bladder cancer is the second most common genitourinary malignancy and the sixth most common cancer in the world . in china ,
the incidence of bladder cancer also increases with age , and the peak is reached approximately at the age of 60 years ; bladder cancer is three times more common in men than in women . among the newly diagnosed cases of transitional cell carcinomas , approximately 75% to 80% of these cases present superficial tumours ; 50% to 70% of these superficial tumours relapse within five years ; and roughly 10% to 20% progress to a more aggressive disease .
bladder cancer is a multifactorial disease mediated by genetic abnormalities , environmental factors , and chronic irritation .
although many individuals are exposed to these risk factors , only a fraction of exposed individuals develop bladder cancer in their lifetime , suggesting that genetic variations may participate in bladder carcinogenesis .
thrombospondin-1 ( tsp-1 ) is an adhesive glycoprotein with a size of 450  kd initially discovered in platelets , where tsp-1 is sequestered in a platelet -granule .
tsp-1 has been implicated in regulating numerous biological activities , including cell adhesion , cell migration , proliferation , angiogenesis , inflammation , and wound healing [ 712 ] .
tsp-1 also elicits different effects on tumour growth and progression depending on the tumour type , leading to cancer progression and inhibition in different instances [ 1118 ] . in bladder cancer , low tsp-1 expression is significantly associated with an increased risk of disease recurrence and decreased overall survival [ 17 , 18 ] .
studies have been conducted to assess the association between polymorphisms in candidate genes and bladder cancer risk . to date , genetic polymorphisms in tsp-1 gene are possibly associated with coronary artery disease and myocardial infarction [ 20 , 21 ] .
the transcriptional initiation site of the tsp-1 gene was identified by laherty et al . .
tsp-1 - 1223 a / g polymorphism is located in the 5 near - gene region of the tsp-1 gene , which may influence the transcriptional activity of tsp-1 .
furthermore , tsp-1 - 1223 a / g polymorphism may participate in the aetiology and development of bladder cancer . in the present study , tsp-1 - 1223 a / g polymorphism ( rs2169830 )
, we genotyped the polymorphism and determined its association with the risk of bladder cancer in our ongoing , hospital - based , case - control study in a chinese population .
the study was approved by the institutional review board of the first affiliated hospital of nanjing medical university , nanjing , china . during recruitment ,
the present study included 609 patients with bladder cancer and 670 age - matched control subjects from han population living in jiangsu and anhui provinces in eastern china .
the patients with bladder cancer were recruited from july 2006 to july 2012 in the department of urology , the first affiliated hospital of nanjing medical university .
the patients were excluded in this study according to the following : previous cancer , metastasised cancer from other or unknown origins , and previously subjected to radiotherapy or chemotherapy .
cancer - free control individuals were frequency matched with the cancer patients in terms of age ( 5 years ) and gender .
all of the control subjects were recruited from healthy individuals who were scheduled for a physical examination in the outpatient department at the same hospital .
the control subjects were excluded if they manifested symptoms of bladder cancer , such as haematuria .
prior to recruitment , all of the subjects were personally interviewed to collect demographic data and clinical characteristics , including age , gender , race , tobacco use , alcohol use , and self - reported family history of cancer . according to the tumour , node , and metastasis classification of cancer stages ( 2002 international union against cancer ) ,
the clinical stage at the time of diagnosis was classified into two subgroups : nonmuscle invasive group ( pta - pt1 ) and muscle invasive group ( pt2-pt4 ) . according to histopathological grade ( who 1973 , grading of urothelial papilloma ) , the patients were classified into three subgroups : grades 1 , 2 , and 3 .
individuals who smoked daily for > 1 year were defined as smokers and the rest were considered as nonsmokers .
individuals who drank alcohol at least three times per week for more than 6 months were defined as drinkers and the rest were considered as nondrinkers . for survival analysis ,
260 patients were followed up . among these follow - up cases , 24 ( 24/260 , 9.2% )
survival time was calculated from the date of confirmed diagnosis until the date of the last follow - up or recurrence .
the date of recurrence was obtained from inpatient and outpatient records or from patients ' families via follow - up telephone calls .
