treatment of malignant gliomas.