Genetic Variation in DROSHA 3’UTR Regulated by hsa-miR-27b Is Associated with Bladder Cancer Risk

Purpose miRNAs can regulate the biological processes, including differentiation, proliferation and apoptosis. DICER and DROSHA are two members of RNase III family, playing pivotal roles in the pathway of miRNAs biogenesis. In this study, we hypothesized that genetic variations of the DICER and DROSHA genes were associated with the bladder cancer risk. Experimental Design We performed a case-control study of 685 bladder cancer cases and 730 controls to investigate the association between the seven functional SNPs of DICER and DROSHA genes and bladder cancer risk. We then evaluated the functionality of the important SNPs. Results We found that rs10719T>C polymorphism located in 3’ untranslated region (UTR) of DROSHA gene was associated with the increased risk of bladder cancer. Stratified analysis suggested that rs10719TC/CC genotype can increase risk of bladder cancer among male patients (Adjusted OR = 1.34, 95% CI = 1.05-1.70, P = 0.018), and ever smokers (1.56, 1.14-2.14, 0.006), compared with TT genotype. Furthermore, DROSHA rs10719T>C polymorphism was predicted to regulate the binding activity of hsa-miR-27a/b. Luciferase reported gene assay confirmed that rs10719 T to G substitution disrupted the binding site for hsa-miR-27b, resulting the increased levels of DROSHA protein. Conclusions Taken together, these findings suggested that DROSHA rs10719T>C polymorphism may be associated with bladder cancer risk in a Chinese population, and hsa-miR-27b can influence the expression of DROSHA protein by binding with 3’UTR.


Introduction
Bladder cancer accounts for approximately 2% of all human malignancies with an estimated 72,570 new cases and 15,210 deaths in the USA in 2013 alone [1]. In China, the overall registered bladder cancer incidence was 7.49/100,000 in 2008, and the incidence of bladder cancer was rising during 1998-2008 (average growth rate per year, 4.60%) [2]. More than 90% of the bladder cancer is transitional cell carcinoma. The incidence of bladder cancer is generally high in the USA and Europe, but low in Asia. Like other common cancers, bladder cancer is a complex disease caused by both genetic and environmental risk factors. Cigarettes smoking, occupational and environmental exposures are wellestablished known risk factors for bladder cancer [3]. It has been reported that FGFR3 mutation was associated with the low bladder tumor grade and stage, and the mutations of TP53 and FGFR3 showed an inverse relationship [4][5][6]. Recently, several genome-wide association studies (GWAS) with replications have identified that the common genetic variations are associated with susceptibility to bladder cancer [7][8][9][10][11]. Tang et al. also identified that an uncommon coding variant of UGT1A locus (GWAS related) can affect UGT1A mRNA expression and decrease the risk of bladder cancer [12], However, the exact mechanisms of the bladder cancer did not be clarified.
MicroRNAs (miRNAs) are a class of small non-coding RNA molecules of ~22 nucleotides, which regulate gene expression at the post-transcriptional level through binding the 3' untranslated region (UTR) of target genes mRNA [13]. miRNAs are generated in a two-stepwise processing pathway mediated by two major enzymes (DICER and DROSHA): In the nucleus, longer precursors are processed into primary RNAs (pri-miRNAs) by the RNase II and then pri-miRNAs are processed by the RNase enzyme (DROSHA) into precursors (pre-miRNAs) with a stem-loop structure [14,15]. The pre-miRNAs are exported from the nucleus to the cytoplasm by the exportin-5 protein. In the cytoplasm, pre-miRNAs are processed into mature miRNAs by another RNase enzyme (DICER). The mature miRNAs play roles by incorporating into the RNA-induced silencing complex (RISC) [16]. It has been suggested that miRNAs are predicted to regulate 30% of human genes [17]. Recently, several studies showed that miRNAs could act as oncogenes and tumor suppressors by targeting 3'UTR of important genes [18,19] and the genetic variants in 3'UTR of the miRNA target genes would affect miRNA-mediated gene regulation, eventually resulting the increased risk of cancer [19,20]. It is worth to note that DICER and DROSHA play the crucial role in carcinogenesis. Accumulated evidences have shown that imbalance DICER and DROSHA expression levels are associated with bladder cancer risk [21][22][23]. Recently, Han and his colleagues also found that DICER and DROSHA expression levels were upregulated in bladder cancer tissues compared to the matched normal bladder tissues, and silencing DICER or DROSHA can inhibit cell proliferation and induce cell apoptosis [24]. Here, we propose that it is warranted to investigate the roles of the DICER and DROSHA in the susceptibility to bladder cancer.
