Role of Specific miRNA Expression and Nuclear Kappa B Gene Polymorphism as Potential Diagnostic Markers for Chronic Myeloid Leukemia

Abstract

Objective

This study assesses microRNA-21 (miR-21) and microRNA-302 (miR-302) levels and examines their association with a nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) gene single nucleotide polymorphism (SNP) in chronic myeloid leukemia (CML) patients, aiming to identify these miRNAs as potential diagnostic biomarkers. The findings could contribute to improved diagnostic and treatment approaches.

Subject and Methods

This study involved 65 patients with CML at Al-Basheer Hospital, Amman, Jordan, and 30 healthy controls. Hematological parameters were analyzed via complete blood count. Gene expression was assessed by real-time PCR to analyze miRNA and NF-KB SNPs in patients’ plasma.

Results

A highly significant difference (p < 0.0001) was observed, indicating that miR-21 expression was higher in CML patients compared to the controls. miR-302 levels were lower in CML patients. The NF-κB Del/Del genotype was associated with a higher white blood cell count compared to the Ins/Ins and Ins/Del genotypes. Platelet counts varied among the CML patients with three polymorphisms.

Conclusion

miR-21 is elevated in CML patients, suggesting an oncogenic role. Our results suggest that miR-302 may serve as a prognostic and diagnostic biomarker. The NF-κB1 gene rs28362491 Del/Del genotype may be associated with an increased risk of CML. Identification of these biomarkers can contribute significantly to improve both diagnosis and treatment strategies.

Highlights of the Study

• microRNA-21 was significantly elevated in chronic myeloid leukemia patients compared with healthy controls, suggesting it may function as an oncogenic factor.

• As a tumor suppressor gene, microRNA-302 could serve as a prognostic and diagnostic biomarker.

• There may be a connection between the nuclear factor kappa B gene rs28362491 deletion/deletion genotype and a higher risk of chronic myeloid leukemia.

Introduction

Cancer significantly affects global health and the quality of life. It is considered to result from gene mutations that change cell function, and the chemical substances may play a role in causing mutations and thus resulting in cancer cells [1]. Uncontrolled cell proliferation causes various cancers, damages tissues, disrupts the balance of healthy cells, and spreads to organs. The five main cancer types are carcinoma, sarcoma, myeloma, leukemia, and lymphoma, based on histological features [2]. Leukemia is a hematologic cancer caused by abnormal proliferation of leukocytes at different stages. It can be primary or secondary, based on abnormal leukocyte production, proliferation rate, and the cell origin. The disease is classified as acute (precursor cells) or chronic (mature cells) and as myeloid or lymphoid [3].

CML is a myeloproliferative neoplasm (MPN) characterized by uncontrolled myeloid cell proliferation at various stages of maturation, usually caused by a translocation between chromosomes 9 and 22 involving the proto-oncogene Abelson. This results in the BCR-ABL fusion oncoprotein (Philadelphia chromosome), which activates tyrosine kinase signaling and promotes clonal expansion of myeloid cells [4].

NF-κB proteins form a family of transcription factors in eukaryotic cells that regulate essential genes, trigger inflammation, stimulate carcinogenesis, and promote cancer progression [5, 6]. NF-κB signaling is crucial in leukemogenesis, regulating over 150 genes related to inflammation, survival, and growth, including cytokines like IL-1β and TNF-α [7]. Functional polymorphisms in the NFKB1 gene, particularly the -94 ins/del ATTG variant (rs28362491), can influence NF-κB expression and activity [8]. This may contribute to malignant transformation and impact miRNA regulation, such as miR-21 [9].

