| Glioblastoma is a highly aggressive primary tumor,which could not undergo the resection completely.The effect of radiotherapy and chemotherapy are not good.The recurrence rate and mortality rate of patients are both very high.Therefore,it is necessary to further study the etiology and pathogenesis of glioblastoma.Previous researches have shown that HCMV plays an important role in the occurrence and development of glioblastoma,and HCMV genes and their products are expressed in different levels of glioblastoma tissues.Other studies have shown that HCMV infection can affect the expression of TOP2 A,which plays an important role in DNA synthesis,transcription and chromosome separation.It is highly expressed specifically in a variety of tumors,promoting tumor cell proliferation and inhibiting apoptosis.TOP2 A could be a potential therapeutic target.In our study,we detected the relationship between HCMV infection and TOP2 A expression in clinical pathological specimens and patient clinical data.Moreover,the function of TOP2 A on the occurrence and development of glioblastoma was explored by detecting cell proliferation,apoptosis,cell invasion and cell migration.To further elucidation,TOP2 A could be used as a potential target gene.The target gene prediction website Taget Scan7.1 was used to predict the mi RNA that could be combined with TOP2 A.Moreover,mi R-144-3p could directly target TOP2 A,which was detected by using Luciferase reporter assays.Finally,we detected the effects of mi R-144-3 on cell proliferation,apoptosis,cell invasion and cell migration.Take together,we aimed to study the role of mi R-144-3p in HCMV-positive glioblastoma using TOP2 A as a point,so as to explore the mechanism of the occurrence and development of glioblastoma from a new perspective.Object: 1.To detect the relationship between HCMV-infected and TOP2 A expression in glioblastoma clinical specimens and glioblastoma cell lines.To analyze the correlation between the TOP2 A expression and gender,WHO grading,KPS score,age,the overall survival rate and the survival rate without tumor after surgery.2.TOP2 A expression was detected in glioma cell lines with HCMV infection,and the relationship between HCMV IE1 and TOP2 A expression was detected.3.Cell proliferation,apoptosis,invasion and cell migration were detected in glioma cell lines transfected with si TOP2 A or si NC,and to investigate the molecular mechanism of HCMV-infected induced the expression of TOP2 A and its role in the development of glioma metastasis.4.mi RNAs bounds to TOP2 A was predicted by bioinformatics tools analyzing,and m R-144-3p was directly targeted to TOP2 A by luciferase assay.5.The relationship between HCMV-infected and mi R-144-3p was detected in clinical glioblastoma specimens and glioma cell lines,and the relationship between TOP2 A and mi R-144-3p was analyzed.6.Cell proliferation,cell migration and cell clone-formation in glioma cell lines transfected with mi R-144-3p were detected,and to investigate the molecular mechanism of HCMV-infected induced the expression of mi R-144-3p and its role in the development of glioma metastasis.Methods: 1.Glioblastoma tissue samples with HCMV-infected were detected by immunohistochemistry and q PCR,and the expression of TOP2 A was detected by q PCR and Western-blot.Moreover,the relationship between TOP2 A expression and HCMVinfected was analyzed.Furthermore,the correlation between the TOP2 A expression and WHO grading,KPS score,age,the overall survival rate,as well as the survival rate without tumor after surgery in patients with clinical data was statistically analyzed.2.The expressions of IE1 and TOP2 A in glioma cell lines with HCMV-infected were detected by q PCR and western-blot assay.3.RNA and protein were extracted from glioblastoma cell lines transfected with si TOP2 A and si NC.The expresion of TOP2 A and IE1 were detected by q PCR and Western-blot analysis.Cell growth was detected by CCK-8 assay.Cell apoptosis was detected by TUNEL assay.Cell invasion was detected by wound healing.Cell migration was detected by Transwell assay.4.Target Scan7.1 was used to predict the mi RNA that could bind to TOP2 A,and Luciferase assay was used to detect the possibility of binding TOP2 A to mi R-144-3p.5.q PCR was used to detect the relationship between HCMV and mir-144-3p expression in clinical glioblastoma specimens and glioblastoma cell lines.The relationship between TOP2 A and mi R-144-3p expression was analyzed by q PCR and Western-blot.6.Cell growth,cell invasion and cell clone-formation were detected respectively in glioblastoma cell lines with transfected mi R-144-3p mimics or mi R-con.Moreover,the mice model was established.The mice were divided into the following 2 groups: mi Rcon group and mi R-144-3p group.A total of 3×10~6 cells were subcutaneously injected into the right armpits of 4-week-old BALB/c mice.During this experiment,the mice were weighed,and tumors were measured with calipers every week.After 4 weeks,the mice were euthanized using a subcutaneous injection of sodium pentobarbital(50 mg/kg),after which the tumor tissue volume was measured.The tumor volume was calculated by the following formula: V(mm~3)=0.5×length×(width)~2.Results: 1.Glioblastoma tissue samples were analyzed by immunohistochemistry and q PCR.The results showed that there were 29 HCMV-positive cases and 11 HCMV-negative cases in the 40 samples.TOP2 A expression in HCMV-positive samples were significantly higher than HCMV-negative samples(P<0.01).The results were consistent with immunohistochemistry(P<0.01)and Western-blot(P<0.01).The relative expression of TOP2 A was significantly up-regulated in cells with HCMV-infected(P<0.01).The relative expression of TOP2 A showed no significant change in the HCMV-uninfected group.2.In this study,the m RNA and protein expression levels of TOP2 A were increased at different times in glioblastoma cells with HCMV-infected,and there was a significantly increased at 48 hours(P<0.01).The Glioma cell lines with HCMV-infected and HCMVuninfected were transfected with si TOP2 A or si NC,respectively.The expressions of TOP2 A and IE1 in glioma cell lines with transfected were detected by q PCR analysis and western-blot analysis.The transfection efficiency of si RNA was statistically significant,and TOP2 A si RNA inhibited the expression of IE1(P<0.01).3.Compare to si NC group,TOP2 A knockdown significantly reduced cell growth and enhanced apoptosis(P<0.01),and TOP2 A knockdown could restrain cell invasion and migration(P<0.01).4.We found that TOP2 A could potentially be targeted by mi R-144-3p by directly binding to TOP2 A.5.The expression of mi R-144-3p were detected by q PCR in 40 clinical samples.We found that the expression of mi R-144-3p in the HCMV-positive samples was significantly lower than that samples with HCMV-negative,and these results were consistent with the results in vitro(P<0.01).We detected the expression of mi R-144-3p and TOP2 A in glioma cells transfected with mi R-144-3p mimics or mi R-con.The results showed that there was a negative correlation between the expression of TOP2 A and mi R-144-3p(P<0.01).6.We detected the function of glioma cells transfected with mi R-144-3p or mi R-con,the high expression of mi R-144-3p could inhibit cell proliferation(P<0.01),the high expression of mi R-144-3p could inhibited cell invasion(P<0.01),the high expression of mi R-144-3p inhibited cell clon-formation(P<0.01).Moreover,the mice model was established.The tumor weight was weighted,the tumor volume was measured and calculated.We found high expression of mi R-144-3p inhibited tumor growth in vivo(P<0.05). |
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