Abstract
Background:
A candidate gene for hereditary prostate cancer (PC), recently identified is the RNASEL gene on the chromosome loci 1q25. This gene mediates the apoptotic and antiviral activities of interferon. In some studies, a significant relationship has been reported between the chromosome 1q24-25 (HPC1) and prostate cancer risk, while some other studies did not approve.Objectives:
The aim of this study was to determine the association between R462Q mutation and prostate cancer in a cross-sectional study.Patients and Methods:
One hundred twenty one samples from 51 patients with sporadic PC and 70 patients with non-cancerous prostate were screened for the R462Q mutation. All samples were formalin-fixed and paraffin embedded. The samples were investigated by the use of amplification refractory mutation system (ARMS) PCR, followed by gel electrophoresis. To analyze the data the Fisher’s exact and Chi-square tests were used. Statistical analyses were performed, using SPSS 17 software program.Results:
The present study findings showed that RR, RQ and QQ genotypes compromised 82, 14, and 4% of the cancerous samples and 87, 13 and 0 % of the non-cancerous samples, respectively.Conclusions:
We did not find any association between the RNASEL Arg462Gln polymorphism and prostate cancer. Based on these results the RNASEL Gln/Gln genotype does not play an important role in the etiology of sporadic prostate cancer, in the general population. However, additional studies with bigger sample sizes are needed to more clearly explain the role of RNASEL mutations in hereditary prostate cancer.Keywords
1. Background
The first defensive line against pathogens (viruses, bacteria, fungi, and parasites) is the innate immune system. Pathogens must cross physical barriers, including skin and mucosal tissues in the first step. If the pathogens cross these barriers, they will be recognized by the innate immune system. Then active anti-microbial responses are generated and pro-inflammatory cytokines and interferons (IFNs) are produced at the cellular level. A subset of genes, called IFNs stimulated genes (ISGs), mediate IFN effects of. The ISGs are implicated in anti-angiogenic, apoptotic, cell cycle inhibitory and anti-viral effects (1, 2). After the viral attack, innate immune responses by producing interferon- induced 2΄-5΄ oligo adenylate synthetase, which activates an endoribonuclease, called RNASEL. The RNASEL is expressed by the RNASEL gene localized on the chromosome 1q24-25 (HPC-1), a region for hereditary prostate cancer (3). In some studies a significant relationship has been demonstrated between the chromosome 1q24-25 (HPC1) and prostate cancer risk (4). RNASEL, a role playing gene in regulating cell proliferation, apoptosis and tumor-suppressor cell, is one the candidate genes within the HPC1 region (5). According to the close linkage of HPC1 to the RNASEL, it seems that RNase L could suppress one or more steps in prostate tumorigenesis and/or metastasis, directly or indirectly. One of the most frequently occurring cancers among men is prostate cancer. The highest rates are reported in the United States, Sweden, Australia, Canada and France (4). The rate of prostate cancer incidence in Iran is 9.6 (3.2 to 16.0) per 100000 and is significantly less than those in developed countries. The incidence rate is similar to the Eastern Mediterranean regions (6). Prostate cancer may appear in familial or sporadic versions. There are usually mutations in genes, like ElaC2, MSR1 and RNase L, in familial cancer, however, hereditary prostate cancer has a low prevalence (7). In relation to sporadic and familial prostate cancer, several variants in RNASEL (E262X, 471delAAAG, Arg462Gln, Asp541Glu) have been detected. A common missense variant, causing threefold reduction in enzymatic activity, is substitution of glutamine (Gln) by Arginine (Arg) at codon 462 (a G to A nucleotide transition at base pair 1385) (3).
2. Objectives
As found in the epidemiological studies, there were diverse associations between Arg462Gln polymorphism and prostate cancer in sporadic and familial prostate cases (3, 5, 8-10). Therefore, in this study we evaluated the association between this polymorphism and prostate cancer in a cross-sectional study.
