The Impact of PPARD and PPARG Polymorphisms on Glioma Risk and Prognosis

Recent studies showed that peroxisome proliferator-activated receptors (PPARs) had effects on the progression of multiple tumors, but the role of PPARD and PPARG in glioma remains poorly understand. We conducted a case-control study to investigate the association of polymorphisms in PPARD and PPARG with glioma risk and prognosis in the Chinese Han population. Seven polymorphisms (PPARD: rs2016520, rs67056409, rs1053049 and rs2206030; PPARG: rs2920503, rs4073770 and rs1151988) were genotyped using the Agena MassARRAY system in 568 glioma patients and 509 healthy controls. The odd ratios (OR) and 95% confidence interval (CI) were calculated to assess the association of PPARD and PPARG polymorphisms with glioma risk. The Multifactor dimensionality reduction (MDR) method was used to analysis interactions of genetic polymorphisms on glioma risk. Then, we conducted log-rank test, Kaplan-Meier analysis and Cox regression model to evaluate the relationship of PPARD and PPARG polymorphisms with glioma prognosis. We found PPARD polymorphisms (rs2016520, rs67056409, rs1053049) were significantly associated with glioma risk in multiple models (P < 0.05). Stratified analysis showed rs2016520, rs67056409, rs1053049 of PPARD significantly decreased risk of glioma in the subgroup of age > 40 and astrocytoma (P < 0.05). For male, PPARD rs1053049 had a strong relationship with glioma risk in allele (P = 0.041), dominant (P = 0.040) and additive (P = 0.040) models. The effect of PPARG rs2920503 on glioma risk was related to glioma grade (P < 0.05). MDR showed that a seven-locus model was the best polymorphisms interaction pattern. Moreover, surgery and chemotherapy had strongly impact on overall survival and progression free survival of glioma patients. Our findings suggested that PPARD and PPARG polymorphisms were associated with glioma risk and prognosis in the Chinese Han population, and further studies are need to confirm our results.


Snp selection and genotyping.
Combined previously studies, we selected four SNPs of PPARD (rs2016520, rs67056409, rs1053049 and rs2206030) and three SNPs of PPARG (rs2920503, rs4073770 and rs1151988), with minor allele frequencies (MAF) greater than 5% in the HapMap Chinese Han Beijing population. We extracted DNA from peripheral blood samples using the blood DNA kit (GoldMag Co. Ltd., Xiʹan, China). SNP genotyping was performed in the Agena MassARRAY system (Agena, San Diego, CA, USA). Primers for polymerase chain reaction (PCR) amplification and extension were designed by the Agena MassARRAY Assay Design 3.0 Software (San Diego, CA USA). PCR primers of selected SNPs were listed in Supplemental Table 1. In addition, we used Agena Typer 4.0 Software (San Diego, CA, USA) to manage and analyze data.

Statistical analysis.
We conducted all statistical analysis using Microsoft Excel and SPSS version 21.0 software (SPSS, Chicago, IL, USA). Student's t-test and chi-square test were used to compare the differences in age and sex between glioma patients and healthy controls. The Hardy-Weinberg equilibrium (HWE) was checked for controls with Fisher's exact test. We assessed the association of PPARD and PPARG polymorphisms with glioma risk by calculating odd ratios (OR) and 95% confidence intervals (CI) using logistic regression. Multifactor dimensionality reduction (MDR, version 3.0.2) was used to analyze SNP-SNP interactions on glioma risk. Then, we plotted patient survival curves by the Kaplan-Meier method and log-rank test. The association of PPARD and PPARG polymorphisms with OS and progression free survival (PFS) of glioma patients was evaluated by calculating hazard ratios (HR) and 95%CI using univariate and multivariate analysis. In multivariable survival analysis, we assessed the associations of PPARD and PPARG polymorphisms with glioma prognosis adjusted by age, sex, WHO grade, surgery, radiotherapy and chemotherapy. All tests were two-sided, and P < 0.05 was regarded as statistical significance. Additionally, our results were adjusted for multiple comparison using false discovery rate (FDR) correction.

Results
characteristics of study population. The characteristics of 568 glioma patients and 509 healthy controls were presented in Table 1. The mean ages of the cases and controls were 39.68 ± 16.96 and 41.32 ± 15.69 years old, respectively. No significant variation in age or sex was found between the two groups (age: P = 0.102, sex: P = 1.000). Among glioma patients, 438 (77%) people were astrocytoma. According to WHO grading standards, 35 (6%) patients were grade I, 320 (56%) patients were grade II, and others are in high-grade glioma (III + IV). In addition, surgery method, radiotherapy and chemotherapy of patients were shown in Table 1.
Association of PPARD and PPARG polymorphisms with glioma risk. In Table 2, PPARD and PPARG polymorphisms were accord with HWE in controls (P > 0.05).