the patients who did not suffer from recurrence on the last follow - up date were considered as non - recurrent .
genomic dna of each individual was extracted from 150  l of edta - anticoagulated peripheral blood samples by using a dna extraction kit ( tiangen biotech , beijing , china ) according to the manufacturer 's instructions .
the tsp-1 - 1223 a / g polymorphism was genotyped using taqman single nucleotide polymorphism ( snp ) genotyping assay ( applied biosystems , foster city , ca , usa ) .
the primers , probes , and reaction conditions of each snp are available upon request . for quality control ,
four negative controls were included in each plate , and 5% of the samples were randomly selected for repeated genotyping to verify the results ; all of the results were 100% consistent .
total rna from 66 bladder cancer tissues was extracted using trizol reagent ( invitrogen , carlsbad , ca ) according to the manufacturer 's protocol .
total rna ( 1  g ) was used for cdna synthesis with oligo - dt primers ( invitrogen , karlsruhe , germany ) and superscript ii reverse transcriptase ( takara bio , shiga , japan ) .
pcr was performed using pcr master ( roche , mannheim , germany ) with the following primers : for tsp-1 mrna , 5-actgtccattccattacaacccagc-3 ( forward ) and 5-tgtcacactgatctccaaccccatcca-3 ( reverse ) ; and for -actin , 5-actggaacggtgaaggtgac-3 ( forward ) and 5-agagaagtggggtggctttt-3 ( reverse ) .
fold changes were normalised based on -actin expression , and each assay was conducted in a 384-well abi 7900ht real - time pcr system ( applied biosystems , foster city , ca , usa ) .
the frequency distributions of the selected demographic variables as well as each allele and genotype of the tsp-1 - 1223 a / g polymorphism between the cases and the control subjects were evaluated using -type distribution .
hardy - weinberg equilibrium ( hwe ) was determined using a goodness - of - fit  test .
unconditional univariate and multivariate logistic regression analyses were conducted to calculate the crude and adjusted odds ratios ( ors ) and 95% confidence intervals ( cis ) to determine the risk of bladder cancer .
interaction was investigated using a multiplicative interaction term included in the multivariate model . to investigate potential interactions between the polymorphism and tobacco smoking , we assessed a multiplicative gene - environment interaction by logistic regression analysis , including the main effect variables and their product terms . hazard ratios ( hrs ) and 95% cis of the hrs were derived from univariate and multivariate cox proportional hazard models .
survival curves were generated using the kaplan - meier method and compared using the log - rank - mantel - cox test .
the frequency distributions of the selected characteristics of the cases and the control subjects are shown in table 1 .
the cases and the control subjects were adequately matched in terms of age and gender ( p = 0.486 for age and p = 0.091 for gender ) .
a higher number of smokers were found among the cases than the control subjects ( 47.9% versus 36.4% ; p < 0.001 ) .
no significant difference in drinking status was found between the cases and the control subjects ( p = 0.196 ) .
in addition , the frequency of the first - degree relatives with cancer was higher in the cases than in the control subjects ( 27.8% versus 7.2% ; p < 0.001 ) .
these variables were further adjusted in the multivariate logistic regression analysis to assess the main effect of the tsp-1 - 1223 a / g polymorphism on bladder cancer risk .
the genotype and allele frequency distributions of the tsp-1 - 1223 a / g polymorphism among the cases and the control subjects as well as their associations with the risk of bladder cancer are presented in table 2 .
the genotype frequencies in the control subjects are consistent with those of hwe ( p = 0.851 ) .
the frequencies of aa , ag , and gg genotypes were 47.1% , 40.2% , and 12.6% among the cases and 43.9% , 44.5% , and 11.6% among the control subjects , respectively ( p = 0.307 ) .
no correlation was observed between tsp-1 - 1223 a / g polymorphism and the risk of bladder cancer .
we further assessed the effect of tsp-1 - 1223 a / g polymorphism on the risk of bladder cancer risk stratified by age , gender , smoking status , drinking status , self - reported family history of cancer , and tumour grade and tumour stage ( tables 3 , 4 , and 5 ) .
logistic regression analysis also showed no association between tsp-1 - 1223 a / g polymorphism and bladder cancer risk .
