- Email: [email protected]
Tagging SNPs in the HOTAIR gene are associated with bladder cancer risk in a Chinese population
Accepted Manuscript Tagging SNPs in the HOTAIR gene are associated with bladder cancer risk in a Chinese population
Xiang Wang, Wenying Wang, Qiang Zhang, Damin Gu, Ke Zhang, Yuqiu Ge, Haiyan Chu, Mulong Du, Bin Xu, Meilin Wang, Xu Lv, Zhengdong Zhang, Lin Yuan, Weida Gong PII: DOI: Reference:
S0378-1119(18)30408-6 doi:10.1016/j.gene.2018.04.039 GENE 42763
To appear in:
Gene
Received date: Revised date: Accepted date:
30 October 2017 6 April 2018 13 April 2018
Please cite this article as: Xiang Wang, Wenying Wang, Qiang Zhang, Damin Gu, Ke Zhang, Yuqiu Ge, Haiyan Chu, Mulong Du, Bin Xu, Meilin Wang, Xu Lv, Zhengdong Zhang, Lin Yuan, Weida Gong , Tagging SNPs in the HOTAIR gene are associated with bladder cancer risk in a Chinese population. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2018.04.039
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ACCEPTED MANUSCRIPT Tagging SNPs in the HOTAIR gene are associated with bladder cancer risk in a Chinese population Xiang Wanga,1 , Wenying Wangb,1 , Qiang Zhangc,d,1 , Damin Gua, Ke Zhanga, Yuqiu Gec,d, Haiyan Chuc,d, Mulong Duc,d, Bin Xue, Meilin Wangc,d, Xu Lvf, Zhengdong
Department of Anesthesiology, Yixing People’s Hospital, Yixing, China;
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a
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Zhangc,d,* , Lin Yuang,h,* , Weida Gonge,*
b
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Department of Urology, Beijing Friendship Hospital affiliated to Capital Medical
University, Beijing, China;
Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer
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c
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Biomarkers, Prevention and Treatment, Collaborative Innovation Center For Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China; d
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Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of
China; e
Department of Urology, Affiliated Zhongda Hospital of Southeast University ,
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Nanjing , China. f
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Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing,
Department of General Surgery, Yixing Tumor Hospital, Yixing, China;
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Department of Urology, Jiangsu Province Hospital of TCM, Nanjing, China;
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Department of Integrated traditional Chinese and Western medicine Tumor Research
Lab, Nanjing, China. 1
These authors contributed equally to this work.
*Correspondence to: Zhengdong Zhang, Department of Environmental Genomics,
ACCEPTED MANUSCRIPT School of Public Health, Nanjing Medical University, Nanjing 211166, China. Email: [email protected]; Lin Yuan, Department of Urology, Jiangsu Province Hospital of TCM, Nanjing 210029, China. Email: [email protected]; Weida Gong, Department of General Surgery, Yixing Tumor Hospital, Yixing, 214200, China.
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Email: [email protected].
ACCEPTED MANUSCRIPT Abstract Background: The HOX transcript antisense intergenic RNA (HOTAIR) is a well-known long noncoding RNA (lncRNA) that plays a critical role in biological processes in most cancers. However, the function of HOTAIR in bladder cancer
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remains largely unknown. In this study, we hypothesize that tag single nucleotide
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polymorphisms (tagSNPs) in HOTAIR are associated with bladder cancer (BCa) risk.
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Methods: We performed a hospital-based case-control study of 1,050 cases and 1,407 controls to investigate the associations between tagSNPs and the risk of BCa in a
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Chinese population.
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Results: We found that individuals with the rs874945 AG/AA genotype had a significantly increased risk of BCa compared with those carrying the GG genotype,
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with an odds ratio (OR) of 1.23 [95% confidence interval (CI) = 1.04-1.46, P = 0.014].
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The subsequently stratified analyses showed that the increased risk was more pronounced in subgroups of older subjects (age > 60 years), never smokers and subjects without pack-years of smoking. Interactive analysis showed that there was no
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interaction effect between smoking status and rs874945. Conclusion: Our study showed that rs874945 in HOTAIR was associated with BCa risk in a Chinese population.
ACCEPTED MANUSCRIPT A case-control study was performed to investigate the association. HOTAIR rs874945 may contribute to the susceptibility to bladder cancer. AG/AA genotype has an increased risk of bladder cancer compared with GG genotype.
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Key words: Bladder cancer; HOTAIR; TagSNP; Case-control study
ACCEPTED MANUSCRIPT 1. Introduction Bladder cancer (BCa) is one of the most frequently diagnosed urologic cancers in the world, with an estimated 72,570 new cases and 15,210 deaths in the USA alone in 2013 (Siegel et al., 2013). In China, the incidence of BCa has increased steadily over
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recent years (Chen et al., 2016). Data from the International Agency for Research on
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Cancer showed that there were 55,486 cases and 26,820 deaths in China in 2012.
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(http://globocan.iarc.fr). Like many other cancers, environmental factors are among the major causes of BCa development; these factors include tobacco smoking and
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occupational exposure to chemical carcinogens (Hainaut and Pfeifer, 2001). However,
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although many people have been exposed to these same risk factors, only some of them develop BCa, suggesting that genetic susceptibility may also play a central role
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in BCa.
