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Indian Journal of Clinical Biochemistry logoLink to Indian Journal of Clinical Biochemistry
. 2024 Feb 24;40(4):576–587. doi: 10.1007/s12291-024-01186-z

Genetic Variants of AGO1*rs595961 and AGO2*rs4961280 with Susceptibility to Bladder Carcinoma

Rami M Elshazli 1,✉, Hamed M Kassab 2, Afrah F Salama 2, Kamal M Okasha 3
PMCID: PMC12420549  PMID: 40937387

Abstract

The microRNA (miRNA) biogenesis mechanism remains elusive, yet various genetic variants may play a role in predicting the biological function of this machinery system and its association with the advancement of specific tumors. This study aimed to investigate the relationship between AGO1 and AGO2 gene variants with the elevated risk of bladder carcinoma (BLCA) using genetic, biochemical, and bioinformatic approaches. This retrospective study comprised of 252 participants [122 BLCA patients and 130 cancer-free controls], matched for age and gender. The AGO1*rs595961 and AGO2*rs4961280 genetic variants were characterized using TaqMan genotyping assay. A bioinformatics framework, stratification analysis, and multivariate regression were employed. The genetic distribution of AGO1*rs595961 was associated with an increased risk of bladder carcinoma under allelic (OR = 1.50, p = 0.026) and recessive (OR = 2.01, p = 0.010) models. In contrast, the AGO2*rs4961280 variant demonstrated no considerable association with bladder carcinoma progression under various genetic models (p > 0.05). An in-depth examination of AGO1 and AGO2 protein revealed their involvement in gene silencing by miRNA, post-transcriptional gene silencing, regulatory RNA binding, and positive regulation of mRNA catabolic processes. The AGO1*rs595961 genetic variant was correlated with an elevated risk of bladder carcinoma, whereas the AGO2*rs4961280 variant was not. Further research is warranted to understand the underlying molecular mechanisms and potential clinical implications.

Keywords: AGO1 (rs595961), AGO2 (rs4961280), Genetic variants, Bladder carcinoma

Introduction

Bladder carcinoma (BLCA) represents the second most frequent genitourinary malignancy and is a substantial contributor to mortality within the urinary bladder [1–3]. The management of BLCA is challenging due to various heterogeneous attributes that contribute to the worldwide expansion in mortality rates [4, 5]. In Egypt, over 10,655 new cases and 6170 deaths were reported [6, 7]. Approximately 79% of diagnosed bladder tumors involved males, and 21% involved females [8, 9]. Additionally, the estimated numbers of prevalent cases among Egyptian males were 21,481 (17.5%), while those among Egyptian females were 5505 (3.5%) [10, 11]. The variability in BLCA incidence and prevalence across ethnic groups may be attributed to a multitude of environmental and genetic variables [12, 13].

MicroRNA (miRNA) is a small, non-coding, and regulatory single-stranded RNA consisting of 20–23 nucleotides, thought to play a vital function in gene expression, regulation, and phenotype development [14–16]. The miRNA biogenesis mechanism originates in the nucleus and is processed within the cytosol [16, 17]. Additionally, XPO5, RanGTP, Dicer, GEMIN3, GEMIN4, and AGO1/2 genes have crucial roles in cytoplasmic regulation of multiple miRNAs [16, 18–20]. Cytoplasmic processing of miRNA components is initiated when the miRNA precursor (pre-miRNA) is exported from the nucleus to the cytoplasm by the XPO5/RanGTP complex through the nuclear membrane [20, 21]. The ribonuclease Dicer, along with TRBP2 and PACT cofactors, can cleave the hairpin loop of the pre-miRNA, producing mature double-stranded miRNA [16, 19]. Notably, mature miRNA interacts with Argonaute RISC Components (AGO), which trigger the biological activities of the RNA-Induced Silencing Complex (RISC) [14]. The passenger strand then detaches from the RISC complex, leaving the guide strand complexed with AGO1/2, TRBP2, GEMIN3, and GEMIN4 to recognize the target mRNA [14, 17, 21]. Ultimately, the AGO-miRNA within the RISC complex interacts with the 3'UTR of mRNA, leading to gene silencing through inhibition and degradation of mRNA [18, 22]. While the AGO1 gene has potential impacts on the post-transcriptional regulatory processes of mRNAs, the AGO2 gene may exert an inhibitory function, leading to the decay of target mRNAs and cancer advancement [23, 24].

Interestingly, the argonaute RISC component 1 (AGO1; ENSG00000092847) gene is located on chromosome 1 at 1p34.3, while the argonaute RISC catalytic component 2 (AGO2; ENSG00000123908) gene is positioned on chromosome 8 at 8q24.3 [17, 25, 26]. Several single nucleotide variants (SNV) have been studied within these potential genes, including the rs595961 variant in the tenth intron of the AGO1 gene [27, 28], and the rs4961280 variant in the promoter region of the AGO2 gene [27, 29]. Moreover, these two variants may alter the regulatory processes of mRNA and increase susceptibility to various cancers, including breast cancer [30], prostate cancer [31], lung cancer [29], bladder cancer [32], and renal cell carcinoma [33]. Based on these previous discussions, our team was motivated to study the association of AGO1 (rs595961; c.1264-25A > G) and AGO2 (rs4961280; g.140637315C > A) variants with bladder carcinoma susceptibility among Egyptian subjects using genetic, biochemical, and bioinformatics approaches.