Growing evidence indicates that mis-regulated miRNAs are a key factor in hematologic malignancies through their role in gene regulation [10, 11]. Research shows miRNA-21 is often upregulated in tumors, promoting growth and invasiveness through multiple pathways [12]. Recent studies confirm its oncogenic potential and increased expression in aggressive cancers [13, 14]. miR-21 is a diagnostic biomarker and an oncogene in multiple cancers, highlighting its role in early diagnosis and as a therapeutic target [15, 16]. Members of the miR-302/367 cluster, including miR-302a-d and miR-367, target genes involved in neoplastic diseases, aiding immune evasion, proliferation, angiogenesis, invasion, and metastasis. Most of these act as tumor suppressors [17]. Bioinformatic tools (TargetScan, miRDB, and miRTarBase) identified high-confidence target genes of miR-21 and miR-302, including apoptotic regulators such as PTEN, PDCD4, TPM1, RECK, and PI3K/AKT regulators such as miR-21 and miR-302. The miR-302 cluster is predicted to regulate cell cycle and stemness genes, such as CDK2, CDK4, E2F1, and BMI1 [18, 19]. These interactions support the biological relevance of our findings, indicating that changes in miR-21 and miR-302 may influence proliferative and survival pathways in CML [5, 17, 20–22]. Although miR-21, miR-302, and NF-κB are all associated with cancer biology, their combined diagnostic and prognostic roles in CML, especially regarding the rs28362491 polymorphism, remain unclear. The study of this polymorphism could yield new insights into the molecular processes underlying disease progression and treatment resistance [23]. Altered miRNA expression may impact CML development and treatment. Although miR-21 and miR-302 have been studied separately in various cancers, their combined analysis with the NFKB1-94 ATTG (rs28362491) polymorphism has not been done in CML. This study is the first to correlate miR-21 and miR-302 expression with NFKB1 genotypes in CML patients, offering a novel perspective on how deregulation of the NF-kB pathway influences the expression of oncogenic and tumor-suppressive miRNAs.

This study aims to measure the expression of miRNA-21 and miRNA-302, as well as the NF-kB SNP, in CML patients. It investigates the correlation between miRNA-21 and miRNA-302 levels and the SNP in the NF-kB gene in CML patients. Additionally, the study also examines the diagnostic efficiency of miRNA-21 and miRNA-302 in CML.

Materials and Methods

Patient Cohort, Criteria, and Design

This is a prospective, cross-sectional, controlled study, which included 95 Jordanian participants (30 healthy controls and 65 CML patients receiving treatment at Al Basheer Hospital’s hematology and oncology clinic in Amman, Jordan). The participants were randomly selected and included both men and women. Two blood samples (2–3 mL) were collected from each patient using EDTA anticoagulant tubes. The samples were kept at 0–4°C during transport to the Molecular Biology Laboratory at Al-Ahliyyah Amman University. One sample underwent a complete blood count (CBC) on the same day using Sysmex XP-300. The sample was centrifuged at 15,000 rpm for 10 min, and plasma was stored at −80°C for future gene expression analysis using qPCR. Gene expression studies used miRNA and SNP NF-κB assays.

Ethical Consideration

The study, approved by the Faculty of Pharmacy and Allied Medical Sciences at Al-Ahliyya Amman University (IRB: AAU/3/8/2022-2023), was conducted in accordance with the Declaration of Helsinki. Informed written consent was obtained and an Arabic questionnaire was used to collect medical, sociodemographic, and behavioral data after explaining the research aims, procedures, duration, and benefits to the participants. All the information related to patients remained confidential, accessible only to investigators, and was stored electronically only on the investigator’s computer.

Complete Blood Count

CBC analysis was performed immediately after sample collection using the Sysmex XP-300 automated hematology analyzer. Parameters measured included white blood cell count (WBC), lymphocyte count, neutrophil count, and the platelet count.

Plasma Separation

Whole blood was centrifuged at 1,500 g for 10 min at 4°C. Plasma was transferred to RNase-free tubes and centrifuged again at 12,000 g for 10 min to remove residual cells. Aliquots were stored at −80°C until RNA and DNA extraction.

miRNA Extraction and Isolation

miRNA was extracted from blood samples using the miRNeasy Serum/Plasma Advanced Kit (cat No. 217204, QIAGEN, Venlo, The Netherlands), following the manufacturer’s instructions. A synthetic spike-in control (UniSp6) was added to monitor extraction efficiency. It was evaluated using a NanoDrop spectrophotometer to determine the purity and concentration of the extracted miRNA. In accordance with the manufacturer’s protocol, the miRNeasy Plasma Advanced Kit was used to extract high-quality, pure miRNA, including miR-16-5p, miR-142-3p, and miR-144-3p. This kit employs spin-column technology with a silica membrane, in which the sample binds to total RNA upon application to the column.

