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. 2021 Mar 3;40(13):2382–2394. doi: 10.1038/s41388-021-01672-1

Identification of low-frequency variants of UGT1A3 associated with bladder cancer risk by next-generation sequencing

Rui Zheng 1,2,#, Mulong Du 1,3,#, Yuqiu Ge 1,2,#, Fang Gao 2,4, Junyi Xin 1,2, Qiang Lv 5, Chao Qin 5, Yao Zhu 6, Chengyuan Gu 6, Mengyun Wang 6, Qiuyuan Zhu 1,2, Zheng Guo 1,2, Shuai Ben 1,2, Haiyan Chu 1,2, Dingwei Ye 6, Zhengdong Zhang 1,2,✉, Meilin Wang 1,2,7,8,✉
PMCID: PMC8016664  PMID: 33658628

Abstract

Although genome-wide association studies (GWASs) have successfully revealed many common risk variants for bladder cancer, the heritability is still largely unexplained. We hypothesized that low-frequency variants involved in bladder cancer risk could reveal the unexplained heritability. Next-generation sequencing of 113 patients and 118 controls was conducted on 81 genes/regions of known bladder cancer GWAS loci. A two-stage validation comprising 3,350 cases and 4,005 controls was performed to evaluate the effects of low-frequency variants on bladder cancer risk. Biological experiments and techniques, including electrophoretic mobility shift assays, CRISPR/Cas9, RNA-Seq, and bioinformatics approaches, were performed to assess the potential functions of low-frequency variants. The low-frequency variant rs28898617 was located in the first exon of UGT1A3 and was significantly associated with increased bladder cancer risk (odds ratio = 1.50, P = 3.10 × 10−6). Intriguingly, rs28898617 was only observed in the Asian population, but monomorphism was observed in the European population. The risk-associated G allele of rs28898617 increased UGT1A3 expression, facilitated UGT1A3 transcriptional activity, and enhanced the binding activity. In addition, UGT1A3 deletion significantly inhibited the proliferation, invasion, and migration of bladder cancer cells and xenograft tumor growth. Mechanistically, UGT1A3 induced LAMC2 expression by binding CBP and promoting histone acetylation, which remarkably promoted the progression of bladder cancer. This is the first targeted sequencing study to reveal that the novel low-frequency variant rs28898617 and its associated gene UGT1A3 are involved in bladder cancer development, providing new insights into the genetic architecture of bladder cancer.

Subject terms: Bladder cancer, Cancer genomics, Gene regulation, Genetic association study, Sequencing

Introduction

Bladder cancer is a common urological malignancy worldwide [1, 2], and in 2019, an estimated 80,470 new bladder cancer cases and 17,670 deaths occurred in the United States [3]. In China, the incidence rates of bladder cancer have continued to increase in recent years, particularly in males [4, 5]. Investigations aiming to discover the mechanisms underlying bladder cancer are crucial for improving the screening and diagnosis of bladder cancer. Convincing evidence has revealed that occupational exposure and smoking increase the risk of bladder cancer [6, 7]. Importantly, familial and twin studies have reported that genetic factors also play a central role in the etiology of bladder cancer [8, 9]. Currently, genome-wide association studies (GWASs) are a powerful tool for identifying dozens of single nucleotide polymorphisms (SNPs) associated with the risk of bladder cancer [10, 11].

To date, we and other groups have conducted GWASs to identify a subset of common variants (minor allele frequency (MAF) > 5%) associated with bladder cancer [12, 13]. However, GWASs have mainly investigated common proxy variants associated with the risk of complex diseases, and these identified common variants together explain less than 10% of disease heritability [14, 15]. The unexplained heritability of diseases has increasingly been reported to be attributable to low-frequency (with a MAF 0.5–5%) or rare variants (MAF < 0.5%) variants [16–18], and a large number of low-frequency variants have been detected in the human genome [19]. Emerging evidence has revealed that low-frequency variants are associated with prostate [20], lung [18], ovarian [21], and breast cancers [22], but the role of low-frequency variants in the bladder cancer risk remains unclear. Although our previous study described an exome chip analysis and showed that the rare variant rs35356162 in UHRF1BP1 increases the bladder cancer risk [23], the sequencing depth and economic efficiency are inferior to targeted next-generation region sequencing. In addition, targeted sequencing of known risk-associated regions to identify the effects of low-frequency or rare variants on disease has been recognized as a useful approach to help elucidate a further proportion of disease heritability [16, 24, 25].

In this study, we first conducted targeted sequencing of known bladder cancer GWAS loci and validated them using a large two-stage population. Subsequent in vitro and in vivo experiments were utilized to comprehensively characterize the biological genetic effect of low-frequency variants on the risk of bladder cancer.