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Long noncoding RNAs (lncRNAs), a novel kind of RNA of at least 200 nucleotides (Costa, 2010), are transcribed pervasively in the genome (Ponting et al., 2009; Jeggari et al., 2012). Recent studies have shown that lncRNAs play various functions in
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carcinogenesis processes, including transcriptional, post-transcriptional and epigenetic regulation of cancer-related genes (Huarte and Rinn, 2010). HOTAIR, as a paradigm for lncRNA function in cancer, has been widely focused on, and it is transcribed from the HOXC cluster in an antisense manner (Chu et al., 2011). It was found that HOTAIR is involved in the pathogenesis of a variety of cancers (Gupta et al., 2010; Kogo et al., 2011). The expression level of HOTAIR is upregulated in primary and metastatic breast cancer. It is a powerful biomarker for the prognosis of breast cancer.
ACCEPTED MANUSCRIPT High HOTAIR expression in breast cancer is significantly associated with a worse prognosis (Sorensen et al., 2013). So far, studies have discovered that HOTAIR mainly regulates the transcription of a 40 kb region of HOXD. However, few studies have mentioned the molecular mechanisms of HOTAIR in BCa.
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Single nucleotide polymorphisms (SNPs) have been confirmed to participate in
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carcinogenesis of tumors, through influencing expression or function of specific genes
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(Gaffney, 2013). Currently, the known effects of SNPs have been extended to include functional lncRNAs from cancer-related protein coding genes. Mounting evidence
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indicates that SNPs in lncRNAs are associated with cancer risk. Rs7763881 in the
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lncRNA HULC may contribute to decreased susceptibility to hepatocellular carcinoma (Liu et al., 2012), and lncRNA PCGEM1 polymorphisms are associated
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with prostate cancer (Xue et al., 2013). TagSNPs in the HOTAIR gene region are
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associated with the risk of colorectal cancer (Xue et al., 2015). This was the first evidence that variants in HOTAIR are associated with cancer risk. Based on this new
BCa.
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finding, we hypothesized that genetic variants in HOTAIR may also modify the risk of
In the present study, we evaluated the association between HOTAIR tagSNPs and BCa risk in a hospital-based case-control study in a Chinese population. As a result, we found that rs874945, located in the 3’-UTR of the HOTAIR gene, was significantly associated with the risk of BCa.
2. Materials and methods
ACCEPTED MANUSCRIPT 2.1 Study population The hospital-based case-control study consisted of 1,050 BCa cases and 1,403 controls. Detailed recruitment information was described previously (Wang et al., 2016). All patients were recruited from the First Affiliated Hospital of Nanjing
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Medical University, the Huai an Affiliated Hospital of Nanjing Medical University
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and Jiangsu Province Hospital of TCM. The controls were randomly selected from the
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same hospital and matched with BCa cases by age and sex. Patients were divided into never smokers and ever smokers according to their smoking history. Those who
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smoked daily for more than 1 year were treated as ever smokers. Furthermore, of the
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ever smokers, those who had quit smoking for more than 1 year were defined as former smokers, and the remaining were defined as current smokers. Pathological
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diagnosis of tumor stage in all cases was done according to the 2002 International
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Union Against Cancer (UICC) tumor-nodes-metastasis classification. The study was approved by the Institutional Review Board of Nanjing Medical University. 2.2 SNP selection and genotyping
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The SNP selection process was introduced in detail in our previous study (Xue et al., 2015). The list of selected tagSNPs is shown in Table S1. Genomic DNA of all the cases and controls was extracted from peripheral blood lymphocytes using the Qiagen Blood Kit (Qiagen). An ABI 7900HT Real-time PCR System (Applied Biosystems, Foster City, CA, USA) was used for the TaqMan SNP genotyping assay. Sequences of primers and probes for each SNP are listed in Table S2. Genotyping was carried out by two people independently and in a blinded fashion. Approximately 10% of the
ACCEPTED MANUSCRIPT samples were selected randomly for confirmation, and the results were 100% concordant. 2.3 Statistical analysis The frequency distributions of selected demographic variables and genotypes of
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HOTAIR polymorphisms between cases and controls were estimated by Chi-square
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(χ2) test. A goodness-of-fit χ2 test was performed to calculate the Hardy-Weinberg
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equilibrium of the genotype distribution among the controls. Unconditional univariate and multivariate logistic regression analyses were conducted to analyze the
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associations between HOTAIR tagSNPs and the risk of BCa by odds ratios (ORs) and
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their 95% confidence intervals (CIs). Interactions were tested using a multiplicative interaction term included in the multivariate model. All ORs were adjusted for age
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and sex. The stratification analyses were performed according to age, sex, smoking
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status and pack-years of smoking. All analyses were conducted with SAS software (version 9.1; SAS Institute, Inc., Cary, NC, USA) with two-sided P values, and a P
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value of < 0.05 indicated statistical significance.
3. Results
3.1 Characteristics of the study population Characteristics of the 1,050 BCa patients and 1,403 control subjects are summarized in Table 1. The cases and controls were matched adequately for age (P = 0.320) and sex (P = 0.544). There were more ever smokers (52.7%) among the cases than among the controls (47.3%). Significant differences were shown in smoking
ACCEPTED MANUSCRIPT status and pack-years of smoking between cases and controls (P < 0.001 for both). Of the 1,050 cases, approximately 64.4% of patients were in the non-muscle invasive stage, and 34.2% were in the invasive stage. 3.2 Effects of SNPs in HOTAIR and BCa risk
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Frequencies of the rs874945 and rs7958904 genotypes among the controls were in
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Hardy-Weinberg equilibrium (P = 0.718 for rs7958904 and P = 0.764 for rs874945);
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however, this was not true for rs4759314. The genotype distribution of rs874945 and rs7958904 and their associations with BCa risk are shown in Table 2. ORs and 95%
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CIs were used to assess the associations of the two SNPs with BCa risk in additive,
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co-dominant, recessive and dominant models. The results show that carriers with the AG genotype of rs874945 have a markedly increased risk of BCa (adjusted OR = 1.28,
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95% CI = 1.08-1.52, P = 0.005) compared to those carrying the GG genotype. When
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we combined the AG and AA genotypes to construct a dominant model, a significant increased risk was also found in the AG/AA genotypes, with an adjusted OR of 1.23 (95% CI = 1.04-1.46, P = 0.014). Additionally, no notable association was observed
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between rs7958904 and BCa risk.
