Abstract

Introduction: Major β-thalassemia is a severe hereditary anemia caused by defective β-globin production and characterized by a permanent transfusion requirement, which leads to an overload of systemic iron and subsequent liver dysfunction. MicroRNA-451 (miR-451) is an erythroid-specific microRNA that plays a critical role in erythropoiesis, oxidative stress regulation, and iron metabolism. Nevertheless, there is still insufficient research on the connection between β-thalassemia major and liver function.

Aim: The study aimed to evaluate the expression of miR-451 in Iraqi patients with β-thalassemia major and correlate it with liver function parameters.

Materials and methods: This case-control study involved 50 patients diagnosed with β-thalassemia major and 50 healthy individuals serving as controls. Total RNA was extracted from whole blood samples, and the expression levels of miR-451 were quantified using quantitative real-time PCR (qRT-PCR). U6 small nuclear RNA was utilized as the endogenous control for normalization. Hematological indices and liver function parameters, specifically ALT, AST, and total serum bilirubin, were assessed and analyzed statistically.

Results: The expression of miR-451 was upregulated significantly in patients in comparison with the controls (≈260-fold increase). The patients had significantly lower levels of hemoglobin and elevated platelet counts (p≤0.05). The liver enzymes, ALT and AST, and the total serum bilirubin were significantly increased in the patient group (p≤0.01). This study found a positive association between the overexpression of miR-451 and hepatic dysfunction markers.

Conclusion: In β-thalassemia major, miR-451 is significantly upregulated, suggesting it could be a biomarker for disease severity and liver function.

Keywords

major β-thalassemia, liver functions, microRNA-451, gene expression

Introduction

Beta-thalassemia major is a severe hereditary anemia that requires frequent blood transfusions due to a lack of β-globin production brought on by a mutations in the HBB gene.‌^[1,2] Persistent transfusion therapy causes systemic iron overload^[3-5], which damages hepatocellular structures and compromises the liver function (iron build-up damages hepatic enzymes)^[6-8].

MicroRNAs (miRNAs) are small (~22 nucleotides) non-coding RNAs that regulate genes post-transcriptionally.^[9] By modulating the expression of genes related to erythropoiesis, hemoglobin synthesis, and cellular stress responses, they play a significant regulatory role in β-thalassemia major. One of the miRNAs that is most readily expressed in erythroid cells, miR-451, is essential for iron metabolism, erythropoiesis, and oxidative stress regulation.^[10-12]

Clinically, individuals with β-thalassemia or β-thalassemia/HbE have been found to have elevated plasma miR-451 levels, which correlate with hemolysis and erythropoietin activity markers like reticulocyte and platelet counts. ^[13-15] Furthermore, miR-451 may provide protection against the renal damage associated with β-thalassemia, hence enhancing its potential as a biomarker.^[16] However, the association between the expression of miR-451 and liver function parameters in patients with β-thalassemia major is still insufficiently examined, especially in Iraqi populations.

Aim

The objective of this study was to evaluate the levels of gene expression of miR-451 in β-thalassemia major patients from Iraq and to investigate their link with liver function parameters.

Materials and methods

The research was carried out at the University of Baghdad’s Institute of Genetic Engineering and Biotechnology from February 2024 to February 2025. The samples for the study were provided by the laboratories at Yarmouk Hospital in Bagdad. The study used 100 blood samples, 50 from patients with major beta-thalassemia and 50 from healthy individuals without major beta-thalassemia or other disorders. Consultant hematologists diagnosed all of the patients.

This study measured the hematological parameters, containing white blood cell count (WBC), hemoglobin (Hb), and platelet count, using an automated hematology analyzer. Liver function parameters (ALT, AST, and TSB) were determined using an automated biochemical analyzer.

Total RNA was extracted from whole blood samples following the manufacturer’s protocol.^[17] RNA concentration and purity were measured spectrophotometrically. cDNA Synthesis SuperMix (EasyScript , Cat No. E-003S, TransGen Biotech) and the One-Step gDNA Removal were used for synthesis of cDNA. The reaction of cDNA synthesis contained the following components: 10 μl of reaction mix, 1 μl of random primers, 1 μl of anchored oligo dT, 1 μl of genomic DNA remover, 1 μl of E-mix reverse transcriptase, 3 μl of RNase-free water, and 5 μl of total RNA. Thermal cycling consisted of an initial denaturation at 25°C for 10 min, reverse transcription at 42°C for 15 min, and enzyme inactivation at 85°C for 5 seconds.