a significantly increased risk was observed in smokers with aa / ag genotypes compared with nonsmokers exhibiting aa / ag genotypes ( p = 0.001 ; ors = 1.58 ; 95% ci = 1.20 to 2.10 ; table 6 ) .
we then evaluated whether or not an interaction between smoking status and tsp-1 - 1223 a / g polymorphism status occurs .
we did not observe a multiplicative interaction effect between polymorphism and smoking status ( p = 0.823 ; table 6 ) .
we further investigated whether or not tsp-1 - 1223 a / g polymorphism is associated with the recurrence of bladder cancer .
the demographic and clinical characteristics at primary diagnosis and the genotype distribution in patients with bladder cancer are shown in table 7 .
the median follow - up duration for the 236 patients was 19 months ( range , 1 month to 68 months ) .
the mean time - to - recurrence was 20.8 months ( 95% ci = 17.2 to 24.3 months ) .
kaplan - meier curves showed statistical difference between tsp-1 - 1223 a / g polymorphism and time - to - recurrence in bladder cancer ( aa 20.5 months , 95% ci = 14.7 to 26.3 ; ag 22.2 months , 95% ci 15.6 to 28.7 ; gg 18.8 months , 95% ci 12.725.0 ; log - rank test , p = 0.017 , figure 2 ) . compared with aa + ag genotypes ,
the gg genotype exhibited a significant recurrence risk of bladder cancer ( hr = 2.07 , 95% ci = 1.23 to 3.49 , p = 0.006 ) , and the recurrence risk was more prominent among patients with the gg genotype ( hr = 2.63 , 95% ci = 1.43 to 4.83 , p = 0.002 ) .
the cases exhibiting ag + gg genotypes were also associated with an increased recurrence risk compared with the aa genotype ( hr = 1.95 , 95% ci = 1.20 to 3.19 , p = 0.007 ; table 8) . to evaluate the association between tsp-1 - 1223 a / g polymorphism and recurrence of bladder cancer , we examined whether or not the tsp-1 - 1223 a / g polymorphism was associated with an altered tsp-1 mrna expression .
no significant difference was observed in the relative tsp-1 mrna expression level between patients with aa ( n = 23 ) and ag ( n = 28 ) genotypes ( p = 0.103 ) . the patients with the gg genotype exhibited lower tsp-1 mrna expression levels than those with aa genotype ( n = 15 ; p = 0.002 ) , although a significant overlap among the three groups was observed ( figure 1 ) .
in the present study , the association between -1223 a / g polymorphism in tsp-1 gene ( rs2169830 ) and risk of bladder cancer was investigated .
the results do not indicate that the genotypes of the tsp-1 - 1223 a / g polymorphism are associated with an increased bladder cancer incidence . however , the time - to - recurrence was significantly shorter in g allele carriers than that in individuals with a homozygous aa genotype .
patients with the ag / gg genotypes have 1.95 times greater risk of the recurrence of bladder cancer than those with the aa genotype ( p = 0.007 ) .
furthermore , individuals with the gg genotype exhibited a higher risk of recurrence than those with the aa genotype ( or = 2.63 , p = 0.002 ) .
to the best of our knowledge , this study is the first to investigate the function of the tsp-1 - 1223 a / g polymorphism in the aetiology of bladder cancer .
tsp-1 has been implicated in tumour growth and progression by regulating cell adhesion , motility , proliferation , and angiogenesis . as a multifunctional protein involved in tumour growth regulation , tsp-1 elicits an inhibitory effect on the growth of various malignancies such as melanoma , prostate cancer , cutaneous squamous cell carcinoma , and glioblastoma .
strong evidence has also suggested that tsp-1 may stimulate lung cancer   and breast cancer .
a previous study identified that tsp-1 , secreted by normal urothelium cells , is responsible for antiangiogenic activity .
the downregulation of tsp-1 in bladder cancer is considered as a primary factor contributing to specific changes , for instance , from an antiangiogenic to an angiogenic phenotype during cancer development and recurrence [ 17 , 18 , 33 ] .
