| Glioma is the most commom malignancy of the central nervous system,and it is also a high incidence of intracranial malignancies.However,glioma cells have a high degree of malignancy,strong proliferative capacity,migration and invasion ability,anti-apoptosis ability.There are unclear between the tumor tissue and surrounding tissues,Glioma shows invasive growth,which leds to treat very difficult.Although the medical technology is progressing continuously,the therapeutic effect of glioma has not been significantly improved.Despite surgical treatment combined with chemotherapy and radiation therapy,the mortality rate of glioma is high.Therefore,it is important that molecular targets of glioma patients with prevention,diagnosis and therapeutic significance have important scientific and clinical significance for monitoring and improving their malignant biological behavior.In recent years,more and more studies showed that there were human cytomegalovirus(HCMV)infection and its viral DNA and protein expression in glioma patients,it suggested that HCMV may be related to the occurrence and development of glioma.In addition,HCMV gene products activate a variety of cytokines,which could promote tumor progression and angiogenesis.Early infection of IE1 could promote tumor invasion and metastasis and promote the development of glioma.Other studies showed that HCMV could stimulate the overexpression of cell growth factors,activate protooncogenes,inhibit tumor suppressor gene products,regulate cell regulation mechanisms,and increase the expression of malignant features of cells.Thus,it makes an important impact on the occurrence and development of glioma.Based on this,this study detected HCMV DNA and antibodies,and analyzed the expression levels of HCMV immediate early genes 1-72(IE1-72)and phosphorylated glycoprotein 65(pp65)in glioma tissues,and analyzed the relationship between IE1-72,pp65 protein expression level and clinic-pathological features of glioma.And in vitro,we constructed IE1 expression vector,successfully transfected into glioma cell line U251 and increased the expression of IE1 mRNA in cells.The proliferation,migration,invasion and anti-apoptotic ability of U251 cells were detected after IE1 over expression.Objective In this study,we detected the positive rates of HCMV in the tumors and peripheral blood of patients with glioma and primary epilepsy to observe the effect of HCMV IE1 on the biological characteristics of glioma cells,and explored the correlation of HCMV and gliomas.Methods In this study,39 patients were recruited from the neurosurgical ward in the Second People's Hospital of AnhuiProvince,who recently diagnosed with gliomas between January 2011 and December 2014.9 patients that underwent surgery for head trauma hemorrhage were recruited for control group.Both gliomas tissue samples and epilepsy tissue samples were obtained by surgical resection,Peripheral blood samples were obtained from gliomas patients with disposable needles before surgery.The tumor tissues and blood samples of 39 patients with glioma were randomly collected into the experimental group.According to the WTO pathological classification criteria,the tumor tissues were classified into:3 cases,grade I astrocytoma;6 cases,grade II astrocytoma;16 cases,grade III astrocytoma;14 cases,Grade IVastrocytoma.Brain tissue samples and blood samples from 9 patients with non-glioma were collected as control group.(1)Peripheral blood lymphocytes were separated by density gradient centrifugation to detect HCMV pp65 antigenemia,and HCMV IgG and IgM antibodies were detected by ELISA.The positive rates of antigen and antibody in the test group and control group were calculated respectively.(2)HCMV UL55 gene in HCMV DNA from two groups of glioma tissues and peripheral blood,and the copy number of HCMV DNA was detected by real-time PCR.(3)Immunohistochemistry was used to analyze the expression levels of HCMV IE1-72 and pp65 antigen in glioma tissues.(4)Plasmid pCDNA3.0-IE1 carrying HCMV1 was constructed andtransfected into glioma cell line U125,and stable transfected cell lines were screened.The effect of HCMV IE1 on the biological characteristics of glioma cells were observed,including proliferation ability of glioma cells were analyzed by CCK-8 kit,the anti apoptotic ability of glioma cells were analyzed by flow cytometry,the migration and invasion of glioma cells were analyzed by cell scratch test and transwell assay.Results(1)Anti-HCMV IgG,IgM antibody test results:From 39 glioma patients,36(92.3%)were positive for anti-HCMV IgG antibodies and 7(17.9%)were positive for anti-HCMV IgM antibodies.IgG antibody and IgM antibody were negative in 9 cases of the control group.The IgG detection rate of the experimental group is significantly higher than that of the control group(?