Potential Functions of MicroRNA Biomarkers as a Prognostic Factor in Urothelial Bladder Carcinoma in a Saudi Community

Growing knowledge supports the importance of microRNAs in cell growth regulation, differentiation ...


Introduction
Bladder cancer (BCa) is one of the most frequent malignancies worldwide. It has been characterized by a high recurrence rate, thus reflecting a substantial public health burden. In Saudi Arabia, almost 83% of all diagnosed BCa is urothelial bladder carcinoma (UBCa), and one-third of UBCa is described as invasive UBCa with a high risk for distant metastases [1]. Approximately 70% to 80% of patients are diagnosed with non-invasive UBCa (pTa-pT1), and the remaining patients have invasive UBCa that often leads to recurrence [2]. The recurrence rate within five years following the transurethral resection of bladder tumors (TURBT) can be estimated as 75% [3]. Thus, the disease may invade the muscle layer very rapidly. UBCa is not easy to cure because of its high recurrence and metastasis rates, with a five-year survival rate of about 57%. [4]. The great impact of microRNA (miRNA) has revolutionized current cell biology and medical science. miRNAs are a group of highly conserved small noncoding RNA molecules with [18][19][20][21][22][23][24] nucleotides [5]. They can post-transcriptionally regulate gene expression by directly targeting messenger RNAs (mRNAs). miRNAs can bind to the 3` untranslated regions of mRNAs, resulting in the degradation or translational repression of mRNAs. Previous reports revealed that miRNAs are widely involved in various processes of cell proliferation, differentiation, and apoptosis [6][7][8]. However, miRNAs can function as either oncogenes or tumor suppressors in different carcinomas, including BCa [6,[9][10][11][12][13][14][15][16][17]. Reports on candidate genes and their impact on the risk of UBCa are still inadequate in Saudi population. Recently, we investigated associations between combinations of genetic variants of glutathione transferases, cytochrome P450, TP53 and MTHFR, and MTRR genes with the risk of UBCa among Saudi patients [18]. Here, we have extended our work to examine the effect of single nucleotide polymorphisms (SNPs) in miRNAs-the rs2910164 SNP in miR-146a, the rs11614913 SNP in miR-196a2, and the rs3746444 SNP in miR-499 genes-on the risk of UBCa in Saudi patients.
All study participants signed an informed consent form.

Study Population
The  [20]. In the TNM system, 'pTa' is defined as a noninvasive papillary urothelial neoplasm of low malignancy, and the small section of tissue can be easily removed with TURBT. In contrast, 'pT1' describes a tumor that invades the subepithelial connective muscle but does not involve the bladder, and 'pT2' describes a tumor that has spread to the muscle of the bladder wall. The management of the disease-through intravesical bacillus Calmette-Guérin (BCG), immunotherapy, a conservative TURBT-BCG therapy (injection), chemotherapy, chemoradiotherapy, or radical cystectomy-was also recorded. After TURBT was performed, the high-grade tumor cases were treated with BCG. Patients who previously had cancer, radiotherapy, and chemotherapy, or metastasized cancer from non-Saudi or unknown origins were excluded.
Patients (n = 36) with immune disease or any histopathologic diagnosis other than UBCa were also excluded. Healthy individuals (n = 78) having no evidence of any clinical phenotypes of malignancies were selected as controls (ages 44-89 years) in routine follow-up at governmental hospitals in Makkah ( Figure 1).

TaqMan Genotyping Analysis
Genomic DNA was extracted from peripheral blood (200 μL) using the QIAamp DNA blood kit as recommended by the manufacturer (Qiagen, Hilden, GmbH, Germany). In some cases, DNA samples were taken from buccal mucosa using the Oragene.

Statistical Analysis
SNPs from all participants were tested for Hardy-Weinberg equilibrium (HWE) using the χ 2 test, and a P value > 0.05 was considered consistent with HWE. We conducted the statistical analysis considering the interactive models of inheritance-   Figure 2). However, there was a highly significant difference between pathologic tumor stages with response to tumor grades among our cases with UBCa (χ 2 = 30.8, P ≤ 0.001).

Characteristics of the Study Population
The predominant management course for our cases with UBCa was BCG immunotherapy (76.9%; z = 23.8, P < 0.0001). The proportion of cases managed with TURBT-BCG injection (conservative therapy) was higher than the proportions managed with other strategies (74.2% versus 13.6% for radical cystectomy, 7.6% for chemo-radiotherapy, and 4.5% for chemotherapy) ( Table 1).

Figure 2:
Pathological stages and tumor grades in cases with urothelial bladder carcinoma (UBCa). The number of cases with tumors of "high grade" increased from pTa to pT2. There were no cases with tumors of "low grade" linked to pTa or pT2.

HWE of SNPs in miRNA Regions
Three SNPs in the miRNAs' regions-rs2910164 G>C, rs11614913 C>T, and rs3746444 A>G-were successfully genotyped in 66 patients with UBCa and 78 control subjects.
All cases and controls were in HWE at the examined SNPs in the miRNAs (P > 0.05) ( Table 2).

