Abstract
miRNA play role in post transcriptional regulation of genes and serves a range of biological functions such as initiation, development, metastasis etc. which are also hallmarks of cancer. Hence, we evaluated miRNA 181a, miRNA 30c and miRNA 570 in bladder cancer risk association among North Indians. miRNA 570 C/G (rs4143815), miRNA 30c A/G (rs928508) and miRNA 181a C/T (rs12537) single nucleotide polymorphisms (SNPs) were genotyped by allelic discrimination TaqMan assay in 100 bladder cancer (BC) patients and 100 healthy controls. No significant difference was found in the genotype frequencies of the candidate SNPs among cases and controls. However, combined effect of miRNA 570-miRNA 30c (CG + AA) p = 0.005, OR = 0.223, 95% CI and miRNA 570-miRNA 181a (CG + CC) p = 0.003, OR = 0.169, 95% CI conferred association with no risk of BC. miRNA 181a C/T (rs12537), miRNA 30c A/G (rs928508) and miRNA 570 C/G (rs4143815) should be further validated in large sample size to be used as a risk predictor for bladder cancer among North Indians.
Keywords: Bladder cancer, miRNAs, SNPs, Cigarette smoking, Association
Introduction
Bladder cancer is the tenth most common cancer being 4 times more common in men than women globally [1]. Apart from various occupational exposures, smoking poses to be the main risk factor for BC. miRNAs play major role in processes which are hallmarks of cancer, so it is important to check their involvement in the risk prediction of bladder cancer. In a previous study from our lab we studied three SNPs of miRNAs viz. miRNA 146 G/C (rs2910164), miRNA 196a2 C/T (rs11614913) and miRNA 499 T/C (rs3746444) and found no association of any of these SNPs with bladder cancer risk [2]. Based on this, the present study was designed as a pilot study to see association of miRNA 570 C/G (rs4143815), miRNA 30c A/G (rs928508) and miRNA 181a C/T (rs12537) in BC risk.
miRNA 30c belongs to miR-30 family and is involved in a variety of malignant disease [3].
Recent studies have shown that miR-30 family, as tumor suppressor, played important roles in the development of various cancers. For example, Cheng et al. found that miR-30a could inhibit the metastasis and invasion of breast cancer cells by negatively regulating the expression of the vimentin [4]. Braun et al. reported that miR-30 could reduce the invasive potential of mesenchymal anaplastic thyroid carcinoma-derived cells [5]. Zhong et al. reported that the inhibition of miR-30c promoted the invasion of non-small cell lung cancer by promoting EMT process [6]. Furthermore, recent studies have found that miR-30 family could inhibit tumor cell growth, which was related to the change of tumor cell autophagy. Singh et al. found that the restoration of miR-30a weakened the tumorigenesis of medulloblastoma cells, accompanied with a decreased expression in Beclin 1 and the inhibition of autophagy [7]. Moreover, Zhang et al. found that miR-30d could inhibit autophagy of colon cancer cells by directly targeting messenger RNA of autophagy related protein 5 (ATG5), Beclin 1, and phosphoinositide 3-kinase (PI3K), thereby promoting cell apoptosis [8]. Collectively to the contradictory roles of miR-30 family in tumorigenesis, we infer that this may reflect the biological function of miR-30 family, which was related to the different tumor types, the expression level of miR-30 family, and corresponding various target molecules in distinct types of cancers. Therefore, we considered miRNA 30c to be a candidate molecule for risk prediction in bladder cancer. This new class of genes miRNA 181a has recently been shown to play a central role in malignant transformation [9]. miRNA 181a are down regulated in many tumors and thus appear to function as tumor suppressor genes. The mature products miR-181a, miR-181b, miR-181c or miR-181d are thought to have regulatory roles at post-transcriptional level, through complementarity to target mRNAs [10]. MicroRNA 570, are small noncoding RNAs that regulate gene expression by interacting with short complementary seed sequences in the 3-prime UTRs of target mRNAs [11]. Binding of miRNAs to mRNAs can inhibit translation or direct mRNA degradation. Thus, genetic alterations within the candidate molecules would be expected to provoke a deficient signaling, thereby facilitating the development of cancer.