Subjects and Methods

Ethical Approval Statement

Firstly, the scientific ethical committee at the Institutional Review Board of the Faculty of Medicine, Tanta University, Tanta, Egypt authorized this work protocol with code number (33,722/3/20). Furthermore, this study was established based on the declarations of Helsinki and its improvements. Lastly, all the participants were subjected to assign an informed written consent after understanding the advantages of hereditary evaluation in the diagnosis of cancerous disorders.

The Study Participants

This retrospective hospital-based work is carried out based on 252 participants that characterized into two main categories including 122 patients diagnosed with bladder carcinoma (82.0% males and 18.0% females) along with 130 cancer-free controls that collected from the same geographic district (75.4% males and 24.6% females) and matched with age and gender. Concisely, the BLCA patients were recruited from the Outpatient Clinics of the Oncology Department, Tanta University Hospital, Faculty of Medicine, Tanta University, Egypt at the interval ranging from December 2020 up to October 2022. The gold diagnostic approach of bladder carcinoma was initially beginning with flexible fiberoptic cystoscopy technology after excluding other urinary, urethral, and urogenital disorders [9]. Nevertheless, patients with negative cystoscopy findings were subjected for urine cytology and renal function assessments followed by further imaging techniques including computed tomography (CT) urography, magnetic resonance imaging (MRI) urography, and/or retrograde pyelogram [34]. The tumor staging system was accomplished based on the tumor-node-metastasis (TNM) updated criteria [35]. Individuals with a previous history of autoimmune diseases, other forms of malignancies, renal/hepatic illnesses, urogenital disorders, and chronic infections were excluded from this research. The clinical and demographic data of the involved patients were extracted from the electronic medical records.

Sample Collection and Assessment of Laboratory Measurements

Initially, two aliquots of venous peripheral blood from all participants were gathered and subdivided into two different partitions. Furthermore, the first aliquot was collected within vacutainer tubes containing EDTA anticoagulant for genetic analysis and hematological measurements, while the second aliquot was drawn within plain vacutainer gel tubes that subjected for centrifugation to acquire serum for biochemical assessments involving the levels of hepatic aminotransferases, albumin, total bilirubin, alkaline phosphatase, creatinine, sodium, and potassium.

Genomic DNA Extraction and Purification

Generally, the extraction of genomic DNA for all subjects enrolled in this work was performed using the GeneJET Mini Kit for whole blood DNA extraction (Thermo Fisher Scientific, Waltham, MA, USA), as previously described [36]. Additionally, the intensity and concentration of purified genomic DNA were assessed utilized NanoDrop™ ND-1000 Spectrophotometer [37].

Allelic Discrimination of the AGO1*rs595961 and AGO2*rs4961280 Variants

The genotyping of the genetic variants for the AGO1*rs595961 and AGO2*rs4961280 was executed among all study participants using real-time polymerase chain reaction (RT-PCR) technique with applying TaqMan® SNP genotyping assay (Applied Biosystems, Waltham, USA). The AGO1 (rs595961; c.1264-25A > G) variant could be either wild-type (A allele) or mutant type (G allele), while the AGO2 (rs4961280; g.140637315C > A) variant could be either wild-type (C allele) or mutant type (A allele). The reaction protocol for PCR was functioned blindly with duplicates using a final volume of 30 μl containing 20 ng genomic DNA, TaqMan PCR Master Mix, and nuclease-free water. The operation of PCR amplification was carried out utilizing StepOne™ Real-Time PCR System (Applied Biosystems, USA) [38]. The analysis of allelic discrimination for these two variants was processed using the SDS software version 1.3.1 (Applied Biosystems).

Statistical Analysis

In the beginning, the study’s power was evaluated to test the strength of this recognized work using statistical power analysis G*Power software (https://www.gpower.hhu.de/), between the two groups that accredited acquiring a study power (1-β err prob) of 0.88 with an effect size of 0.25, alpha error probabilities of 0.05, and overall sample size of 252 [39]. Furthermore, statistical analysis was performed utilizing the STATA 17.0 software (StataCorp. College Station, TX, USA), and R programming language version # 4.2.2 with RStudio 2022.12.0 build 353. Genotypic/allelic frequencies, and Hardy–Weinberg equilibrium of the AGO1 (rs595961; c.1264-25A > G) and AGO2 (rs4961280; g.140637315C > A) variants were assessed using electronic bioinformatic SNP tool (SNPStats; https://snpstats.net/start.htm, accessed on 12 July 2023) [35]. Several hereditary genetic models were applied for these two genetic variants including allelic, recessive, dominant, homozygote, and heterozygote comparisons [40]. Moreover, continuous variables were tested for normality using the Shapiro–Wilk test and expressed as median with interquartile range, while categorical variables were tabulated using chi-square test [41]. The probability of the statistical measure was settled to be significant with p-value less than 0.05 [42].