3.3 Stratified analysis of rs874945 in HOTAIR Stratified analysis was applied to evaluate the associations of rs874945 and BCa risk in the subgroups based on age, sex, smoking status and pack-years of smoking. As shown in Table 3, compared with the AG/AA genotype, patients with the CC genotype had a significantly increased risk of BCa in the subgroups of older subjects
ACCEPTED MANUSCRIPT (age > 60) (adjusted OR = 1.35, 95% CI = 1.10-1.65, P = 0.004), males (adjusted OR = 1.22, 95% CI = 1.01-1.47, P = 0.039), never smokers (adjusted OR = 1.30, 95% CI = 1.04-1.62, P = 0.022), and subjects without pack-years of smoking (adjusted OR = 1.30, 95% CI = 1.04-1.62, P = 0.022). Nevertheless, no interaction effect between
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rs874945 and smoking status was detected (P = 0.382).
ACCEPTED MANUSCRIPT 4. Discussion Recently, lncRNAs have received widespread attention for their vital function in various diseases, especially cancers. Evidence has shown that genetic variants in lncRNAs may alter individual susceptibility to cancer by modulating the expression
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and function of lncRNAs (Liu et al., 2012; Wang et al., 2013; Wu et al., 2013).
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HOTAIR is pervasively overexpressed in many cancers and is involved in the process
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of tumor progression. However, little attention has been given to the effects of HOTAIR SNPs in human cancer. Recently, the relationship of genetic variants in
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HOTAIR and cancer was first reported in colorectal cancer (Xue et al., 2015).
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HOTAIR is a spliced, polyadenylated transcript of approximately 2.2k nucleotides that is transcribed from the HOXC cluster in an antisense manner. It has been widely
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treated as a functional lncRNA participating in multiple cancers. Evidence has shown
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that HOTAIR could function as a molecular scaffold to link polycomb repressive complex 2 (PRC2) and lysine specific demethylase 1 (LSD1) protein complexes and regulate gene expression by modulating chromatin structure in trans across the 40-kb
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HOXD locus2 (Gupta et al., 2010; Chu et al., 2011), which causes epigenetic silencing of various cancer-related genes, especially the HOXD gene, by H3K27 methylation and H3K4 demethylation (Tsai et al., 2010). These epigenetic changes result in upregulation or downregulation of the expression of genes affecting the proliferation, invasion, apoptosis and migration of cancer cells. Such processes have been shown in breast cancer (Gupta et al., 2010), colorectal cancer (Kogo et al., 2011), pancreatic cancer (Kim et al., 2013), and gastric cancer (Xu et al., 2013).
ACCEPTED MANUSCRIPT A meta-analysis revealed that there was no significant association between SNPs in HOTAIR and cancer risk (Tian et al., 2016). However, other studies have found different results. Additional larger studies incorporating subjects of different ethnic backgrounds combined with re-sequencing of the marked region and functional
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evaluations are needed. It has been reported that genetic variants in lncRNAs
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modulate individual susceptibility to cancer (Liu et al., 2012; Li et al., 2013) and that
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tagSNPs in the HOTAIR gene region are associated with the risk of colorectal cancer. We speculated that genetic variants in HOTAIR might also participate in BCa. The
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association between tagSNPs in the HOTAIR gene region and the susceptibility to
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BCa was assessed in a case-control study. The results showed a remarkable association between an increased risk of BCa and the rs874945 genetic variant.
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Subsequently, we performed stratified analyses of rs874945 according to epidemic
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variables. We found that rs874945 increased BCa risk in a dominant model and was more pronounced in subgroups of older subjects, males, never smokers and subjects without pack-years of smoking. These results indicate that the effects of the HOTAIR
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variant on BCa risk might be modulated by specific demographic factors and environmental exposures. A possible explanation is that some environmental hazards such as tobacco smoking promote BCa risk, which enhances the effects of rs874945 variants. This also provides evidence that carcinogenesis is a complex progress involving both genetic and environmental factors. There are several strengths of this study. First, although the role of HOTAIR in carcinogenesis has been reported in a variety of cancers, this is the first study aimed at
ACCEPTED MANUSCRIPT investigating genetic variants of HOTAIR in the susceptibility to BCa. Our observations contribute to further understanding of the susceptibility to BCa. The relatively large sample size (1,050 BCa cases and 1,403 controls) is another strength, affording sufficient statistical power to our study. However, there were also some
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limitations of our study. First, this hospital-based case-control study was a one-stage
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design, and selection bias was unavoidable. Our study also lacks functional evidence,
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and functional studies are needed in the future.
In conclusion, we are the first to provide evidence that HOTAIR rs874945 may
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contribute to the risk of BCa in a Chinese population. Further well-designed studies in
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multicenter, large, independent populations and functional studies will be performed to validate our findings in the future.
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Author Contributions
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Conceived and designed the experiments: ZZ WG. Performed the experiments: QZ XW. Analyzed the data: MD YG HC. Contributed reagents/materials/analysis tools: WW LY MW DG KZ BX XL. Wrote the paper: XW. Revised the manuscript critically:
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ZZ WG.
