Study On Functions And Regulatory Mechanisms Of WDR1 In Glioblastoma Susceptibility And Progression

Gliomas are the most common primary cancers in the central nervous system,of which glioblastoma with the highest degree of malignancy accounts for the largest percentage.Characterized by rapid proliferation,invasive growth,and resistance to treatment,glioblastoma recurs almost 100% even after surgical resection combined with temozolomide and radiotherapy.Patients with glioblastoma have to face a fatal course with a median overall survival of 12-15 months.Therefore,it is urgent to develop new therapeutic regimens to improve the clinical outcome of glioblastoma patients.Currently,studies have suggested that the tumorigenesis and development of glioblastoma is closely related to the accumulation of gene mutations and dysfunction of signal pathways.Based on the development of big dataomics,a variety of molecular mechanisms involved in the malignant process are being gradually identified,which pave a new way for treating glioblastoma.However,results of most of clinical trials have shown that glioblastoma patients have benefitted little from current molecular targeted therapies.Thus,it is particularly important to find new therapeutic targets that play central roles in the process of cell proliferation,invasion,and chemo-radiotherapy resistance of glioblastoma.Actin interacting protein WDR1 participates in the regulation of actin filament dynamics and actin-dependent cellular biological processes,such as the structure and remodeling of cytoskeletal and intercellular junctions,and plays important roles in cell division and migration.It has been found that WDR1 expression increases significantly in breast cancer,ovarian cancer,and thyroid cancer,and promotes tumor invasion and migration,suggesting WDR1 as a tumor-specific molecule with important functions involved in tumor development and progression.In this study,multi-omics microarray data were used to filter out WDR1 up-regulated in glioblastoma and associated with patient’s prognosis.Then,the function of WDR1,as well as its regulatory signaling,was further studied in glioblastoma cells.Finally,the association between WDR1 genetic variation and the susceptibility of glioblastoma was analyzed.Part Ⅰ: Expression of WDR1 in Glioblastoma and Its Association with Patients’ PrognosisOBJECTIVE: A small sample of gliomas was used to screen prognosis-related genes,which were analyzed in multi-database with glioblastoma.Then,the expression of aimed gene and its prognostic value would be further validated in a large-sample cohort of glioblastoma.METHODS: 14 fresh glioma samples were collected for RNA microarray detecting.Then,a parallel cluster analysis of differentially expressed genes with prognosis was performed based on the length of survival time(cut by 60 months).The gene expression profiles and clinical data of gliomas and normal brains from TCGA and CGGA databases were collected to analyze the expression level of aimed gene in tumors and controls,its association with pathological grades and prognosis through Student-t test,Spearman correlation test,and Kaplan-Meier curve.Further,a total of 100 glioblastomas and 16 normal brain samples were collected to construct tissue microarrays.The expression of WDR1 detected by immunohistochemical staining,as well as its relevance of prognosis was verified by t-test,univariate analysis and multivariate Cox regression.RESULTS: In gene expression array analysis,a total of 83 genes were selected with p-value of less than 0.001 and fold change more than 2.59 up-regulated genes were screened out in the group with poor prognosis.Analysis of all selected genes was carried out in both TCGA and CGGA databases.The results of TCGA database showed that WDR1 expression was significantly up-regulated 2.2 fold in glioblastoma compared with normal brain(p<0.001),and high expression of WDR1 significantly related to shorter overall survival(p=0.017,median survival time=345 days)and progression-free survival(p=0.044,median survival time=186 days).The results of CGGA database showed that the expression of WDR1 was increased with the pathological grade of gliomas(p<0.001)and associated with poor overall survival(p=0.024,median survival time=345 days)and progression-free survival(p=0.046,median survival time=240 days)of glioblastoma patients.Further analysis in our cohort also showed that WDR1 expression was higher in glioblastoma than control(p<0.001).Survival analysis showed that up-regulated of WDR1 was significantly associated short overall survival time(p=0.001,median survival time=10 months)and progression-free survival(p=0.005,median survival time=8 months),and the expression of WDR1 was independent prognosis risk factor for glioblastoma(overall survival time,HR=2.147,p=0.001;progression-free survival time,HR=1.912,p=0.005).Conclusion: The expression of WDR1 in glioblastoma is significantly up-regulated and is positively correlated with the pathological grade.It can work as an independent risk factor for the survival of glioblastoma patients,suggesting that WDR1 may be involved in the malignant process of glioblastoma.Part Ⅱ: The Function and