Complementary Deoxyribonucleic Acid Synthesis

Complementary deoxyribonucleic acid was synthesized using the miRCURY LNA RT Kit (Qiagen, cat. No. 339340) and the Applied Biosystems reverse transcription system, allowing for complementary deoxyribonucleic acid production from serum, plasma, and other biofluids. The RT reactions were performed in 200 μL PCR tubes, following the manufacturer’s guidelines. The reaction mixture consisted of 5x miRCURY RT reaction buffer (2 μL) + Synthetic RNA spike-ins (0.5 μL) + 10x miRCURY RT Enzyme mix (1 μL). The reverse transcription reaction involved incubating at 40°C for 60 min, followed by heating at 95°C for 5 min to inactivate the enzyme, and then storing the mixture at 4°C until the next step.

Relative Quantification of Plasma miRNA Levels

Plasma miRNA levels were quantified using quantitative real-time PCR (qRT-PCR) with Fast SYBR Green and analyzed using the Pfaffl method [24]. When both reference and target DNA double per cycle, the amplification efficiency is 2, with a standard curve slope of −3.32.

SNP Assay

DNA Extraction

Plasma DNA was extracted using PureLink Genomic DNA Kits. Initially, 200 μL of plasma was combined with 200 μL of digestion buffer and 20 μL of proteinase K, and the mixture was incubated at 56°C for 15 min. Subsequently, 200 μL of absolute ethanol was added, and the mixture was centrifuged at 13,000 rpm for 3 min in a PureLink spin column. The collection tubes were then discarded, and the DNA was washed with 500 μL of buffer 1, followed by a second centrifugation at the same speed. The sample was further washed with 500 μL of buffer 2. The supernatant was removed, and the DNA pellet was resuspended in 100 μL of elution buffer.

Determination of DNA Concentration and Purity

The concentration and purity of the extracted DNA were evaluated using a NanoDrop spectrophotometer by measuring optical densities at 260 nm and 280 nm. Afterward, the DNA was stored at −80°C for future use.

Real-Time PCR Procedure

To analyze NF-κB1 (rs28362491; -94 ins/Del CGT​GCT​GCC​TGC​GTT​CCC​CGA​CC[ATTG/-] ATT​GGG​CCC​GGC​AGG​CGC​TTC​CTG), TaqMan^® Universal PCR Master Mix (Thermo Fisher) was used. The assay included VIC- and FAM-labeled probes, two forward primers, and one reverse primer. After thawing the DNA, 5 μL was combined with 10 μL of master mix, 1 μL of primer probe, and 4 μL of nuclease-free water. qPCR was performed with an initial denaturation step at 95°C for 1 min, followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 5 min using the Rotor-Gene Q RT-PCR system (Qiagen).

Results interpretation is based on the colors from the PCR reaction. A green color (FAM) without yellow indicates the presence of the Del/Del allele. A yellow color (VIC) without green suggests the Ins/Ins allele. If both green and yellow are observed, it indicates the presence of the Del/Ins allele.

Statistical Analysis

A one-way ANOVA was used to evaluate group differences, with Dunnett’s post hoc test used for further analysis. Data analysis was performed using SPSS 22, with results reported as means ± SD across 3–4 experiments. GraphPad Prism was used to perform ANOVA comparisons between the control and treatment groups, followed by Dunnett’s post hoc test. Significance was set at p < 0.05, with p < 0.001 considered highly significant.

Results

This study presents the sample demographics, PCR results showing fold changes in miRNA-21 expression, clinical characteristics, and genotype and allele frequencies of the -94 ATTG polymorphism (rs28362491). CML sample expression levels were normalized to the internal control miRNA and compared to the control group’s mean. This approach ensures the fold change accurately reflects expression differences between patients and healthy controls.

Study Sample Characteristics

Table 1 presents the characteristics of the study sample and the PCR results. Among the participants, 55.3% were female, while males accounted for 44.6%.

Table 1.