Materials and Methods
Study Subjects
The bladder cancer patients in this analysis were enrolled from an on-going case–-control study of bladder cancer. All enrolled patients were incident cases of histologically confirmed invasive or superficial bladder cancer and were recruited from the Department of Urology at Sanjay Gandhi Postgraduate Institute of Medical Sciences, a tertiary care center. A total of 100 patients with histologically confirmed transitional urothelial BC (Mean age 58.5 years; 88 Men and 12 Women) were recruited for the study. Those with previous history of other cancer, cancer metastasized to the bladder from another origin, and previous radiotherapy was excluded. Healthy and genetically unrelated individuals visiting the hospital for a routine checkup or health awareness camps and hospital employees were recruited as the controls (n = 100). All the controls were age and sex matched with similar ethnicity and had no evidence of malignancy or chronic disease. The mean age of the controls was 56.8 years, and M: F ratio as 89:11. The disproportionate ratio between male and female bladder cancer in our population could be largely due to increased prevalence in case of males (3:1). Secondly due to social taboos females avoid visiting hospitals/clinics. The participation rate was 100%, and blood samples were available for all subjects. Ethnicity was based on self-report and categorized as North Indian. An epidemiologic questionnaire was designed for study participants to collect data on demographic characteristics, smoking history, occupation history, and other lifestyle factors were employed. At the end of the interview, a 5-ml blood sample was drawn into coded tubes. Informed and written consents were taken from all subjects when interviewing for the demographic details and blood sample collection. The Ethical Review Board of the Institute approved the study and the study was conducted in accordance with the Declaration of Helsinki.
Genotyping
The genomic DNA from peripheral blood was extracted using salting out method [12]. Genotyping of miRNA 570 C/G (rs4143815), miRNA 30c A/G (rs928508) and miRNA 181a C/T (rs12537) was done using Taqman allelic discrimination assay. Genotyping was carried out in a 96 well plate system with ABI 7500HT Fast Sequence Detection System (Applied Biosystems, Foster City, CA). Positive and negative controls were used in each genotyping assay plate, and 10% of the samples were randomly selected and run in duplicates with 100% concordance. The results were reproducible with no discrepancy.
Statistics
Sample size for the study was calculated using Quanto program version 1.1 (https://hydra.usc.edu/gxe). Present study achieved 80% of power. P value < 0.05 was considered as significant. Risk for susceptibility of disease or a trait of disease was expressed as Odds ratio (OR) as risk estimate with their corresponding 95% confidence intervals (CI).
Results
Characteristic of Subjects
A total of 100 controls and 100 cases were recruited for this study. No significant age difference was there between the cases and controls. The cases had significantly higher percentage of smokers than the controls.
Association of miRNA 570 C/G (rs4143815), miRNA 30c A/G (rs928508) and miRNA 181a C/T (rs12537) Gene Polymorphism with Bladder Cancer Risk
The genotype frequencies of both the polymorphisms in control group were in Hardy–Weinberg equilibrium. To find out whether the variant genotypes were associated with BC susceptibility, we have compared the genotype distribution between controls and patients. In miR30c, no significant difference was found between cases and controls. The other two SNPs i.e. miR570 and miR181a did not confer any association or risk to bladder cancer as well. None of the candidate molecules showed any association with the disease neither at genotypic or allelic level. The reason behind this may be the lower sample size as it is a pilot study from North India (Table 1).
Table 1.
Association of miRNA 570 C/G (rs4143815), miRNA 30c A/G (rs928508) and miRNA 181a C/T (rs12537) gene polymorphism with bladder cancer risk
| Genetic model | Genotypes | Controls n = 100 n(%) |
Patients n = 100 n(%) |
p value | OR (95% CI) |
|---|---|---|---|---|---|
| miRNA 570C/G(rs4143815) | |||||
| Additive | CC | 57 (57.0) | 68 (68.0) | Ref | Ref |
| CG | 36 (36.0) | 25 (25.0) | 0.087 | 0.582 (0.313–1.082) | |
| GG | 7 (7.0) | 7 (7.0) | 0.754 | 0.838 (0.278–2.531) | |
| Dominant | CC | 57 (57.0) | 68 (68.0) | Ref | Ref |
| CG + GG | 43 (43.0) | 32 (32.0) | 0.109 | 0.624 (0.350–1.111) | |
| Multiple | C Allele | 150 (75.0) | 161 (80.5) | Ref | Ref |
| G Allele | 50 (25.0) | 39 (19.5) | 0.187 | 0.727 (0.452–1.168) | |
| miRNA 30cA/G(rs928508) | |||||
| Additive | AA | 46 (46.0) | 38 (38.0) | Ref | Ref |
| AG | 42 (42.0) | 48 (48.0) | 0.286 | 1.383 (0.762–2.513) | |
| GG | 12 (12.0) | 14 (14.0) | 0.443 | 1.412 (0.584–3.414) | |
| Dominant | AA | 46 (46.0) | 38 (38.0) | Ref | Ref |
| AG + GG | 54 (54.0) | 61 (61.0) | 0.317 | 1.332 (0.759–2.338) | |
| Multiple | A Allele | 134 (67.0) | 124 (62.0) | Ref | Ref |
| G Allele | 66 (33.0) | 76 (38.0) | 0.296 | 1.244 (0.826–1.876) | |
| miRNA 181aC/T(rs12537) | |||||
| Additive | CC | 40 (40.0) | 33 (33.0) | Ref | Ref |
| CT | 46 (46.0) | 50 (50.0) | 0.376 | 1.318 (0.715–2.426) | |
| TT | 14 (14.0) | 17 (17.0) | 0.370 | 1.472 (0.633–3.424) | |
| Dominant | CC | 40 (40.0) | 33 (33.0) | Ref | Ref |
| CT + TT | 60 (60.0) | 66 (66.0) | 0.380 | 1.294 (0.728–2.301) | |
| Multiple | C Allele | 126 (63.0) | 116 (58.0) | Ref | Ref |
| T Allele | 74 (37.0) | 84 (42.0) | 0.307 | 1.233 (0.825–1.842) | |
OR = Odds ratio; p value < 0.05 significant
Association of Smoking with Bladder Cancer risk
Effect of gene-smoking interaction was evaluated in a case only study. We did not find any modulation of risk of BC in miRNA 570 C/G (rs4143815), miRNA 30c A/G (rs928508) and miRNA 181a C/T (rs12537)(Table 2).