Results

Burden of low-frequency UGT1A3 variants on bladder cancer

A flowchart of the discovery and validation of low-frequency genetic risk variants for bladder cancer is presented in Fig. 1A. We obtained 81 target genes/regions based on 17 loci previously reported in GWAS for bladder cancer (Fig. 1B, Tables S1 and S2) and then conducted next-generation sequencing to capture 45 key genes with low-frequency variants for gene-set analysis (Table S3). The SNP-set kernel association test (SKAT) results showed that a significant low-frequency variant in CBX6 was associated with the bladder cancer risk (P = 0.010) in only the recessive model, whereas UGT1A3 was significantly associated with the bladder cancer risk in two genetic effect models (Pdominant = 0.002 and Padditive = 0.007), and these models were further confirmed by MB and variable-threshold (VT) tests (Fig. 1C and Table S3), indicating that the low-frequency UGT1A3 variant is strongly related to bladder cancer risk.

Fig. 1. The producers of the identified low-frequency variant associated with bladder cancer risk.

Fig. 1

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A Flow chart of selecting the low-frequency variants by targeted sequencing. B Circle Manhattan plot for 17 GWAS-identified loci in bladder cancer. C The P values of significant genes were calculated by gene-set analysis (R: recessive model, D: dominant model, A: additive model, NA: not detected, “-”: the test is not applicable to this genetic model).

In the discovery stage, we found that rs28898617 (MAF = 0.021) was significantly associated with an increased risk of bladder cancer (odds ratio (OR) = 5.02, 95% confidence interval (CI) = 1.60-15.79, Pdominant = 0.006; OR = 3.53, 95% CI = 1.30-9.59, Padditive = 0.013) (Table S4). Due to the small number of low-frequency alleles, we used a dominant model to evaluate the effect of rs28898617 in UGT1A3 on bladder cancer risk. To confirm the effect of low frequency, we performed the first replication set to find a significant association between rs28898617 and bladder cancer risk (OR = 1.46, 95% CI = 1.19-1.79, P = 2.75×10-4). Subsequently, the second replication set showed that rs28898617 was still significantly associated with the bladder cancer risk (OR = 1.46, 95% CI = 1.06-2.02, P = 2.03×10−2). Especially when the results of the discovery and two-stage replication studies combined, the bladder cancer risk caused by the rs28898617 G allele increased by 1.5 times compared with that caused by the AA genotypes without heterogeneity (OR = 1.50, 95% CI = 1.27-1.78, Pmeta = 3.10 × 10−6; Table 1). We further performed a stratified analysis and found that the risk of bladder cancer in males with the G allele increased by 48% compared with the risk in males with the A allele; however, this association was not detected in females (Fig. S1).

Table 1.

Association of rs28898617 in UGT1A3 with the bladder cancer risk in the discovery and replication stages.

Stage Samples Genotypes (AA/AG/GG) MAFc ORd (95% CI) P Phete I2
Cases Controls Cases Controls Cases Controls
Discoverya 112 118 95/16/1 114/3/1 0.080 0.021 5.02 (1.60–15.79) 5.78×10−3
Replication 1 2227 2998 2019/201/7 2801/194/3 0.048 0.033 1.46 (1.19–1.79) 2.75×10−4
Replication 2 1123 1007 1016/103/4 941/63/3 0.049 0.034 1.46 (1.06–2.02) 2.03×10−2
Replication 3350 4005 3035/304/11 3742/257/6 0.048 0.033 1.46 (1.23–1.74) 1.70×10−5 1.000 0.00%
Combinedb 3462 4123 3130/320/12 3856/260/7 0.049 0.033 1.50 (1.27–1.78) 3.10×10−6 0.112 54.30%
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MAF minor allele frequency, OR odds ratio, CI confidence interval.

aA case with no genotyping information from next-generation sequencing.

bThe discovery and two replication stages were combined by meta-analysis.

cMinor allele frequency of the G allele.

dP for dominant model adjusted for age, sex and smoking status in the logistic regression model.

ePhet, P value for heterogeneity.

In addition, we evaluated the association of UGT1A3 rs28898617 with the bladder cancer risk in individuals of European ancestry and found that rs28898617 was monomorphic in the European population. Intriguingly, evidence from the Exome Aggregation Consortium database showed that rs28898617 was polymorphic in only an Asian population (allele frequency = 0.046), but monomorphic in other populations (Table S5). Furthermore, the power analysis results showed that 3,462 cases and 4,123 controls were sufficient (power = 93.1%) to detect a combined OR of 1.50 and α = 0.05 for low-frequency rs28898617, with an MAF of 0.033 in controls (Fig. S2).

Potential regulatory role of rs28898617 in UGT1A3

To evaluate the expression of UGT1A3 in various tissues, we used the cBioPortal and GTEx public databases to find that UGT1A3 was more abundant in bladder tissues than in most other tissues (Figs. S3 and S4). We subsequently observed the expression of UGT1A3 in bladder cancer tissues and their adjacent nontumor bladder tissues based on in-house samples (P = 0.029), and this result was consistent with the evidence from the Gene Expression Omnibus (GEO) (P = 0.006 in GSE7476 and P = 0.034 in GSE3167) and The Cancer Genome Atlas (TCGA) public databases (P = 0.025 in unpaired tissues), showing greater expression of UGT1A3 in bladder cancer tissues than in normal tissues (Fig. 2A–D). In addition, levels of the UGT1A3 protein were significantly increased in bladder cancer tissues compared with adjacent normal tissues (P < 0.001; Fig. 2E and Fig. S5). Moreover, we measured UGT1A3 mRNA expression in cell lines and found that UGT1A3 was had higher expression in bladder cancer cells than in normal 293 A cells (Fig. 2F).

