Controversial data on the association of single-nucleotide polymorphisms (SNPs, rs3787016G>A and rs10773338G>A) in long non-coding RNA (lncRNA) with prostate cancer risk were emerged. Considering possible genetic differences among populations, we conducted the present study to clarify these discrepancies and re-validate these results in an eastern Chinese population and thus provide clues for new therapeutic targets of prostate cancer.
Genotypes of these two SNPs from 1015 ethnic Han Chinese patients with prostate cancer and 1032 cancer-free controls were determined by Taqman assays. Logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for risk associations.
The association of rs3787016 A variant genotypes with a significantly higher prostate cancer risk were found (adjusted OR=1.418, 95% CI=1.090–1.844 for AA vs GG). Stratification analysis indicated that the risk of rs3787016 variant AG/AA genotypes was more evident in younger subjects, ever smoking, patients with Gleason score ⩾7(4+3) and highly aggressive status. All these risks were not present for rs10773338G>A.
These findings suggested that lncRNA SNPs may contribute to prostate cancer risk in an eastern Chinese population. Larger and well-designed studies with different ethnic populations are warranted to validate our findings.
Prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer-related deaths in the western male population.1 In China, the detection rate of prostate cancer is increasing rapidly due to the extension of life expectancy, the change of lifestyles and the improvement of health-care system.2,3 To date, the etiology of prostate cancer remains unclear, although classic risk factors include older age, African–American racial group and family history.4, 5, 6, 7 Extensive basic studies have found that genetic variants, particularly single-nucleotide polymorphisms (SNPs), may also influence susceptibility to prostate cancer.8
Long non-coding RNA (lncRNA) can regulate the expression of genes in close genomic proximity and target distant transcriptional activators or repressors by a variety of mechanisms, such as transcriptional interference, initiation of chromatin remodeling, promoter inactivation by binding to basal transcriptional factors and activation of an accessory protein.9, 10, 11 Therefore, variations in lncRNAs are likely to modify functions of various biological pathways involved in prostate carcinogenesis.
Recently, genome-wide association studies have found a large number of prostate cancer risk loci.12, 13, 14 Furthermore, both in the meta-analysis of two existing prostate cancer genome-wide association studies and then in an additional replication study, rs3787016 and rs10773338 remained significantly association with prostate cancer risk.15 The SNP rs10773338 is located in region of chromosome 12, as well as rs3787016 in a lncRNA spanning 364 kb at 19p13, which two previously published genome-wide association studies have identified as a prostate cancer susceptibility region among Swedish men with hereditary prostate cancer,16 and in young members of families with multiple case of prostate cancer.17 However, the results of association studies may vary among populations owing to inter-population genetic differences, including differences in allele frequencies and linkage disequilibrium structures.18 Therefore, it is more reasonable to evaluate the relationship between SNPs and prostate cancer risk in different cohorts other than the one in which this association was identified.
To further assess the reported associations of rs3787016 and rs10773338 with prostate cancer risk, we conducted a case−control study in an eastern Chinese man. Meanwhile, the associations of these two SNPs with clinical characteristics (for example, age, body mass index (BMI), smoking status, Gleason score, stage of disease and aggressive feature) were also evaluated.
Materials and methods
The study cohort consisted of 1015 ethnic Han Chinese patients with newly diagnosed and histopathologically confirmed prostate cancer from Fudan University Shanghai Cancer Center (FUSCC) between 2009 and 2012. All patients were from eastern China, including Shanghai, Zhejiang, Jiangsu and the surrounding regions, and samples from prostate cancer patients were provided by the tissue bank of FUSCC. An additional 1032 cancer-free controls of ethnic Han Chinese in eastern China were recruited from the Taizhou Longitudinal (TZL) study at the same period, with the selection criteria including no individual history of cancer.19 Controls were frequency matched to the cases on age and sex. Data on demographic characteristics and environmental exposure history of each participant, such as age, sex, ethnicity, BMI, smoking status, were collected. Each participant donated approximately 10 ml of blood, of which 1 ml was used for genomic DNA extraction. Written informed consents from all study participants had been obtained. This study protocol was approved by the institutional review board of FUSCC.
The selected rs3787016G>A and rs10773338G>A of lncRNA were genotyped using genomic DNA, which was isolated from blood samples by utilizing the QIAamp DNA blood maxi kit (Qiagen, Valencia, CA, USA). Genotyping for SNPs rs3787016 and rs10773338 was performed using the Taqman assays (Applied Biosystems, Foster City, CA, USA) with a 7900 HT sequence detector system (Applied Biosystems). For the quality control, four negative controls (without DNA template) and two duplicated samples were included in each 384-plate as recommended by the company. The assays were repeated for 5% of the samples, and the results were 100% concordant.
