The correlation between Aromatase (CYP19A1) and Bladder cancer in Chinese Han Population

Several P450 metabolic enzymes genes have been discovered that affect the genetic susceptibility of bladder cancer. While, there is no report about CYP19A1 gene polymorphism on the susceptibility of bladder cancer. So, we performed a case–control study to investigate the association between polymorphisms in CYP19A1 gene and the susceptibility to bladder cancer in Chinese Han population. We found that CYP19A1 rs4646 “A” allele and rs17601876 “GA” genotype added the risk of bladder cancer (rs4646: OR = 1.28, 95%CI: 1.01 - 1.62, p = 0.043; rs17601876: OR= 1.64, 95%CI: 1.17 – 2.29, p = 0.004 ), while with rs6493487 “G” and rs1062033 “G” allele in CYP19A1 gene reduced the risk of bladder cancer (rs6493487: OR = 0.68, 95%CI: 0.51 - 0.89, p = 0.005; rs1062033: OR = 0.36, 95%CI: 0.28 - 0.47, p = 9.09E-16). WHO grade stratied analysis shown that rs6493487 (OR =0.42, 95%CI: 0.23 – 0.76, p = 0.004) and rs17601876 (OR =0.44, 95%CI: 0.23 – 0.85, p = 0.015) reduced the risk of bladder cancer with WHO III-IV, when compared with the bladder cancer with WHO I-II. Age stratied analysis shown that rs4646 “A”, rs6493487 “G”, and rs1062033 “G” allele were associated with bladder cancer risk. gene polymorphism on the susceptibility of bladder cancer. We performed a case–control study to investigate the association between polymorphisms in CYP19A1 gene and the susceptibility to bladder cancer. The result revealed that CYP19A1 rs4646 “A” allele and rs17601876 “GA” genotype increased the risk of bladder, while, CYP19A1 rs6493487 “G” allele and rs1062033 “G” allele reduced the risk of bladder cancer.


Introduction
Bladder cancer, also known as urothelial cancer of the bladder, is the most common malignancy affecting the urinary system.
Most cases occur in people over 60 years, the incidence of bladder cancer increases with age, the incidence of bladder cancer in males is signi cantly higher than that in females (1,2). Currently, smoke and occupational exposure (aromatic compounds: benzidine and beta-naphthylamine) have been identi ed as the most common environmental pathogens of bladder cancer (3)(4)(5). But still can't explain that some people will never get bladder cancer even if they are exposed to speci c chemicals. Therefore, more and more people realized that the evolution of bladder cancer is a multi-gene, multi-factor participation process, which is the result of complex genetic and environmental factors.
In the human body, a variety of metabolic enzymes play an important role in the activation and inactivation of exogenous chemicals, thus, the genetic polymorphism of metabolic enzymes affects the occurrence of bladder cancer in individuals exposed to pathogenic factors. For example, one meta-analysis on CYP2E1 gene and bladder cancer, found that CYP2E1 gene polymorphisms might be a protective factor against bladder cancer in Asian people (6). In research of Turkish population revealed that the GSTT1 null genotype increased the risk of bladder cancer with OR of 3.08 (7). Two SNPs (rs2472299 and rs2198843) on CYP1A1 gene has been identi ed that increased the risk of bladder cancer (8). On the study of Spanish population provides strong evidence for the role of common CYP1B1 variants as risk factors for BC (9). In addition, the P540 genes that affect the risk of bladder cancer include CYP2D6 (10), CYP2C9 (11), and so on. While, there is no report about CYP19A1 gene polymorphism on the susceptibility of bladder cancer.
Cytochrome P450 19A1 (CYP19A1), also known as aromatase, encoded by the CYP19A1 gene, has a key role in estrogen biosynthesis, irreversible step in the synthesis of estrogens through mediating the conversion of androstenedione and testosterone to estrone and estradiol. Moreover, several studies have shown that expression of aromatase is correlated with tumorigenesis in prostate cancer (12), bladder cancer (13), ect. Thus, the CYP19A1 polymorphism may affect changes in estrogen levels and is associated with risk of bladder cancer. So, we selected ve SNPs on CYP19A1 gene to assess the association between CYP19A1 gene polymorphisms and bladder cancer in Chinese Han population from Hainan Province.