Conflicts of interest
The authors declare no conflicts of interest. Acknowledgments This study was partly supported by National Natural Science Foundation of China (81473050), Jiangsu Provincial “333” project (BRA2012033), National Health and Family Planning Commission Project (W2015PM113), Collaborative research project of Southeast University-Nanjing Medical University (2017DN39), Beijing Municipal Administration of Hospitals’ Youth Programme (QML20150101), Natural Science Foundation of Jiangsu Province (BK20151603) and Nanjing Science and Technology
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Program (201605009), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).
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ACCEPTED MANUSCRIPT Table 1. Frequency distributions of selected variables between the bladder cancer cases and controls Variables
Cases (n = 1,050)
Controls (n = 1,403)
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%
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Age (mean ± SD) 64.8 ± 12.6 65.2 ± 9.3 Sex Male 839 79.9 1107 78.9 Female 211 20.1 296 21.1 Smoking status Never 553 52.7 866 61.7 Ever 497 47.3 537 38.3 Former 225 21.4 179 12.8 Current 272 25.9 358 25.5 Pack-years of smoking 0 553 52.7 866 61.7 ≤ 20 191 18.2 259 18.5 > 20 306 29.1 278 19.8 b Stage Non-muscle invasive (Ta-T1) 676 64.4 Invasive (T2-T4) 359 34.2 Missing 15 1.4 a 2 Two-sided χ test for the frequency distributions of selected variables between the cases and controls. b stage in accordance with the 2004 WHO/ISUP classification of bladder cancer.
Pa 0.320 0.544
< 0.001
< 0.001
ACCEPTED MANUSCRIPT Table 2. Associations of the selected SNPs with bladder cancer risk Variations
Genotypes
N(cases/controls)
P (HWE)
Crude OR (95% CI)
Adjusted OR (95% CI) a
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GG 649/931 AG 370/416 0.764 1.14(0.99-1.32) 1.14(0.99-1.32) AA 29/49 codominant GG 649/931 1.00 1.00 AG 370/416 1.28(1.07-1.52) 1.28(1.08-1.52) AA 29/49 0.85(0.53-1.36) 0.85(0.53-1.36) dominant GG 649/931 1.00 1.00 AG/AA 399/465 1.23(1.04-1.46) 1.23(1.04-1.46) recessive GG/AG 1019/1347 1.00 1.00 AA 29/49 0.78(0.49-1.25) 0.78(0.49-1.25) rs4759314 additive AA 897/1217 AG 129/143 2.26E-23 1.05(0.87-1.27) 1.06(0.87-1.28) GG 19/34 codominant AA 897/1217 1.00 1.00 AG 129/143 1.22(0.95-1.58) 1.23(0.95-1.58) GG 19/34 0.76(0.43-1.34) 0.77(0.44-1.37) dominant AA 897/1217 1.00 1.00 AG/GG 148/177 1.13(0.90-1.44) 1.14(0.90-1.44) recessive AA/AG 1026/1360 1.00 1.00 GG 19/34 0.74(0.42-1.31) 0.76(0.43-1.34) rs7958904 additive AA 551/764 AG 400/531 0.718 1.10(0.97-1.25) 1.10(0.97-1.25) GG 91/97 codominant AA 551/764 1.00 1.00 AG 400/531 1.04(0.88-1.24) 1.05(0.88-1.24) GG 91/97 1.30(0.96-1.77) 1.30(0.96-1.77) dominant AA 551/764 1.00 1.00 AG/GG 491/628 1.08(0.92-1.27) 1.09(0.92-1.28) recessive AA/AG 951/1295 1.00 1.00 GG 91/97 1.28(0.95-1.72) 1.28(0.95-1.72) a Adjusted by age and sex in logistic regression analysis. b 2 Two-sided χ test for the frequency distributions of selected variables between the cases and controls.
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rs874945
Genetic models additive
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0.005 0.499 0.014 0.306 0.579
0.115 0.378 0.274 0.335 0.145
0.612 0.092 0.322 0.107
ACCEPTED MANUSCRIPT Table 3. Stratification analyses between rs874945 and bladder cancer risk Variations
N(cases/controls)
Crude OR(95% CI)
Adjusted OR(95% CI)
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Age ≤ 60 358/381 1.02(0.75-1.39) 1.01(0.75-1.38) > 60 690/1015 1.35(1.11-1.65) 1.35(1.10-1.65) Sex Male 837/1100 1.22(1.01-1.47) 1.22(1.01-1.47) Female 211/296 1.29(0.89-1.87) 1.29(0.89-1.87) Smoking status Never 553/864 1.30(1.04-1.62) 1.30(1.04-1.62） Ever Former 224/175 1.19(0.79-1.78) 1.22(0.81-1.85) Current 271/357 1.07(0.77-1.49) 1.07(0.77-1.49) P c for interaction (multiplicative) Pack-years of smoking 0 553/864 1.30(1.04-1.62) 1.30(1.04-1.62) ≤ 20 190/256 1.36(0.92-2.01) 1.39(0.94-2.07) > 20 305/276 0.99(0.71-1.39) 0.99(0.71-1.39) a Adjusted by age and sex in logistic regression analysis. b Two-sided χ2 test for the frequency distributions of selected variables between the cases and controls. c The multiplicative interaction test between smoking status (never or ever) and genotypes (AA/AG or GG). Adjusted by age and sex in logistic regression model.
