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Introduction

Prostate cancer is the second most common cancer in Iran, after gastric cancer [1]. In the early stages of PCa, symptoms are rarely appearing. There may be no symptoms at the time of diagnosis even in the advanced states [2]. Blood in the urine and semen [3], recurrent pain in the affected area[4], difficulty in urinating[5], sudden or frequent urination [6], unexplained weight loss [7], pain in the pelvis [8], thighs and back [9], pain or abnormal symptoms in the penis [10], poor urination, and bone pain [11] are signs of cancer progression.

While the underlying causes of prostate cancer remain unknown, the risk of developing prostate cancer is increasing [12]. Having one or more risk factors does not mean that one will definitely develop the disease. The risk of PCa in men under the age of 40 is very low [13]. But, the chance of the disease appearance increases rapidly in people over 50 years old [13], while about 6 out of 10 cases of the patients are found in over 65 years old men [14]. Blacks are more likely to develop PCa than men of other races and are more than twice as likely to die from the cancer as white men [15].

Smoking is one of the most effective risk factors for the disease [16]. This cancer seems to be more common in some families, suggesting that in some cases there may be hereditary or genetic factors [16]. A person with a first degree relative with prostate cancer is two to three times as likely to get the disease as other men [17]. People with a strong family history of breast and ovarian cancer are also more likely to develop prostate cancer than people without a family history [18] . A number of genetic changes increase the risk of prostate cancer, but in general they are likely to account for only a small percentage of cases [19]. Screening tests help in diagnosis and treatment the cancer, early or even before symptoms appear [20]. PCa screening is performed based on PSA level measurement, MRI/ ultrasound and/or CT scan, and finally the results of pathology, in Iran. Determining the level of PSA in a blood test is not a specific test for cancer [21]. Even if the serum level of prostate-specific antigen rises above normal, the risk of prostate cancer is not very high are life-threatening or never cause symptoms, but if they are detected by screening tests, the person may be treated for cancer. Complications of prostate biopsy such as false negative results delay treatment [25] and false positive cases undergo more biopsies[26]. The side effects of biopsy are many and also cause anxiety and worry [27]. Abnormal prostate changes do not always indicate prostate cancer. In some cases, an MRI before a biopsy is recommended [28]. Currently the only reliable way to diagnose this disease is a biopsy that shows a person has prostate cancer. Other measures are taken to find out if the cancerous mass has spread from its original location to other parts of the body [29]. CT scan shows the presence of cancer cells in the bone [5]. Post-diagnosis PET scan is used to identify the stage and degree of disease progression and to diagnose relapse after treatment [30]. For some men, immediate treatment is not necessary or may not be appropriate. The therapist can allow the patient to make treatment based on the stage of the cancer. Surgery, radiotherapy, chemotherapy and palliative care are other current treatments [30].

Given the heterogeneous nature of prostate tumors, researchers are trying to find more sensitive and specific tumor markers in the blood to replace solid biopsy with liquid biopsy and to create a chance to differentiate between different stages and grades of disease development by providing specific patterns of genetic changes and gene expression [31]. So far, more than 100 gene loci associated with the disease have been identified [32]. Numerous reports have shown an increased risk of prostate cancer in connection with certain single or combined genetic polymorphisms. The aim of this study was to investigate the relationship between rs1800629 and rs361525 TNF-α gene polymorphisms and gene variants of UGT2B28, UGT2B17 and UGT2B15 genes from glucuronidation pathway with increased risk of prostate cancer in Iranian population.

Materials and Methods

Sampling

In this case-control study, 120 men with healthy prostate (control), 120 patients with BPH, and 120 patients with prostate cancer (PCa) were selected from the individuals referred to the urology department of Shohadaye Tajrish Hospital in Tehran between 2019 and 2020. Demographic information was recorded using a conscious questionnaire. The PCa group included 50-year-old men with or without a family history of the disease, high PSA, urinary symptoms, digital rectal examination, CT scan, ultrasound, biopsy with a definitive diagnosis of prostate cancer by a urologist, based on pathological tests and the presence of neoplastic tissue at any stage or degree. Also, body mass index, height and weight, smoking / hookah use or any other drug, place of residence, use of other drugs, PSA level were recorded. The BPH group included men with disturbing urinary symptoms, PSA levels above 4, benign prostate swelling as shown by TR biopsy (rectal examination), pathologically negative results, and a detected prostate volume of more than 30 ml by radiology. The control group included healthy individuals who showed urinary symptoms, abdominal pain, and age 31-41 years. In addition to PSA measurements, ultrasound and urine culture data, their clinical and paraclinical examinations confirmed prostate health. They had no previous surgery, chemotherapy, or radiation therapy for prostate cancer, had no family history of BPH / PCa, and was considered healthy.