Up to now, several studies have investigated the association between the genetic variants of the DICER and DROSHA genes and risk of diseases. Lin et al. reported that the DICER and DROSHA haplotypes were associated with the altered survival and recurrence of renal cell carcinoma patient in Caucasians [25]. However, the genetic variants of DICER and DROSHA were not associated with the development of renal cell carcinoma [26]. In addition, Yang et al. also observed the similar result of the bladder cancer in Caucasians [27]. Recently, Qin et al. showed that the DICER and DROSHA polymorphisms might modify the risk of abnormal semen parameters and be associated with the Chinese male infertility [28]. Taken together, we hypothesized that the genetic variants of DICER and DROSHA are also be associated with the susceptibility to bladder cancer in a Chinese population.
On the basis of this postulation, we selected seven polymorphisms of DICER (rs12323635CT, rs13078TA,  rs1057035TC,  and  rs3742330AG) and DROSHA (rs2291109AT, rs10719TC, and rs642321CT) to evaluate the association between the genetic variants of DICER and DROSHA genes and risk of bladder cancer. In this study, we found that DROSHA 3'UTR polymorphism rs10719TC can increase the risk of bladder cancer in a Chinese population, which was located near a miRNA binding site. Furthermore, we conducted a series of functional assays on DROSHA 3'UTR polymorphisms to reveal its molecular mechanism.

Study subjects
In the present study, we included 685 histopathologically confirmed bladder transitional cell carcinoma and 730 cancerfree controls. Included study subjects were recruited from the First Affiliated Hospital and Huai-An Affiliated Hospital of Nanjing Medical University, and Jiangsu Province Hospital of Traditional Chinese Medicine (TCM) between January 2003 and January 2010. The detailed method of recruiting study subjects for the study had been described previously [29]. Pathological diagnosis for bladder tumor stage was according to the 2002 International Union Against Cancer tumor-nodesmetastasis classification and the World Health Organization 1973 grading of urothelial papilloma was used to define the bladder cancer grade: well differentiated (grade 1, G1), moderately differentiated (grade 2, G2) or poorly differentiated (grade 3, G3). Bladder cancer patients were excluded, if which had previous cancer, metastasized cancer from other origin, previous radiotherapy or chemotherapy. The cancer-free subjects were recruited from those who were seeking health care in the outpatient departments at the hospital. The cancerfree controls were matched by age (± 5 years) and sex to the cases, which were genetically unrelated to the cases and had no individual history of cancer including melanoma skin cancer. The cancer-free subjects who had symptoms suggestive of bladder cancer, such as hematuria, were excluded. We used a short questionnaire to obtain the demographic and risk factor information from the included subjects. In this study, we defined ever smokers (former and current smokers) based on smoking condition. Subjects who smoked daily for >1 year were defined as ever smokers. Ever smokers who had quit smoking for >1 year were defined as former smokers and the others as current smokers. This case-control study was approved by the institutional review board of Nanjing Medical University. All individuals signed informed consents, and each subject donated 5 ml blood sample for genomic DNA extraction.

