Abstract
Recently N6-Methyladenosine (m6A) has been identified to guide the interaction of RNA-binding protein hnRNP C and their target RNAs, which is termed as m6A-switches. We systematically investigated the association between genetic variants in m6A-switches and bladder cancer risk. A two-stage case–control study was performed to systematically calculate the association of single nucleotide polymorphisms (SNPs) in 2798 m6A-switches with bladder cancer risk in 3,997 subjects. A logistic regression model was used to assess the effects of SNPs on bladder cancer risk. A series of experiments were adopted to explore the role of genetic variants of m6A-switches. We identified that rs5746136 (G > A) of SOD2 in m6A-switches was significantly associated with the reduced risk of bladder cancer (additive model in discovery stage: OR = 0.80, 95% CI 0.69–0.93, P = 3.6 × 10−3; validation stage: adjusted OR = 0.88, 95% CI 0.79–0.99, P = 3.0 × 10−2; combined analysis: adjusted OR = 0.85, 95% CI 0.78–0.93, P = 4.0 × 10−4). The mRNA level of SOD2 was remarkably lower in bladder cancer tissues than the paired adjacent samples. SNP rs5746136 may affect m6A modification and regulate SOD2 expression by guiding the binding of hnRNP C to SOD2, which played a critical tumor suppressor role in bladder cancer cells by promoting cell apoptosis and inhibiting proliferation, migration and invasion. In conclusion, our findings suggest the important role of genetic variants in m6A modification. SOD2 polymorphisms may influence the expression of SOD2 via an m6A-hnRNP C-dependent mechanism and be promising predictors of bladder cancer risk.
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Abbreviations
- SNP:
-
Single nucleotide polymorphism
- GWAS:
-
Genome-wide association study
- m6A:
-
N6-Methyladenosine
- RBP:
-
RNA-binding proteins
- MAF:
-
Minor allele frequency
- HWE:
-
Hardy–Weinberg equilibrium
- LD:
-
Linkage disequilibrium
- qRT-PCR:
-
Quantitative real-time PCR
- TCGA:
-
The Cancer Genome Atlas
- GEO:
-
Gene Expression Omnibus
- GEPIA:
-
Gene Expression Profiling Interactive Analysis
- GTEx:
-
Genotype-Tissue Expression
- ORF:
-
Open reading frame
- RIPA:
-
Radio immunoprecipitation assay
- PMSF:
-
Phenylmethanesulfonyl fluoride
- PVDF:
-
Polyvinylidene fluoride
- CCK8:
-
Cell Counting Kit 8
- DAA:
-
3-Deazaadenosine
- RIP:
-
RNA immunoprecipitation
- meRIP:
-
Methylated RNA immunoprecipitation
- OR:
-
Odds ratio
- CI:
-
Confidence interval
- eQTL:
-
Expression quantitative trait locus
- dbGaP:
-
Database of Genotypes and Phenotypes
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Funding
This study was supported in part by Collaborative Innovation Center for Cancer Personalized Medicine, and Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).
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ZZ, LY and HC conceived and designed the experiments. HL, JG and YJ wrote the paper. QY, GM, MD, YG, YL, ZG, and MW contributed reagents/materials/analysis tools. CQ, QL, GF, and LY recruited samples. All authors reviewed this manuscript.
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204_2020_2911_MOESM1_ESM.tif
Supplementary file1 (TIF 6235 kb) Supplementary Figure S1. The expression of SOD2 was significantly decreased in tumor tissues based on GEPIA2 (https://gepia2.cancer-pku.cn/) online database. GEPIA, Gene Expression Profiling Interactive Analysis.
204_2020_2911_MOESM2_ESM.tif
Supplementary file2 (TIF 12666 kb) Supplementary Figure S2. Disordered SOD2 expression was associated with poor prognosis in multiple tumors patients. Kaplan-Meier survival analysis of OS based on SOD2 expression using the Kaplan-Meier Plotter (https://kmplot.com/analysis/) online bioinformatics tool in 165 patients with rectum adenocarcinoma (A), 304 patients with cervical squamous cell carcinoma (B), 530 patients with kidney renal clear cell carcinoma (C), and 259 patients with sarcoma (D), respectively. OS, overall survival.
204_2020_2911_MOESM3_ESM.tif
Supplementary file3 (TIF 6140 kb) Supplementary Figure S3. SNP rs5746136 (G>A) was an eQTL for SOD2 in heart(A) and esophagus(B) based on GTEx Portal data. GTEx, Genotype-Tissue Expression.
204_2020_2911_MOESM4_ESM.tif
Supplementary file4 (TIF 14951 kb) Supplementary Figure S4. SNP rs5746136 G>A was associated with the increasing of WTAP mRNA level in multiple normal tissues based on GTEx Portal (https://www.gtexportal.org/) online database.
204_2020_2911_MOESM5_ESM.tif
Supplementary file5 (TIF 635 kb) Supplementary Figure S5. Data from the m6AVar (https://m6avar.renlab.org/) online database predicts and visualizes m6A site located in SOD2 transcripts.
204_2020_2911_MOESM6_ESM.tif
Supplementary file6 (TIF 26 kb) Supplementary Figure S6. The m6A modification enzyme WTAP was aberrantly decreased in TCGA data (P = 0.030). TCGA, The Cancer Genome Atlas.
204_2020_2911_MOESM7_ESM.tif
Supplementary file7 (TIF 261 kb) Supplementary Figure S7. EJ and J82 cells were transfected with SOD2-expressing plasmid or NC vector and the over-expression effect was verified at mRNA level by qRT-PCR(A) and protein level by Western blotting (B). All of the experiments were conducted in triplicate. Results were presented as mean ± SD. **P < 0.01. qRT-PCR, quantitative real-time PCR.
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Liu, H., Gu, J., Jin, Y. et al. Genetic variants in N6-methyladenosine are associated with bladder cancer risk in the Chinese population. Arch Toxicol 95, 299–309 (2021). https://doi.org/10.1007/s00204-020-02911-2
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DOI: https://doi.org/10.1007/s00204-020-02911-2