Materials And Methods
This study was approved by the Research Ethics Committee of the Haikou people's Hospital in Haikou, and the study was A case-control study was performed to determine the association between polymorphisms of aromatase (CYP19A1) gene and BC. 217 BC cases (age 64.40 ± 10.99 years) and 550 age-matched healthy controls (age 63.92 ± 6.62 years) were enrolled from Haikou people's Hospital. All BC cases were diagnosed by histological con rmation and were from outpatients of the Department of Urology. Also, we recruited controls from adult health examinations. The case group and the control group were all Han nationality and were living Haikou and surrounding areas, and there was no difference in the distribution of the land. Written informed consent was obtained from all study subjects before a questionnaire interview and biological specimen collection.

SNP selection and genotyping
By reviewing CYP19A1 and urinary system tumor related literature, we selected ve SNPs on CYP19A1 gene (rs4646, rs6493487, rs1062033, rs17601876, and rs3751599) according to the principle that the minimum allele frequency was greater than 0.05, which were associated with tumor. The primers for related sites were designed according to Agena online software. 5 ml venous blood was taken and whole genome DNA was extracted. The single nucleotide polymorphisms rs4646, rs6493487, rs1062033, rs17601876, and rs3751599 were detected in the case group and the control group by MassARRAY time ight mass spectrometry array SNP genotyping platform, which consistent with the method of the previously published article (14-16).

Statistical analysis
Demographic characteristics were counted. The Hardy-Weinberg equilibrium (HWE) was calculated by χ2 test (17). Five genetic models were used to evaluate the association between gene polymorphisms and breast cancer risk. Odds ratios (ORs) and its corresponding 95%CI were estimated using an logistic regression model with adjustments for age and gender through the PLINK software (18). Risk analysis of CYP19A1 and bladder cancer was also performed in different genetic models according the age strati cation. Table 1 shows the basic information of the 767 participants, a total of 217 bladder cancer (64.40 ± 10.99 years) and 550 controls (63.92 ± 6.62) including in our research, and the mean age of the case group and the control group is matched (p = 0.549). Of 217 patients with bladder cancer, 68 were classi ed as WHO I-II and 86 as WHO III-IV, and 63 patients unknown. Table 2 shows the basic information of ve gene polymorphisms on the CYP19A1 gene, including gene, position, alleles, SNP function annotations and HWE results The results of the HWE showed that the genotype frequency distributions of CYP19A1 in the control groups was in line with genetic balance (all p > 0.05), which showed that all of the 5 SNPs were at equilibrium and were representative.

Results
We analysis the in uence of CYP19A1 gene polymorphisms on the risk of bladder cancer by logistic regression ( Then strati ed analysis was conducted according to age (Table 4), and it was found that in the allele model, rs4646 A allele increased the risk of bladder cancer (OR = 1.42, 95%CI: 1.03-1.95, p = 0.033) and rs6493487 G allele decreased the risk of bladder cancer (OR = 0.60, 95%CI: 0.41-0.89, p = 0.011) in less than 65 years old group; while in more than 65 years old group, we did not nd signi cant results. In less than 65 years old group and more than 65 years old group, we also found that rs1062033 G allele decreased the risk of bladder cancer in the allele model (≤ 65 years: OR = 0.38, 95%CI: 0.28-0.54, p = 1.13E-08; > 65 years: OR = 0.35, 95%CI: 0.23-0.52, p = 7.88E-08 ) Then strati ed analysis was conducted according to WHO grade (Table 4)