SNPs selecting and genotyping
In this study, we studied the genetic variants of DICER and DROSHA genes, which play the important roles in the miRNA biogenesis. Here, we focused on studying the single nucleotide polymorphisms (SNPs) spanning these two genes (HapMap Data Release 27), including 2 kb upstream and 2 kb downstream using the Haploview software [30]. The following criteria should be included: (i) SNPs should be located in the 5' flanking region, 5' UTR, 3'UTR, and coding region with amino acid changes, (ii) minor allele frequency (MAF) > 5% in Han Chinese in Beijing (CHB). According to the criteria, four SNPs were identified in DICER (rs12323635, rs13078, rs1057035, and rs3742330) and three SNPs in DROSHA (rs2291109, rs10719, and rs642321).
Genomic DNA was extracted from peripheral blood lymphocytes of the subject. The included seven SNPs were genotyped in all 1415 subjects using the MGB TaqMan probe Assay (7900HT Real Time PCR System, Applied Biosystems, Foster City, USA). About 10% of the samples were randomly selected for repeated genotyping for validation, and the results were 100% concordant. Genotype analysis was performed by two persons independently in a blinded fashion and controls were included in each plate to ensure accuracy of the genotyping. However, several samples failed in genotyping were due to DNA quality, and we would exclude them in the further analyses. Table 1 provided the primary information of the selected seven SNPs.
For detection of the correlation between the DROSHA mRNA levels and rs10719 T>C polymorphism in vivo, a total of 61 bladder tumor tissues with different genotypes (32 for TT, 24 for TC, and 5 for CC genotypes) were subjected to extraction of the total RNA using Trizol Reagent (Invitrogen, CA, USA). Bladder tumor tissues were preserved in liquid nitrogen after being removed from the body. The total RNA was evaluated by both reverse transcriptase reaction and real-time quantitative PCR based on the SYBR-Green method (Toyobo, Tsuruga, Japan). The cDNA was used for the amplification of DROSHA gene and an endogenous control gene GAPDH. The primers information of DROSHA and GAPDH genes were included in the Table 1. Fold changes were normalized by the expression levels of GAPDH and each assay was performed in triplicate.

DROSHA 3'UTR containing rs10719TC reporter gene constructs and Luciferase reporter assays
To construct the luciferase reporter plasmids of DROSHA 3'UTR, DROSHA 3'UTR fragments (937bp) carrying the major rs10719T allele were amplified by PCR. The primers were 5'-ACCTTGGTACCCCAGATGAGACTGAAGACATC-3' (forward) and 5'-ACCTTCTCGAGGCACTCACTATATATTTGCTG-3' (reverse). The PCR products were extracted and separated by agarose gel, which were cloned with TA cloning Kit (Invitrogen, CA, USA). In addition, the fragment containing minor rs10719C allele was conducted using the following primers: 5'-TAGTTTTCCTGCAGACAATGAACGAAGTGTGC-3' (forward) and 5'-TTTATTTCAATGAGCACACTTCGTTCATTGTC-3' (reverse). Finally, the amplified fragment carrying T or C allele was inserted downstream of the luciferase gene in a pGL3promoter plasmid and then the plasmid containing T or C allele was conducted, which were confirmed by sequencing.
For luciferase reporter assay, T24 and J82 cells were placed in 24-well plates (1×10 5 cells per well) and then cotransfected with pGL3-DROSHA 3'UTR-T or pGL3-DROSHA 3'UTR-C and pRL-SV40 (50:1). The mimics and inhibitors of hsa-miR-27a/b and their negative controls (GenePharma, Shanghai, China) were cotransfected with the reporter plasmids at a final concentration of 20nmol/μl. Forty-eight hours after transfection in T24 and J82 cells, luciferase activity in lysates was measured with a Dual-Luciferase Reporter Assay System (Promega, WI, USA) and normalized against the activity of the pRL-SV40. Assays were followed by the manufacture's suggestions. Independent triplicate experiments were performed for each plasmid construct.