Fig. 2. The expression pattern of UGT1A3 and allele-specific effect of rs28898617.

Fig. 2

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The mRNA expression levels of UGT1A3 were examined with in-house datasets (17 paired bladder cancer tissues) (A) and public databases (the GEO database: GSE7476 and GSE3167 (B and C) and TCGA database (D)). E The levels of the UGT1A3 protein relative to β-Actin were measured in 19 paired bladder cancer tissues. F The expression levels of the UGT1A3 mRNA were analyzed in human cell line (HEK 293 A) and bladder cancer cell lines (UMUC3, T24, EJ and J82). G An eQTL analysis of the association of UGT1A3 expression and rs28898617 was determined using 39 bladder cancer tissues (left panel) and 36 normal tissues (right panel). H The transcriptional activity of the constructs with the rs28898617 A or G allele in EJ and J82 cells by using dual-luciferase reporter assays. I EMSA with biotin-labeled rs28898617 A or G probes and EJ nuclear extracts. 100x and 200x represent 100-fold and 200-fold excess amounts of an unlabeled probe over that of the labeled probe. The arrow indicates a DNA-protein complex. P values were estimated using Student’s t test.

We next performed an expression quantitative trait locus (eQTL) analysis to investigate the genetic effect of rs28898617 on UGT1A3 expression and found that the G risk allele of rs28898617 significantly increased UGT1A3 expression in both bladder cancer tissues (P = 0.027) and normal tissues (P = 0.008; Fig. 2G). We then detected the genotype of rs28898617 in bladder cancer cell lines using Sanger sequencing and found that four bladder cancer cell lines had the same UGT1A3 genotype (rs28898617[A], Fig. S6). Consistent with the results from the eQTL analysis described above, we performed a dual-luciferase reporter assay and found significantly higher relative luciferase expression in the EJ and J82 cell lines expressing the risk G allele construct (P = 0.021 in EJ cells and P = 0.044 in J82 cells) than the A allele construct (Fig. 2H). Moreover, we conducted electrophoretic mobility shift assays (EMSAs) to evaluate the differences in transcription factor binding affinity between the rs28898617 A and G alleles and determined that the probe containing the G allele displayed a stronger binding signal than the probe containing the A allele (Fig. 2I, lane 7 versus lane 2). The competitive EMSA confirmed that the binding activity of the G allele was significantly weakened with the addition of a 100/200-fold excess of unlabeled probe containing the G allele (Fig. 2I, lane 8 and lane 9). In addition, a 200-fold excess of unlabeled probe containing the A allele did not exert an analogous effect on the binding affinity of the G allele (Fig. 2I, lane 10). We next explored the genetic effect of rs28898617 on UGT1A3 mRNA stability and the protein sequence, and found that rs28898617[G] reduced the decay of UGT1A3 mRNA (Fig. S7), whereas it did not regulate UGT1A3 protein, as it is located in the signal peptide of UGT1A3 protein (Fig. S8). Moreover, we explored 500 kb upstream and downstream of rs28898617, and found that rs28898617 was also located in the intron of 7 members of the UGT1A gene family, including UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 (Fig. S9A). We then conducted an eQTL analysis and observed that rs28898617 did not regulate the expression of these genes in both bladder cancer tissues and normal tissues (Fig. S9B). These findings illustrated that the low-frequency variant rs28898617 G allele could significantly affect the transcriptional activity and expression level of UGT1A3.

Functional analyses of UGT1A3 in bladder cancer cellular phenotypes

After transient transfection, overexpression of UGT1A3 in both the EJ and J82 cell lines significantly promoted cell proliferation, clone formation, invasion and migration (Fig. S10), whereas the knockdown of UGT1A3 remarkably inhibited biologically malignant behavior (Fig. S11). Furthermore, we performed CRISPR/Cas9 system deletion of exon 1 containing UGT1A3 to confirm this effect (Fig. 3A). The expression of UGT1A3 was significantly reduced in cells after CRISPR/Cas9-mediated knockout of UGT1A3 according to qRT-PCR and Western blot analyses (Fig. 3B). Similarly, the deletion of UGT1A3 remarkably inhibited cell proliferation (Fig. 3C) and clonogenicity (Fig. 3D), impeded the invasion and migration abilities (Fig. 3E), and arrested the cell cycle at the S phase (Fig. 3F). Consistent with the results obtained from the populations, UGT1A3[G] substantially increased bladder cancer cellular phenotypes, such as proliferation, clone formation, migration and invasion abilities, compared with UGT1A3[A] or empty vector (Fig. S12). Taken together, UGT1A3 rs28898617 may promote the progression of bladder cancer in vitro.

Fig. 3. Roles of UGT1A3 knockout in the bladder cancer cellular phenotype.