Table 2.
Effect of Smoking on miRNA 570 C/G (rs4143815), miRNA 30c A/G (rs928508) and miRNA 181a C/T (rs12537) Genotype with Bladder Cancer risk
| Genotype | Patients non smokers n = 29 n(%) |
Patients smoker n = 55 n(%) |
p value | OR (95% CI) |
|---|---|---|---|---|
| miRNA 570C/G(rs4143815) | ||||
| CC | 18 (62.1) | 41 (74.5) | Ref | Ref |
| CT | 8 (27.6) | 12 (21.8) | 0.437 | 0.659 (0.230–1.886) |
| TT | 3 (10.3) | 2 (3.6) | 0.199 | 0.293 (0.045–1.905) |
| miRNA 30cA/G(rs928508) | ||||
| CC | 13 (44.8) | 23 (41.8) | Ref | Ref |
| CT | 12 (41.4) | 24 (43.6) | 0.805 | 1.130 (0.428–2.985) |
| TT | 4 (13.8) | 8 (14.5) | 0.862 | 1.130 (0.285–4.491) |
| miRNA 181aC/T(rs12537) | ||||
| AA | 13 (44.8) | 18 (32.7) | Ref | Ref |
| AG | 10 (34.5) | 29 (52.7) | 0.152 | 2.094 (0.761–5.766) |
| GG | 6 (20.7) | 8 (14.5) | 0.954 | 0.963 (0.269–3.451) |
OR = Odds ratio; p value < 0.05 significant
Gene–Gene Interaction
To check the combinatorial effect of SNPs on one another we further performed gene–gene interaction study. We had three combinations viz. miR570C/G (rs4143815) and miR30cA/G (rs928508), miR30cA/G (rs928508) and miR181aC/T (rs12537), and miR570C/G (rs4143815) and miR181aC/T (rs12537). Combined effect of miRNA 570 and miRNA 30c (CG + AA) p = 0.005, OR = 0.223, 95% CI (Table 3) and miRNA 570 and miRNA 181a (CG + CC) p = 0.003, OR = 0.169, 95% CI (Table 4) conferred association with no risk of BC whereas, miR30cA/G (rs928508) and miR181aC/T (rs12537) showed no association (Table 5).
Table 3.
Gene–Gene interaction between miR570C/G (rs4143815) and miR30cA/G (rs928508)
| Gene combination | Controls | Patients | p value | OR (95% CI) |
|---|---|---|---|---|
| CC + AA | 23 (23.0) | 31 (31.0) | Ref | Ref |
| CC + AG | 29 (29.0) | 27 (27.0) | 0.335 | 0.691 (0.326–1.465) |
| CC + GG | 5 (5.0) | 10 (10.0) | 0.520 | 1.484 (0.446–4.934) |
| CG + AA | 20 (20.0) | 6 (6.0) | 0.005 | 0.223 (0.077–0.642) |
| CG + AG | 10 (10.0) | 16 (16.0) | 0.725 | 1.187 (0.456–3.090) |
| CG + GG | 6 (6.0) | 3 (3.0) | 0.191 | 0.371 (0.084–1.641) |
| GG + AA | 2 (2.0) | 1 (1.0) | 0.430 | 0.371 (0.032–4.343) |
| GG + AG | 3 (3.0) | 5 (5.0) | 0.786 | 1.237 (0.268–5.708) |
| GG + GG | 2 (2.0) | 1 (1.0) | 0.430 | 0.371 (0.032–4.343) |
OR = Odds ratio; p value < 0.05 significant
Significant values are shown in bold
Table 4.