3. Patients and Methods
3.1. Tissue Specimens
We used a cross-sectional design, using samples of cancerous and non-cancerous prostate tissue at the baseline. All human samples used in this study were retrieved from the archive of the Pathology Service of the Shafa Hospital, Jundishapur University of Medical Science, Ahvaz, IR Iran and were related to Khuzestan province. These formalin-fixed and paraffin embedded samples had been collected from 2010 to 2011. The ethical approval code was 55.14.3.2011. Age, clinical parameters and geographical locations of samples were recorded but unfortunately it was not feasible to separate the familial cases from the sporadic cases, based on the available data. Our study population consisted of 121 males, divided into two groups of individuals, previously diagnosed with prostate cancer by histopathological examination (n = 51) and control individuals with non-cancerous prostate (prostatitis, hyperplasia) (n = 70). Pathologically confirmation of prostate cancer was considered as inclusion criteria, whereas samples from patients with cancers related to the other parts of the body were excluded from study.
3.2. Molecular Analysis
Each paraffin block was cut at 10μm and collected in 1.5 mL autoclaved plastic micro tubes) (9). Ten sections were placed in each micro tube. Deparaffinization was performed with heating procedure as follow: a total of 300 μL universal buffer solution (H3BO3, diethyl barbituric acid, citric acid and KH2PO4 28.6 mM for all ) at pH 9 was added to each micro tube, containing tissue sections and heated at 120˚C, using autoclave (Iran Tolid-110N) for 20 minutes. Then the samples were cooled for five minutes. The solid paraffin wax ring, formed above the buffer, was removed (10). DNA was extracted by DNA extraction kit (Cinnagen, Iran). The RNASEL G1385A mutation analysis was carried out, using the amplification refractory mutation system (ARMS). ARMS, also known as allele specific PCR (ASPCR) or PCR amplification of specific alleles (PASA), is used for detection of known single-base substitutions or micro deletions/insertions (11). Primers described by Casey et al. were used in this study (Table 1). PCR was performed in a final volume of 15μL for control gene containing: Taq polymerase (Cinnagen, Iran) (1 U), PCR buffer 10 x (1.5 μL), 10 mM dNTPs (0.3 μL), 50 mM MgCl2 (0.5 μL), 10 ρmol/μL forward primer (0.3 μL), 10 ρmol/μL reverse primer (0.3 μL) and the sample DNA (1 μL) and in a final volume of 15 μL for RNASEL gene containing: Taq polymerase (1 U), PCR buffer 10 x (1.5 μL), 10 mM dNTPs (0.3 μL), 50 mM MgCl2 (0.5 μL), 10 ρmol/uL forward primer (0.5 μL), 10 ρmol/uL reverse primer (0.5 μL) and round 1 PCR product (2 μL). Modified programs for control gene and RNASEL gene is described as follows, respectively:
Cycles: Seven minutes at 95°C; (30 sec 95°C, 30 sec 65°C and 40 sec 72°C) × 30 cycles; 10 minutes at 72°C.
Cycles: Five minutes at 95°C; (30 sec 94°C, 30 sec 58°C, 40 sec 72°C) × 30 cycles; 10 minutes at 72°C.
Electrophoresis was performed using 8% polyacrylamide gel (3.2 mL acrylamide 30%, 1.2 mL TBE 10X, 7.6 mL water, 200 μL APS 10% and 10 μL tetramethylethylenediamine [TEMED]) and 2% agarose. Two PCR products were sent to Bioneer Company for sequencing, for result accuracy investigation.
Lists of the Used DNA Primer Sequences
| Gene | Product Size | Primer Sequence | Primer Name |
|---|---|---|---|
| RNASEL | 123 bp | 5’-GTGGAAAATGAGGAAGATGAATTTGCCAG-3’ | 1385G |
| 5’-GTGGAAAATGAGGAAGATGAATTTGCCAA-3’ | 1385A | ||
| 5’-ATTGGGGACTCACCTATTAAGATGTTTTG-3’ | 1385R | ||
| Control gene | 393 bp | 5’-CCCACCTTCCCCTCTCTCCAGGCAAATGGG-3’ | ARMS-A |
| 5’-GGGCCTCAGTCCCAACATAGGCTAAGAGGTG-3’ | ARMS-B |
3.3. Statistical Analysis
Statistical analyses were performed, using SPSS 17 software program. Correlation between RNASEL gene type and prostate cancer was analyzed, using Fisher’s exact test and Chi-square.