Fig. 3

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A CRISPR/Cas9-mediated knockout of UGT1A3 in T24 cells (boxes 1–5: exons 1–5). B The expression of UGT1A3 at the mRNA and protein levels was detected by qRT-PCR and Western blot in T24 cells before (NC) or after CRISPR/Cas9-mediated knockout of UGT1A3 (UGT1A3-KO). C Cell proliferation was measured using a CCK-8 assay (OD450 absorbance). D The colony formation ability of NC (upper) and UGT1A3-KO cells (lower) was measured by a colony formation assay. The colonies were counted and captured. E Representative images of invasion (upper) and migration (lower) assays of NC (left) and UGT1A3-KO cells (right). The number of cells was counted. Scale bar, 100 μm. F The cell cycle of NC (left) and UGT1A3-KO cells (right) was analyzed by flow cytometry. *P < 0.05 according to Student’s t test.

Effect of AR regulating UGT1A3 in bladder cancer

Based on the results of the stratified analyses showing the significant risk effect of rs28898617 among males, we assumed that androgen receptor (AR) would serve as an important factor leading to sex differences in bladder cancer risk. Using the GTEx database, we observed a significant increase in AR expression in bladder tissues compared with many other tissues (Fig. S13). Moreover, a positive correlation between the UGT1A3 and AR expression levels was found in bladder cancer tissues (Fig. S14A–D). In addition, we performed the JASPAR database to observe the possible regulatory sites of AR on UGT1A3 rs28898617 with a relatively high prediction score, suggesting that the expression of UGT1A3 could be activated by AR (Fig. S14E and Table S6). We interfered with AR expression in cell lines to further evaluate the association between AR and UGT1A3 (Fig. S15) and found that overexpressed AR increased UGT1A3 expression, while knockdown of AR inhibited UGT1A3 expression (Fig. S16); these results were consistent with the correlation pattern. We subsequently evaluated the mechanism of AR in regulating UGT1A3 expression after transfecting si-AR cells with UGT1A3 overexpression plasmids. As presented in Fig. S17, knockdown of AR significantly diminished the biochemical cellular processes of bladder cancer, and the tumor suppressive function could be reversed after UGT1A3 overexpression. In addition, 28 AR binding sites were predicted in the region harboring rs28898617 using Find Individual Motif Occurrences (FIMO), and the A > G mutation at rs28898617 destroyed the AR binding motif in the DNA sequence (PA allele = 0.044; PG allele = 0.072; Fig. S18). These data indicate that AR may accelerate the expression of UGT1A3.

LAMC2 is an effector of UGT1A3 oncogenic action in bladder cancer

We conducted an RNA-Seq assay from UGT1A3-KO and NC bladder cancer cell lines to explore the mechanism by which UGT1A3 promotes bladder cancer progression and detected 7,170 genes (Padj < 0.05) (Fig. 4A). Based on these genes, we excluded 1,585 genes in the present study for the following reasons: genes with no protein coding ability (n = 822), non-autosome (n = 337) or reads ≤10 (n = 426) (Fig. S19). Exclusion of these genes resulted in the inclusion of 5,585 genes, which included 140 downregulated genes and 23 upregulated genes after knocking out UGT1A3 (fold change >4) (Fig. 4B). The KEGG enrichment analysis results revealed that UGT1A3 knockout activated a subset of differentially expressed genes (DEGs) associated with cancer pathways, the PI3K-Akt signaling pathway and focal adhesion pathways (Fig. 4C and Table S7). Because the PI3K-Akt signaling pathway has been widely reported to be related to the progression of bladder cancer [26], we preferentially focused on 12 DEGs enriched in this pathway, including LAMC2, FN1, VEGFA, LAMA4, THBS2, TNC, DDIT4, ITGB3, LPAR3, COL4A4, RELN, and KITLG. LAMC2 and DDIT4 were expressed at high levels in bladder cancer tissues, whereas COL4A4, RELN, and KITLG displayed low expression in bladder cancer tissues (Fig. S20), and the results were consistent with RNA-Seq. Considering that UGT1A3 may act as an oncogenic role in bladder cancer, we then investigated LAMC2 and DDIT4 mRNA and protein expression levels in UGT1A3 knockout and negative control bladder cancer cell lines. LAMC2 and DDIT4 expression levels were significantly reduced in UGT1A3 knockout cell lines, consistent with the RNA-Seq results (Figs. 4D and S21). Because LAMC2 has previously been reported to be involved in bladder cancer progression [27], we preferentially selected it to explore whether UGT1A3 regulated LAMC2 expression to promote the biological malignant behavior of bladder cancer cells. LAMC2 was also enriched in bladder tissues (Fig. S22).

Fig. 4. The effect of LAMC2 regulation by UGT1A3 on the bladder cancer cellular phenotype.