Statistical analysis
The Hardy-Weinberg equilibrium of the genotype distribution among the controls was applied by using a goodness-of -fit χ 2 test. The frequency distributions of selected demographic variables between the cases and controls were tested using χ 2 test. Genotype-specific odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated by unconditional univariate and multivariate logistic regression analyses. The multivariate adjustment included the age, sex and smoking status (never and ever smoking). In addition, Kruskal-Wallis one-way ANOVA tests were used for analyzing the results of DROSHA mRNA expression in vivo. In this study, the relative luciferase reporter gene expression for T or C allele was calculated separately. Student's t test was used to evaluate the differences in the expression levels of luciferase reporter gene among subgroups. All tests were two-sided using the SAS software (version 9.1; SAS Institute, Inc, Cary, NC, USA) and P < 0.05 was considered statistically significant.

Association study between the polymorphisms of DICER and DROSHA and bladder cancer risk
The characteristics of the 685 bladder transitional cell carcinoma patients and 730 controls are summarized in Table  2. Herein, we did not observe statistical difference in the distribution of age (P = 0.144) and sex (P = 0.825) between patients and controls. However, there were more ever smokers (55.6%) among the patients more than among the controls (38.4%), and this difference was statistically significant (P < 0.001). These variables were adjusted for the subsequent multivariate logistic regression analysis. Of the 685 cases, there were 315 tumor grade 1 (46.0%), 261 tumor grade 2 (38.1%) and 109 tumor grade 3 (15.9%) patients. In addition, 435 (63.5%) had superficial tumors and remaining 250 (36.5%) had invasive tumors.
The seven SNPs genotype frequencies among the control were in agreement with the Hardy-Weinberg equilibriums (P > 0.05; Table 1). As shown in Table 3, we observed that subjects with the DROSHA 3'UTR rs10719C allele (TC and CC genotypes) had a 1.24-fold increased risk of bladder cancer (Adjusted OR = 1.25, 95% CI = 1.01-1.55, P = 0.041) compared with the rs10719TT genotype. Meanwhile, we observed that the distribution of rs10719TC genotypes between the cases and controls showed significant difference (P = 0.017). However, we did not observe any significant differences in genotype distribution of the DICER rs12323635CT, rs13078TA, rs1057035TC, rs3742330AG and DROSHA rs2291109AT, rs642321CT polymorphisms between the cases and controls (all P >0.05, Table 3). We further evaluated the effect of included seven polymorphisms on bladder cancer risk by stratifying by sex and smoking status. As shown in Table S2 in File S1, we found that rs10719TC polymorphism can increase risk of bladder cancer among male patients (Adjusted OR = 1.34, 95% CI = 1.05-1.70, P = 0.018), and ever smokers (Adjusted OR = 1.56, 95% CI = 1.14-2.14, P = 0.006).

DROSHA 3'UTR rs10719TC affects DROSHA expression by regulating the hsa-miR-27b binding
In order to explore the possible mechanism of the DROSHA 3'UTR in the bladder cancer risk, we performed the functional assays. Based on bioinformatics analysis, the DROSHA 3'UTR was predicted a binding site for hsa-miR-27a/b ( Figure 1A). Here, we demonstrated that rs10719TC was located 46bp downstream of the hsa-miR-27a/b binding site in the DROSHA 3'UTR.
As shown in Figure S1 in File S1, real-time quantitative RT-PCR assay suggested that endogenous expression levels of hsa-miR-27a/b in J82 cell line were more significantly higher than other cells (T24, EJ, and 5637) (P < 0.001). Here, we chose J82 and T24 cell lines in the further luciferase assay. Then, we used the plasmids for transient co-transfection with the T24 cells [stable negative control (NC) miRNA: stable NC or hsa-miR-27a/b mimics] and J82 cells (inhibitor NC or hsa-miR-27a/b inhibitor). As shown in Figure 1B (T24 cell), hsa-miR-27a/b suppressed luciferase expression in the presence of rs10719T allele, comparing with NC (P < 0.05), but not the rs10719C allele (P > 0.05). Additionally, we also found that the inhibition of hsa-miR-27a/b expression can increase luciferase expression efficiently for the rs10719T-containing plasmid rather than the rs10719C-containing plasmid in J82 cells (P < 0.05; Figure 1C). However, we also found that a significant decrease in luciferase expression, when we added hsa-miR-27a inhibitor to allele C in J82 cells. The similar result did not be observed in adding hsa-miR-27b inhibitor to allele C in J82 cells. Maybe, hsa-miR-27a did not directly affect DROSHA luciferase expression. Our data suggested that rs10719 T to C substitution acts as a loss-of-function mutation and would affect DROSHA luciferase expression by hsa-miR-27b target.