4. Results
We investigated whether the prostate cancer incidence is higher among carriers of the R462Q polymorphism of RNASEL. In total, 51 prostate cancer samples and 70 non-cancerous prostate samples were screened by the ARMS assay to detect the presence of R462Q polymorphism in RNASEL. Most of the patients with cancer were70-79 years old (mean age: 70.88) and patients with non-cancerous prostate were 60-69 years old (mean age: 64.83). The incidence of RR (wild-type), RQ (heterozygous) and QQ (homozygous) variants presence in cancerous samples were 42 (82%), 7 (14%) and 2 (4%), respectively and 61 (87%), 9 (13%) and 0 (0%) in non-cancerous samples, respectively. The allelic frequency for R462Q in cancerous and non-cancerous patients was determined to be 0.108 and 0.064, respectively. It is the same as the results of a study by Shea et al. (12) and lower in comparison to other prostate cancer studies (the allel frequency of R462Q, 0.25 and 0.38) (9, 10, 13, 14). Prostate cancer was not found to be significantly associated with the R462Q polymorphism of RNASEL (P > 5% Fisher’s exact test) (Table 2). Odds ratio was less than one, which means that this mutation was not related to prostate cancer.
5. Discussion
The third most common cancers among male population in Iran is prostate cancer (6). One factor with a probable role in the etiology of prostate cancer is chronic inflammation in prostate (3). Epidemiological studies indicated that prostatitis and sexually transmitted infections increase the risk of prostate cancer, while anti-inflammatory agents like aspirin decrease the risk of this disease (3). Some studies show relation of the 462Q/Q RNASEL genotype with viral infections and cancers of prostate (15). Studies of Casey et al. demonstrated reasonable evidence for the existence of prostate cancer susceptibility gene at the 1 q24-25 (11). In their study, a common polymorphism, namely Arg462Gln, was found to be associated with hereditary prostate cancer The studied RNASEL variant had three times less enzymatic activity than the wild type (11). An endoribonuclease, a member of the interferon regulated 2-5A system, is encoded by this gene. Some studies suggest that the RNASEL gene may function as a tumor suppressor gene, although some similar studies are not in agreement with this hypothesis (16). We, in our study investigated the relationship between RNASEL Arg462Gln gene polymorphism and sporadic prostate cancer risk; however a small proportion of cases with family prostate cancer history were included, because it is not feasible for us to separate the familial cases from the sporadic cases based on the available data (recorded information was defective). In this investigation no association was found between the RNASEL Arg462Gln polymorphism and prostate cancer. Review of some published linkage studies, including a nested case-control study in the USA (3), a population-based study conducted in Sweden (8), a meta-analysis study by Wei et al. (17), a German population study (18), a study of prostate cancer in Poland (19) and an analysis of the RNASEL in familial and sporadic prostate cancers by Wang et al. (5) did not show any association between the Arg462Gln polymorphism and sporadic prostate cancer risk. In a study by Meyer et al. mutation R462Q was only associated with an increased risk in the pre-prostate-specific antigen era and they suggested that this association may be mediated through inflammation (20). Larson et al. found that prostate cancer in patients with the R462Q mutation is not associated with more aggressive pathological or clinical features (21). Surprisingly, in an Asian study they found a decreased risk for familial prostate cancer in a Japanese population with the genotype Gln/Gln (22).
Krüger et al. investigated a variation at codon 462 of RNASEL in hereditary non-polyposis colorectal cancer. In their study the frequency of these codons did not differ between patients and controls; while sequence variation at RNASEL codon 462 was associated with age of onset in patients with cancer in a dose-dependent way (23). One of the reasons that we did not find any association between the Arg462Gln polymorphism and sporadic prostate cancer was the number of our samples. This is a pilot study and further studies are suggested with larger sample sizes. In other studies, like a study by Casey et al. (11) and the study of Alvarez-Cubero et al. in Spain (24), an association was found between this polymorphism and the sporadic prostate cancer risk. In the study of Casey et al. this relationship was also found among Caucasians and African Americans. The strongest the relationship between RNASEL and prostate cancer has been proven to be with the hereditary and familial prostate cancers and not with the sporadic forms (5, 11). The study by Robbins et al. showed RNASEL may play a role, however minor, in prostate cancer risk among African American men (25). To sum up, in the studied population, we found two cases with Gln/Gln genotype of codon 462. These cases were related to patients with prostate cancer, but these results were not statistically significant. Therefore, it is suggested that the Arg462Gln polymorphism in RNASEL does not play a significant role in the etiology of prostate cancer, in the population studied. However additional studies are essential to confirm the consequences of the RNASEL gene presence on the prostate cancer.