P
b
0.929 0.004 0.039 0.185 0.022 0.345 0.692 0.382 0.022 0.102 0.964
ACCEPTED MANUSCRIPT Abbreviations: HOTAIR, The HOX transcript antisense intergenic RNA; lncRNA, long noncoding RNA; TagSNP, tagging single nucleotide polymorphisms; BCa, bladder cancer; OR, odds ratio;
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CI, confidence interval; MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium
Xiang Wang, Wenying Wang, Qiang Zhang, Damin Gu, Ke Zhang, Yuqiu Ge, Haiyan Chu, Mulong Du, Bin Xu, Meilin Wang, Xu Lv, Zhengdong Zhang, Lin Yuan, Weida Gong PII: DOI: Reference:
S0378-1119(18)30408-6 doi:10.1016/j.gene.2018.04.039 GENE 42763
To appear in:
Gene
Received date: Revised date: Accepted date:
30 October 2017 6 April 2018 13 April 2018
Please cite this article as: Xiang Wang, Wenying Wang, Qiang Zhang, Damin Gu, Ke Zhang, Yuqiu Ge, Haiyan Chu, Mulong Du, Bin Xu, Meilin Wang, Xu Lv, Zhengdong Zhang, Lin Yuan, Weida Gong , Tagging SNPs in the HOTAIR gene are associated with bladder cancer risk in a Chinese population. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2018.04.039
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ACCEPTED MANUSCRIPT Tagging SNPs in the HOTAIR gene are associated with bladder cancer risk in a Chinese population Xiang Wanga,1 , Wenying Wangb,1 , Qiang Zhangc,d,1 , Damin Gua, Ke Zhanga, Yuqiu Gec,d, Haiyan Chuc,d, Mulong Duc,d, Bin Xue, Meilin Wangc,d, Xu Lvf, Zhengdong
Department of Anesthesiology, Yixing People’s Hospital, Yixing, China;
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a
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Zhangc,d,* , Lin Yuang,h,* , Weida Gonge,*
b
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Department of Urology, Beijing Friendship Hospital affiliated to Capital Medical
University, Beijing, China;
Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer
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c
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Biomarkers, Prevention and Treatment, Collaborative Innovation Center For Cancer Personalized Medicine, Nanjing Medical University, Nanjing, China; d
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Department of Genetic Toxicology, The Key Laboratory of Modern Toxicology of
China; e
Department of Urology, Affiliated Zhongda Hospital of Southeast University ,
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Nanjing , China. f
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Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing,
Department of General Surgery, Yixing Tumor Hospital, Yixing, China;
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Department of Urology, Jiangsu Province Hospital of TCM, Nanjing, China;
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Department of Integrated traditional Chinese and Western medicine Tumor Research
Lab, Nanjing, China. 1
These authors contributed equally to this work.
*Correspondence to: Zhengdong Zhang, Department of Environmental Genomics,
ACCEPTED MANUSCRIPT School of Public Health, Nanjing Medical University, Nanjing 211166, China. Email: [email protected]; Lin Yuan, Department of Urology, Jiangsu Province Hospital of TCM, Nanjing 210029, China. Email: [email protected]; Weida Gong, Department of General Surgery, Yixing Tumor Hospital, Yixing, 214200, China.
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Email: [email protected].
ACCEPTED MANUSCRIPT Abstract Background: The HOX transcript antisense intergenic RNA (HOTAIR) is a well-known long noncoding RNA (lncRNA) that plays a critical role in biological processes in most cancers. However, the function of HOTAIR in bladder cancer
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remains largely unknown. In this study, we hypothesize that tag single nucleotide
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polymorphisms (tagSNPs) in HOTAIR are associated with bladder cancer (BCa) risk.
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Methods: We performed a hospital-based case-control study of 1,050 cases and 1,407 controls to investigate the associations between tagSNPs and the risk of BCa in a
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Chinese population.
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Results: We found that individuals with the rs874945 AG/AA genotype had a significantly increased risk of BCa compared with those carrying the GG genotype,
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with an odds ratio (OR) of 1.23 [95% confidence interval (CI) = 1.04-1.46, P = 0.014].
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The subsequently stratified analyses showed that the increased risk was more pronounced in subgroups of older subjects (age > 60 years), never smokers and subjects without pack-years of smoking. Interactive analysis showed that there was no
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interaction effect between smoking status and rs874945. Conclusion: Our study showed that rs874945 in HOTAIR was associated with BCa risk in a Chinese population.
ACCEPTED MANUSCRIPT A case-control study was performed to investigate the association. HOTAIR rs874945 may contribute to the susceptibility to bladder cancer. AG/AA genotype has an increased risk of bladder cancer compared with GG genotype.
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Key words: Bladder cancer; HOTAIR; TagSNP; Case-control study
ACCEPTED MANUSCRIPT 1. Introduction Bladder cancer (BCa) is one of the most frequently diagnosed urologic cancers in the world, with an estimated 72,570 new cases and 15,210 deaths in the USA alone in 2013 (Siegel et al., 2013). In China, the incidence of BCa has increased steadily over
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recent years (Chen et al., 2016). Data from the International Agency for Research on
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Cancer showed that there were 55,486 cases and 26,820 deaths in China in 2012.
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(http://globocan.iarc.fr). Like many other cancers, environmental factors are among the major causes of BCa development; these factors include tobacco smoking and
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occupational exposure to chemical carcinogens (Hainaut and Pfeifer, 2001). However,
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although many people have been exposed to these same risk factors, only some of them develop BCa, suggesting that genetic susceptibility may also play a central role
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in BCa.
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Long noncoding RNAs (lncRNAs), a novel kind of RNA of at least 200 nucleotides (Costa, 2010), are transcribed pervasively in the genome (Ponting et al., 2009; Jeggari et al., 2012). Recent studies have shown that lncRNAs play various functions in
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carcinogenesis processes, including transcriptional, post-transcriptional and epigenetic regulation of cancer-related genes (Huarte and Rinn, 2010). HOTAIR, as a paradigm for lncRNA function in cancer, has been widely focused on, and it is transcribed from the HOXC cluster in an antisense manner (Chu et al., 2011). It was found that HOTAIR is involved in the pathogenesis of a variety of cancers (Gupta et al., 2010; Kogo et al., 2011). The expression level of HOTAIR is upregulated in primary and metastatic breast cancer. It is a powerful biomarker for the prognosis of breast cancer.