most of the tsp-1 snps have only been observed in cardiac disease [ 20 , 21 ] ; no studies have been shown to provide a relationship between tsp-1 gene polymorphism and cancer . in our study ,
the gg / ag genotypes of the tsp-1 - 1223 a / g polymorphism in bladder cancer were associated with a shorter time - to - recurrence than the aa genotype .
individuals carrying the lower production genotype of tsp-1 - 1223 a / g polymorphism possibly possess an enhanced ability to promote the recurrence of bladder cancer .
using the program tfsearch , we found that the g allele of tsp-1 - 1223 a / g polymorphism was located in the consensus dna sequence tgtggt .
the dna sequence was a potential transcription regulation region , which can be recognised by aml1-a [ 35 , 36 ] .
however , any transcriptional factor was not bound to the sequence tgtgat containing the a allele of the tsp-1 - 1223 a / g polymorphism .
furthermore , aml1-a participates as a transcription inhibitor by suppressing the transcriptional activation of aml1-b .
thus , we cautiously speculated that aml1-a binds to the consensus sequence ( tgtggt ) of the g allele in the tsp-1 - 1223 a / g polymorphism , thereby suppressing the transcription of tsp-1 .
a previous study revealed that tsp-1 can suppress vegf mobilisation from the extracellular matrix by inhibiting the activity of mmp-9 .
the possible mechanism between angiogenesis and bladder cancer recurrence has been related to an increased vegf expression level , which serves as a major factor in angiogenesis .
the increased expression level of vegf was implicated in the pathogenesis of bladder cancer recurrence by promoting the growth and implantation of a bladder cancer cell via angiogenesis .
conversely , decreased tsp-1 may be associated with an increased risk of bladder cancer recurrence via the inhibitory effect of tsp-1 on vegf - mediated tumour angiogenesis . in the current study ,
the expression level of tsp-1 mrna in bladder cancer samples was lower in patients with heterozygous g allele than those with homozygous a allele .
the expression level of tsp-1 mrna was the lowest in individuals with homozygous g allele .
as such , the shorter time - to - recurrence in patients with bladder cancer exhibiting a g allele may be attributed to a decreased tsp-1 expression , which has been associated with an increased risk of disease recurrence and a decreased overall survival by promoting tumour neovascularization [ 17 , 18 , 34 ] .
our results partly supported our hypothesis ; however , further functional experiments should be conducted to validate the specific mechanism .
first , our sample size was relatively small , which may limit the statistical power of our study , particularly for gene - environment interaction analyses .
second , our study was a hospital - based case - control study ; hence , we can not rule out the possibility of selection bias for subjects who may have been associated with a particular genotype . in conclusion
, our study shows that g allele in tsp-1 - 1223 a / g polymorphism may modulate the risk of recurrence in bladder cancer . a lack of association between tsp-1 - 1223 a / g polymorphism and risk of bladder cancer was observed in our population .
nevertheless , a possible function of this polymorphism in other cancers or in other bladder cancer populations should be considered .
hence , additional studies should be conducted to confirm the specific function of tsp-1 - 1223 a / g polymorphism in bladder cancer development .
 
 backgrounds . tsp-1 is a glycoprotein that functions in the biology of bladder cancer . 
 we investigated the relationship between the distribution of tsp-1 - 1223 a / g polymorphism ( rs2169830 ) and the clinical characteristics of bladder cancer . 
 materials and methods . 
 taqman assay was performed to determine the genotype of 609 cases and 670 control subjects in a chinese population . 
 logistic regression was used to assess the association between the polymorphism and the risk of bladder cancer . 
 quantitative real - time polymerase chain reaction was performed to determine tsp-1 mrna expression . 
 survival curves were generated using the kaplan - meier method . 
 results . 
 no significant differences were detected in the genotype frequencies of healthy control subjects and patients with bladder cancer . by contrast , the time until the first recurrence differed significantly between genotypes ( p = 0.017 ) . 
 the expression of tsp-1 mrna in bladder cancer tissues was lower in patients with an ag genotype than in those with an aa genotype . 
 the lowest expression was observed in patients with a gg genotype . 
 conclusions . in conclusion 
 , tsp-1 - 1223 a / g polymorphism may contribute to the recurrence of bladder cancer in chinese population . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion