The results presented suggest a risk effect for age, male gender, and cigarette-smoking in etiology of UBC. With respect to age, UBC is regarded as a disease that mostly afflicts middle-aged or elderly people. The recorded age range at diagnosis of UBC is 60–69 years [13]. The present UBC age median was within this range, and the sixth decade of age is considered as a crucial risk factor for UBC. Male gender is a further risk factor, and most studies agree that UBC occurs more frequently in men than in women [5]. Two important explanations may justify the male-gender dominancy in UBC; males are at a greater chance to be exposed to environmental risk factors, and a potential for sex steroid hormone regulation has also been suggested to play an active role in UBC development and progression [14]. Cigarette-smoking is also a further well-established risk factor for UBC worldwide [15]. Interestingly, smoker-men were reported to have UBC more frequently than smoker-women, and accordingly, cigarette-smoking and male gender have been regarded as co-risk factors for UBC [16]. Emerging data suggest that nicotine exposure may enhance tumor growth and metastasis, and there is a growing body of evidence depicting that nicotine promotes cell proliferation, angiogenesis, and epithelial-to-mesenchymal transition through nicotinic acetylcholine receptors found in the urinary bladder, leading to enhanced tumor growth and metastasis [17].
Assessment of TLR7 serum level in UBC patients revealed that such serum marker was downregulated. Accordingly, the risk of UBC is suggested to be associated with a decreased serum level of TLR7, and its downregulation may serve as important biomarker for the progression of UBC. However, the potential-risk of TLR7 in UBC has not been well-investigated. So far, the expression of TLR7 has been determined in six human cancers, liver, cervix, pancreas, and lung cancers, as well as chronic lymphocytic leukemia and multiple myeloma. In agreement with the present study, downregulation of TLR7 expression has been demonstrated in these malignancies. In one study, the expression of TLR7 in cancerous and non-cancerous liver tissue from 87 patients with hepatocellular carcinoma was investigated, and the results demonstrated that TLR7 is significantly downregulated in neoplastic hepatocytes, especially in patients with hepatitis B virus infection [18]. TLR7 was also investigated in a human cell line of pancreatic adenocarcinoma (BxPC-3 cells). The cells were treated with TLR7 agonist (gardiquimod), and then proliferation, migration, cell cycle, and apoptosis of these cells were analyzed. It was demonstrated that TLR7 activation inhibited proliferation and migration, and induced apoptosis in pancreatic cancer cells [19]. Accordingly, agonists to TLR7 have been developed as a strategy for anti-tumor therapy. Among these are imidazoquinolines, which are suggested to have anti-tumor effect in urothelial cell carcinoma of the bladder. These agonists have a potent direct activity against UBC cells by decreasing cell viability and inducing cytokine production and apoptosis [8]. Further investigators formulated an immune-stimulating TLR7 agonist (TMX-202) in the liposomes and examined its immune activating potential in immune cells that included blood-derived monocytes, myeloid dendritic cells (DCs), and plasmacytoid DCs. These cells exhibited potent TLR7-specific secretion of some cytokines that have anti-cancer effects (IL-12p70, IFN-α, and IFN-γ) [20]. Based on these findings, two formulations of TLR7 agonists (TMX-101 and TMX-202) were investigated to determine their therapy potential for urothelial carcinoma in an orthotopic bladder cancer rat model. The results showed a lower number of tumor-positive rats after therapy with TLR7 agonists [21]. In line with these findings, MMC and BCG therapies were associated with an increased serum level of TLR7 compared to untreated patients. Although the differences were not significant, it is possible to suggest that both therapies enhanced production of TLR7. There is no direct evidence to support such findings, but it has been demonstrated that under treatment with MMC, urinary bladder cells showed increasing apoptosis through upregulation of TLR6 and connective tissue growth factor under hydrostatic pressure stimulation [22]. In a further study, it has been suggested that TLR8 expression can be upregulated in macrophages after exposure to BCG [23].
For a further understanding of TLR7 in etiology and pathogenesis of UBC, four gene SNPs were investigated. Disease-association studies suggested that SNPs of TLR genes are associated with an increased risk to develop a wide range of different malignancies [24]. Among these are TLR7 SNPs, and studies have suspected their potential in progression of different malignancies, and moreover, these SNPs are suggested to predict the response outcome to anti-cancer therapies [25]. In line with these suggestions, the present study examined an intron region of TLR7 gene in UBC and UTI patients to seek for novel SNPs that may have a role in predisposition to develop UBC and/or UTI in samples of Iraqi patients. DNA-sequence analysis revealed the existence of four informative SNPs that have minor allele frequencies greater than 10%, rs179018, rs179019, rs179020, and rs179021. Searching the PubMed for these SNPs came-up with five publications for rs179019 and one publication for rs179020, while rs179018 and rs179021 SNPs have not been investigated in any human disease.
For rs179019 SNP, the results suggest a susceptibility role for C allele in UBC male patients (OR = 3.23), while A allele may have a protective effect. No available evidence confirms or refutes these findings, but five previous studies evaluated the risk effect of rs179019 SNP in hepatitis C virus [26] and enterovirus 71 [27] infections and two autoimmune diseases, systemic lupus erythematosus (SLE) [28, 29] and Graves’ disease [30]. No clear effect was presented regarding viral infections or Graves’ disease, while contradicting results were found in SLE. However, among UTI patients, rs179019 SNP was associated with susceptibility for UTI but through A allele. Therefore, it is possible to suggest that C and A alleles of rs179019 SNP may have different predisposing effects in UBC and UTI.
For rs179020, it may also be a potential-risk SNP for UBC, and male patients having G allele were at a greater risk to develop UBC but the difference was not significant. This SNP appeared in one study, which investigated its association with vitiligo (a chronic disease characterized by distinctive lightening of the skin), and a strong association with the entire disease was observed; moreover, male, sporadic, and late onset vitiligo maintained a corrected significant association [31].
The presented findings highlighted the importance of an intron region in TLR7 gene in predisposition and risk of UBC, and the SNP effect in this context cannot be underestimated but the available evidence is limited. In one published study, the relation between a promoter TRL7 gene SNP (rs179008; not investigated in present study) and response to imiquimod (TLR7 agonist) was explored in cutaneous basal cell carcinoma patients. The results demonstrated that patients carrying at least one SNP allele (T allele) are at an increased risk to be resistant to such therapy [32]. Therefore, TLR7 gene may harbor important functional SNPs that not just influence susceptibility to malignancies, but may also influence response to therapies. The results of present study agree with such conclusion, although some of the observed findings were statistically not significant. In terms of statistics, the sample size influences the level of significance effectively and represents an important limitation of present study.