The resulting cDNA was prepared for subsequent RT-qPCR analysis. The primer sequences (Table 1) used in this study were recommended by Macrogen Company, Korea, in a lyophilized form, where Nuclease Water (NFW) was used to dissolve them. Thus, a 100 picomolar stock solution was obtained, and NFW was added to achieve a concentration of 10 picomoles by preparing a work solution.^[18]

Table 1.

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Primers designed in the study

Primer	Sequence (5'→3' direction)	Primer size bp	References	t°C
miR-451 (gene expression)
Forward	AAACCGTTACCATTACTGAGTT	21	Designed	60
miR U6 F.P.	AGAGAAGATTAGCATGGCCCCT	22	19	60
miRNA-universe R.P.	GCGAGCACAGAATTAATACGAC	22	20	58
Universal R. transcription p	GAGGTCCAGTTTTTTTTTTTTTTTVN	26	21	58

The expression levels of miR-451 and the endogenous control U6 were estimated using qRT-PCR with SYBR Green. The resulting data were then amplified and utilized to normalize the miR-451 levels.^[19]

The reaction was carried out with a volume of 20 μL, following the manufacturer’s instructions. The amount of Super Mix named TransStart® Top Green qPCR (Cat No. AQ131-01, TransGen Biotech Company, Beijing, China) needed to prepare the necessary amount for reactions was calculated to be 10 μL. A quantity of 3 μL of cDNA as a template, 1 μL each of the forward and reverse primers, and, finally, 5 μL of nuclease-free water were added to complete the final volume of the reaction.^[20,21] The stages and temperature of gene expression are shown in Table 2. U6 are small nuclear RNA (snRNA) that act as a housekeeping gene. Gene expression levels were quantified for both patient and control groups using quantitative real-time PCR (qRT-PCR). Cycle threshold (Ct) values were determined for each target gene and a housekeeping gene. Data analysis was performed using two established methods: the ΔCt method and the ΔΔCt method for fold-change calculation.^[22,23]

Table 2.

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Stages and temperatures of qRT PCR for U6 and miRNA-451 genes

Stage		Temperature °C	Time (seconds)	Cycle
Stage 1	Denaturation	94	60	1
Stage 2	Denaturation	94	5	35
	Annealing	58	15
	Extension	72	20
Stage 3	Dissociation	65–95	1	1

The SAS software (version 9.6) was used in the statistical analysis. Data were expressed as mean ± standard error (SE). Patients and controls differences were assessed using t-tests (independent sample). The statistically significant was signed as p-value ≤0.05.^[24]

Results

Table 3 shows the distribution of study samples by WBC, Hb, and platelets in major β-thalassemia patients and control groups.

Table 3.

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Distribution of study samples by WBC, Hb, and platelets in major β-thalassemia patients and control groups

Groups	WBC Mean ± SE	Hb Mean ± SE	Platelets Mean ± SE
Control	7.13±0.23	14.29±0.10	253.6±7.85
Patients	7.90±0.27	10.68±0.19	424.2±114.9
T-test	−2.147	16.035	−1.481
p-value	0.060 NS	0.017^*	0.011^*

While the mean WBC count was only slightly higher in patients than controls without reaching statistical significance (p=0.060), mean hemoglobin (Hb) was markedly and significantly reduced in patients compared to controls (p=0.017). Furthermore, platelet count was significantly higher in patients than in the control group (p=0.011).

Table 4 shows how study samples were distributed in patients and control groups based on ALT, AST, and TSB levels. Patients’ ALT levels were significantly higher than those of controls (p=0.0001). AST levels were significantly higher in patients than in controls (p=0.0001). TSB levels were significantly higher in patients compared to controls (p=0.0001).

Table 4.

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Distribution of study samples by ALT, AST, and TSB in major β-thalassemia patients and control groups

Groups	ALT Mean ± SE	AST Mean ± SE	TSB Mean ± SE
Control	19.24±0.82	21.52±0.79	0.40±0.02
Patients	23.60±2.88	31.70±3.45	0.81±1.29
T-test	−1.45	−2.87	−3.137
P-value	0.0001^**	0.0001^**	0.0001^**

Molecular study

Comparison miRNA-451 gene expression between study groups

Table 5 compares the expression of miRNA-451 between patients and control groups using the ΔΔCt method. In the control group, the mean Ct of miRNA-451 was 36.91, while in the patient group, the mean Ct of miRNA-451 was 28.65, showing markedly lower Ct compared to controls and indicating higher expression. The relative expression (2^-ΔΔCt) reached ~250, which is higher than in the control group. This clearly shows that thalassemia patients have much higher miRNA-451 levels than healthy controls.