Acknowledgements
References
-
1.
Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons. Annu Rev Biochem. 1998;67:227-64. [PubMed ID: 9759489]. https://doi.org/10.1146/annurev.biochem.67.1.227.
-
2.
Silverman RH. Implications for RNase L in prostate cancer biology. Biochemistry. 2003;42(7):1805-12. [PubMed ID: 12590567]. https://doi.org/10.1021/bi027147i.
-
3.
Daugherty SE, Hayes RB, Yeager M, Andriole GL, Chatterjee N, Huang WY, et al. RNASEL Arg462Gln polymorphism and prostate cancer in PLCO. Prostate. 2007;67(8):849-54. [PubMed ID: 17407163]. https://doi.org/10.1002/pros.20537.
-
4.
Li H, Tai BC. RNASEL gene polymorphisms and the risk of prostate cancer: a meta-analysis. Clin Cancer Res. 2006;12(19):5713-9. [PubMed ID: 17020975]. https://doi.org/10.1158/1078-0432.CCR-05-2799.
-
5.
Wang L, McDonnell SK, Elkins DA, Slager SL, Christensen E, Marks AF, et al. Analysis of the RNASEL gene in familial and sporadic prostate cancer. Am J Hum Genet. 2002;71(1):116-23. [PubMed ID: 12022038]. https://doi.org/10.1086/341281.
-
6.
Mousavi SM. Toward prostate cancer early detection in Iran. Asian Pac J Cancer Prev. 2009;10(3):413-8. [PubMed ID: 19640184].
-
7.
Karimpur-Zahmatkesh A, Farzaneh F, Pouresmaeili F, Hosseini J, Azarghashb E, Yaghoobi M. A2 allele polymorphism of the CYP17 gene and prostate cancer risk in an iranian population. Asian Pac J Cancer Prev. 2013;14(2):1049-52. [PubMed ID: 23621184].
-
8.
Wiklund F, Jonsson BA, Brookes AJ, Stromqvist L, Adolfsson J, Emanuelsson M, et al. Genetic analysis of the RNASEL gene in hereditary, familial, and sporadic prostate cancer. Clin Cancer Res. 2004;10(21):7150-6. [PubMed ID: 15534086]. https://doi.org/10.1158/1078-0432.CCR-04-0982.
-
9.
Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR. XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. Proc Natl Acad Sci U S A. 2009;106(38):16351-6. [PubMed ID: 19805305]. https://doi.org/10.1073/pnas.0906922106.
-
10.
Shi SR, Cote RJ, Wu L, Liu C, Datar R, Shi Y, et al. DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH. J Histochem Cytochem. 2002;50(8):1005-11. [PubMed ID: 12133903].
-
11.
Casey G, Neville PJ, Plummer SJ, Xiang Y, Krumroy LM, Klein EA, et al. RNASEL Arg462Gln variant is implicated in up to 13% of prostate cancer cases. Nat Genet. 2002;32(4):581-3. [PubMed ID: 12415269]. https://doi.org/10.1038/ng1021.
-
12.
Shea PR, Ishwad CS, Bunker CH, Patrick AL, Kuller LH, Ferrell RE. RNASEL and RNASEL-inhibitor variation and prostate cancer risk in Afro-Caribbeans. Prostate. 2008;68(4):354-9. [PubMed ID: 18189233]. https://doi.org/10.1002/pros.20687.
-
13.
Danielson BP, Ayala GE, Kimata JT. Detection of xenotropic murine leukemia virus-related virus in normal and tumor tissue of patients from the southern United States with prostate cancer is dependent on specific polymerase chain reaction conditions. J Infect Dis. 2010;202(10):1470-7. [PubMed ID: 20936978]. https://doi.org/10.1086/656146.
-
14.