Fig. 4

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The UGT1A3 expression plasmid was transfected into UGT1A3-KO cells (UGT-KO), which were designated UGT-KO + UGT over, and then the LAMC2 knockdown plasmid was transfected into UGT-KO + UGT over cells, which were designated UGT-KO + UGT over+ si-LAM. A Differential expression levels of genes between UGT-KO and NC cell lines are displayed by a heat map. B Volcano plots of differentially expressed gene expression. The green or red plots are the DEGs (fold change >4 and Padj < 0.05). C DEGs were analyzed by KEGG pathway enrichment analysis. D The expression of LAMC2 at the mRNA and protein levels was detected by qRT-PCR and Western blot in the UGT-KO and NC cell lines. E The expression of LAMC2 at the mRNA and protein levels was detected by qRT-PCR and Western blot in the UGT-KO, UGT-KO + UGT over and UGT-KO + UGT over+ si-LAM cell lines. F Cell viability was detected using a CCK-8 assay (OD450 absorbance). G The colony formation ability was measured using a colony formation assay. The colonies were counted and captured. H Representative images of invasion (upper) and migration assays (lower). The number of cells was counted. Scale bar, 180 μm. I The cell cycle was measured by flow cytometry. *P < 0.05 according to Student’s t test.

To further investigate the mechanism of UGT1A3 in regulating the expression of LAMC2, we transfected an UGT1A3 expression plasmid into UGT1A3 knockout cell lines, and then si-LAMC2 was transfected into the above cell lines. UGT1A3 upregulated the LAMC2 levels and si-LAMC2 rescued the tumor-promoting function of high UGT1A3 expression (Figs. 4E-I and S23A). We then detected the relationships between AR and LAMC2 expression and observed that the LAMC2 expression levels were positively correlated with the AR expression levels in bladder cancer cell lines (Fig. S23B). Furthermore, a recent study has reported that CREB-binding protein (CBP) promotes LAMC2 transcription by binding to the promoter region and mediating histone acetylation [28]; therefore, we hypothesized that UGT1A3 binds to CBP to co-regulate LAMC2 expression. Using co-immunoprecipitation (Co-IP) assays, we showed that UGT1A3 bound CBP (Fig. S24A and B). Then, using a luciferase reporter assay, we observed that the deletion of UGT1A3 or CBP significantly decreased the luciferase activity of the plasmid harboring the LAMC2 promoter, suggesting that both UGT1A3 and CBP regulated the expression of LAMC2 at the transcriptional level (Fig. S24C and D). We further conducted chromatin immunoprecipitation (ChIP) assays and observed less enrichment of CBP and acetylated histone H3 on lysine 27 (H3K27ac) at the promoter region of LAMC2 following UGT1A3 deletion, whereas the enrichment of the negative control MyoD did not change (Fig. S24E). Collectively, these findings suggest that UGT1A3 binds CBP to form a complex that promotes the transcription of LAMC2 by increasing H3K27ac activity at the LAMC2 promoter.

Effect of UGT1A3 on bladder cancer tumors in vivo

We injected UGT1A3 knockout (UGT1A3-KO) or NC cell lines into eight humanized NCG mice to further elucidate the biological effect of UGT1A3 in vivo. In line with the in vitro findings, the growth of xenograft tumor tissues in the UGT1A3 knockout group significantly decreased compared with that in the control group (Fig. 5A–D). As shown in Fig. 5E and F, UGT1A3 knockout significantly attenuated the expression levels of UGT1A3 and LAMC2. Furthermore, hematoxylin and eosin (H&E) and immunohistochemical (IHC) analyses showed that UGT1A3 knockout remarkably reduced the Ki67 and LAMC2 expression levels, whereas no significant difference in the AR expression levels was detected (Fig. 5G). Collectively, these results provide evidence that UGT1A3 promotes bladder cancer development by increasing LAMC2 expression levels during oncogenesis.

Fig. 5. Effect of UGT1A3 knockout on tumors in xenograft models.

Fig. 5

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UGT1A3 knockout (UGT1A3-KO) or negative control (NC) cell lines were injected into humanized NCG mice. A The eight burdened NCG mice in the UGT1A3-KO and NC groups. Red arrows show the position of tumors. B The harvested tumor tissues in the UGT1A3-KO and NC groups. The mean weight of excised tumor tissues (C) and the average volume of xenografts (D) were detected in the UGT1A3-KO and NC groups. The expression of UGT1A3 (E), and LAMC2 (F) in tumors was detected using qRT-PCR. G Images of H&E staining and IHC staining for Ki67, AR, and LAMC2 expression in tumor tissues. Scale bar, 120 μm. H The graphic illustration of our findings. *P < 0.05 according to Student’s t test.

Discussion

In this study, we first conducted targeted sequencing based on GWAS-identified loci and validated the results in two independent replicate samples of bladder cancer. Here, we identified a low-frequency variant, UGT1A3 rs28898617 on chromosome 2q37.1, with effects on increasing the risk of bladder cancer (Fig. 5H). Further analysis revealed that the risk allele G of rs28898617 increased the transcriptional activity and expression of UGT1A3. Moreover, we found that the expression of UGT1A3 was upregulated in both bladder cancer tissues and cell lines and that UGT1A3 knockout suppressed the malignant behavior of bladder cancer, including the proliferation, clonogenicity, and invasion/migration abilities. The biological function of UGT1A3 was verified by CRISPR/Cas9 genomic editing in mouse models. Furthermore, using RNA-Seq assays, pathway analysis, Co-IP experiments and ChIP assays, we found that UGT1A3 may increase the expression of LAMC2 by promoting the enrichment of CBP and H3K27ac at the LAMC2 promoter.

Our group united multiple scientific research units to perform the first GWAS in a Chinese cohort and revealed CWC27 rs2042329 as a new susceptibility locus for bladder cancer [13]. Moreover, GWASs of bladder cancer have shown great success in the identification of a subset of common variants in populations of other ethnicities. However, the identified common variants, which are estimated in GWAS, account for only a small portion of bladder cancer heritability. To address this limitation, the targeted sequencing technology used in the present study specifically contributed to capturing low-frequency variants in the targeted region of recombination hotspots according to GWAS-identified loci. The rationale for choosing the targeted sequencing technology is that this platform is a powerful and cost-effective strategy to explore low-frequency variants involved in complex diseases [29, 30]. In addition, we focused on the 100 kbp regions flanking the known bladder cancer GWAS loci as recombination hotspots to find targeted regions for the following reasons: (i) unlike other regions in the genome, genetic variants in these regions around GWAS loci have been estimated to confer cancer risk, and (ii) low-frequency variants at previous GWAS-identified loci regions play a pivotal role in multiple diseases [24, 31–33].

Previous studies have reported that UGT1A3 rs28898617 is mainly responsible for individual drug metabolism. Iwai et al. found that the UGT1A3 missense mutation, including rs28898617 (p.Gln6Arg, Q6R) which caused amino acid substitutions, was capable of affecting individual differences in adverse drug effects and altering the ability of estrogen metabolism [34]. In addition, Chen et al. have also revealed that rs28898617 is one of UGT1A3 variants and could induce human susceptibility to flavonoid exposure [35]. However, the association between UGT1A3 rs28898617 and tumor susceptibility has not been reported to date. In the current study, for the first time, we revealed that the low-frequency variant rs28898617 in UGT1A3 was significantly associated with an increased risk of bladder cancer in Chinese patients. Interestingly, we also explored the genotype frequencies of rs28898617 in multiple ethnicities and found that the rs28898617 polymorphism was present in only the Asian population, indicating that rs28898617 has specificity in populations of different ethnicities. Subsequent eQTL analyses, dual-luciferase reporter assays, and EMSAs indicated that carriers with the risk rs28898617 G allele presented significantly increased expression and transcriptional activity of UGT1A3. However, we only observed the AA genotype of rs28898617 in bladder cancer cell lines, which may be due to the low frequency of the rs28898617 variant. Furthermore, CRISPR/Cas9 system will be performed to generate rs28898617 genotypes, thereby further demonstrating the effect of genotype on bladder cancer.

Uridine 5’-diphosphate (UDP)-glucuronosyltransferase (UGT) is an important member of phase II drug metabolism enzymes in humans and conducts a pivotal process in endobiotic and xenobiotic metabolism [36–38]. Human UGT enzymes have been systematically classified into the UGT1 and UGT2 families and three subfamilies, including UGT1A, UGT2A, and UGT2B [39]. UGT1A3 is a functional isoform encoded by the UGT1A family [40]. Moreover, the UGT family mainly catalyzes the glucuronidation of a series of endogenous and exogenous compounds, such as steroid hormones, polycyclic hydrocarbons, heterocyclic amines and therapeutic drugs, many of which have been identified as human carcinogens [41–43]. Emerging studies have revealed that UGT1A3 is involved in the pathogenesis of several cancers, including pancreatic [44], liver [45] and lung cancer [46]. However, the role of UGT1A3 in human bladder cancer has not been revealed. Here, we found for the first time that UGT1A3, which has higher expression in bladder cancer tissues than in adjacent normal tissues, is present not only in public databases but also in our in-house samples. Consistently, UGT1A3 was significantly enriched in bladder cancer cell lines. On account of these results, we then integrated plasmid overexpression, RNA interference and CRISPR/Cas9-mediated knockout to demonstrate that UGT1A3 may function as an oncogene by promoting bladder cancer cell phenotypes. Moreover, the finding from xenograft models was consistent with the in vitro results, revealing that UGT1A3 ultimately contributed to xenograft growth in a bladder cancer model.