Results

Characteristics of the Study Population

The present study involved 252 participants, including 122 BLCA patients and 130 cancer-free controls. The median age (IQR) for cancer patients was 61.0 (49.0–67.0) years, while that of cancer-free controls was 57.0 (54.0–62.0) years. No statistically significant differences were observed regarding clinical, chemical, and hematological parameters between these two groups (p > 0.05), Table 1. Moreover, the correlation matrix was executed using Pearson’s correlation test to evaluate the conventional possibilities for the clinical, biochemical, and hematological parameters among BLCA patients (Fig. 1).

Table 1.

The main demographic, clinical, and laboratory measurements of the study participants

Variable Levels Cancer-free controls BLCA patients P-value
I. Demographic and clinical characteristics (n = 130) (n = 122)
1. Age, years Median (IQR) 57.0 (54.0–62.0) 61.0 (49.0–67.0) 0.076
2. Age groups (≤ 55/ > 55) n (%)/n (%) 43 (33.1)/87 (66.9) 45 (36.9)/77 (63.1) 0.597
3. Gender (male/female) n (%)/n (%) 98 (75.4)/32 (24.6) 100 (82.0)/22 (18.0) 0.223
4. Tumor grade Grade I, n (%) – 58 (47.5) NA
Grade II, n (%) – 23 (18.8)
Grade III, n (%) – 41 (33.7)
5. T stage T0, n (%) – 14 (11.5) NA
T1, n (%) – 27 (22.1)
T2, n (%) – 38 (31.1)
T3, n (%) – 39 (32.0)
T4, n (%) – 4 (3.3)
6. N stage NX, n (%) – 34 (27.9) NA
N0, n (%) – 22 (18.0)
N1, n (%) – 26 (21.3)
N2, n (%) – 25 (20.5)
N3, n (%) – 15 (12.3)
7. M stage M0, n (%) 99 (81.1) NA
M1, n (%) – 23 (18.9)
II. Biochemical measurements
1. ALT, U/L Median (IQR) 21.0 (14.0–27.0) 17.5 (12.0–28.0) 0.341
2. AST, U/L Median (IQR) 18.0 (15.0–23.0) 20.0 (15.0–27.0) 0.060
3. Albumin, g/l Median (IQR) 41.0 (35.0–47.0) 40.0 (37.0–43.0) 0.210
4. ALP, U/L Median (IQR) 83.0 (67.0–98.0) 80.5 (63.0–99.0) 0.569
5. Total bilirubin, mg/dl Median (IQR) 0.61 (0.53–0.67) 0.50 (0.40–0.80) 0.071
6. Creatinine, mg/dl Median (IQR) 1.03 (0.84–1.27) 1.00 (0.81–1.20) 0.338
7. Sodium levels (Na), mmol/l Median (IQR) 139.0 (137.0–141.0) 137.0 (135.0–140.0) 0.628
8. Potassium levels (K), mmol/l Median (IQR) 4.10 (3.80–4.50) 4.20 (3.90–4.50) 0.598
III. Hematological parameters
1. RBCs, × 1012/L Median (IQR) 4.62 (4.31–5.11) 4.70 (4.41–5.20) 0.513
2. Hematocrit (HCT), % Median (IQR) 39.3 (36.4–42.2) 39.9 (36.2–43.5) 0.654
3. Hemoglobin, g/dl Median (IQR) 13.1 (12.2–14.1) 13.3 (11.9–15.2) 0.227
4. MCV, fL Median (IQR) 84.1 (79.7–86.6) 83.6 (79.6–87.9) 0.931
5. MCH, pg Median (IQR) 27.9 (26.5–29.3) 28.7 (26.2–30.2) 0.095
6. MCHC, g/dl Median (IQR) 33.3 (32.8–33.8) 33.9 (32.7–34.7) 0.119
7. WBCs, × 109/L Median (IQR) 8.01 (6.20–9.51) 7.50 (6.40–9.72) 0.857
8. Neutrophils, % Median (IQR) 63.2 (56.0–71.5) 62.0 (55.3–69.0) 0.219
9. Absolute Neutrophils, × 109/L Median (IQR) 5.01 (3.70–6.41) 4.67 (3.70–5.94) 0.523
10. Lymphocytes, % Median (IQR) 29.0 (22.2–36.2) 28.9 (22.1–34.6) 0.921
11. Absolute Lymphocytes, × 109/L Median (IQR) 2.10 (1.50–2.62) 2.37 (1.58–2.84) 0.221
12. Platelet count, × 109/L Median (IQR) 225.0 (181.0–270.0) 237.0 (189.0–310.0) 0.138
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Data are expressed as numbers (%) or median (IQR). Fisher’s exact and two-sample Wilcoxon rank-sum tests were applied

IQR interquartile range, BLCA bladder carcinoma, ALT alanine transaminase, AST aspartate transaminase, ALP alkaline phosphatase, RBCs red blood cells, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, WBCs white blood cells

Fig. 1.

