Study On The Relationship Between Methylation Status And Expression Level Of CTSZ And Prognosis Of High Grade Glioma Patients

Objective High-throughput profiling techniques for gene expression and epigeneticmodification have provided new thinking of subtype classifications and some targetmolecules for gliomas. These target molecules, which are related to cellular biologicalcharacteristics and prognosis of glioma may have potential prognostic and predictivevalues. We had performed genome-wide DNA methylation profiling of44III gradegliomas and42GBM patients from Department of Neurosurgery, Beijing TiantanHospital, aiming to identify epigenetically altered gene(s) which may affect highgrade patient outcome. Cathepsin Z(CTSZ) gene which is differentially methylated inthese patients has been assumed as the best candidate gene for further study. Thisstudy is to explore the methylation level, prognostic value and functional role ofCTSZ in glioma patients,Methods：1, DNA extraction: Genomic DNA was isolated from frozen tumor tissuesusing the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol.DNA concentration and quality were measured using the NanoDrop ND-1000spectrophotometer (NanoDrop Technologies, Houston, TX).2, Genome-wide DNAmethylation profiling: A series of86samples (44III grade and42GBM) weremeasured by methylation microarray. The Illumina Infinium Human Methylation27Bead-Chips (Illumina Inc.) were used.. The BeadChip contains27,578highlyinformative CpG sites covering more than14,000human Ref Seq genes. Bisulfitemodification of DNA, chip processing and data analysis were performed followingthe manufacturer’s manual at the Wellcome Trust Centre for Human Genetics Genomics Lab, Oxford, UK. The array results were analyzed with the BeadStudiosoftware (illumina).3, In vitro function study: we treated glioblastoma cell linesU251with SiRNA and detected cell biological behaviors. Western blotting was usedto verify CTSZ protein expression and MTT test to analyze cell proliferation,transwell assay to analyze cell invasiveness.4, Statistical analysis: SAM (significanceanalysis of microarrays) was used for genes significantly differently methylatedbetween44III grade gliomas and42GBM. Cox-regression was performed usingMatlab2009b software. Survival curves were calculated according to theKaplan–Meier method, and differences between curves were assessed using thelog-rank test. All statistical analysis were carried out using the software GraphPadPrism (GraphPad Software, La Jolla, CA) and SPSS version16.0(SPSS, Chicago, IL,USA). P <0.05was considered significant.Results：.The methylation level of CTSZ were relative to glioma grade progression andinversely correlated with overall survival in high-grade glioma patients (anaplasticgliomas and glioblastomas) The relationship is consistent with the result of TCGAdata. Functional analyses in U251cells revealed that CTSZ was involved in cellinvasion and proliferation.Conclusions：The expression of CTSZ is relative to glioma grade progression andconfers a poor prognosis via promoting cellular invasion and proliferation inhigh-grade glioma patients.