Fischer N, Hellwinkel O, Schulz C, Chun FK, Huland H, Aepfelbacher M, et al. Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer. J Clin Virol. 2008;43(3):277-83. [PubMed ID: 18823818]. https://doi.org/10.1016/j.jcv.2008.04.016.
-
15.
Martinez-Fierro ML, Leach RJ, Gomez-Guerra LS, Garza-Guajardo R, Johnson-Pais T, Beuten J, et al. Identification of viral infections in the prostate and evaluation of their association with cancer. BMC Cancer. 2010;10:326. [PubMed ID: 20576103]. https://doi.org/10.1186/1471-2407-10-326.
-
16.
Chen H, Griffin AR, Wu YQ, Tomsho LP, Zuhlke KA, Lange EM, et al. RNASEL mutations in hereditary prostate cancer. J Med Genet. 2003;40(3). ee21. [PubMed ID: 12624150].
-
17.
Wei B, Xu Z, Ruan J, Zhu M, Jin K, Zhou D, et al. RNASEL Asp541Glu and Arg462Gln polymorphisms in prostate cancer risk: evidences from a meta-analysis. Mol Biol Rep. 2012;39(3):2347-53. [PubMed ID: 21656378]. https://doi.org/10.1007/s11033-011-0985-x.
-
18.
Maier C, Haeusler J, Herkommer K, Vesovic Z, Hoegel J, Vogel W, et al. Mutation screening and association study of RNASEL as a prostate cancer susceptibility gene. Br J Cancer. 2005;92(6):1159-64. [PubMed ID: 15714208]. https://doi.org/10.1038/sj.bjc.6602401.
-
19.
Cybulski C, Wokolorczyk D, Jakubowska A, Gliniewicz B, Sikorski A, Huzarski T, et al. DNA variation in MSR1, RNASEL and E-cadherin genes and prostate cancer in Poland. Urol Int. 2007;79(1):44-9. [PubMed ID: 17627168]. https://doi.org/10.1159/000102913.
-
20.
Meyer MS, Penney KL, Stark JR, Schumacher FR, Sesso HD, Loda M, et al. Genetic variation in RNASEL associated with prostate cancer risk and progression. Carcinogenesis. 2010;31(9):1597-603. [PubMed ID: 20576793]. https://doi.org/10.1093/carcin/bgq132.
-
21.
Larson BT, Magi-Galluzzi C, Casey G, Plummer SJ, Silverman R, Klein EA. Pathological aggressiveness of prostatic carcinomas related to RNASEL R462Q allelic variants. J Urol. 2008;179(4):1344-8. [PubMed ID: 18289577]. https://doi.org/10.1016/j.juro.2007.11.078.
-
22.
Nakazato H, Suzuki K, Matsui H, Ohtake N, Nakata S, Yamanaka H. Role of genetic polymorphisms of the RNASEL gene on familial prostate cancer risk in a Japanese population. Br J Cancer. 2003;89(4):691-6. [PubMed ID: 12915880]. https://doi.org/10.1038/sj.bjc.6601075.
-
23.
Kruger S, Silber AS, Engel C, Gorgens H, Mangold E, Pagenstecher C, et al. Arg462Gln sequence variation in the prostate-cancer-susceptibility gene RNASEL and age of onset of hereditary non-polyposis colorectal cancer: a case-control study. Lancet Oncol. 2005;6(8):566-72. [PubMed ID: 16054567]. https://doi.org/10.1016/S1470-2045(05)70253-9.
-
24.
Alvarez-Cubero MJ, Saiz M, Martinez-Gonzalez LJ, Alvarez JC, Lorente JA, Cozar JM. [RNASEL study of genetics of prostate cancer and its relation to clinical staging]. Actas Urol Esp. 2012;36(5):306-11. [PubMed ID: 22464196]. https://doi.org/10.1016/j.acuro.2011.12.002.
-
25.
Robbins CM, Hernandez W, Ahaghotu C, Bennett J, Hoke G, Mason T, et al. Association of HPC2/ELAC2 and RNASEL non-synonymous variants with prostate cancer risk in African American familial and sporadic cases. Prostate. 2008;68(16):1790-7. [PubMed ID: 18767027]. https://doi.org/10.1002/pros.20841.