The Study Of Tumor Suppressor Gene RUNX3 Expression And Promoter Aberrant Methylation In Glioma

Brain glioma is a common primary intracranial tumor,gradeⅡ-Ⅳare regarded as malignant and prognosis remains dismal.Although surgical treatment combined with radiotherapy and/or chemotherapy were taken, the curative effect is still not satisfied.RUNX3 gene is a member of runt domain-containing family of transcription factors and plays an important role in axon guidance,cell proliferation and induction of apoptosis.Previous studies indicate that RUNX3 expression is down-regulated in many human tumors and may be a putativ tumor suppressor gene.But RUNX3 expression and its characteristics in glioma are still unknown,its mechanism of transcriptional expression needs further investigation.We first use IHC to detect RUNX3 protein expression in normal brain and glioma and analyze its relationship with clinical data.Then we use MSP to detect RUNX3 promotor methylation status in glioma specimens and reveal the regulatory mechanism of RUNX3 gene.Finally,5-azaC was used to reverse methylation status in U-251 glioma cell line and invasive ability and apoptosis of tumor cell were also investigated.RUNX3 maybe a new therapeutic target gene. Part 1 The expression and clinical significance of RUNX3 protein in gliomaObjective:To investigate the expression of RUNX3 protein and its potential clinical significance in human brain and glioma.Methods:RUNX3 protein expression was determined in 8 normal brains and 40 gliomas paraffin embedded specimens by immunohistochemical method and the differences of RUNX3 protein expression among clinical pathological groups were also investigated.Results:In all normal brain tissues,the RUNX3 protein expressions were either positive or strong positive,but in 42.5%(17/40)of glioma specimens were weak positive or negative,the expression of RUNX3 protein was significantly down-regulated in glioma(P=0.024).Compared among different pathological groups,RUNX3 expression was higher in low-grade glioma(IRS=7.70±3.20)than in high-grade glioma (IRS=4.75±2.88),which is statistically significant different.The increase of RUNX3 correlated negatively with tumor grade(γ=-0.478,P=0.002). No relationship was observed between the expression of RUNX3 protein and sex,age,pathological types,tumor location.Conclusion:The expression of RUNX3 protein was down-regulated in gliomas when compared with normal brain tissues and its increase correlated negatively with tumor grade.RUNX3 may be functioned as a tumor suppressor gene in the occurrence and development of glioma.Part 2 The experimental study of transcriptional expression and promoter methylation status of RUNX3 gene in gliomaObjective:To detect the promoter methylation status of RUNX3 gene and investigate the molecular mechanism of RUNX3 gene down-regulation in glioma.Methods:Total RNA and protein were extracted from 35 human gliomas and 10 normal brain tissue specimens.RT-PCR,Western blot and MSP were used to detect the RUNX3 mRNA expression,RUNX3 protein expression and promoter methylation status.In the same time,the correlations between clinic pathological parameters and the expression and methylation status of RUNX3 gene were analyzed.Results:Relative expression of RUNX3 mRNA and protein in 35 cases of glioma specimens were 0.303±0.231 and 0.401±0.271 respectively,but in 10 normal brain tissues were 0.767±0.047 and 0.753±0.042 respectively,the difference of expression was significant between normal brain and glioma(P＜0.01).Relative expression of RUNX3 mRNA and protein in low-grade glioma were 0.428±0.241 and 0.553±0.196 respectively,in high-grade glioma were 0.208±0.175 and 0.287±0.154 respectively.The decreased expression of RUNX3 mRNA and protein correlated positively with tumor grade(r_mRNA=-0.598, P_mRNA＜0.01;r_protein=-0.703,P_proteinin＜0.01)and the RUNX3 mRNA and protein were positively associated.The prevalence of RUNX3 promoter methylation in glioma tissue was 42.8%(15/35),while in normal brain tissue no methylaiton was detected,the difference between glioma and normal brain was statistically signiflcant(P＜0.01).RUNX3 promoter methylation status was also statistically different between low-grade(20%) and high-grade(60%)glioma tissues(P=0.037).Expression of RUNX3 mRNA and protein in 15 cases of RUNX3 promoter methylation glioma were 0.113±0.098 and 0.213±0.105 respectively,in 20 cases of non-methylation glioma were 0.445±0.197and 0.612±0.169 respectively. RUNX3 promoter methylation was statistically associated with the down-regulation of RUNX3 mRNA and protein.Conclusion:Aberrant RUNX3 gene promoter methylaiton was found in glioma and epigenetic silencing of RUNX3 gene expression by promoter hypermethylation could play an important role in glioma. RUNX3 promoter hypermethylation was more common in high-grade glioma. Part 3 5-azacytidine induced RUNX3 re-expression and impacts on apoptosis and invasive ability of tumor cellsObjective:To explore the transcription expression and promoter methylation status of RUNX3 gene in U-251 cell line in vitro exposed to the specific demethylation agent--5-azacytidine.Tumor cellular growth suppression and invasive potential in vitro were also observed.Methods:MTT colorimetric assay was performed to detect the inhibition effect on U-251 cellular growth after treated with 5-azacytindine after 12hrs,24hrs,48hrs and 72hrs respectively.Apoptosis was analyzed by flow cytometry through PI stain.Transwell chamber in vitro invasive assay was used to examine the effects of inhibiting tumor cell invasive abilities.RT-PCR,Western blot and MSP were used to detect RUNX3 mRNA,protein and promoter methylation status before and after treatment with 5-azacytidine.Results:Inhibitory effects of 5-azacytidine on proliferation of U-251 cells observed by MTT colorimetric survival assay,was that treatment with 5-azacytidine inhibited growth of U-251 cells in a time and dose-dependent manner.5-azacytidine-induced apoptosis in a dose-dependent manner was determined by flow cytometry.Compared with control group,the apoptosis ratio of U-251 cell raise from 1.32±0.21%to 39.21±1.32%,which is treated with 50μmol/L 5-azacytidine,significant difference was noticed when compared with control group(P＜0.01).The result of Transwell assay exhibited that cell number through the artificial basal membrane became less compared with the control group,from 51.3±2.6 to 29.2±2.3.By using RT-PCR,Western blot and MSP techniques,we found out that RUNX3 gene promoter was methylated,RUNX3 mRNA and protein expression were absent in U-251 cell line before 5-azacytidine treatment,but after treated with 5-azacytidine,RUNX3 mRNA and protein were re-expressed and RUNX3 promoter methylation status were partially reversed.Conclusion:5-azacytidine can inhibit proliferation and invasive ability and increase apoptosis of U-251 tumor cell line.5-azacytidine can reactivate RUNX3 expression by demethylation RUNX3 promoter methylation status.