In this study, we evaluated the differences in the frequencies of genotypes as well as demographic and other covariates between the cases and controls by using X2-test. The Hardy–Weinberg equilibrium of genotype distributions in the controls was estimated by the good-of-fit X2-test. Crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate associations between the genotypes and risk of prostate cancer with and without adjustment for and stratified by age, BMI, smoking status, Gleason score, stage of disease and aggressive status employing univariate and multivariate logistic regression models, respectively. The homogeneity tests were performed to detect the difference in risk estimates among subgroups. Because the selected SNPs appear to be in the same block, the haplotype analysis was not performed. Haplotype frequencies and individual haplotypes were estimated and analyzed using Statistical Analysis Software PROC HAPLOTYPE.
All statistical analyses were conducted with SAS software (version 9.1; SAS Institute, Cary, NC, USA), and all tests were two-sided, and P-values <0.05 were considered statistically significant.
Characteristics of the study cohorts
The current study included 1015 cases of prostate cancer and 1032 controls of cancer-free population, whose characteristics are presented in Table 1. There were no significantly statistical differences in the distributions of age and smoking status between cases (mean age of 69.13±8.15, and 60% of ever smoking) and controls (mean age of 68.75±8.97, and 60.66% of ever smoking). However, a higher proportion of BMI ⩾25 in controls than in cases was presented (39.15% vs 24.9%, P<0.0001). These variables were further adjusted for in the subsequent multivariate logistic regression analyses. Additionally, PSA (1.6% of PSA⩽4, 16.2% of 4–10, 19.2% of 10–20, 54.4% of >20 and 8.7% of missing), Gleason score (31.2% of Gleason score ⩽7(3+4), 59.7% of ⩾7(4+3) and 9.1% of missing) and TNM stage (0.5% of TNM stage I, 42.8% of II, 14.0% of III, 35.1% of IV and 7.7% of missing) of the cases were listed.
Associations of selected SNPs with prostate cancer risk
The allele and genotype distributions of the two selected SNPs in cases and controls are summarized in Table 2. The observed genotype frequencies among the controls were consistent with the Hardy–Weinberg equilibrium (P=0.064 for rs3787016 and P=0.579 for rs10773338). The genotype distribution between the cases and controls was significantly different for rs3787016 (P=0.0285), but not for rs10773338 (P=0.7325). The rs3787016 A allele was significantly more frequent in cases than in controls (P=0.0121), whereas the rs10773338 A allele was less frequent in cases than in controls with no significantly statistical different (P=0.4428). When age, smoking and BMI were adjusted in multivariate logistic regression analysis and the rs3787016 GG genotype was used as reference, genotype AA was associated with a significantly increased risk of prostate cancer (adjusted OR=1.418, 95% CI=1.090–1.844, P=0.0093), whereas genotypes AG and AG/AA were borderline significantly associated with a higher risk of prostate cancer (adjusted OR=1.125, 95% CI=0.925–1.367, P=0.2377 and adjusted OR=1.191, 95% CI=0.989–1.434, P=0.0653, respectively). In the recessive model, the increased risk of prostate cancer associated with rs3787016 homozygous AA genotypes was still significant (adjusted OR=1.320, 95% CI=1.044–1.669, P=0.0205), compared with the genotypes (GG/AG). Nevertheless, no significant associations with prostate cancer risk were found for variant genotypes of rs10773338 (Table 2).
As shown in Table 3, the associations of the two selected SNPs with prostate cancer risk were further evaluated by stratified analysis using age, BMI, smoking, Gleason score, TNM stage and aggressive status. The increased prostate cancer risk associated with rs3787016 variant AG/AA genotypes was more evident in younger subjects (adjusted OR=1.366, 95% CI=1.055–1.767), ever smoking (adjusted OR=1.285, 95% CI=1.011–1.633), Gleason score ⩾7(4+3) (adjusted OR=1.339, 95% CI=1.076–1.667) and highly aggressive status (adjusted OR=1.244, 95% CI=1.019–1.520), comparing with the GG genotype. These results indicated potential interactions among age, smoking, Gleason score and aggressive status in the etiology of prostate cancer. However, further heterogeneity tests suggested nonsignificant differences in risk estimates among these strata, except for Gleason score. Additionally, nonsignificant relationships were found between the genotype of rs10773338 and prostate cancer risk in the stratification analysis. And there was no statistical evidence for interactions between these variables and the variant genotypes on the risk of prostate cancer (data not shown).
The current study, which mainly evaluated the relationships between two selected SNPs of lncRNA and the risk of prostate cancer, is, to the best of our knowledge, the first research in an eastern Chinese population.