Fig. 1

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Correlation matrix for the AGO1 (rs595961) and AGO2 (rs4961280) genetic variants with the clinical, biochemical, and hematological measurements among the BLCA patients. Pearson’s correlation test was applied

Analysis of the AGO1*rs595961 and AGO2*rs4961280 Genetic Variants

The allelic and genotypic frequencies of the AGO1 (rs595961; c.1264-25A > G) and AGO2 (rs4961280; g.140637315C > A) variants were compared between BLCA patients and cancer-free controls, and the results were summarized in Table 2. The genetic distributions of the AGO1*rs595961 and AGO2*4,961,280 variants were consistent with the Hardy–Weinberg equation (HWE) among cancer-free controls (p = 0.056 and 0.569, respectively). The minor allele frequency (MAF) of the AGO1*rs595961 variant (G allele) was 0.48 in cancer-free controls and 0.58 in cancer patients. In contrast, the MAF of the AGO2*rs4961280 variant (A allele) was 0.33 in cancer-free controls and 0.40 in cancer patients (Table 2).

Table 2.

The genotypic, and allelic frequencies of the AGO1*rs595961 and AGO2*rs4961280 variants

Genetic polymorphisms All subjects Cancer-free controls BLCA patients OR (95% CI) P-value
1. AGO1 (rs595961; c.1264-25A > G)
Genotypic frequencies n (%) 252 n (%) 130 n (%) 122
A/A 74 (29.4) 41 (31.5) 33 (27.1) 1.0
A/G 91 (36.1) 54 (41.5) 37 (30.3) 0.85 (0.46–1.58) 0.637
G/G 87 (34.5) 35 (27.0) 52 (42.6) 1.84 (0.98–3.46) 0.059
HWE χ2 = 19.2, p < 0.001 χ2 = 3.64, p = 0.056 χ2 = 17.46, p < 0.001
Allelic frequencies n (%) 504 n (%) 260 n (%) 244
A allele 239 (47.4) 136 (52.3) 103 (42.2) 1.0
G allele 265 (52.6) 124 (47.7) 141 (57.8) 1.50 (1.06–2.13) 0.026
2. AGO2 (rs4961280; g.140637315C > A)
Genotypic frequencies n (%) 252 n (%) 130 n (%) 122
C/C 102 (40.5) 59 (45.4) 43 (35.2) 1.0
C/A 116 (46.0) 55 (42.3) 61 (50.0) 1.52 (0.89–2.60) 0.136
A/A 34 (13.5) 16 (12.3) 18 (14.8) 1.54 (0.71–3.37) 0.322
HWE χ2 = 0.01, p = 0.911 χ2 = 0.32, p = 0.569 χ2 = 0.23, p = 0.638
Allelic frequencies n (%) 504 n (%) 260 n (%) 244
C allele 320 (63.5) 173 (66.5) 147 (60.2) 1.0
A allele 184 (36.5) 87 (33.5) 97 (39.8) 1.31 (0.91–1.89) 0.165
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Bold values indicate the P-value < 0.05

Data are expressed as numbers with percentages. The Chi-square test was applied

BLCA bladder carcinoma, HWE Hardy–Weinberg equilibrium, OR odds ratio, CI confidence intervals

Impact of AGO1*rs595961 and AGO2*rs4961280 Variants on Cancer Risk

BLCA patients demonstrated a significant association with the AGO1 (rs595961; c.1264-25A > G) variant compared to cancer-free controls under allelic (57.8% vs. 47.7%; OR = 1.50, 95%CI 1.06–2.13, p = 0.026), and recessive (42.6% vs. 27.0%; OR = 2.01, 95%CI 1.17–3.39, p = 0.010) models, Table 3. However, no statistically significant difference was observed between BLCA patients and cancer-free controls concerning the (rs4961280; g.140637315C > A) variant under all hereditary genetic models (p > 0.05) Fig. 2.

Table 3.

