Microarray Analysis Of Aberrant MicroRNA Expression Pattern In Different Grade Gliomas

Glioma is the most common intracranial tumor with malignant biological characteristics of invasive growth, high rate of relapse and rich vascularity. Despite all clinical effort, patients with glioma still carry a bad prognosis. With the development of molecular genetics and epigenetics, numbers of researches involved in molecular and cellular mechanisms improve our understanding of gliomas, as well as provide effective targets and valuable insight to improve patient outcomes. MicroRNAs have the ability to negatively regulate gene expression at the post-transcriptional level. Aberrant expression of mi RNAs has been found in a wide variety of human tumors, including brain gliomas, which were also found to be involved in multiple crucial biological processes such as proliferation, apoptosis, invasion, differentiation and angiogenesis. Emerging studies focus on important roles of mi RNA in brain gliomas. However, both the expression pattern of mi RNAs in different grades and some mi RNAs involved in malignant progression of gliomas are poorly understood. In the present study, we used miRNA microarray to investigate aberrant mi RNA expression patterns in gliomas compared with the matched normal tissues for finding special mi RNAs involved in tumorigenesis, malignant progression and pathological grading in human gliomas. Isocitrate dehydrogenase 1 gene mutations and MGMT methylation occur frequently in patients with glioma, which are strong predictors of a more favorable prognosis. Some study indicated that the combination of IDH1 mutations and MGMT methylation outperforms either IDH1 mutations or MGMT methylation alone in predicting survival of glioblastoma patients; IDH1 mutants was associated with increased histone methylation. Whether IDH1 mutations and MGMT methylation are regulated by mi RNAs or not and exist potential link. Investigating the correlation of IDH1 mutation and MGMT gene promoter methylation in brain glioma patients and further study the mechanism may provided a useful clue for diagnosis and treatment of the patients.METHODS:In part I, we used a microRNA microarray assay to investigate the mi RNAs expression profiling in gliomas compare to matched normal tissues. The microarray data was validated by quantitative RT-PCR assay. Some of the online softwares were used for target prediction of the mi RNAs.In part II, we collected 133 surgical-based and pathologically confirmed glioma samples, then detected the status of IDH1 mutation by Direct sequencing.In part III, nested methylation-specific PCR(n MSP) and methylation-specific PCR(MSP) combined with denaturing high performance liquid chromatography(DHPLC) was used to detect the status of MGMT gene promoter methylation.In part IV, relevancy of IDH1 mutation and MGMT gene promoter methylation was analyzed by chi-squared test.RESULTS:1. The expression pattern of mi RNAs in gliomas(1) Microarray is a reliable method to find biomakers for gliomas.(2) In total, we found 13 differentially expressed mi RNAs between 9 gliomas and their matched surrounding tissues by mi RNA microarray assay. Among them, 12 mi RNAs were up-regulated and one was down-regulated.(3) Totals of 8, 9, 15 aberrantly expressed mi RNAs were detected in WHO grade II, III and IV gliomas respectively.(4) The down-regulated mi R-4489 may play a important role in the tumorigenesis which was found to be related to gliomas first time.(5) Mi R-106b-25 cluster may play an assistant role in gliomas progression and result in greater proliferation ability and anti-apoptosis effects in tumors with up-regulated mi R-17-92 clusters.(6) Predicted target genes of 13 differently miRNAs were obtained by Bioinformatics. Gene Ontology(GO) and Pathway analysis showed that the 926 target genes were significantly enriched in biological process and signaling pathways associated with nerve system and tumor.2. IDH1 mutation in gliomas(1) 62 cases(46.62%) were confirmed with IDH1 mutations, which mainly involved in WHO II,WHO III grade glioma and second gliomblastoma.(2) There is correlation between IDH1 mutation and patient’ age(p=0.001,R=0.296).3. MGMT gene promoter methylation in gliomas(1) 66.17%(65/98) and 65.71%(23/35) cases were confirmed with MGMT gene promoter methylation by n MSP and MSP-DHPLC respectively.(2) There is no significant difference between nMSP and MSP-DHPLC significant difference in detection rate( X2=0.004,p=0.948).(3) Correlation was not found both in MGMT gene promoter methylation and the grade of glioma(p>0.05) and patients’ age(p=0.654).4. The correlation of IDH1 mutation and MGMT gene promoter methylation(1) Relevance was confirmed in IDH1 mutation and MGMT gene promoter methylation(X2=6.57,P=0.01)) by chi-square test.(2) IDH1 and MGMT is not in the 926 predicted genes, which means IDH1 and MGMT may not be regulated by the 13 mi RNAs directly.CONCLUTIONS:1. Microarray is a reliable method to find biomakers for gliomas. 2. We described expression profiles of dysregulated mi RNAs in gliomas. The result may contribute to a useful clue for finding sensitive and special miRNA biomarkers for early diagnosis, progression detection and pathological grading in gliomas. 3. MiR-4489 down dysregulated in gliomas. 4. IDH1 mutation is associated with MGMT gene promoter methylation, which implys that there may be mutual regulation mechanism that remains to be further studied. 5. IDH1 and MGMT may not be regulated by the 13 mi RNAs directly. The dysregulated mi RNAs whether regulated IDH1 and MGMT or not and the mechanism need to be further investigated.