Informed consent was received from each patient

DNA extraction from peripheral blood

In this study, 5 ml of peripheral blood of each patient was collected in a tube containing EDTA. Blood samples were taken from the control subjects in the same way and then DNA extraction was performed (DiatomTM DNA prep kit and in some samples using salting out method). The PCR conditions were based on previously works [33, 34]. The forward and reverse primers for examining the studied copy number variations and the gene polymorphisms are shown in Table 1.

Enzyme digestion and gel electrophoresis

After the amplification of rs361525 promoter snp and UGT2B15 D85Y polymorphism, the first variation PCR products were digested by MspI and the second polymorphism products were cut by Sau3AI restriction enzymes (Fermentas, Hanover, MD, USA) and separated by polyacrylamide gel electrophoresis (PAGE, 12%).

Amplification of rs1800629

Tetra-ARMS-PCR technique was used to identify rs1800629 polymorphism. Based on this method, a pair of non-specific primers (Forward outer and Reverse outer) that amplify the entire sequence containing this variant and a pair of specific primers (Forward inner and Reverse inner) that identify and amplify a specific sequence of the interest region were used.

Results

PCR based experiments results were the same as our previous work [33, 34]. The expected fragment of PCR for UGT2B15 D85Y polymorphism was a 215 bp band, which after restriction digest, could produce two bands of 28 and 187 bp (figure1). The PCR products with YY genotypes showed unique fragments of 215 bp, those with DD genotypes showed two different fragments with lengths of 187 and 28 bp, and DY genotypes demonstrated three bands of 215, 187, and 28 bp on gel electrophoresis (Figure 1).

The pattern of UGT2B17 PCR fragments on 2.5% agarose gel is shown in Figure 2. Checking for the presence or absence of the gene required two pairs of primers, one pair for exon1 amplification which could create a 173 bp band to confirm the definite presence of the allele and a second pair of primers which could amplify a region of 893 base pairs when the gene was deleted but could produce two fragments of 173 and 893 base pairs in heterozygotes of the deleted allele.

The pattern of UGT2B28 PCR fragments on 2.5% agarose gel is shown in Figure 3. Checking for the presence or absence of the gene required two pairs of primers, one pair for exon1 amplification which could create a 324 bp band to confirm the definite presence of the allele and a second pair of primers which could amplify a region of 450 base pairs in homozygutes of the deletion, but could produce two fragments of 324 and 450 base pairs in heterozygote samples.

The amplification of TNF-α promoter rs361525 polymorphism produced a fragment of 152 bp on 2% agarose gel, while after PCR-RFLP and digestion by MspI restriction enzyme was digested into a 152 bp, a 132bp, and a 20 bp fragments in GA heterozygotes and into two fragments of 132bp and 20 bp in GG homozygotes (Figures 4 and 5).

For rs1800629 PCR products were electrophoresed directly and without enzymatic digestion on 2% agarose gel. Individuals with the G / G genotype had two fragments of 304 and 197 bp, G/A heterozygotes showed to have three-bands of 304, 197, and 162 bp, and two fragments of 304 and 162 bp. were resulted from A / A homozygotes (Figure 6).

Exact test showed a Hardy-Weinberg Equilibrium in the studied alleles of the target population (p value > 0.05) (Table 2). The examined groups were compared in terms of genotypes to determine a possible allelic relationship in the genes (Table 3). There was a codominant, dominant, and/ or recessive, or Log-Additive model of allelic relationship between the variants of UGT2B17 and BPH (p value<0.05). UGT2B17 showed to be associated with BPH in comparison with the control group (P value = <0.0001) (Del versus INS: OR (95% CI) = 2.08 (1.41-3.08). Also, an allelic codominant, dominant, recessive, or Log-Additive model was anticipated for UGT2B15 and BPH (p value < 0.05).

UGT2B15 was associated with BPH in comparison to the control group (P value = 0.007) (G versus T: OR (95% CI) = 1.64 (1.14-2.37). For other examined variants, there was no significant relationship between allelic models and disease etiology (Table 3).

Since, the understudy genes UGT2B28, UGT2B17 and UGT2B15 were located on 4q13.2, different combinations of their alleles were considered as haplotypes. There was a significant relationship between G score and UGT2B15 in PCa group, so that 90% of patients with GG genotype were found among those with G score ≤ 6 group (Table4).

The frequency of each haplotype was compared using SNP Stats in three groups. Genotypes of three UGT2B28- UGT2B17- UGT2B15 genes in BPH group as Ins-Del-G allelic combination was significantly higher than the frequency of this allelic combination in control subjects (p value= 0.011; OR (95%CI) (Table 5). A chi-squared test was used to derive p-Values.

