Study On The Difference Of Clinical Characteristics And Gene Expression Of Gliomas Between Uygur And Han Patients In Xinjiang

Objective: Summarize the clinical characteristics of glioma patients of Han andUygur ethnicity in Xinjiang to analyze the distribution of different clinical factors.Perform survival analysis, according to follow-up study. By analyzing the relationshipbetween P53, MGMT, PTEN protein and Ki-67expression and clinical characteristics, theeffect of P53, MGMT, PTEN, Ki-67protein expression in glioma development is exploredbetween Han and Uygur patients. For the first time, mRNA gene expression profiling aswell as whole-genomemethylation difference detection are performed on glioblastomapatients of Han and Uygur ethnicity in Xinjiang. Differential expression genes arescreened and explored to examine their importance on the pathogenesis and prognosticoutcome of glioblastoma patients. Methods:1)985cases of adult glioma patient werecollected from Department of Neurosurgery in Affiliated Tunor Hospital of XinjiangMedical University and First Affiliated Hospital of Xinjiang Medical University. Amongthese cases,843were from Han or Uygur ethnicity. The rest of142cases were fromKazak, Hui, and Mongolian ethnicity. The patient’s general information, clinicalmanifestations, imaging features, clinical and pathological features were gathered andcompared. The distribution of clinical features was compared and analyzed usingstatistical approach. In addition, telephone interview and follow up screening wereconducted among111glioma patients from Tumor Hospital Affiliated to Xinjiang MedicalUniversity, all in the period of Jan2010to May2013.80of them were from Han or Uygurethnicity, and31of them were from other ethnic groups. P53, MGMT, PTEN and Ki-67protein expression were examined using immunohistochemical SP method. Therelationship between P53, MGMT, PTEN, Ki-67protein expression and the patients’ clinical characteristics was studied.2) Using Illumina HT-12mRNA expression profilingmicroarray, gene expression from6cases of glioblastomas (3Uygur patients and3Hanpatients) was examined. Differentially expressed genes were screened. Total RNA wasextracted, reverse transcribed into cDNA, and hybridized with gene expression microarrayto obtain the final data and images. All thesamples passed category6qualitycontrol.Immunohistochemical method was used to detect tumor-associated molecularmarkers. Comprehensive analysis and verification were performed with screeneddifferentially expressed genes.3) Illumina Methylation450K genome-wide methylationmicroarray was used to examine6cases of glioblastoma (Han, Uygur,3cases each).Differentially methylated sitesand their corresponding gene expression were screened.DNA was extracted. After bisulfite treatment, and hybridization, data and images wereobtained. Allsamples passed category11internal quality control. Screened differentialgene corresponding to the differentially methylated sites and screened differential genesfrom mRNA gene expression were subjected to joint analysis of hypermethylation lowexpression and hypomethylation high expression. Differentially expressed genes wereidentified. Results:1) In all enrolled patients,481cases were male and362cases werefemale. The age was from1～79. The average age was40.14±18.03years, of which342were Uygur patients and501were Han patients. The distribution difference based onWHO classification was statistically significant. Astrocytoma accounted for the highestpercentage (65.72%), up to554cases. The rest are: oligodendrocytes tumors (n=50,5.93%), oligodendrocytes astrocytomas (n=12,1.42%), ependymal tumors (n=149,17.67%), choroid plexus tumors,(n=7,0.83%), mixed neuronal neurons (n=11,1.30%),embryonal tumors (n=54,6.41%), and pineal tumors (n=6,0.71%). From the80followed-up patients, WHO grade, taken radiotherapy or chemotherapy, total section ornot are the risk factors affect the prognosis of glioma patients. P53, MGMT, PTEN andKi-67-positive rate was66.25%(53/80),65%(52/80),45%(36/80) and47.5%(38/60)respectively among80patients. MGMT, P53, and Ki-67expression were not statisticallysignificant (P＞0.05) in the groups of different age, gender, ethnicity, tumor location,tumor size, invasion near the Lobar. Statistical significance was found in the case ofHistological grade. PTEN is not statistically significant in the groups of different age,gender, ethnicity, tumor size, or invasion near the lobar. However, histological grading andtumor region were statistically significant factors (P＜0.05).2) Import the microarrayscanning images into Illumina Genome Studio software. Standardizing the corrected datato analysis the differential gene expression using the language of R Lumi (P＜0.05); using Web Gestalt software, GO and KEGG analysis of differential gene (P＜0.05).Glioblastomas in Uygur and Han gene mRNA expression profiles comparative analysisscreen out1475significant differentially expressed genes, which contained669(44.84%)up-regulated as well as807(55.16%) down-regulated (of which there are1genes STRCcorresponding to2transcripts,1transcripts up-regulated expression of1transcriptsdown-regulated), mainly involved in the metabolic process, biological regulation, inresponse to stimuli, multicellular organic process. The genes contains a sort of functionalnode, such as small GTP enzyme regulation signal pathway and Ras signal pathway andneuronal response protein regulation, central nervous system myelin formation. There arealso involved in multiple signaling pathways associated with tumor occurrence, such as:metabolic pathway and cancer pathways, MAPK signaling pathway and TGF-β signalingpathway, neurotrophic factor signaling pathway, mTOR signaling pathway.3) Themicroarray hybridization scanned images were imported into Illumina GenomeStudiosoftware. After calibration, data was standardized. Illumina custom was used to identifydifferentially expressed sites. Results were obtained after multiple times of correction andscreening. Using WebGestalt software, corresponding genes were annotated with GO andKEGG function (P＜0.05). Comparing the genome-wide methylation profiling,1903genes corresponding to6406differentially expressed sites was identified.1953sitesshowed up side (30.49%) and4453sites showed down side (69.51%) They are mainlyinvolved in biological regulation, metabolic process, multi-cell organic process, responseto stimuli, developmental processes, etc, including small GTP enzyme regulating signalingpathways, Ras signaling pathways, neuron-reactive protein regulation, CNS myelinationand other functional nodes. They are also participated in a number of tumor-associatedsignaling pathways, Such as: neural differentiation, neuronal development, MAPKsignaling pathway, TGF-β signaling pathway, neurotrophic factor signal transductionpathway, mTOR signaling pathways.4) Through the association analysis of mRNAexpression profiling and methylation, compare the upward or downward condition ofdifferentially expressed genes which are corresponding to the mRNA expression profileand methylation sites. There are12genes Hypermethylation of low expression, whichcontain ADARB2, ATP11C, ATPGD1, C1orf59, GPR62, PIK3C2B, PIR, PLLP, SLC5A11,ZFYVE27, ZNF415, ZNF536; There are12genes High expression of low methylation,which contain HSD17B10, MRPL3, ZBTB5, ZNF662. Conclusions:1) Comparing toglioma patient of Han ethnicity, Uygur patients have the trend of following characteristics:younger, more male patients and more likely to have low-grade glioma and astrocytoma type. Nation, WHO grade, taken radiotherapy or chemotherapy, total section or not may bethe factors affect the prognosis of glioma patients. Joint detection of MGMT, P53, PTENand Ki-67expression in gliomas provides a reference for determining the biologicalbehavior and prognosis of glioma.2) The differential genes of glioblastomas betweenUygur and Han patients in Xinjiang screened by mRNA expression profiling microarrayand genome-wide methylation microarray mainly involved in metabolic processes,biological regulation, response to stimuli, multicellular organic process, developmentalprocesses, etc. Small GTP enzyme regulating signaling pathways, Ras signaling pathways,neuronal response protein regulation, and central nervous system myelin formation areincluded. In addition, differential genes are also involved in a number of tumor-relatedsignaling pathways, such as: metabolic pathways, tumor pathways, neural differentiation,neuronal development, MAPK signaling pathway, TGF-β signaling pathway, neurotrophicfactor signal transduction pathway, mTOR signaling pathways, et al.3) The correlationanalysis between mRNA expression profiles and methylation status reveals the following:There are12common genes between the corresponding genes to high methylation sites(upregulation) from genome-wide methylation microarray screening and the lowexpressed (downregulated) differential genes from mRNA expression profiles, namely,ADARB2, ATP11C, ATPGD1, C1orf59, GPR62, PIK3C2B, PIR, PLLP, SLC5A11,ZFYVE27, ZNF415, ZNF536. There are4common genes between the correspondinggenes to low methylation sites (downregulation) from genome-wide methylationmicroarray screening and the highly expressed (upregulated) differential genes frommRNA expression profiles, namely, HSD17B10, MRPL3, ZBTB5, and ZNF662. Furtherstudy of the above genes will be help to reveal differences in the pathogenesis of gliomapatients of Uygur and Han ethnicity in Xinjiang in the gene level, and also provide a newbasis for further research and treatment of glioma in Xinjiang.