ACCEPTED MANUSCRIPT High HOTAIR expression in breast cancer is significantly associated with a worse prognosis (Sorensen et al., 2013). So far, studies have discovered that HOTAIR mainly regulates the transcription of a 40 kb region of HOXD. However, few studies have mentioned the molecular mechanisms of HOTAIR in BCa.
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Single nucleotide polymorphisms (SNPs) have been confirmed to participate in
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carcinogenesis of tumors, through influencing expression or function of specific genes
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(Gaffney, 2013). Currently, the known effects of SNPs have been extended to include functional lncRNAs from cancer-related protein coding genes. Mounting evidence
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indicates that SNPs in lncRNAs are associated with cancer risk. Rs7763881 in the
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lncRNA HULC may contribute to decreased susceptibility to hepatocellular carcinoma (Liu et al., 2012), and lncRNA PCGEM1 polymorphisms are associated
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with prostate cancer (Xue et al., 2013). TagSNPs in the HOTAIR gene region are
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associated with the risk of colorectal cancer (Xue et al., 2015). This was the first evidence that variants in HOTAIR are associated with cancer risk. Based on this new
BCa.
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finding, we hypothesized that genetic variants in HOTAIR may also modify the risk of
In the present study, we evaluated the association between HOTAIR tagSNPs and BCa risk in a hospital-based case-control study in a Chinese population. As a result, we found that rs874945, located in the 3’-UTR of the HOTAIR gene, was significantly associated with the risk of BCa.
2. Materials and methods
ACCEPTED MANUSCRIPT 2.1 Study population The hospital-based case-control study consisted of 1,050 BCa cases and 1,403 controls. Detailed recruitment information was described previously (Wang et al., 2016). All patients were recruited from the First Affiliated Hospital of Nanjing
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Medical University, the Huai an Affiliated Hospital of Nanjing Medical University
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and Jiangsu Province Hospital of TCM. The controls were randomly selected from the
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same hospital and matched with BCa cases by age and sex. Patients were divided into never smokers and ever smokers according to their smoking history. Those who
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smoked daily for more than 1 year were treated as ever smokers. Furthermore, of the
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ever smokers, those who had quit smoking for more than 1 year were defined as former smokers, and the remaining were defined as current smokers. Pathological
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diagnosis of tumor stage in all cases was done according to the 2002 International
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Union Against Cancer (UICC) tumor-nodes-metastasis classification. The study was approved by the Institutional Review Board of Nanjing Medical University. 2.2 SNP selection and genotyping
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The SNP selection process was introduced in detail in our previous study (Xue et al., 2015). The list of selected tagSNPs is shown in Table S1. Genomic DNA of all the cases and controls was extracted from peripheral blood lymphocytes using the Qiagen Blood Kit (Qiagen). An ABI 7900HT Real-time PCR System (Applied Biosystems, Foster City, CA, USA) was used for the TaqMan SNP genotyping assay. Sequences of primers and probes for each SNP are listed in Table S2. Genotyping was carried out by two people independently and in a blinded fashion. Approximately 10% of the
ACCEPTED MANUSCRIPT samples were selected randomly for confirmation, and the results were 100% concordant. 2.3 Statistical analysis The frequency distributions of selected demographic variables and genotypes of
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HOTAIR polymorphisms between cases and controls were estimated by Chi-square
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(χ2) test. A goodness-of-fit χ2 test was performed to calculate the Hardy-Weinberg
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equilibrium of the genotype distribution among the controls. Unconditional univariate and multivariate logistic regression analyses were conducted to analyze the
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associations between HOTAIR tagSNPs and the risk of BCa by odds ratios (ORs) and
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their 95% confidence intervals (CIs). Interactions were tested using a multiplicative interaction term included in the multivariate model. All ORs were adjusted for age
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and sex. The stratification analyses were performed according to age, sex, smoking
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status and pack-years of smoking. All analyses were conducted with SAS software (version 9.1; SAS Institute, Inc., Cary, NC, USA) with two-sided P values, and a P
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value of < 0.05 indicated statistical significance.
3. Results
3.1 Characteristics of the study population Characteristics of the 1,050 BCa patients and 1,403 control subjects are summarized in Table 1. The cases and controls were matched adequately for age (P = 0.320) and sex (P = 0.544). There were more ever smokers (52.7%) among the cases than among the controls (47.3%). Significant differences were shown in smoking
ACCEPTED MANUSCRIPT status and pack-years of smoking between cases and controls (P < 0.001 for both). Of the 1,050 cases, approximately 64.4% of patients were in the non-muscle invasive stage, and 34.2% were in the invasive stage. 3.2 Effects of SNPs in HOTAIR and BCa risk
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Frequencies of the rs874945 and rs7958904 genotypes among the controls were in
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Hardy-Weinberg equilibrium (P = 0.718 for rs7958904 and P = 0.764 for rs874945);
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however, this was not true for rs4759314. The genotype distribution of rs874945 and rs7958904 and their associations with BCa risk are shown in Table 2. ORs and 95%
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CIs were used to assess the associations of the two SNPs with BCa risk in additive,
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co-dominant, recessive and dominant models. The results show that carriers with the AG genotype of rs874945 have a markedly increased risk of BCa (adjusted OR = 1.28,
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95% CI = 1.08-1.52, P = 0.005) compared to those carrying the GG genotype. When
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we combined the AG and AA genotypes to construct a dominant model, a significant increased risk was also found in the AG/AA genotypes, with an adjusted OR of 1.23 (95% CI = 1.04-1.46, P = 0.014). Additionally, no notable association was observed
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between rs7958904 and BCa risk.