Genetic association models of the AGO1*rs595961 and AGO2*rs4961280 variants with the risk of bladder carcinoma

Model Genotypes Cancer-free controls BLCA patients Crude OR (95% CI) P-value Adjusted OR (95% CI) P-value
1. AGO1 (rs595961; c.1264-25A > G) n (%) 130 n (%) 122
Codominant A/A 41 (31.5) 33 (27.1) 1.0 0.028 1.0 0.030
A/G 54 (41.5) 37 (30.3) 0.85 (0.46–1.58) 0.83 (0.44–1.55)
G/G 35 (27.0) 52 (42.6) 1.85 (0.99–3.46) 1.80 (0.96–3.38)
Dominant A/A 41 (31.5) 33 (27.1) 1.0 0.430 1.0 0.490
A/G + G/G 89 (68.5) 89 (73.0) 1.24 (0.72–2.14) 1.21 (0.70–2.10)
Recessive A/A + A/G 95 (73.1) 70 (57.4) 1.0 0.009 1.0 0.010
G/G 35 (27.0) 52 (42.6) 2.02 (1.19–3.42) 2.01 (1.17–3.39)
Log-additive –- –- –- 1.38 (1.01–1.89) 0.044 1.36 (0.99–1.87) 0.054
2. AGO2 (rs4961280; g.140637315C > A) n (%) 130 n (%) 122
Codominant C/C 59 (45.4) 43 (35.2) 1.0 0.260 1.0 0.201
C/A 55 (42.3) 61 (50.0) 1.52 (0.89–2.60) 1.58 (0.92–2.71)
A/A 16 (12.3) 18 (14.8) 1.54 (0.71–3.37) 1.66 (0.75–3.66)
Dominant C/C 59 (45.4) 43 (35.2) 1.0 0.101 1.0 0.073
C/A + A/A 71 (54.6) 79 (64.8) 1.53 (0.92–2.54) 1.60 (0.96–2.67)
Recessive C/C + C/A 114 (87.7) 104 (85.2) 1.0 0.570 1.0 0.490
A/A 16 (12.3) 18 (14.8) 1.23 (0.60–2.54) 1.29 (0.62–2.69)
Log-additive –- –- –- 1.31 (0.91–1.89) 0.140 1.36 (0.94–1.97) 0.101
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Bold values indicate the P-value < 0.05

Data are expressed as numbers with percentages. Adjusted by age and gender. The Chi-square test was applied

BLCA bladder carcinoma, OR odds ratio, CI confidence intervals

Fig. 2.

Fig. 2

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Genetic association models for the AGO1*rs595961 and AGO2*rs4961280 genetic variants among BLCA patients compared to cancer-free controls. (1) Genetic frequencies of the AGO1 (rs595961; c.1264-25A > G) variant among cancer patients compared to healthy controls, and (2) Genetic frequencies of the AGO2 (rs4961280; g.140637315C > A) variant among cancer patients compared to healthy controls

Association of AGO1*rs595961 and AGO2*rs4961280 Variants with the Clinical, and Laboratory Variables

Both AGO1 (rs595961; c.1264-25A > G) and AGO2 (rs4961280; g.140637315C > A) variants showed no significant difference among BLCA patients in relation to clinical, biochemical, and hematological parameters (p > 0.05) Table 4.

Table 4.

Genotypic frequencies of AGO1*rs595961 and AGO2*rs4961280 variants stratified by the clinical and laboratory variables among BLCA patients

Parameter* AGO1 (rs595961; c.1264-25A > G) AGO2 (rs4961280; g.140637315C > A)
A/A (n = 33) A/G (n = 37) G/G (n = 52) p-value C/C (n = 43) C/A (n = 61) A/A (n = 18) p-value
I. Demographic and clinical characteristics
1. Age, years Median (IQR) 58.0 (44.0–66.0) 63.0 (54.0–68.0) 59.5 (51.0–68.0) 0.246 63.0 (54.0–67.0) 58.0 (49.0–66.0) 63.0 (46.0–68.0) 0.752
2. Age groups (≤ 55/ > 55) n (%)/n (%) 15 (45.5)/18 (54.5) 11 (29.7)/26 (70.3) 19 (36.5)/33 (63.5) 0.395 13 (30.2)/30 (69.8) 25 (41.0)/36 (59.0) 7 (38.9)/11 (61.1) 0.525
3. Gender (Male/Female) n (%)/n (%) 24 (72.7)/9 (27.3) 32 (86.5)/5 (13.5) 44 (84.6)/8 (15.4) 0.263 39 (90.7)/4 (9.30) 47 (77.1)/14 (22.9) 14 (77.8)/4 (22.2) 0.181
II. Biochemical measurements
1. ALT, U/L Median (IQR) 20.0 (11.0–29.0) 18.0 (13.0–30.0) 16.0 (10.0–26.5) 0.291 17.0 (11.0–27.0) 19.0 (12.0–29.0) 23.0 (13.0–34.0) 0.667
2. AST, U/L Median (IQR) 22.0 (15.0–29.0) 23.0 (17.0–27.0) 18.0 (14.0–26.5) 0.224 18.0 (14.0–26.0) 20.0 (17.0–27.0) 20.5 (15.0–34.0) 0.444
3. Albumin, g/l Median (IQR) 41.0 (38.0–44.0) 41.0 (36.0–43.0) 40.0 (37.0–43.0) 0.419 41.0 (38.0–44.0) 41.5 (38.0–43.0) 37.5 (33.0–42.0) 0.167
4. ALP, U/L Median (IQR) 76.0 (59.0–90.0) 85.0 (67.0–108.0) 82.0 (62.5–100.5) 0.436 83.0 (58.0–108.0) 81.0 (66.0–97.0) 73.5 (65.0–89.0) 0.817
5. Total bilirubin, mg/dl Median (IQR) 0.40 (0.30–0.90) 0.60 (0.40–0.80) 0.55 (0.40–0.80) 0.424 0.50 (0.40–0.90) 0.60 (0.40–0.80) 0.43 (0.40–0.70) 0.541
6. Creatinine, mg/dl Median (IQR) 1.00 (0.80–1.10) 0.90 (0.80–1.20) 1.00 (0.80–1.30) 0.832 1.10 (0.80–1.30) 0.90 (0.80–1.10) 1.00 (0.80–1.70) 0.063
7. Sodium levels (Na), mmol/l Median (IQR) 137.0 (136.0–140.0) 136.0 (134.0–139.0) 138.0 (136.0–141.0) 0.204 138.0 (135.0–140.0) 137.0 (135.0–140.0) 137.5 (134.0–140.0) 0.883
8. Potassium levels (K), mmol/l Median (IQR) 4.20 (3.80–4.30) 4.10 (3.90–4.40) 4.30 (3.90–4.70) 0.057 4.30 (3.90–4.60) 4.20 (3.90–4.40) 4.01 (3.70–4.30) 0.103
III. Hematological parameters
1. RBCs, × 1012/L Median (IQR) 4.80 (4.40–5.10) 4.70 (4.30–5.20) 4.79 (4.40–5.28) 0.756 4.70 (4.50–5.20) 4.80 (4.35–5.24) 4.71 (4.20–5.01) 0.729
2. Hematocrit (HCT), % Median (IQR) 40.3 (35.3–42.7) 39.3 (36.9–43.9) 40.4 (35.8–43.3) 0.909 40.3 (36.7–43.5) 39.9 (36.7–44.0) 39.3 (35.1–42.5) 0.642
3. Hemoglobin, g/dl Median (IQR) 13.3 (11.6–15.0) 13.3 (12.5–15.3) 13.2 (11.7–14.8) 0.948 13.3 (11.9–14.8) 13.2 (12.0–15.2) 13.4 (11.6–14.3) 0.793
4. MCV, fL Median (IQR) 82.5 (79.2–86.0) 84.3 (81.4–87.8) 83.4 (79.3–88.6) 0.554 83.5 (79.2–87.2) 84.4 (80.4–88.9) 81.7 (77.9–86.0) 0.264
5. MCH, pg Median (IQR) 28.4 (26.5–30.0) 29.1 (27.5–30.2) 28.5 (25.6–29.8) 0.488 28.4 (25.9–29.7) 29.0 (26.2–30.6) 28.7 (26.6–29.4) 0.519
6. MCHC, g/dl Median (IQR) 34.1 (32.9–34.7) 33.9 (33.1–34.6) 34.0 (32.1–34.7) 0.749 33.9 (32.5–34.6) 34.0 (32.9–34.7) 33.8 (32.6–34.9) 0.801
7. WBCs, × 109/L Median (IQR) 7.40 (6.10–9.30) 7.80 (6.20–9.30) 7.60 (6.55–10.1) 0.625 7.50 (6.40–9.50) 7.51 (6.01–9.30) 7.65 (7.30–10.5) 0.667
8. Neutrophils, % Median (IQR) 60.0 (55.7–66.1) 62.0 (55.7–66.6) 63.1 (54.8–70.7) 0.709 61.6 (51.0–69.0) 61.3 (56.4–70.4) 63.2 (60.0–65.9) 0.366
9. Absolute Neutrophils, × 109/L Median (IQR) 4.50 (3.54–5.70) 4.74 (3.64–5.89) 4.71 (3.79–6.54) 0.659 4.48 (3.57–5.88) 4.76 (3.74–6.07) 4.99 (4.25–6.74) 0.554
10. Lymphocytes, % Median (IQR) 29.5 (23.6–34.0) 29.0 (21.1–34.4) 27.4 (22.0–35.6) 0.846 29.7 (22.5–37.9) 29.0 (21.1–34.0) 27.9 (25.0–32.0) 0.436
11. Absolute Lymphocytes, × 109/L Median (IQR) 2.41 (1.64–2.84) 2.38 (1.63–2.72) 2.16 (1.56–2.97) 0.945 2.41 (1.67–2.96) 2.18 (1.58–2.73) 2.37 (1.28–2.84) 0.682
12. Platelet count, × 109/L Median (IQR) 270.0 (177.0–326.0) 239.0 (199.0–291.0) 222.0 (176.0–300.5) 0.491 261.0 (197.0–321.0) 222.0 (172.0–311.0) 246.5 (186.0–301.0) 0.243
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Data are expressed as numbers (%) or median (IQR). Chi-square and Kruskal–Wallis tests were applied