Gene–Gene interaction between miR30cA/G (rs928508) and miR181aC/T (rs12537)
| Gene combination | Controls | Patients | p value | OR (95% CI) |
|---|---|---|---|---|
| AA + CC | 28 (28.0) | 29 (29.0) | Ref | Ref |
| AA + CT | 15 (15.0) | 6 (6.0) | 0.084 | 0.386 (0.131–1.137) |
| AA + TT | 3 (3.0) | 3 (3.0) | 0.967 | 0.966 (0.180–5.193) |
| AG + CC | 8 (8.0) | 2 (2.0) | 0.088 | 0.241 (0.047–1.237) |
| AG + CT | 31 (31.0) | 43 (43.0) | 0.410 | 1.339 (0.668–2.683) |
| AG + TT | 3 (3.0) | 3 (3.0) | 0.967 | 0.966 (0.180–5.193) |
| GG + CC | 4 (4.0) | 2 (2.0) | 0.421 | 0.483 (0.082–2.849) |
| GG + CT | NA | 1 (1.0) | NA | NA |
| GG + TT | 8 (8.0) | 11 (11.0) | 0.596 | 1.328 (0.465–3.788) |
OR = Odds ratio; p value < 0.05 significant; NA = Not available
Table 5.
Gene–Gene interaction between miR570C/G (rs4143815) and miR181aC/T (rs12537)
| Gene combination | Controls | Patients | p value | OR (95% CI) |
|---|---|---|---|---|
| CC + CC | 17 (17.0) | 28 (28.0) | Ref | Ref |
| CC + CT | 30 (30.0) | 30 (30.0) | 0.214 | 0.607 (0.276–1.334) |
| CC + TT | 10 (10.0) | 10 (10.0) | 0.358 | 0.607 (0.210–1.759) |
| CG + CC | 18 (18.0) | 5 (5.0) | 0.003 | 0.169 (0.053–0.538) |
| CG + CT | 15 (15.0) | 14 (14.0) | 0.239 | 0.567 (0.220–1.458) |
| CG + TT | 3 (3.0) | 6 (6.0) | 0.801 | 1.214 (0.268–5.504) |
| GG + CC | 5 (5.0) | NA | NA | NA |
| GG + CT | 1 (1.0) | 6 (6.0) | 0.250 | 3.643 (0.403–32.913) |
| GG + TT | 1 (1.0) | 1 (1.0) | 0.730 | 0.607 (0.036–10.356) |
OR = Odds ratio; p value < 0.05 significant; NA = Not available
Significant values are shown in bold
Discussion
It is biologically plausible that the combined effects of single nucleotide polymorphisms in micro RNA genes and exposure to environmental factors such as tobacco may influence an individual’s risk of cancer. This investigation was aimed to analyze the association between SNPs in miRNA genes and BC predisposition. Genetic variations in microRNAs might affect individual bladder cancer risk, thus we investigated the prevalence of annotated microRNA polymorphisms in a set of 100 DNA samples from bladder cancer patients as well as 100 healthy individuals in a North Indian population. To the best of our knowledge, this is the first case–control molecular epidemiologic study that has investigated the association of miRNA variants and risk of BC development and progression from India.
Understanding the role of differentially expressed miRNAs, as well as their molecular targets, in bladder cancer will provide an effective and promising strategy for miRNA-based therapeutics for the treatment of bladder cancer.
Previous studies suggest a significant role of miRNAs in variety of malignancies viz. bladder cancer in American population [13], breast cancer in Japanese population [14], gastric cancer in Chinese population [15] Song et al. [16] showed downregulation of miR30c in prostate cancer. Our study did not reveal significance with bladder cancer in any of the three candidate SNPs. Individual polymorphisms are likely to confer modest effects to the risk of BC, we examined the effects of multiple miRNA polymorphisms by performing gene–gene interaction analyses among all 3 SNPs. Additional research work in a larger cohort is required to investigate the functional effect of these SNPs.
In summary, the clinical relevance of miRNA polymorphism in bladder cancer susceptibility remains questionable and various multicentric association studies in large cohort of bladder cancer patients should be further investigated in different ethnic populations for appropriate evaluation.
Acknowledgement
The study was funded by Council of Science and Technology [CST/D1269], Uttar Pradesh, India.
Author's Contribution
The study was conceived and supervised by RDM. Manuscript writing, and experimental work was performed by AV under the guidance of RDM.
Compliance with Ethical Standards
Conflict of interest
None.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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