Mechanistically, we further investigated which upstream and downstream factors are involved in UGT1A3-regulated carcinogenesis in bladder cancer. Previous studies reported that males have a greater risk of bladder cancer than age-matched females [47]. AR is an important factor leading to sex differences in the risk of bladder cancer [48, 49]. Importantly, AR plays a critical role in bladder cancer progression by regulating the expression of the UGT family [50, 51]. Initially, we observed a positive association between AR and UGT1A3 expression in both tissues and cell lines. Then, we found that AR knockdown suppressed the malignant biological behavior of bladder cancer. Moreover, when UGT1A3 was overexpressed in si-AR bladder cancer cell lines, the inhibitory effect on malignant tumor phenotypes was reversed. Based on this feature, we adopted in silico analysis to observe that rs28898617 maps within the binding motif of AR, indicating that rs28898617 may moderate UGT1A3 expression by altering the binding activity with the transcriptional factor AR. Further functional investigations are needed to confirm these results. In addition, we performed CRISPR/Cas9 genome editing, RNA-Seq assays and pathway analysis to detect UGT1A3-regulated genes, which revealed that UGT1A3 could positively moderate the expression of LAMC2. After knockdown of LAMC2 in UGT1A3-overexpressing cell lines, the results showed that si-LAMC2 could abrogate the carcinogenic effect caused by UGT1A3 in bladder cancer cell lines, and this finding was further supported by in vivo observations. Our results, in concert with previous studies of associating LAMC2 with bladder cancer progression, might indicate that high LAMC2 expression promotes bladder cancer both in vitro and in vivo [27, 52]. Based on the results of RNA-Seq assays, UGT1A3 might regulate DDIT4 to promote bladder cancer growth, but the function of DDIT4 remains to be further investigated. Furthermore, the independent effects of AR and LAMC2 in vivo must be separately investigated in further studies. In addition, the biological effects of LAMC2 on bladder cancer cell proliferation and colony formation ability was not completely consistent due to different experimental principles. Moreover, similar results were reported by Wang et al. [53]. and Huang et al. [54]. Notably, we also observed that AR could increase the expression of LAMC2. Therefore, these findings suggest that AR increases the expression of UGT1A3, thereby contributing to bladder oncogenesis through activation of LAMC2. In summary, we highlighted the association of the novel low-frequency variant UGT1A3 rs28898617 on chromosome 2q37.1 with bladder cancer. In addition, UGT1A3 promoted bladder tumorigenesis partly by mediating LAMC2. Our findings not only bring us new insights into how low-frequency variants contribute to explaining the unidentified heritability in bladder cancer, but also provide an integrative research strategy to help uncover more biomarkers that may be beneficial for preventing bladder cancer.

Materials and methods

Study populations

To systematically assess the association between low-frequency variants and bladder cancer risk, we performed a discovery analysis and a two-stage replication study. In the discovery stage, 113 bladder cancer cases and 118 healthy controls were used for next-generation sequencing. In the first replication, we recruited 2,227 bladder cancer cases and 2,998 healthy controls from Nanjing Medical University Affiliated Hospital. The subjects in the second replication included 1,123 bladder cancer cases and 1,007 healthy controls from Fudan University Shanghai Cancer Center. All bladder cancer cases were histopathologically diagnosed at the hospital, and all healthy controls were individuals without tumors in the surrounding area. Moreover, healthy controls were frequency-matched to the bladder cancer cases in terms of age and sex. The demographic characteristics of all individuals are summarized in Table S8. All individuals were of the Han Chinese population. In addition, we collected 5,930 bladder cancer cases and 5,468 controls of European ancestry from the database of Genotypes and Phenotypes (dbGaP, phs000346.v2.p2) [18, 55, 56]. This study was approved by the Institutional Review Board of Nanjing Medical University (approval No. 2015-243) and other relevant ethical committees, and written informed consent was obtained from all subjects.

Targeted region selection

In the discovery stage, the bladder cancer risk GWAS loci were extended 100 kbps upstream and downstream to explore recombination hotspots. Based on Asian population data from the 1000 Genomes Project (Phase 1, March 2012), PHASE v2.1 was utilized to calculate the recombination rate. Next, recombination hotspots were verified by selecting a likelihood ratio greater than 12 as the cutoff threshold using SequenceLDhot software. Subsequently, the exons (±25 bp flanking), 5′-UTR (adding 2 kbps proximal) and 3′-UTR of candidate genes (coding genes, long noncoding RNA and microRNA) from recombination hotspots were selected as the target genes/regions based on the University of California Santa Cruz (UCSC) Genome Browser. Then, next-generation sequencing analysis (Genesky Biotechnologies, China) for target genes/regions was conducted and was based on targeted sequencing technology with the Agilent SureSelect Target Enrichment System Kit (Agilent Technologies, CA) and Illumina HiSeq 2000 platform (Illumina, CA) [57, 58].

Next-generation sequencing

All 231 samples (113 bladder cancer cases and 118 controls) and obtained a mean coverage of 74.57X for cases and 81.71X for controls. The average target rates with at least 30X, 20X, 10X and 2X coverage were 75.95%, 82.66%, 89.34% and 95.53%, respectively (Fig. S25 and Table S9). The raw data were filtered to obtain clean data by removing reads carrying adapters, low-quality reads or undetected reads. Each read in the clean data was aligned to hg19 (February 2009 GRch37/hg19) from the UCSC Genome Browser by using Burrows-Wheeler Aligner (BWA) with the default parameters. The Picard Tool and Genome Analysis Toolkit (GATK) 1.6 Tool was utilized to remove duplicate reads and perform local realignment and base quality recalibration, and VarScan software was used to test SNPs/indels. The variant quality score recalibration method was utilized to evaluate a true genetic variant. Then, the variants and their assigned genes from targeted sequencing were annotated by using the UCSC Genome Browser, the Single Nucleotide Polymorphism database (dbSNP), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) annotation databases. Subsequently, the common variants were removed, and a call rate > 90% and nonsynonymous mutations (missense, splicing, and nonsense) were selected for quality control of low-frequency variants.