IQR interquartile range, BLCA bladder carcinoma, ALT alanine transaminase, AST aspartate transaminase, ALP alkaline phosphatase, RBCs red blood cells, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, MCHC mean corpuscular hemoglobin concentration, WBCs white blood cells

In Silico Data Analysis of the AGO1*rs595961 and AGO2*rs4961280 Variants

The functional role of the argonaute family sheds light on their mechanisms in gene silencing of target messenger RNAs. The AGO1 gene (OMIM #606,228) is located on the short arm of chromosome 1p34.3 along the plus strand, while the AGO2 gene (OMIM #606,229) is positioned within the elongated arm of chromosome 8q24.3 along the reverse strand (Fig. 3). Analysis of AGO1 and AGO2 gene expression indicated that AGO1 showed lower distribution among tumor tissues compared to normal tissues (TNMplot database, https://tnmplot.com/analysis/). Additionally, the AGO1 gene is traversed about 60,772 bases [Chr.1: 35,869,761–35,930,532], while the AGO2 gene is spanned about 122,158 bases [Chr.8: 140,520,156–140,642,313]. According to the human genome assembly GRCh38.p13, the AGO1 gene comprises eight splice variants, with the main transcript (ENST00000373204.6) consisting of nineteen exons and eighteen introns. In contrast, the AGO2 gene contains ten splice variants, with the main transcript (ENST00000220592.10) involving nineteen exons and eighteen introns. The AGO1*rs595961 (c.1264-25A > G) variant is situated in the tenth intron, while the AGO2*rs4961280 (g.140637315C > A) variant is in the promoter region.

Fig. 3.

Fig. 3

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Computational bioinformatic frameworks of the AGO1 and AGO2 genes. a Differential gene expression analysis for AGO1 indicated lower expression among tumor tissues compared to normal tissues. b AGO1 expression analysis among different tumor stages. c Differential gene expression analysis for AGO2 among tumor tissues compared to normal tissues. d AGO2 expression analysis among different tumor stages. e–f Correlation analysis for AGO1 expression compared to AGO3 and AGO4. g Protein–protein interactions of the AGO1 and AGO2 proteins utilizing STRING database. h Gene–gene interactions of the AGO1 and AGO2 genes using GeneMANIA database. i Subcellular localization of the AGO1 protein, with darker colors indicating more abundance. j Subcellular localization of the AGO2 protein, with darker colors indicating more abundance. k The crystalline structure of human AGO1 protein using Protein Data Bank. l The crystalline structure of human AGO2 protein using Protein Data Bank. m Survival analysis with high and low AGO1 expression. n Survival analysis with high and low AGO2 expression. [Data source: TNMplot database, Ensembl.org, NCBI database, STRING version 11.5, GeneMania database, Compartment database, Protein Data Bank, and Kaplan–Meier plotter database]

Protein–protein interactions of the AGO1/2 family have essential functions in siRNA loading on RISC involved in RNA interference, miRNA-mediated inhibition of translation, pre-miRNA processing, gene silencing by miRNA, miRNA biogenesis pathway, RISC-loading complex, competing exogenous RNAs, and negative regulation of gene expression [Data source: STRING database, version 11.5]. Gene–gene analysis indicated that AGO1/2 genes are involved in gene silencing by miRNA, post-transcriptional gene silencing, regulatory RNA binding, and positive regulation of mRNA catabolic processes [Data source: GeneMANIA database]. The subcellular localizations of AGO1 and AGO2 proteins were found to be abundant in the nucleus, cytosol, and extracellular space [Data source: Cellular compartment database]. Additionally, the crystal structures of AGO1/AGO2 proteins revealed their binding with target miRNAs [Data source: Protein Data Bank]. Lastly, the Kaplan–Meier plotter represented the survival rate of AGO1 and AGO2 in high and low expression over time (p = 0.006 and 0.380, respectively) [Data source: Kaplan–Meier Plotter database].

Discussion

The mechanism of canonical miRNA biogenesis plays a crucial role in deciphering the enigmatic pathway of miRNA components and the progression of various cancerous disorders, particularly bladder carcinoma [17, 32]. Generally, the activation of the RNA-induced silencing complex (RISC) with the help of argonaute proteins can catalyze specific factors that have an essential function in the destabilization and silencing of target mRNAs [43, 44]. Numerous articles have investigated the association of canonical AGO1/2 genes with susceptibility to different cancerous diseases, but with inconclusive results [31, 45, 46].