Characteristics of the study sample and PCR results

​	Patient	Control	χ2 test
	n (%)	n (%)	p value
Sex
F	36 (55.3)	18 (58.0)	0.6728
M	29 (44.6)	12 (38.7)	​
PCR
Homozygous VIC (yellow) Ins/Ins	16 (34.8)	3 (12)	​
Heterozygous Ins/Del	11 (23.9)	15 (60)	​
Homozygous FAM (green) Del/Del	19 (41.3)	7 (28)	​

PCR results show that only 23.9% of the patients had a heterozygous Del/Ins, 34.7% had a homozygous VIC (yellow) Ins/Ins, and 41.3% had a homozygous FAM (green) Del/Del. In contrast, 60% of the control group had a heterozygous Del/Ins, 12% had a homozygous VIC (yellow) Ins/Ins, and 25% had a homozygous FAM (green) Del/Del.

miRNA Expression Folds and Clinical Characteristics

Table 2 presents the fold changes in miRNA expression in patients and control subjects. Both miRNA-21-5p and miRNA-302 showed significant differences in patients with CML compared with the control group. Data are presented in Table 2 as the mean ± SD for both the patient and control groups. The data were analyzed using an unpaired t test.

Table 2.

miRNA expression fold in patients and the control group

​	Patients	Control group	p value
	N (65)	N (30)
miRNA 21-5p	(70,763.5±87.8)	(1,741.617±131.55)	<0.0001****
miRNA 302	(0.003±0.1)	(89,955.9±19.55)	<0.0001****

Asterisks indicate the degree of significance where necessary.

WBC Counts in the Patient and Control Groups

Figure 1 illustrates the CBC parameters. The findings indicate that the white blood cell (WBC) count in the patient cohort is significantly elevated compared to that in the control group. Nonetheless, the analysis showed no notable difference in lymphocyte counts between patients and controls. Furthermore, neutrophil counts also do not differ significantly between the two groups. Consistent with the WBC results, platelet counts differ significantly between the patient cohort and the control group.

Fig. 1.

Number of WBC, lymphocyte, neutrophil, and platelet counts between the patient and the control groups. Results are shown as mean ± SD. t test was used for statistical analysis. Asterisks indicate the degree of significance where necessary. **p < 0.005 compared to the control group.

Genotype and Allele Frequencies of -94 ATTG Polymorphism (rs28362491)

Table 3 presents the genotypic and allelic frequencies of the -94 ATTG polymorphism (rs28362491) in the patient and control groups. To enhance clarity, values shown in brackets in Table 3 indicate the percentage of patients within each genotype category.

Table 3.

Hematological parameters among CML patients according to NFKB1 rs28362491 genotype

Genotypes	Patients %	Control group %	p value
	N (65)	N (30)
Homozygous Ins/Ins	16 (34.7)	3 (12)	​
Heterozygous Ins/Del	11 (23.9)	15 (60)	0.0061**
Homozygous Del/Del	19 (41.3)	7 (25)	0.4805
Ins allele frequency	27 (47)	18 (45)	​
Del allele frequency	30 (52.6)	22 (55)	0.8391

Values outside brackets represent the absolute number of patients, while the numbers inside brackets indicate the percentage (%) within each genotype group. Asterisks indicate the degree of significance where necessary.

These findings indicate a significant difference in genotype distribution between patients and the control group; however, there was no notable variation in allele frequencies for the -94 ATTG Ins/Ins and Del/Del polymorphisms (rs28362491) compared to the Ins/Del polymorphism.

Association between -94 ATTG Polymorphism (rs28362491) and Plasma miRNA

In contrast, an association was detected between the -94 ATTG polymorphism (rs28362491) and plasma miRNA 21-5p levels in patients (Table 4). The study demonstrated significant differences between the heterozygous Ins/Del allele and the homozygous Del/Del and Ins/Ins alleles in 21-5p levels. Table 4 also indicates that homozygous Del/Del allele is the predominant polymorphism in the patient group. The data related to association between the -94 ATTG polymorphism (rs28362491) and serum 302 levels in patients are also presented in Table 4. The results showed no significant difference in the serum levels among the Ins/Del, Del/Del, and Ins/Ins alleles. However, a significant difference was observed between the homozygous Del/Del allele and the homozygous Ins/Ins allele in terms of serum levels of 302 levels.

Table 4.

Association of the −94 ATTG polymorphism (rs28362491) with miRNA-21 (A) and miRNA-302 (B) expression folds in patients

Genotypes	Plasma miRNA 21-5p expression fold (mean±SD)	p value vs. Ins/Ins	p value vs. Ins/Del
(A)
Homozygous Ins/Ins	4.3±0.5	​	ns
Heterozygous Ins/Del	3.07±0.4	<0.0001***	​
Homozygous Del/Del	76.9±0.5	<0.0001***	<0.0001***

Genotypes	Serum miRNA 302 expression fold (Mean±SD)	p value vs. Ins/Ins	p value vs. Ins/Del
(B)
Homozygous Ins/Ins	1.22±0.5	​	ns
Heterozygous Ins/Del	1.13±0.4	0.1657 ns	​
Homozygous Del/Del	0.8±0.5	0.0077**	0.1805 ns

Values are presented as mean ± SD. Genotypes include Ins/Ins (homozygous insertion), Ins/Del (heterozygous), and Del/Del (homozygous deletion). Statistical comparisons were performed using one-way ANOVA followed by Dunnett’s post hoc test: “p value vs. Ins/Ins” indicates comparison with the Ins/Ins reference group. “p value vs. Ins/Del” indicates comparison with the Ins/Del reference group.

ns, not significant.

**p < 0.01; ***p < 0.001.

Association between -94 ATTG Polymorphism (rs28362491) and Different Blood Parameters

Figure 2 compares WBC counts among the three NFKB1 rs28362491 genotypes (Ins/Ins, Ins/Del, and Del/Del) in CML patients. Each bar illustrates the average WBC count for individuals in each genotype group, with error bars representing standard deviation. Notably, the Ins/Ins genotype exhibits significantly higher WBC levels compared to the Ins/Del and Del/Del genotypes. These results demonstrate that a significant difference existed between the homozygous Del/Del and both homozygous Ins/Ins and heterozygous Ins/Del of the -94 ATTG (rs28362491) in relation to lymphocyte counts in patients. This indicates that the polymorphism affected lymphocyte counts in these patients as lymphocyte counts were significantly lower in the Del/Del group than in the Ins/Ins and Ins/Del groups.

Fig. 2.

WBC counts were analyzed across patients with the three genotypes of the NFKB1 rs28362491 polymorphism: Ins/Ins, Ins/Del, and Del/Del. The Ins/Ins genotype showed notably higher WBC counts than the Ins/Del and Del/Del genotypes. Results are shown as mean ± SD. One-way ANOVA was used for statistical analysis. A p value <0.05 was considered statistically significant. Asterisks indicate the degree of significance where necessary.

There was no significant difference among the CML patients with the three polymorphisms (Del/Ins, Del/Del, and Ins/Ins) of the -94 ATTG (rs28362491) polymorphism in plasma neutrophil counts indicating that these polymorphisms did not affect neutrophil counts in the CML patients. In contrast, there was a significant difference among the three polymorphisms (Del/Ins, Del/Del, and Ins/Ins) of the -94 ATTG (rs28362491) polymorphism regarding plasma platelet counts in CML patients, suggesting that these polymorphisms may influence the platelet counts in these patients.

Discussion

miRNA-21 is widely recognized for its role in promoting cancer progression. It helps regulate cell proliferation, apoptosis (cell death), and inflammation, often resulting in abnormal cell growth or survival. The higher levels of miRNA-21 in the patient group suggest that it may contribute to the disease process. In these conditions, miRNA-21 can act as an oncogene, encouraging cell survival and contributing to a pro-inflammatory environment [25].

miRNA-302 generally helps regulate cell differentiation and is linked to maintaining the stem cell characteristics and controlling the cell cycle [26]. Lower levels of miRNA-302 in the patient group could suggest that the disease suppresses pathways involved in normal cell differentiation and repair. This reduction may be associated with a loss of control over cell growth and differentiation, a common feature of diseases like cancer, in which cells have difficulty differentiating properly [27].

This study examined the relationship between variations in NF-κB rs28362491 genotypes and allele frequencies, specifically the Ins/Ins, Ins/Del, and Del/Del variants of the NF-κB1 gene, miR-21-5p, and miR-302 levels in patients with CML from the Jordanian population. The frequency of the -94 ATTG polymorphism (rs28362491) alleles in the control group is important because it provides a baseline for comparison with CML patients. Knowing how Ins and Del alleles are distributed in healthy individuals can help in determining whether specific alleles or genotypes are more prevalent in patients, suggesting a potential link to the disease risk.

In this study, the control group showed a relatively even distribution of Ins and Del alleles. The absence of significant differences in allele frequencies between patients and controls suggests that this allele alone might not directly influence the risk of CML. Instead, differences in genotype distribution, such as the higher Del/Del frequency among CML patients, could indicate functional effects of the polymorphism, including changes in NF-κB expression that may play a role in disease development.

Our findings align with those of other studies on miR-21 across various cancer types. Hematological malignancies have been found to aberrantly express miRNAs, with distinct profiles compared to their normal counterparts. An attempt has been made to explain how various miRNAs may participate in the pathophysiology of these malignancies by modifying cancer-associated genes, acting directly as oncogenes or tumor suppressor genes, or acting as spectators or controllers of epigenetic pathways in cancer, as well as through the use of functional and target analyses and animal models [28]. Additionally, it has been demonstrated that miRNAs influence medication resistance and the prognosis of different subtypes of blood malignancies. The finding that nearly 50% of miRNA genes are located within genomic regions associated with cancer or fragile sites led researchers to hypothesize that miRNAs may contribute to the pathophysiology of cancer [29].

To contextualize our results within the broader oncology context, we compiled summary findings from earlier studies on miR-21, miR-302, and the NFKB1 rs28362491 polymorphism across different cancer types (Tables 5–7). These comparative statistics underscore the specific value of the current study, particularly regarding the coordinated dysregulation of these biomarkers in CML.

Table 5.

Summary of reported miR-21 findings across human cancers

Cancer type (mentioned in the article)	Expression pattern	Mechanisms/targets	Relevance to CML (from this article)	Key references
Glioblastoma	Upregulated	General oncogenic activity via multiple pathways	miR-21 is broadly oncogenic; this underpins its role in hematologic malignancies like CML	[13]
Colorectal cancer	Upregulated	miR-21 is linked to tumor development	Supports miR-21’s overall oncogenic profile; used as a comparative background	[14]
Prostate cancer	Upregulated	The role of a diagnostic biomarker is cited	Shows the biomarker potential of miR-21, similar to the argument used for CML	[15]
Lung cancer (NSCLC)	Upregulated	The NF-κB/miR-21/PTEN axis is implicated in other cancers, including NSCLC, and affects cisplatin sensitivity	The NF-κB → miR-21 → PTEN/PI3K/AKT pathway is a plausible mechanism in CML, based on analogous evidence	[21]
Breast cancer	Upregulated	miR-21 influences proliferation and apoptosis; PDCD4, involved in hematopoiesis and AML, is a target	Supports miR-21’s oncogenic role; this was associated with decreased PDCD4 and activated AKT signaling in CML	[30]
AML — NPM1-mutant	Upregulated	miR-21 is overexpressed in AML blasts, leading to downregulation of PDCD4	Direct hematologic precedent: similar miR-21 overexpression patterns support findings in CML	[31]
Other solid tumors (general statement)	miR-21 is “elevated in all solid cancers,” citing Volinia et al. [13] and reviews	PTEN and PDCD4 suppression lead to PI3K/AKT activation, a common mechanism	A generalized oncogenic mechanism to account for the elevated miR-21 levels in CML, as shown in their data	[10]

Table 7.

Summary of NFKB1 rs28362491 (−94 ATTG) polymorphism across cancers

Cancer type	Risk/association	Functional impact	Notes	Key references
Breast cancer	Mixed	Alters NF-κB1 transcription	Del allele → higher promoter activity	[5]
Lung cancer	Del allele ↑ risk	Chronic inflammation	NF-κB hyperactivation	[21]
Gastric cancer	Del/Del ↑ risk	Increased NF-κB1 activation	Supports carcinogenesis	Meta-analyses
Leukemia (general)	Limited data	Unclear	Under-studied	–
CML (current study)	Del/Del is overrepresented in patients	Higher miR-21, reduced miR-302, altered WBC counts	First study to show genotype-miRNA relationship	Current study

Table 6.