3.3 Stratified analysis of rs874945 in HOTAIR Stratified analysis was applied to evaluate the associations of rs874945 and BCa risk in the subgroups based on age, sex, smoking status and pack-years of smoking. As shown in Table 3, compared with the AG/AA genotype, patients with the CC genotype had a significantly increased risk of BCa in the subgroups of older subjects
ACCEPTED MANUSCRIPT (age > 60) (adjusted OR = 1.35, 95% CI = 1.10-1.65, P = 0.004), males (adjusted OR = 1.22, 95% CI = 1.01-1.47, P = 0.039), never smokers (adjusted OR = 1.30, 95% CI = 1.04-1.62, P = 0.022), and subjects without pack-years of smoking (adjusted OR = 1.30, 95% CI = 1.04-1.62, P = 0.022). Nevertheless, no interaction effect between
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rs874945 and smoking status was detected (P = 0.382).
ACCEPTED MANUSCRIPT 4. Discussion Recently, lncRNAs have received widespread attention for their vital function in various diseases, especially cancers. Evidence has shown that genetic variants in lncRNAs may alter individual susceptibility to cancer by modulating the expression
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and function of lncRNAs (Liu et al., 2012; Wang et al., 2013; Wu et al., 2013).
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HOTAIR is pervasively overexpressed in many cancers and is involved in the process
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of tumor progression. However, little attention has been given to the effects of HOTAIR SNPs in human cancer. Recently, the relationship of genetic variants in
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HOTAIR and cancer was first reported in colorectal cancer (Xue et al., 2015).
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HOTAIR is a spliced, polyadenylated transcript of approximately 2.2k nucleotides that is transcribed from the HOXC cluster in an antisense manner. It has been widely
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treated as a functional lncRNA participating in multiple cancers. Evidence has shown
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that HOTAIR could function as a molecular scaffold to link polycomb repressive complex 2 (PRC2) and lysine specific demethylase 1 (LSD1) protein complexes and regulate gene expression by modulating chromatin structure in trans across the 40-kb
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HOXD locus2 (Gupta et al., 2010; Chu et al., 2011), which causes epigenetic silencing of various cancer-related genes, especially the HOXD gene, by H3K27 methylation and H3K4 demethylation (Tsai et al., 2010). These epigenetic changes result in upregulation or downregulation of the expression of genes affecting the proliferation, invasion, apoptosis and migration of cancer cells. Such processes have been shown in breast cancer (Gupta et al., 2010), colorectal cancer (Kogo et al., 2011), pancreatic cancer (Kim et al., 2013), and gastric cancer (Xu et al., 2013).
ACCEPTED MANUSCRIPT A meta-analysis revealed that there was no significant association between SNPs in HOTAIR and cancer risk (Tian et al., 2016). However, other studies have found different results. Additional larger studies incorporating subjects of different ethnic backgrounds combined with re-sequencing of the marked region and functional
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evaluations are needed. It has been reported that genetic variants in lncRNAs
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modulate individual susceptibility to cancer (Liu et al., 2012; Li et al., 2013) and that
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tagSNPs in the HOTAIR gene region are associated with the risk of colorectal cancer. We speculated that genetic variants in HOTAIR might also participate in BCa. The
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association between tagSNPs in the HOTAIR gene region and the susceptibility to
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BCa was assessed in a case-control study. The results showed a remarkable association between an increased risk of BCa and the rs874945 genetic variant.
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Subsequently, we performed stratified analyses of rs874945 according to epidemic
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variables. We found that rs874945 increased BCa risk in a dominant model and was more pronounced in subgroups of older subjects, males, never smokers and subjects without pack-years of smoking. These results indicate that the effects of the HOTAIR
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variant on BCa risk might be modulated by specific demographic factors and environmental exposures. A possible explanation is that some environmental hazards such as tobacco smoking promote BCa risk, which enhances the effects of rs874945 variants. This also provides evidence that carcinogenesis is a complex progress involving both genetic and environmental factors. There are several strengths of this study. First, although the role of HOTAIR in carcinogenesis has been reported in a variety of cancers, this is the first study aimed at
ACCEPTED MANUSCRIPT investigating genetic variants of HOTAIR in the susceptibility to BCa. Our observations contribute to further understanding of the susceptibility to BCa. The relatively large sample size (1,050 BCa cases and 1,403 controls) is another strength, affording sufficient statistical power to our study. However, there were also some
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limitations of our study. First, this hospital-based case-control study was a one-stage
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design, and selection bias was unavoidable. Our study also lacks functional evidence,
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and functional studies are needed in the future.
In conclusion, we are the first to provide evidence that HOTAIR rs874945 may
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contribute to the risk of BCa in a Chinese population. Further well-designed studies in
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multicenter, large, independent populations and functional studies will be performed to validate our findings in the future.
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Author Contributions
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Conceived and designed the experiments: ZZ WG. Performed the experiments: QZ XW. Analyzed the data: MD YG HC. Contributed reagents/materials/analysis tools: WW LY MW DG KZ BX XL. Wrote the paper: XW. Revised the manuscript critically:
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ZZ WG.
Conflicts of interest
The authors declare no conflicts of interest. Acknowledgments This study was partly supported by National Natural Science Foundation of China (81473050), Jiangsu Provincial “333” project (BRA2012033), National Health and Family Planning Commission Project (W2015PM113), Collaborative research project of Southeast University-Nanjing Medical University (2017DN39), Beijing Municipal Administration of Hospitals’ Youth Programme (QML20150101), Natural Science Foundation of Jiangsu Province (BK20151603) and Nanjing Science and Technology
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Program (201605009), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Public Health and Preventive Medicine).