In the present study, we also performed RT-PCR assay to explore whether hsa-miR-27b affected DROSHA expression by degrading mRNA or suppressing mRNA post-translational translation ( Figure S1 in File S1). A total of 61 bladder tumor tissues with different genotypes of the DROSHA rs10719TC polymorphism were used to assess the expression of DROSHA mRNA. No significant difference levels of DROSHA mRNA among individuals with the TT, TC and CC genotypes was observed (P > 0.05). Taken together, hsa-miR-27b may affect DROSHA expression by regulating protein translation.

Discussion
In the present study, we investigated the association between seven polymorphisms of DICER and DROSHA genes and bladder cancer risk in a Chinese population, and identified that rs10719TC polymorphism adjacent to the hsa-miR-27b Table 3. Genotype frequencies of the DICER and DROSHA SNPs among bladder cancer cases and controls and their association with bladder cancer risk. binding site in DROSHA 3'UTR was associated with significantly increased the risk of bladder cancer. Functional assays indicated that DROSHA rs10719 T to C substitution can decrease the binding activity of hsa-miR-27b with DROSHA 3'UTR. DROSHA is a member of RNase III superfamily and is an important nuclease that executes the initial step in miRNA processing by cutting pri-miRNA to pre-miRNA [31]. RNA interference of DROSHA resulted in accumulation of pri-miRNA and reduction of pre-miRNA and mature RNA [31]. Up to now, several groups have studied the role of DROSHA in cancer [32,33]. It has reported that DROSHA rs644236 TT genotype and rs7737174 AA genotype were associated with breast cancer risk in postmenopausal women [32]. DROSHA 3'UTR rs10719 is in strong linkage disequilibrium with rs644236 based on the one thousand Genomes data (r 2 = 0.88) [33]. In the present association study, we found that DROSHA 3'UTR rs10719TC polymorphism was associated with the risk of bladder cancer. Stratified analysis demonstrated that rs10719TC/CC genotypes can significantly increase the risk of bladder cancer, especially among males and smokers. One possible explanation is that cigarette smoking has been established as the most important risk factor in the development of bladder cancer, which contains hundreds of chemicals, such as polycyclic aromatic hydrocarbons [34,35]. Thus, ever smokers may be prone to be cancer. Another reason might be that compared with female, the male people are more likely to expose to accumulated environmental risk factors involved in the etiology of bladder cancer such as cigarette smoking, occupational exposures (i.e. dyestuff manufacture, leather work). Additionally, we have the relatively small sample size of female people. Therefore, we can not detect the significant association in female people. Weng et al. also proposed that rs10719TC polymorphism was related with malignant peripheral nerve sheath tumor risk, which supported our findings [33]. Over expression of DROSHA was shown to effect cell proliferation and predicted poor prognosis in esophageal cancer [36], ovarian cancer [37], breast cancer [38], and cervical cancer [39]. Previous study had also revealed that over expression of DROSHA can promote cell proliferation and inhibit cell apoptosis in bladder cancer [24]. Therefore, we hypothesized that DROSHA 3'UTR rs10719C allele can increase the risk of bladder cancer, mainly through regulating the expression of DROSHA.
