The Role And Mechanism Of JMJD3 In Tumorigenesis And Progression Of Glioma

【Background】 In china,about 7500 people died of cancer every day.Only in 2015,about 2,814,000 Chinese died of cancer and 61,000 Chinese died of central nervous system malignant tumor which ranking ninth in all tumors.Among the malignant tumors of the central nervous system,the incidence of glioma is the highest.The World Health Organization(WHO)classification system divides glioma into four levels(I,II,III and IV)according to biological behavior.The grade I gliomas tend to grow locally and can be cured only by surgery.The grade II,III and IV gliomas grow invasively.In the light of the fact that the central nervous system is the vital center,aggressive gliomas of central nervous system can not be extensively resected and completely removed.Even with radiation and chemotherapy,gliomas tend to recur,and some recurrent cases evolve from a low grade tumour(grade II or III)to a high grade tumour(grade III or IV).At present,the prognosis of glioma is still not optimistic,and further research is needed to find a new target for treatment.The jumonji domain-containing protein 3(JMJD3),also known as KDM6 B,is the demethylation enzymes for the twenty-seventh lysine of histone H3.JMJD3 located at 17p13.1 and near the tumor suppressor gene TP53,suggests that it may play an important role in tumorigenesis.The previous research found that JMJD3 was up-regulated in renal cell carcinoma,breast cancer,prostate cancer,and acted as proto-oncogene roles;JMJD3 was down-regulated in follicular lymphoma,large B cell lymphoma,pancreatic cancer,and played the role of tumor suppressor gene.It can be seen that the role of JMJD3 in tumor is very complex and may be tissue specific.Our previous studies found that the expression of JMJD3 in glioma was lower than that in normal brain tissue,suggesting that JMJD3 expression was deregulated in glioma.The down-regulation of JMJD3 expression may promote the development and progression of glioma.However,there is very few literature on JMJD3 in gliomas,and the role of JMJD3 in gliomas remains unclear.Our study investigated the role and mechanism of down-regulation of JMJD3 in tumorigenesis and development of gliomas.【Objectives】 1.To study the expression of JMJD3 in normal human tissues,and provide the foundation for further study;2.To investigate the relationship between the expression of JMJD3 in glioma and the clinicopathological features and prognosis of gliomas;3.To study the changes of JMJD3 expression and the molecular classification related genes status in the process of glioma recurrence,and to lay the foundation for exploring the role of JMJD3 in glioma recurrence;4.To investigate the effect of JMJD3 overexpression on the proliferation,invasion and migration of glioma;5.To observe the effect of JMJD3 on the cell cycle of glioma,and to investigate the mechanism of JMJD3 in tumor suppression;6.To explore the possible mechanism of down-regulation of JMJD3 in gliomas.【Methods】 This study involved the methods of histopathology,molecular biology and cell biology: 1.The expression of protein JMJD3 in 33 kinds of normal human tissue and 152 cases of glioma was detected by the immunohistochemistry;2.The relationship between the expression of JMJD3 and the OS of patients with glioma was explored by Kaplan-Meier survival analysis;The Cox risk regression model was used to investigate whether JMJD3 was an independent prognostic factor for OS of patients with astrocytic or oligoastrocytic tumors;The correlation between clinicopathological features and JMJD3 expression in patients with astrocytic or oligoastrocytic tumors was analyzed by chi square test;3.The expression of protein JMJD3 and related protein(EZH2,H3K27me3)in 47 paired primary and recurrent astrocytic tumors was detected by the immunohistochemistry;The mutations in the molecular classification related genes of 47 paired primary and recurrent astrocytic tumors were detected by polymerase chain reaction(PCR)and Sanger sequencing;4.The protein expression level of JMJD3,EZH2 and H3K27me3 as well as the mutation status of molecular classification related genes duiring the tumor recurrence were detected by Mc Nemar test and agreement test;5.The prognostic significance of JMJD3,EZH2 and H3K27me3 as well as the molecular classification related genes for the PFS of patients with astrocytic tumors were investigated by the Kaplan-Meier method and the Cox risk regression model;6.The protein expression of JMJD3 in U251、U87 and Hs683 cell lines was detected by Western Blot;7.The U87 and U251 cell lines were infected by lentivirus,and the m RNA and protein expression of JMJD3 in U87 and U251 cell lines were detected by real-time quantitative PCR and Western Blot respectively;8.The effects of JMJD3 expression on the invasion and migration of glioma cells were detected by Transwell technique.The effects of JMJD3 expression on the proliferation of glioma cells were detected by CCK-8 and plate cloning experiments.The effect of JMJD3 on the cell cycle of U87 cells was detected by flow cytometry;9.The methylation of JMJD3 promoter in glioma was detected by methylation specific PCR(MS-PCR).