~2=33.2,P<0.01).There was a clear correlation between the positive rate of anti-HCMV IgM and high viral DNA copy numbers in glioma tissues and peripheral blood(r=0.76,P<0.01).And IE-72 and pp65 in glioma tissues were also detected in all anti-HCMV IgM antibodies positive patients,the HCMV IgG-negative patients had negative HCMV DNA results in the blood and tumor tissues.(2)HCMV DNA test results:In the experimental group,25 cases(25/39,64.1%)of patients detected HCMV UL55 gene,of which2/3(66.7%)for grade I astrocytomas,3/6(50.0%)for grade II astrocytomas,11/16(68.8%)for grade III astrocytomas and 9/14(64.3%)for grade IV glioblastoma.GI1-72 of 23 glioma tissues in 25 gene-positive patients Both pp65 protein antigen and pp65 protein antigen were positive,and the other 2 glioma tissues positive for HCMV DNA were negative for IE1-72.Five other IE1-72 positive specimens did not detect HCMV DNA.HCMV DNA was also not detected in the brain tissue of 9 patients in the control group.Nested PCR was used to detect glioma in 20 patients(20/39,51.3%).HCMV DNA was positive in peripheral blood.The positive rates of grade I,II,III and IV astrocytoma were 66.7%(2/3),33.3%(2/6),50%(8/16)57.1%(8/14)respectively.The positive rate of HCMV DNA in peripheral blood was not significantly correlated with the detection of HCMV in glioma tissues.Five patients had negative HCMV DNA in peripheral blood,while HCMV DNA in glioma tissues was positive.DNA sequencing confirmed that the sequence of all PCR amplification products was identical to HCMV UL55.The average log10 HCMV DNA copy number in galangoma per 500 ng of tissue and peripheral blood was(3.75±1.60),(2.88±1.20),respectively,there were(1.66±0.19)and(1.54±0.12)in the control group,respectively.The experimental group was significantly higher than the control group(P<0.01)and HCMV DNA copy number was lower than typical activity in glioma tissues and peripheral blood.HCMV DNA copy number at infection(mean log10 HCMV DNA copy number is 9.507).(3)Immunohistochemistry results:In the experimental group,30 cases(30/39,76.9%)of glioma patients were positive for HCMV IE1-72,including grade I,II,III,IV astrocytoma,the positive rates were 66.7%(2/3),83.3%(5/6),75.0%(12/16),and78.6%(11/14),respectively.26(26/39,66.7%)cases of glioma patients were positive for HCMV pp65,the positive rates of grade I,II,III and IV astrocytoma were 66.7%(2/3),66.7%(4/6)and 56.3%(9/16),78.6%(11/14)respectively.Among them,26 cases of pp65-positive specimens were positive for IE1-72,and 4 of 30 specimens with IE1-72 positive were pp65-negative.In the control group,the HCMV IE72 and pp65antigens were negative in the brain tissue of 9 patients.(4)The effect of HCMV IE1 on the biological characteristics of glioma cells:After transfecting HCMV IE1 gene into U251 cells,the successfully transfected cell lines were screened.The results of CCk-8assay showed that the proliferation ability of U251 cells after transfection of HCMV IE1 gene were significantly enhanced,while the proliferation ability of the empty plasmid control group and the cell control group showed no significant difference.Flow cytometry analysis the apoptosis level of HCMV IE1 group,empty plasmid control group and cell control group showed that the anti-apoptotic ability of U251 cells transfected with HCMV IE1 gene was significantly enhanced;there was no significant difference in the cells of the control group.Transwell assay analysis of the migration and invasion ability of HCMV IE1 group,empty plasmid control group and cell control group.The migration and invasion ability in HCMV IE1 group was significantly enhanced compared with the empty plasmid control group and the cell control group(P<0.01),while the empty plasmid control group and cell control group had no significant difference.The scratch test results showed that U251 cell line migration ability was significantly enhanced after HCMV-IE1 transfection(P<0.01).Conclusion:These findings support previous reports of the presence of HCMV infection in glioma tissues.HCMV local infection may play an active role in glioma development and pathogenesis through multiple molecular signaling pathways such as IE1. |
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