Discussion
Clinical Implications Several P450 metabolic enzymes genes have been discovered that affect the genetic susceptibility of bladder cancer. While, there is no report about CYP19A1 gene polymorphism on the susceptibility of bladder cancer. We performed a case-control study to investigate the association between polymorphisms in CYP19A1 gene and the susceptibility to bladder cancer. The result revealed that CYP19A1 rs4646 "A" allele and rs17601876 "GA" genotype increased the risk of bladder, while, CYP19A1 rs6493487 "G" allele and rs1062033 "G" allele reduced the risk of bladder cancer.
Aromatase is the last step-limiting enzyme in the synthesis of estrogen. Its quantity and activity directly determine the content of androgen and estrogen in the body. It belongs to the cytochrome P450 family and has the function of catalyzing the synthesis of estrogen. The aromatase is encoded by the CYP19A1 gene, the structure and activity of the aromatase protein can be affected by genetic variation, which can reduce, inactivate, or increase the enzyme activity. In 95 patients with breast cancer who received letrozole, patients with the CYP19A1 rs4646 A allele were found to be less effective than those with the C allele (19). In a study on the adverse metabolic effects of thiazides on hypertensive patients, CYP19A1 rs6493487 G allele association with glucose response to hydrochlorothiazide in meta-analysis of African-Americans (20). In our research, we found that CYP19A1 rs4646 "A" allele promoted the risk of bladder cancer, and rs4646 located on 3'UTR. SNPs located in the 3′-UTRs of genes may affect the expression of the gene by reinforcing, weakening or disrupting the miRNA-mRNA interaction, and thereby confer the individual's disease risk (21). Nguyen DP et al. found that Aromatase expression was signi cantly associated with bladder cancer tumor stage, and Cox regression analysis demonstrated that aromatase expression was associated with a more than 2-fold risk of cancer recurrence (HR = 2.37) (13). After this, Shulin Wu et al. also found that in 88 bladder cancer cases examined, high aromatase expression was present in 37.5% and 73.9% of Tumor epithelium (TE) and tumor related stroma (TS) respectively, and high aromatase expression in TS was signi cantly associated with poorer overall survival (22). Those remind us aromatase (CYP19A1) plays an important role in the development of bladder cancer. And the risk allele of rs4646 may in uence the expression of aromatase (CYP19A1), and increased the risk of bladder cancer. However, its speci c molecular mechanism is unknown, and our subsequent further research will clearly de ne the mechanism and provide a basis for early diagnosis of bladder cancer.
rs17601876 "GA" genotype increased the risk of bladder. In rectal cancer, GG genotype of rs17601876 increased the mortality risk (HR = 2.6) (23). Besides that rs6493487 G allele and rs1062033 G allele in CYP19A1 gene reduced the risk of bladder cancer.
These sites are located in the intron region of the CYP19A1 gene. Introns can increase transcript levels by affecting the rate of transcription, nuclear export, and transcript stability. Moreover, introns can also increase the e ciency of mRNA translation (24).
However, the speci c mechanism of rs17601876, rs6493487 and rs1062033 on the CYP19A1 gene affecting bladder cancer is unclear.

Study Limitations
In summary, our research report that CYP19A1 rs4646 and rs17601876 increased the risk of bladder, and CYP19A1 rs6493487 and rs1062033 reduced the risk of bladder cancer. The interaction between genes and the environment and the molecular mechanisms of genes in bladder cancer have not been studied, which will be the focus of our follow-up research.

Conclusion
In summary, our research report that CYP19A1 rs4646 and rs17601876 increased the risk of bladder, and CYP19A1 rs6493487 and rs1062033 reduced the risk of bladder cancer. The interaction between genes and the environment and the molecular mechanisms of genes in bladder cancer have not been studied, which will be the focus of our follow-up research.

Declarations
Ethics approval and consent to participate Not applicable

Not applicable
Availability of data and material Data sharing not applicable to this article as no data-sets were generated or analyzed during the current study.

Competing interests
The authors declare that they have no competing interests.  Tables   Table 1 The information of all participants   Variable Cases ( Table 4 Risk analysis of CYP19A1 and bladder carcinoma in different genetic models according the age stratification