【Results】 1.Among the 33 kinds of normal tissue contained in the tissue microarray,15 kinds of normal tissue expressed JMJD3.Only the brain tissue showed strong staining of JMJD3;lymph node,spleen,tonsil,liver,lung,salivary duct epithelial,thymus,pancreas,testis,gastric epithelium,intestinal epithelium,colonic epithelium,esophageal and skin squamous epithelium were stained weakly positive;the remaining 18 kinds of tissues including the ovary,adrenal gland,thyroid,breast,prostate,bone marrow,myocardium,endometrium tissue did not show positive staining.2.In 152 cases of glioma,29 cases were stained positive for JMJD3.Among the 29 cases,17 cases were low grade gliomas(WHOⅠand WHOⅡ)and the positive rate of low grade glioma was 26.98%(17/63).The remained 8 cases were high grade gliomas(WHO Ⅲ and WHO Ⅳ)and the positive rate of high grade glioma was 13.48%(12/89).The difference between low grade glioma and high grade glioma was statistically significant(P=0.037).In the 107 cases of astrocytic and oligoastrocytic tumors,24 cases were positive for JMJD3 and the positive rate was 22.43%(24/107).There were 45 cases of oligoastrocytoma,of which 5 cases were positive for JMJD3,and the positive rate was 11.11%(5/45).The JMJD3 positive rate of astrocytoma and oligoastrocytoma was slightly higher than that in oligodendrocytes,but the difference was not statistical significant(P>0.05).3.In the 107 cases of astrocytic and oligoastrocytic tumor,the expression level of JMJD3 protein were not correlated with patient age and IDH1 mutations(P>0.05),while the positive rate of JMJD3 decreased significantly with tumor grade(P=0.021)and proliferation index(P=0.045).4.The analysis by univariate survival test found that the prognosis of patients with JMJD3 positive gliomas was better(P=0.006).The analysis by COX multivariate survival test found that JMJD3 expression was an independent good prognostic factor for astrocytic tumors and oligoastrocytic tumor(P=0.031).5.In the 47 paired primary and corresponding recurrent astrocytic tumors,using the Mc Nemar test and the agreement test,we found that key molecules of astrocytes—IDH1,TP53 and TERTp mutation status kept stable during the procession of recurrence.MGMT and ATRX expression level in recurrent tumor were moderate consistent with the corresponding primary tumors.Although no significant differences in JMJD3,EZH2 and H3K27me3 expression level during tumor recurrence were found(P>0.05),but the consistencies of JMJD3,EZH2 and H3K27me3 expression level were low.6.Univariate survival analysis showed that JMJD3,EZH2,MGMT,ATRX,TERTp,IDH1,WHO grade,age and Ki67 labeling index were correlated with PFS(P<0.05),while gender,H3K27me3 expression and TP53 mutation status were not correlated with PFS(P>0.05).Multivariate analysis revealed that JMJD3 was not an independent prognostic factor of PFS in astrocytic tumors,while IDH1,MGMT and Ki67 were independent prognostic factors.The three biomarkers were combined and the 47 astrocytic tumours were classified into 3 groups based on IDH1 mutation status and MGMT and Ki-67 expression level without considering histological grade.Group 1 patients(IDH1MUT regardless of Ki67 index or MGMT expression level)had the longest PFS,whereas Group 3 patients(IDH1WT but not in Group 2)had the shortest PFS.Group 2 patients(IDH1WT,low Ki67 index and MGMT protein loss)had an intermediate PFS between that of Groups 1 and 3.The AUC of the comprehensive classifier at the first year was greater than IDH1,Ki67 or MGMT alone in our dataset.7.The result of Western Blot showed the JMJD3 protein level was low in the 3 kinds of glioma cell lines investigated.8.Transwell experiments showed that the number of JMJD3 overexpressed U87 and U251 cells through the artificial matrix membrane were significantly less than the corresponding control group(P<0.05).CCK-8 showed that JMJD3 overexpressed U87 cells and JMJD3 overexpressed U251 cells grew slower than the control group(P<0.05);palet cloning experiment showed that the clone numbers of JMJD3 overexpressed U87 cells and JMJD3 overexpressed U251 cells were significantly less than the control group(P<0.05).9.The ratio of G0/G1 phase of JMJD3 overexpressed U87 cells increased significantly compared with empty vector transfected U87 cells(66.21±2.27 vs.57.14±1.17,P<0.05),while the proportion of JMJD3 overexpressed U87 cells in S phase was significantly decreased(17.18±0.49 vs.25.75±1.45,P<0.05).The proportion of the cells in G2/M phase were no statistically difference(16.17±0.95 vs.16.59±0.77,P>0.05).10.Using MS-PCR to detect the Cp G island methylation of JMJD3 promoter,we found that the consistency between promoter methylation and JMJD3 protein expression was low.【Concusions】 1.The expression of JMJD3 in human glioma tissues was studied.It was found that the expression level of JMJD3 decreased with the grade and proliferation index of glioma.2.JMJD3 exprssion can be an independent prognostic factor for OS of patients with astrocytic and oligoastrocytic tumors;3.JMJD3,EZH2 and H3K27me3 exprssion showed low consistency during tumor recurrence,suggesting that the level of JMJD3,EZH2 and H3K27me3 expression may change with tumor recurrence and progression.4.The statuses of IDH1,TP53 and TERTp in recurrent tumor were highly consistent with the corresponding primary tumors,indicating that the mutations of IDH1,TP53 and TERTp are early events of glioma tumorigenesis.MGMT and ATRX expression level in recurrent tumor were moderate consistent with the corresponding primary tumors.5.IDH1 mutation combined with MGMT protein expression level and Ki67 index is a better prognostic factor than single marker for PFS in patients with astrocytic tumours.6.JMJD3 overexprssion can inhibit the invasion,migration and proliferation of glioma cell lines,suggesting that JMJD3 may play an important role in the process of glioma recurrence and evolution.7.The results suggest that over expression of JMJD3 leads to G0/G1 cell cycle arrest in U87 cells.8.The promoter methylation is one of the mechanisms of the downregulation of JMJD3,and there may be other mechanisms involved in regulation of JMJD3 expression.To sum up,we demonstrate the fact that JMJD3 might act as a potential tumor suppressor gene in the tumorigenesis of glioma,and preliminary discussed the possible mechanism of its down-regulation.