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ACCEPTED MANUSCRIPT Table 1. Frequency distributions of selected variables between the bladder cancer cases and controls Variables
Cases (n = 1,050)
Controls (n = 1,403)
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%
%
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Age (mean ± SD) 64.8 ± 12.6 65.2 ± 9.3 Sex Male 839 79.9 1107 78.9 Female 211 20.1 296 21.1 Smoking status Never 553 52.7 866 61.7 Ever 497 47.3 537 38.3 Former 225 21.4 179 12.8 Current 272 25.9 358 25.5 Pack-years of smoking 0 553 52.7 866 61.7 ≤ 20 191 18.2 259 18.5 > 20 306 29.1 278 19.8 b Stage Non-muscle invasive (Ta-T1) 676 64.4 Invasive (T2-T4) 359 34.2 Missing 15 1.4 a 2 Two-sided χ test for the frequency distributions of selected variables between the cases and controls. b stage in accordance with the 2004 WHO/ISUP classification of bladder cancer.
Pa 0.320 0.544
< 0.001
< 0.001
ACCEPTED MANUSCRIPT Table 2. Associations of the selected SNPs with bladder cancer risk Variations
Genotypes
N(cases/controls)
P (HWE)
Crude OR (95% CI)
Adjusted OR (95% CI) a
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GG 649/931 AG 370/416 0.764 1.14(0.99-1.32) 1.14(0.99-1.32) AA 29/49 codominant GG 649/931 1.00 1.00 AG 370/416 1.28(1.07-1.52) 1.28(1.08-1.52) AA 29/49 0.85(0.53-1.36) 0.85(0.53-1.36) dominant GG 649/931 1.00 1.00 AG/AA 399/465 1.23(1.04-1.46) 1.23(1.04-1.46) recessive GG/AG 1019/1347 1.00 1.00 AA 29/49 0.78(0.49-1.25) 0.78(0.49-1.25) rs4759314 additive AA 897/1217 AG 129/143 2.26E-23 1.05(0.87-1.27) 1.06(0.87-1.28) GG 19/34 codominant AA 897/1217 1.00 1.00 AG 129/143 1.22(0.95-1.58) 1.23(0.95-1.58) GG 19/34 0.76(0.43-1.34) 0.77(0.44-1.37) dominant AA 897/1217 1.00 1.00 AG/GG 148/177 1.13(0.90-1.44) 1.14(0.90-1.44) recessive AA/AG 1026/1360 1.00 1.00 GG 19/34 0.74(0.42-1.31) 0.76(0.43-1.34) rs7958904 additive AA 551/764 AG 400/531 0.718 1.10(0.97-1.25) 1.10(0.97-1.25) GG 91/97 codominant AA 551/764 1.00 1.00 AG 400/531 1.04(0.88-1.24) 1.05(0.88-1.24) GG 91/97 1.30(0.96-1.77) 1.30(0.96-1.77) dominant AA 551/764 1.00 1.00 AG/GG 491/628 1.08(0.92-1.27) 1.09(0.92-1.28) recessive AA/AG 951/1295 1.00 1.00 GG 91/97 1.28(0.95-1.72) 1.28(0.95-1.72) a Adjusted by age and sex in logistic regression analysis. b 2 Two-sided χ test for the frequency distributions of selected variables between the cases and controls.
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rs874945
Genetic models additive
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0.005 0.499 0.014 0.306 0.579
0.115 0.378 0.274 0.335 0.145
0.612 0.092 0.322 0.107
ACCEPTED MANUSCRIPT Table 3. Stratification analyses between rs874945 and bladder cancer risk Variations
N(cases/controls)
Crude OR(95% CI)
Adjusted OR(95% CI)
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Age ≤ 60 358/381 1.02(0.75-1.39) 1.01(0.75-1.38) > 60 690/1015 1.35(1.11-1.65) 1.35(1.10-1.65) Sex Male 837/1100 1.22(1.01-1.47) 1.22(1.01-1.47) Female 211/296 1.29(0.89-1.87) 1.29(0.89-1.87) Smoking status Never 553/864 1.30(1.04-1.62) 1.30(1.04-1.62） Ever Former 224/175 1.19(0.79-1.78) 1.22(0.81-1.85) Current 271/357 1.07(0.77-1.49) 1.07(0.77-1.49) P c for interaction (multiplicative) Pack-years of smoking 0 553/864 1.30(1.04-1.62) 1.30(1.04-1.62) ≤ 20 190/256 1.36(0.92-2.01) 1.39(0.94-2.07) > 20 305/276 0.99(0.71-1.39) 0.99(0.71-1.39) a Adjusted by age and sex in logistic regression analysis. b Two-sided χ2 test for the frequency distributions of selected variables between the cases and controls. c The multiplicative interaction test between smoking status (never or ever) and genotypes (AA/AG or GG). Adjusted by age and sex in logistic regression model.
P
b
0.929 0.004 0.039 0.185 0.022 0.345 0.692 0.382 0.022 0.102 0.964
ACCEPTED MANUSCRIPT Abbreviations: HOTAIR, The HOX transcript antisense intergenic RNA; lncRNA, long noncoding RNA; TagSNP, tagging single nucleotide polymorphisms; BCa, bladder cancer; OR, odds ratio;
AC C
EP T
ED
MA
NU
SC
RI
PT
CI, confidence interval; MAF, minor allele frequency; HWE, Hardy-Weinberg equilibrium