Summary of reported miR-302 findings across cancers

Cancer type (mentioned in the article)	Expression pattern	Mechanisms/targets	Relevance to CML (from this article)	Key references
Liver cancer	Downregulated	miR-302 is recognized as a tumor suppressor that inhibits growth and induces apoptosis in liver cancer (cited)	In CML, miR-302 is significantly decreased (patient mean 0.003 vs. 89,955.9), indicating loss of tumor-suppressive function	[17]
Breast cancer	Downregulated	miR-302 acts as a tumor suppressor in breast cancer (manuscript statement)	It was suggested that low miR-302 levels in CML may indicate downregulation of tumor suppressors and serve as diagnostic or prognostic biomarkers	[17]
Ovarian cancer	Downregulated	miR-302 has been reported to have tumor-suppressive functions	Reduced miR-302 expression in CML may indicate a loss of tumor-suppressor function, as seen in other cancers	[32]
Colon cancer	Downregulated	miR-302 has been reported to perform tumor-suppressive functions	Reduced miR-302 in CML may indicate loss of tumor-suppressor function, as seen in other cancers	[17]
Stem cell/pluripotency context	Highly expressed	The miR-302 cluster plays a role in reprogramming and regulating stemness	Altered miR-302 may affect hematopoietic differentiation processes and chronic myeloid leukemia (CML)	[32]
CML (this study)	Patients have lower circulating miR-302 than controls, especially with the rs28362491 Del/Del genotype, which shows even lower levels than Ins/Ins (p = 0.0077)	p < 0.0001 for the overall difference in miR-302 between patients and controls; genotype comparison p = 0.0077 (Del/Del vs. Ins/Ins)	miR-302 acts as a tumor suppressor in CML and could be a useful diagnostic or prognostic biomarker. Additionally, the association with rs28362491 suggests that NF-κB genotypes may affect miR-302 expression	–

These comparative studies indicate that miR-21 levels increased and miR-302 levels decreased are reported across diverse solid tumors. However, the combined evaluation of the two miRNAs and the functional polymorphism NFKB1 rs28362491 has never been performed in CML. According to our results, NF-κB-miRNA axis dysregulation may be an otherwise undetected contributing factor to CML pathogenesis. It could be used in the future as a multifactorial biomarker panel in clinical settings. The way miR-21 functions as an oncogene could be linked to its influence on the p53 pathway, a well-established gene crucial in cancer development [33]. Moreover, the results matched other findings in patients with AML nucleophosmin 1 gene (NPM1) − mutant as miR-21 was found to have overexpression [31].

The expression of programmed cell death 4 (PDCD4), a target of miRNA-21, and the levels of miRNA-21 during typical hematopoietic differentiation and in AMLs were examined; the results showed that miRNA-21 expression is significantly increased during normal granulocyte/monocytic differentiation, while PDCD4 protein levels are concurrently downregulated. Moreover, miRNA-21 is often overexpressed in AML blasts, accompanied by a marked reduction in PDCD4 protein levels. These findings collectively suggest that dysregulation of miR-21 expression may contribute to disease development in AMLs [31].

Additionally, the study found that the relationship between the -94 ATTG polymorphism (rs28362491) and serum miRNA 21-5p levels in patients showed significant differences. Specifically, levels of 21-5p varied notably between heterozygous Ins/Del, homozygous Del/Del, and homozygous Ins/Ins alleles. It has been reported that NF-kB regulates miRNA-21 expression by binding to the miRNA-21 promoter region, which causes an increase in miRNA-21 expression [21].

The primary reasons the miR-302 cluster has been studied are its roles in reprogramming pluripotent stem cells and maintaining stemness in somatic cells. It also prevents growth and apoptosis in various cancers, such as liver, breast, ovarian, and colon cancers [32]. Furthermore, the inquiry supports the conclusions of the current study, which indicate that miR-302 levels in CML patients were significantly lower than in the control group. This work emphasizes miR-302’s role as a tumor suppressor. Results also strongly suggest that circulating miR-302 can serve as a powerful, noninvasive marker for prognosis, therapy, and diagnosis. It also provides fresh perspectives on the modifications caused by SNPs in miRNA targets.