It has been proposed that some of the 3'UTR polymorphisms may be in the miRNA binding site or in the vicinity of binding site and may interfere with miRNA function leading to differential gene expression to affect the development of cancer [19,20]. Our luciferase reported gene assays indicated that DROSHA rs10719 T to C substitution disrupted a binding site for hsa-miR-27b, resulting the increased levels of DROSHA 3'UTR luciferase expression. Thus, the C allele is associated with an increased risk of bladder cancer, potentially through increased DROSHA expression, which was consistent with the previous finding [24]. Furthermore, no significant difference of the DROSHA mRNA expression level among different rs10719TC genotypes was observed in bladder tumor tissues using RT-PCR assay. These results suggest that the SNP does not affect mRNA expression, however, given that miRNA binding to mRNAs does not always lead to transcript cleavage, and sometimes it leads to translation repression, it is possible that the SNP leads to a change in DROSHA protein. However, we did not test this in our study, and therefore remains a possibility, but not proven. These data suggested that hsa-miR-27b may affect DROSHA expression by regulating protein translation. It was worth to note that our functional findings were in agreement with the results of a case-control study.
In the present study, hsa-miR-27b was firstly reported to regulate the expression of DROSHA in bladder cancer. Hsa-miR-27b had been studied widely, which had been identified to be correlation with many kinds of cancers, such as colorectal cancer [40], neuroblastoma [41]. Recently, hsa-miR-27b was found to inhibit colorectal tumor progression and angiogenesis, through targeting VEGFC [40]. Lee et al. indicated that hsa-miR-27b could act as a tumor suppressor to inhibit cell growth, tumor progression and inflammatory response by targeting PPARγ 3'UTR in neuroblastoma cells [41]. In the present study, our data proposed that hsa-miR-27b may affect DROSHA expression by regulating protein tranlation. Further functional assays should be conducted to reveal the exact mechanism and pathway in bladder cancer.
Some limitations should be proposed in the present study. First, due to the lack of the genitourinary related co-morbidity data information, we were not able to investigate the association between SNPs and co-morbidity adequately. Results need to be confirmed in larger studies with more detailed genitourinary related co-morbidity data information. Second, we predicted DROSHA 3'UTR-related miRNA through several website, and considered that the combination of multiple prediction approaches would greatly reduce the possibility of false positive. At last, hsa-miR-27a/b was chosen. In the present study, rs10719TC was associated with bladder cancer risk and it was adjacent to the hsa-miR-27a/b binding site in DROSHA 3'UTR. Therefore, we performed luciferase assay to explore whether rs10719 T to C substitution can affect the binding activity of hsa-miR-27a/b with DROSHA 3'UTR. However, we also found that rs72547276 SNP laid in a predicted DROSHA binding site for miR-27b. Unfortunately, rs72547276 was not eligible for SNP inclusion criteria (MAF< 5% in CHB). Although this SNP located in the binding site, we did not do more work on this SNP. In the future, we should study this SNP possibly functional effect.
In conclusion, we identified the risk allele of rs10719TC located in DROSHA 3'UTR and rs10719 T to C substitution can affect DROSHA protein expression by hsa-miR-27b target, which provided the possible mechanism in bladder cancer risk. Our study revealed a new insight into bladder carcinogenesis. Furthermore, the association and functional study are warranted to validate our findings.

Supporting Information
File S1.
Supporting figure and tables. Figure S1, Quantitative real-time PCR was used to measure levels of hsa-miR-27a/b expression and DROSHA mRNA expression. (A, B) The levels of hsa-miR-27a/b expression were measured in four cell lines. In (A), relative hsa-miR-27a expression level was higher in J82 cells than in others. In (B), relative hsa-miR-27b expression level was higher in J82 cells than in others. Fold changes are normalized to the expression levels of U6. (C) Association between rs10719TC and DROSHA mRNA expression in bladder cancer tissues. 61 bladder cancer tissues were used to assess the expression of DROSHA mRNA by Quantitative real-time PCR. The frequencies of TT, TC and CC genotypes were 32, 24 and 5, respectively. P value was calculated by non-parametric Kruskal-Wallis H test of three genotypic groups. The fold change was normalized against GAPDH. Table S1, Primer information of Real-time PCR. Table  S2, Stratification analyses between DICER and DROSHA polymorphisms and risk of bladder cancer. (DOC)