Mechanism of WDR1 in Glioblastoma CellsObjective: In vitro experiments were performed to determine the role of WDR1 in the glioblastoma progression,investigate the mechanism of regulation of WDR1 expression,and explore potential downstream signaling pathways involved in WDR1.METHODS: Plasmids of WDR1 overexpression and knockdown were constructed and packaged by lentivirus to infect T98 G and U87 cell lines.Cell proliferation was detected by CCK-8 assay and Ed U staining and Transwell assay was used to detect cell invasion.STAT3 overexpression vector was further constructed and transfected into glioma cells together with IL-6,the expression of WDR1 m RNA was detected by real-time quantitative PCR.The luciferase reportor system was carried out to investigate the regulation between STAT3 and the promoter of WDR1 in 293 FT cell line.Meanwhile,bioinformatics analysis was performed to preliminarily study potential downstream pathways underlying WDR1 based on glioblastoma expression profiles in TCGA and CGGA databases.RESULTS: Plasmids of WDR1 overexpression and sh RNAs were demonstrated to work efficiently.Compared with the control,overexpression of WDR1 significantly increased the proliferation rate and invased cell number,while depleted WDR1 inhibited the abilities of proliferation,and invasion with statistical significance.Additionally,WDR1 expression was elevated in glioma cells with overexpressing STAT3 in the presence of IL-6,and the wild-type WDR1 promoter had a significantly higher fluorescence value than the mutant and control groups.Bioinformatics analysis showed that WDR1 might regulate focal adhension,ECM-receptor interaction,and PI3K-Akt signaling pathway to promote glioblastoma progression.Conclusions: WDR1 is involved in the regulation of the proliferation and invasion of glioblastoma,and STAT3 regulates WDR1 gene expression,suggesting that STAT3-WDR1 pathway plays an important role in the development and progression of glioblastoma.Part Ⅲ: The association between genetic variations of WDR1 and risk of gliomaObjective: Molecular epidemiological study was performed to analysis the association between the single nucleotide polymorphisms of WDR1 and the risk of glioma.METHODS: Peripheral blood samples were collected from 530 glioma patients(208 glioblastomas)and 524 normal healthy persons.The basic epidemiological information(gender,age,and so on)were recorded,and blood sample DNA was extracted for SNP genotyping.Tag-SNPs were selected based on the Hap Map database.Student t-test was carried out to compare the mean value of continuity variable.χ2 test was performed to assess the distribution of discrete variable.Unconditional logistic regression analysis was used to analyze the association between SNPs of WDR1 and the risk of glioma.RESULTS: The mean age was 50.83±10.54 years old in 530 cases and 50.83±10.54 years old in 524 controls.The males accounted for 58.7% in cases and 60.3% in control group.2.3% cases were in grade I,37.9% cases were in grade II,20.6% cases were in grade III,and 39.2% cases were in grade IV.12 tag SNPs were selected in the region spanned from 2K basepairs of WDR1 upstream to 2K basepairs of downstream of WDR1 with the criteria of minimum allele frequency(MAF)≥ 0.05 and r2 ≥ 0.8.rs9926 located in 3’UTR,and rs13441 located in exon,and other SNPs located in introns.χ2 test showed that the genotypes of the rs3756230,rs734122,and rs13441 were distributed differently between the glioma group and the control group with P values of 0.03,0.02,and 0.02,respectively.Single-SNP analysis showed that the GG genotype of rs3756230 could significantly increase the risk of glioma in the co-dominant model(p=0.04,adjusted OR=1.976),and(CG+GG)genotype could significantly increase the risk of glioma(p=0.045,adjusted OR=1.304)compared with the CC genotype in the dominant model.Trend analysis showed that glioma risk increased with the increase of the number of G allele(p=0.019,adjusted OR=1.303).In the co-dominant model,the rs13441 TT genotype had a protected effect for glioma risk compared to wild-type TT(p=0.002,adjusted OR=0.541).Trend analysis showed that glioma risk decreased with the increase of the number of T allele(p=0.007,adjusted OR=0.779).The results of stratified analysis of gender showed that the CT genotype(p=0.043,adjusted OR=0.615)and TT genotype(p=0.001,adjusted OR=0.341)could significantly decrease the risk of female patients(p=0.001,adjusted OR=0.589)compared to CC genotype of rs13441 in the co-dominant model,and(CT+TT)genotype of rs13441 could significantly reduced the risk of female patients(p=0.006,adjusted OR=0.534)compared to CC genotype in dominant model.The results of stratified analysis of pathology showed that TT genotype of rs13441 could significantly decrease the risk of glioblastoma patients(p=0.029,adjusted OR=0.537)compared to CC genotype in the co-dominant model,Trend analysis showed that glioblastoma risk significantly decreased with the increase of the number of T allele(p=0.027,adjusted OR=0.768).Conclusion: WDR1 genetic polymorphisms are associated with glioma susceptibility,suggesting that the genetic background of WDR1 gene may play a role in the development of glioma.