Genotyping associations of miRNAs and tumor grades
The interactive genotypic models of the selected SNPs in miRNAs with the response of tumor grades after adjusting for age and gender are shown in Table 3. There was a significant difference

Discussion
This hospital-based case-control study presents the first evidence that common SNPs in miRNAs regions may be used as candidate genetic markers for UBCa vulnerability in a Saudi population. Our results show that the miR-499 rs3746444 SNP, but not the miR-164a rs2910164 or miR-196a2 rs11614913 SNP, is associated with risk for UBCa. Meta-analyses have been inconclusive regarding the association between the rs2910164, rs11614913, and rs3746444 SNPs in the miRNA genes and cancer risk [22][23][24]. Several reports have provided no evidence of an association between rs2910164 and overall cancer risk in diverse populations [22,23,25]. In our Saudi cases, the lack of association between the rs2910164 SNP and UBCa is consistent with previous results in Caucasian and Asian populations [26][27][28]. In addition, for the rs2910164C allele, reduced risks have been found for various types of cancer and in variable ethnic populations: cervical cancer in Chinese populations; [29] prostate in Iranian; [30] liver in Chinese, [31][32][33][34] Egyptian, [35] and Turkish [36]; colorectal cancer in Czech [37] and South Korean [38]; and gastric in Japanese [39] and Caucasian [40].
In contrast, the rs2910164C variant was found to be associated with increased risks of developing lung cancer, [41] and of developing head/neck cancer among Caucasian populations [42].
These discrepancies cannot be explained by variable ethnicity, but instead are likely due to organ-specific effects as well as different living environments, diets, climates, and lifestyles. Some previous results have suggested that rs11614913 and rs3746444 SNPs confer susceptibility to UBCa risk among different ethnic populations. However, consistent with our study, Mittal et al. [43] reported no association between the rs11614913 SNP and BCa.
For rs11614913, some previous studies reported a significantly increased risk of certain cancers when T/T for C/C or combined C/C-C/T was present: breast cancer, [44] gliomas, [45] prostate cancer, [46] lung cancer, [47] colorectal cancer, [48] and liver cancer in patients infected with hepatitis B virus [49]. In agreement with our study, a meta-analysis found significant associations between allele frequency or different genotypic models within miR-499 rs3746444 SNP and risk of BCa in studies among Chinese populations [23].
In contrast, several reports could not find any associations between the rs3746444 SNP and BCa, [50] but did find associations with other types of cancer, including colorectal, liver, gallbladder, and breast cancer [38,[51][52][53]. Based on the Human Gene Mutation Database, [54] human related-disease genes can be categorized into cancer disease genes and Mendelian disease genes. Several miRNAs have been found to regulate cancer migration, invasion, metastasis, and growth, and could be the targets for cancer therapy in about 30% of cancer genes [15,24,[55][56][57][58]. Unfortunately, few articles have dealt with the regulation of BCa cells by these oncogene-targeted miRNAs, including miR-940/INPP4A, miR-940/GSK3b, miR-146a-3p/PTTG1, and miR-145/PAK1 [59][60][61]. However, Xiang et al.60 found that overexpression of miR-146a-3p could inhibit BCa progression by targeting PTTG1 (OMIM 604147)-an oncogene that is expressed in many tumors and is correlated with tumor size, TNM stage, lymphatic invasion, and distant metastasis of BCa.

Limitations
Meta-analysis has revealed that ethnicity could affect the association of miRNA polymorphisms with cancer risk Qiu et al. [67] reported that the significance of the sample size (P = 0.02), replication of our results through larger, multicentric investigations will be of interest. Also, the literature includes other pathological types of cancer (e.g., squamous carcinoma or adenocarcinoma) in addition to UBCa, which might have influenced the outcomes of associations of the genetic markers with BCa. However, the present study focused only on UBCa, which consequently strengthens the reliability of our outcomes of associations [68,69].

Conclusion
Our results present the first evidence that miRNAs have great potential for clinical use as diagnostic biomarkers for UBCa susceptibility in a Saudi population. Our data show that the miR-499 rs3746444 A>G SNP contributes to an increased susceptibility to UBCa, as compared with the miR-164a rs2910164 G>C or the miR-196a2 rs11614913 C>T SNPs. Furthermore, increased expression of miR-499 rs3746444 is significantly associated with a stage of UBCa with high-grade tumors. Our results regarding the rs3746444 SNP support roles for homozygosity and heterozygosity in affecting risk for UBCa, as one copy of the allelic variant rs3746444G could be necessary to produce a considerable damaging effect. In contrast, each copy of the T allele in rs11614913 can double the protective effect on individuals. Even though we found no association between the variant genotypes of miR-146a and UBCa risk in our Saudi population, miR-146a could exert a prominent role in carcinogenesis via marginal significance of heterozygosity in the debate of Asian and Caucasian ethnic studies. The inconclusive studies of cancer risk and the susceptibility conferred by the examined miRNAs may be due not only to admixture ethnicities and different living environments, diets, climates or lifestyles but also to organ-specific effects. Based on the results of previous clinical trials, some miRNAs under investigation can be used as biomarkers for risk prevention and therapeutic intervention. Ongoing analyses of UBCa patient DNA using whole-exome sequencing are being performed to discover more cancer genes that are targeted predominantly by several miRNAs. Thus, the miRNA targets on cancer genes analyzed will help enhance the knowledge UBa cancer genes and therapeutic improvements in treating cancer.

Acknowledgment
The authors would like to thank all subjects for their participation in this study. We wish to thank the Faculty of Biotechnology, Modern Sciences and Arts University (MSA), October City, Giza, for allowing the MSc student (ENE) to participate in the molecular genetic analyses.