Discussion
In this case-control study, we examined the association of PPARD and PPARG polymorphisms with glioma risk and prognosis in the Chinese Han population. After FDR correction, we found that PPARD polymorphisms were significantly associated with glioma risk, and the effects were dependent on age (P < 0.05, FDR-P < 0.05). Moreover, surgery method and chemotherapy had strongly effects on glioma prognosis (Log-rank P < 0.05, P < 0.05, FDR-P < 0.05).
PPARs are involved in the regulation of metabolic homeostasis, whose activity are controlled by fatty acid ligands 34 . After activation, PPARs heterodimerize with retinoid X receptors (RXRs) to affect the expression of www.nature.com/scientificreports www.nature.com/scientificreports/ downstream genes. It is reported that PPARs might had a functional crosstalk concerning the control of their expression 35 . Previous studies on the role of PPARs signaling in cancer mainly based on the availability of PPARs agonists and antagonists 36 . In brain tumor stem cells, PPARγ agonists inhibit cell growth and induce cell cycle arrest 37 . In mice, expression of PPARδ is related to prognosis and metastatic ability of breast cancer cells 38 . Polymorphisms of PPARD and PPARG are associated with risk and prognosis of many diseases, including cardiovascular disease, diabetes, brain diseases, medulloblastoma and other cancers [39][40][41] . In our study, we firstly observed that PPARD polymorphisms (rs2016520, rs67056409 and rs1053049) were significantly associated with glioma risk. Similar association has been reported in colorectal cancer 39 . It suggests that PPARD polymorphisms could be involved in the susceptibility of glioma development. And, stratified analysis showed the effects  www.nature.com/scientificreports www.nature.com/scientificreports/ of PPARD polymorphisms on glioma risk were age-dependent. It provides a scientific basis on individualized treatment of glioma. The effects of PPARD polymorphisms on glioma risk might related to SiPhy cons, Promoter histone marks, Enhancer histone marks, DNAse, Motifs changed, GRASP QTL hits, NHGRI/EBI GWAS hits, Selected eQTL. However, our results should be confirmed in further studies, including next-generation technology, PCR, western-blot analysis, etc.
Glioma is likely to have unfavorable prognosis caused by rapid proliferation and diffuse brain invasion. Despite surgery, chemotherapy and radiotherapy treatments improve, the prognosis of glioma remains poor 42 . Recent studies reported that some lipophilic molecules have antiproliferation and/or differentiation effects on glioma cells, and PPARs mediated some activities of these processes 43 . PPARγ has been observed in transformed neural cells of human and PPARγ agonist interferes with glioma growth and malignancy [43][44][45] . In this study, we  www.nature.com/scientificreports www.nature.com/scientificreports/ firstly confirmed the effects of surgery method and chemotherapy on prognosis of glioma patients. Then, we explored the association of PPARD and PPARG polymorphisms with OS and PFS of glioma patients. No significant associations were observed by univariate and multivariate analysis. It demonstrated that PPARD and PPARG polymorphisms might not contribute the prognosis of glioma.
There are some limitations in the present study. First, we selected and genotyped several polymorphisms of PPARD and PPARG, more genetic polymorphisms should be studied in the future. Second, we could not evaluate more factors on the association of genetic polymorphisms and glioma risk due to the limited sample size and information. Third, the molecular mechanisms of PPARD and PPARG on glioma risk and prognosis are not elucidated in our study.

conclusion
In conclusion, we found genetic polymorphisms of PPARD were associated with glioma risk in the Chinese Han population, which suggests the role of PPARD in the carcinogenesis of glioma.
It provided information on exploring the mechanism and targeted therapy of glioma, it also promotes the development of precision medicine on glioma. Further studies in larger samples with more ethnic groups are needed to validate our results and explore the mechanism of PPARD and PPARG in glioma.   Table 6. The impact of clinical factors on glioma patient OS and PFS. OS, overall survival; PFS, progression free survival; SR, survival rate; HR, hazard ratio; CI, confidence interval; WHO, World Health Organization; STR, sub-total resection; NTR, near-total resection; GTR, gross-total resection; FDR, false discovery rate. Bold values indicate statistical significance (P < 0.05).