After thorough literature review of published reports examining the involvement of AGO1 (rs595961; c.1264-25A > G) and AGO2 (rs4961280; g.140637315C > A) variants in tumorigenesis, our team observed that this work is the first to explore the potential mechanism of these two variants concerning susceptibility to bladder carcinoma among Egyptian subjects. Moreover, this study revealed a significant correlation between the AGO1 (rs595961; c.1264-25A > G) variant with elevated risk of bladder carcinoma compared to cancer-free controls under allelic (OR = 1.50, p = 0.026), and recessive (OR = 2.01, p = 0.010) models. Conversely, BLCA patients carrying the AGO2 (rs4961280; g.140637315C > A) variant did not show a statistically significant difference compared to cancer-free controls under various hereditary association models (p > 0.05). Recently, two eligible meta-analysis reports examined the susceptibility of AGO1*rs595961 and AGO2*rs4961280 variants with cancer development in different ethnic populations but reached unresolved conclusions [17, 45]. In a meta-analysis study exploring the contribution of different miRNA biogenesis genes to increased carcinoma risk among various ethnic subjects, the authors found no evidence associating AGO1*rs595961 and AGO2*rs4961280 variants with increased carcinoma risk among overall populations. After subgroup stratification based on ethnicity, they discovered a significant association between the elevated risk of AGO1 (rs595961; c.1264-25A > G) variant and cancer progression under allelic (OR = 1.44, and p = 0.002) model among Asian subjects. On the other hand, the authors demonstrated a statistically significant correlation between the AGO2 (rs4961280; g.140637315C > A) variant and protection against cancer development under allelic (OR = 0.79, and p = 0.048) model among European subjects [17]. Another meta-analysis report examined the involvement of AGO1*rs595961 and AGO2*rs4961280 variants concerning susceptibility to cancer risk among different ethnic subjects. They found no clear correlation between these two variants and increased carcinoma risk among overall subjects (p > 0.05). In contrast, the AGO1 (rs595961; c.1264-25A > G) variant conferred protection against lung carcinoma development under allelic and recessive models (OR = 0.69 and 0.65, respectively) [45].

Due to the inconsistency in these findings, our team was inspired to conduct a small-scale meta-analysis comparison of all pertinent studies examining the relationship between the AGO1*rs595961 and AGO2*rs4961280 variants with susceptibility to cancer risk across various ethnic populations (Fig. 4). In addition to our primary research, this comparison encompassed studies on various types of cancer, including lung carcinoma [28, 29], chronic lymphatic leukemia [46], acute lymphatic leukemia [47], renal cell carcinoma [33], prostate carcinoma [31], non-Hodgkin lymphoma [48], nasopharyngeal carcinoma [49], and bladder carcinoma [32]. Interestingly, this comparison revealed a significant association of the AGO1 (rs595961; c.1264-25A > G) variant with elevated risk of carcinoma among overall subjects (OR = 1.43, and p = 0.023) under recessive model. On the contrary, our team could not establish a clear correlation between the AGO2 (rs4961280; g.140637315C > A) variant and carcinoma progression among overall subjects (OR = 0.95, and p = 0.346) under dominant model. In line with our results, we found a single study that reported a statistically significant difference between the AGO1*rs595961 variant and increased risk of acute lymphatic leukemia (OR = 2.35, and p = 0.047) [47]. On the other hand, numerous reports showed no significant association of the AGO2*rs4961280 variant and the susceptibility to cancer risk among different ethnic populations [29, 31–33, 48, 49].

Fig. 4.

Fig. 4

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Pooled results for the association between AGO1*rs595961 and AGO2*rs4961280 variants stratified by different cancerous disorders under a The recessive model of the AGO1 (rs595961; c.1264-25A > G) variant, and b The dominant model of the AGO2 (rs4961280; g.140637315C > A) variant

Additionally, our team identified a single report studying the involvement of the AGO1*rs595961 and AGO2*rs4961280 variants with the progression of bladder carcinoma, and they observed no substantial association with these variants and increased risk of bladder carcinoma among Chinese subjects (p > 0.05) [32]. Based on these prior findings, we could consider that the AGO1 (rs595961; c.1264-25A > G) variant might contribute to an increased risk of bladder carcinoma, while the AGO2 (rs4961280; g.140637315C > A) variant could not implicate a significant correlation with bladder carcinoma. Lastly, concrete limitations were raised during this study, including the relatively small sample size and the lack of data concerning AGO1 and AGO2 gene expressions.

Conclusion

In summary, this current research showed a significant association with the AGO1*rs595961 variant among patients with bladder carcinoma compared with cancer-free controls under allelic, and recessive models. On the other hand, the AGO2*rs4961280 variant did not indicate significant correlation with elevated risk of bladder carcinoma among Egyptian subjects.

Authors contribution

RME: Conceptualization, Methodology, Software, Formal analysis, Data curation, Validation, Investigation, Writing – review & editing. HMK: Methodology, Investigation, Writing – review & editing. AFS: Data curation, Formal analysis, Methodology, Validation, Writing – review & editing. KMO: Project administration, Formal analysis, Methodology, Writing – original draft, Data curation.

Data availability

The dataset used in the preparation of this study will be available from the corresponding author upon reasonable request.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethical Approval and Consent to Participate

All procedures accomplished in this study were approved with the ethical guidelines of the research committee and with the 1964 Helsinki declarations and its guidelines. Informed consent was acquired from all the participants that enrolled in this study.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The dataset used in the preparation of this study will be available from the corresponding author upon reasonable request.

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