LncRNA has increasingly attracted the attention of the scientific community based on the discovery that a major of genome is transcribed but not translated, leading to the formation of non-protein-coding transcripts (for example, short non-coding RNA and lncRNA), which were involved in a variety of critical biological processes. Briefly, lncRNA could be very different in size (ranging from 340 nucleotides of 7SK to 118 kb of Airn), could be transcribed by RNA polymerase II or III, could be either spliced or not and could be localized either in the nucleus or in the cytoplasm.20 Based on these features, lncRNAs could be further categorized into long intergenic non-coding RNA, long intronic non-coding RNA, natural antisense transcript, promoter-associated long RNA, promoter upstream transcript, repetitive element-associated non-coding RNA, transcribed pseudogene, transcribed ultraconserved region, enhancer-like non-coding RNA.21 Furthermore, increasing evidence have indicated that lncRNAs play a significant role in transcription, splicing, translation, protein localization, cellular structure integrity, imprinting, cell cycle and apoptosis, stem cell pluripotency and reprogramming and heat-shock response.20 Meanwhile, it has been indicated that lncRNAs may regulate cancer progression and development of many other human diseases.22,23 All of these observations have shown clearly that variation in lncRNA regions may contribute to the etiology of disease.
In the current study, we found that the lncRNA rs3787016 AA genotype was associated with an elevated prostate cancer risk. However, the marginally significant relationship between genotype AG and AG/AA and the higher risk of prostate cancer was noted. The results from our study supported some studies12,13,15 and simultaneously opposed other studies.24 The inconsistent results among these studies might be explained by differences in sample size and ancestral backgrounds.25,26 In the study with contrary results,24 106 controls, 257 patients with BPH and 261 patients with prostate cancer from Serbian population were included, which were less than the number of subjects in other researches. On this occasion, our study with the larger number of subjects was timely conducted to further study the association of previously reported risk loci with prostate cancer risk.
In the stratified analysis, risk effect of the rs3787016 AG/AA genotype was more evident in younger subjects, ever-smokers, and patients with Gleason score ⩾7(4+3) and highly aggressive status. Such findings might be interpreted as follow. Cigarette smoking-related carcinogens might induce a variety of DNA damage which might lead to mutations and thus initiate carcinogenesis,27 such as prostate carcinogenesis.28, 29, 30, 31, 32 Therefore, the effect of genetic variants on the risk of cancer might be augmented by smoking-related damage. In addition, the fact is that younger patients with prostate cancer were prone to have higher Gleason score and highly aggressive status. These results suggested potential interactions among age, smoking, Gleason score and aggressive status in the etiology of prostate cancer. However, non-significant relationships were found between the genotype of rs10773338 and prostate cancer risk. One of the reasons might be that the number of subjects in our study was still not large enough to provide statistical power to detect any association. Owing to cancer is a complex and multifactorial disease, a single genetic variant is insufficient to predict the overall risk. Therefore, future studies should have larger number of subjects and include more SNPs in lncRNAs or in other related gene which may be involved in the etiology of prostate cancer.
Several limitations of our study need to be addressed. First, it was a hospital-based case−control study with patients from FUSCC and controls from TZL study, and thus bias from selection of the non-representative population would not be completely eliminated. Fortunately, potential confounding bias might be minimized by frequency-matching cases and controls on age, areas of residence and further adjustment for possible confounding factors in final analysis. Second, reliable and sufficient information about exposure data were not available due to the nature of retrospective study design. Third, although our study was relatively large, the small sample size in stratified analysis may have limited statistical power to detect significant associations for each of the strata and assess gene−gene or gene−environment interactions adequately. Finally, only two SNPs of lncRNA were genotyped in this study, which did not cover all variants of lncRNA and might miss some important genetic variations within the gene. Larger, well-designed and prospective population-based studies in the future would conquer these limitations and confirm our findings.
In conclusion, our study provided statistical evidence that rs3787016G>A, but not rs10773338G>A, was associated with prostate cancer risk in an eastern Chinese population, particularly for younger man, ever smoker, patients with Gleason score ⩾7(4+3) and highly aggressive status. Before the findings will contribute to clinical decision-making, larger and more in-depth molecular studies for validating the role of rs3787016G>A in the etiology of prostate cancer are warranted.
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Financial support for our study from the National Natural Science Foundation of China (Grant number: 81302213), 985-III Cancer Research Project at Fudan University (Grant number: 985III-YFX0102) and Hospital-level fund at Fudan University Shanghai Cancer Center (Grant number: YJ201206) is gratefully acknowledged.
The authors declare no conflict of interest.
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