Discussion

Prostate cancer is the second most common cancer of men in the world including Iran [35-37]. Numerous studies have examined the effect of functional polymorphisms of UGT2B28, UGT2B17 and UGT2B15 enzymes on androgen metabolism [38], including single-nucleotide substitutions (SNPs and variable number of copies (CNV) [33-40]. The copy number variations (CNVs) of UGT2B17 and UGT2B28 genes are an important source of variation in their expression which affects the accumulation of dihydrotestosterone [41]. Genetic studies in recent years have shown that in cancer patients where both alleles of the UGT2B17 gene have been deleted, the amount of 3-alpha-diol glucuronidate in circulation is decreased by 42% [42]. However, Habibi and colleagues did not find any association between the null genotype (del/del) of UGT2B17 and UGT2B28 genes with prostate cancer risk in Iranian PCa patients [33]. The present study showed that UGT2B17 Del allele possibly is associated to the disease development and increase risk of BPH (P value = <0.0001). As we considered, this probable association could be in either mode of codominance, dominance, recessiveness, and/or in log-Additive models which could depend on locus neighboring genes in the form of haplotype.

In vitro studies have identified two enzymes, UGT2B15 and UGT2B17, as the major enzymes involved in glucuronidation of androgens [43]. UGT2B15 protein level decreases in prostate cancer compared to benign hyperplasia and the protein is further reduced in hormone resistant form of prostate cancer and is as low as to be measured in metastasis state [44]. D85Y polymorphism (rs1902023) in UGT2B15 gene has been shown to be significantly correlated with prostate cancer in some studies ]45 ,33[and is a malignant polymorphism at gene codon 85 [33] where thymine replaces guanine base and causes the amino acid aspartic acid to be converted to tyrosine. The occurrence of this substitution in the N-terminal region of the enzyme, affects the protein activity in the second binding domain to the substrate [45]. UGT2B15 D85Y polymorphism does not appear to alter the specificity of substrates, but this polymorphism causes the maximum rate (Vmax) of glucuronidation to almost double and may play a role in individual differences in glucuronidation [46].

Some studies have reported that the TT genotype of UGT2B15 D85Y is twice as active as the GG homozygotes, so it glucuronates the hormone dihydrotestosterone more rapidly, further protecting the prostate against high levels of androgens, thereby reducing the risk of the disease [47]. Lower activity of the enzyme variant increases the risk of cancer due to the accumulation of dihydrotestosterone in the prostate [48, 49]. Hajdinjak and colleagues showed that the frequency of homozygous G is high in patients with prostate cancer and the frequency of homozygous T is high in controls [45]. A 2013 study by Grant et al. [48], confirmed the association of D85Y polymorphism with prostate cancer, and in this study, as in previous studies by MacLeod et al. 2000 [50]; Hajdinjak et al. In 2004, a homozygous form of G was shown to be associated with an increased risk of prostate cancer [45]. However, some studies, such as a 2002 study by Gsur et al., found no association between this polymorphism and an increased risk of prostate cancer [51].The results of current study showed that UGT2B15 is associated with BPH in either models of Codominance, Recessive, and Log-Additive (P value = 0.007) and its D85Y, rs1902023 GG genotype is most frequent in individuals with developing PCa (P value = 0.011). This is consistent with the previous reports we pointed here.

Also, the haplotype Ins Del G of the target genes was more common in BPH compared to the control group, one could anticipate that this achievement is an indication of the effect of the association of these three alleles on the etiology of the disease which in turn introduces this genotype as a probably important marker for predisposition a person to prostate hyperplasia.

Recent reports suggest that genetic polymorphisms of cytokines, including tumor necrosis factor-alpha (TNF-α), a proinflammatory molecule, are associated with increased inflammation, cytokine production, and possibly an increased risk of prostate cancer .]52[ It is shown that promoter polymorphisms in the TNF-α gene can directly affect TNF-α production, thus causing interpersonal differences in the immune response that may affect susceptibility to prostate cancer ]53[, and/or malignant tumors like gastric cancer, breast cancer, and hepatocellular carcinoma .]54-56[ We revealed that rs361525 TNF-α polymorphism is not associated with prostate cancer, but rs1800629 may increase the risk of PCa. Also, at least in this experimental study, there was no individual with AA genotype which shows that either for this particular polymorphism, the population of Iran is not in Hardy-Weinberg Equation or A allele has a low frequency in Iranian population, and probably has low influence on the incidence of the disease.

According to the results of this study and similar studies, finding functional polymorphisms as probable new biomarkers in the pathogenesis of cancers including prostate cancer encourages us to conduct more extensive research in this area with a larger population. A similar study with a larger population could confirm the results of this work.

Acknowledgements

The authors thank the medical staff who assisted in preparing the samples. We also thank the patients and people who willingly participated in this study by donating their blood samples at the same time as the pain or problems in life.

This project was based on grants numbers 7698 and 15139 that were supported by Shahid Beheshti University of Medical Sciences Research Council.

Conflict of interest

Authors have no conflict of interests.