Due to its constitutive activation, NF-κB plays a crucial role at various stages of carcinogenesis and in the development of therapy resistance. The -94 ATTG deletion polymorphism (rs28362491) in the NFKB1 promoter decreases NF-κB1 transcription, leading to dysregulated NF-κB signaling. In CML, the Del/Del genotype is more common and associated with higher miR-21 levels. This polymorphism may boost NF-κB’s oncogenic signaling by promoting a more active form of transcription factor. NF-κB binds to the miR-21 promoter, increasing its transcription. Del/Del carriers may experience sustained NF-κB activation and miR-21 overexpression, which suppresses tumor suppressors such as PTEN and PDCD4, strengthening PI3K/AKT activation and other survival pathways that support leukemia.

This study demonstrated that patients with CML exhibit significantly elevated levels of miR-21-5p in comparison to healthy individual controls, suggesting a possible role in the disease’s aggressiveness and prognosis. Thus, the rs28362491 Del/Del genotype likely promotes miR-21’s oncogenic role by activating NF-κB, explaining its association with increased CML risk. Consequently, miR-21 has potential as a clinically valuable biomarker for early detection, risk assessment, and monitoring treatment responses in CML and different hematologic cancers [34].

Regarding miR-302 expression levels, a significant difference was observed between the homozygous Del/Del and Ins/Ins alleles. The homozygous Del/Del genotype exhibited significantly lower miR-302 expression than the Ins/Ins genotype, suggesting a correlation between miR-302 expression and rs28362491 genotype. Del/Del individuals may have a higher risk of CML due to their miR-302 expression levels.

This study demonstrated that miR-302 levels in CML patients were notably lower than those in the control group. This indicates that miR-302 functions as a tumor suppressor and could serve as a valuable prognostic and diagnostic biomarker. Additionally, compared to other genotypes, the NFKB1 gene rs28362491 Del/Del genotype was associated with a notably lower level of miR-302 expression. Most patients had the homozygous Del/Del genotype, suggesting this genotype may confer an increased risk of CML.

Furthermore, this study evaluated various blood parameters related to the -94 ATTG polymorphism (rs28362491). The results showed that the homozygous Ins/Ins polymorphism had significantly higher (p < 0.0001) WBC counts than both the homozygous Del/Del and heterozygous Ins/Del polymorphisms. Additionally, the findings indicate that the polymorphisms affected lymphocyte counts in CML patients. On the other hand, the three polymorphisms (Del/Ins, Del/Del, and Ins/Ins) of the -94 ATTG (rs28362491) polymorphism showed no notable difference regarding neutrophil counts in patients. This suggests that the polymorphisms did not impact neutrophil counts in these patients. However, platelets appear to be significantly influenced by the three polymorphisms (Del/Ins, Del/Del, and Ins/Ins) at the -94 ATTG (rs28362491) locus. To our knowledge, no previous report has documented such a correlation.

Conclusion

This study represents the first comprehensive investigation into the concurrent analysis of miR-21, miR-302, and the NF-κB1 rs28362491 polymorphism in CML, revealing a novel genotype-dependent regulation of these microRNAs. It provides preliminary evidence that the Del/Del genotype correlates with significantly elevated miR-21 levels, reduced miR-302 levels, and specific hematological changes, thereby indicating a previously unrecognized pathway involving NF-κB and microRNAs in CML pathogenesis.

Acknowledgments

We thank Al-Ahliyya Amman University and the Cell Therapy Center at the University of Jordan.

Statement of Ethics

This study was approved by the Scientific Research Ethics Committee of the Faculties of Pharmacy and Allied Medical Sciences at Al-Ahliyya Amman University (IRB: AAU/3/8/2022-2023).

Conflicts of Interest Statement

The authors declare no conflicts of interest.

Funding Sources

Financial support was provided by Al-Ahliyya Amman University in Jordan.

Author Contributions

Jehad F. Alhmoud conceived the idea for the manuscript. Jehad F. Alhmoud and Moath Alqaraleh drafted the initial draft and revised subsequent drafts. Jehad F. Alhmoud, Moath Alqaraleh, and Futoon Abedrabbu Al-Rawashdea reviewed and edited the manuscript before submission. Issa I. Dababneh and Nada L. Odeh provided patient samples. Nirmeen Elzogheir and Dana A. Alqudah provided technical assistance. Sarah N. Dala-Ali and Leen S. Alhiary handled laboratory work. All authors have read and approved the final manuscript version.

Funding Statement

Financial support was provided by Al-Ahliyya Amman University in Jordan.

Data Availability Statement

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.