SNP genotyping

In the two replication stages, variants were genotyped by TaqMan assays with the ABI 7900HT Real-time PCR System (Applied Biosystems, USA), which was previously described in detail [59]. The TaqMan primer sequences are presented in Table S10.

CRISPR/Cas9-mediated knockout of UGT1A3

A pair of sgRNAs for T24 cell lines were designed and synthesized to recognize the whole region of exon 1 of UGT1A3 (Biotechnology, China) (Table S10). The Cas9 protein and sgRNAs were introduced into T24 cells by using Lipofectamine 2000 reagent (Invitrogen, USA). Twenty-four hours after transfection, puromycin (Sigma, USA) was added to the cell medium for a two-day treatment to select for transfected cells, and subsequently, single clones were chosen by serial dilution. Then, the knockout of UGT1A3 was validated through quantitative RT-PCR (qRT-PCR), Sanger sequencing, and Western blot.

Functional experiments

Relative gene expression was detected using qRT-PCR and calculated using the 2-ΔCT method. The primer sequences used in this study are shown in Table S10. An eQTL analysis was performed to evaluate the association between rs28898617 and the expression of the UGT1A gene family in bladder cancer tissues and normal tissues. The dual-luciferase reporter assay was conducted to examine the transcriptional activity of UGT1A3 rs28898617, and the method was confirmed to be accurate by Sanger sequencing (Fig. S26). The binding of UGT1A3 rs28898617 to transcription factors was detected using EMSAs (Table S10). The AR-binding sites were predicted with JASPAR 2018 [60]. FIMO was employed to predict the difference in the AR motif and binding sites affected by the low-frequency variant rs28898617 [61]. A series of assays was performed to investigate the biological effects of UGT1A3, AR, and LAMC2 on malignant behaviors, including gene overexpression, RNA interference, Western blot, cell proliferation, colony formation, invasion, migration, and cell cycle assays. A detailed description of the above methods is available in the Supplementary Materials and Methods.

RNA sequencing array and pathway analyses

Based on the total RNA isolated from UGT1A3 knockout or wild-type bladder cancer cells, whole-transcriptome sequencing (RNA-Seq) was performed by Novogene Co., LTD. (Beijing, China) on the NovaSeq 6000 platform. KEGG pathway enrichment analyses were conducted to evaluate the biological pathways in which DEGs were enriched using the National Institutes of Health Database for Annotation, Visualization, and Integrated Discovery (DAVID) 6.8 software (http://david.abcc.ncifcrf.gov/).

Xenograft models

The female humanized NCG mice were randomly divided into a control and UGT1A3 knockout group (4 weeks old, eight mice per group). UGT1A3 knockout or wild-type bladder cancer cells (1×107 cells per mouse) suspended in 200 μl of PBS were injected subcutaneously into the right flanks of mice. The tumor diameter was examined every 3 days. All mice were sacrificed after one month, and their tumors were measured. Then, H&E and IHC staining were utilized to retain tumors. H&E staining was used to select representative areas, and IHC was applied to confirm the expression of the proliferation markers Ki67 (anti-Ki67, ab15580, Abcam), AR (anti-AR, 22089-1-AP, Proteintech), and LAMC2 (anti-LAMC2, USB-144830, US Biological). We were not blinded to the group allocation in animal experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University (IACUC-1804033).

Statistical analysis

The PS Power and Sample Size Calculations software (V.3.1; Nashville, Tennessee, USA) was used to evaluate whether the sample size in this study could effectively assess the association between low-frequency variants and bladder cancer risk [29]. Pearson’s χ2 test, Student’s independent t test or paired t test was utilized to compare the characteristics of the two groups. In vitro experiments were replicated three times and data are presented as means ± standard deviations. Three gene set analyses of low-frequency variants were performed, including the SKAT [62], simple burden test (T1/T5/MB) [55], and VT test [56]. The OR and 95% CI for the risk of bladder cancer were estimated using multivariate logistic regression analysis to assess the association between UGT1A3 rs28898617 and bladder cancer risk with adjustment for age, sex and smoking status. The meta-analysis was carried out to combine the OR from each stage with the heterogeneity using Cochran’s Q statistics and I2. A two-sided P < 0.05 was regarded as statistically significant. The statistical analyses were performed with PLINK (version 1.90) and R software (version 3.6.1).

Supplementary information

Supplementary (5MB, pdf)

Acknowledgements

This study was supported in part by the National Natural Science Foundation of China (81673264) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine). We are grateful to all the people who helped us accomplish this project. We thank Genesky Biotechnologies Inc. (Shanghai, China) for providing technical assistance.

Author contributions

MW and ZZ designed the study and provided supervision. QL, CQ, YZ, CG, MW, and DY recruited study subjects. RZ, MD, YG, and JX performed statistical analyses and summarized results. FG, QZ, ZG, and SB performed functional experiments. RZ, MD, and HC prepared the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: R. Zheng, M. Du, Y. Ge

Contributor Information

Zhengdong Zhang, Email: drzdzhang@njmu.edu.cn.

Meilin Wang, Email: mwang@njmu.edu.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s41388-021-01672-1.

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