Table 5.

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Comparison of miRNA-451 gene expressions between study groups

Group	Mean Ct MiRNA-451	Mean Ct U6 gene	Δct	Δct calibration	ΔΔct	2^-ΔΔct	Experimental/ control group	Fold change
Control	36.91	23.25	13.66	14.6	−0.94	1.918	1.918/1.918	1
Patient	28.65	23.00	5.64	14.6	−8.96	497.99	497.99/1.918	259.64

Discussion

The significant reduction in Hb among patients reflects the chronic anemia associated with thalassemia, which is caused by ineffective erythropoiesis and accelerated hemolysis. These findings are consistent with those of Cappellini et al. and Bajwa, who found persistently low Hb levels in thalassemia patients, necessitating frequent blood transfusions.^[25,26]

The fact that there were no significant differences between the two groups (patients and controls) suggests that thalassemia has a less direct effect on WBC count. However, some studies, such as Weatherall’s^[27], have found that recurrent infections or transfusion-related complications can occasionally affect WBC levels, though this was not observed in the current cohort.

Patients’ platelet counts were found to be significantly elevated. This could be due to hypersplenism or, more commonly, a splenectomy, which is common in thalassemia patients to reduce transfusion needs. The current study supported the findings of Khawaji et al.^[28], who found elevated levels of platelet count. Taher et al.^[29] have extensively documented elevated platelet counts following splenectomy, which are associated with an increased risk of thromboembolic events.

The significant increase in liver enzymes and bilirubin suggests hepatic stress and potential hepatocellular injury. The current study supported the findings of Bashi and Fathi^[30] and Luaibi and Mohammed^[31,32]. These abnormalities are most likely the result of iron overload from repeated transfusions, as well as ongoing hemolysis. Borgna-Pignatti et al.^[33] found that iron deposition in the liver is a major complication in thalassemia, frequently resulting in hepatic dysfunction and elevated transaminases. Elevated bilirubin levels contribute to the occurrence of enhanced hemolysis in these patients.

According to the current study, thalassemia patients significantly overexpressed miRNA-451 compared to the control group. One of the most common erythroid-specific microRNAs is miRNA-451. It is necessary for controlling erythroid differentiation, red blood cell maturation, and oxidative stress defense. According to previous research by Papapetrou et al., miRNA-451 expression increases during erythropoiesis and is required for final red cell formation.‌^[34] This upregulation is consistent with that study. Thalassemia is characterized by elevated oxidative stress from free α-globin chains and inefficient erythropoiesis. miRNA-451 overexpression in patients may be a compensatory strategy to improve erythroid maturation and prevent oxidative damage. Mustafa et al. found that miRNA-451 is elevated in red blood cells during oxidative stress, which is consistent with this.^[35]

Elevated miRNA-451 levels in thalassemia could serve as a biomarker for hemolysis and erythroid activity. Numerous studies, including Wang et al.’s study from 2025, have suggested that miRNA-451 could be used as a predictive and diagnostic marker for hematological diseases such as sickle cell anemia and thalassemia.^[36] The patients with thalassemia showed a significant (~260-fold) increase in miRNA-451 expression, indicating that the gene may play a role in the illness’s pathogenesis. Chronic overexpression may be a compensatory response to oxidative stress and anemia, but it could also indicate a disruption in erythropoiesis-regulating circuits.^[37]

Limitations

There are several limitations to this study. Serum ferritin or liver iron concentration as iron accumulation parameters were not directly measured, and the sample size was small. Furthermore, causal interpretation is limited by the cross-sectional design. Large-scale, multicenter research is required to confirm these findings.

Conclusion

The patients’ hematological profile revealed a significant decrease in hemoglobin, indicating anemia, and a significantly increased level of platelets, indicating reactive thrombocytosis. The white blood cell count was slightly higher in comparison to the control group, but the difference was not statistically significant.

The findings indicate that these patients may have hepatic stress and hepatocellular injury, possibly as a result of iron overload from chronic transfusion therapy and ongoing hemolysis. Increased bilirubin levels also indicate increased hemolysis, emphasizing the importance of closely monitoring liver function and effectively managing iron in thalassemia patients.

According to the study, miRNA-451 is significantly upregulated in thalassemia patients compared to controls, which is consistent with its established role in regulating oxidative stress and erythropoiesis. This suggests that miRNA-451 could be a biological indicator of disease severity.

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Additional information

Ethical statement

The authors declared that no clinical trials were used in the present study.
The authors declared that no experiments on humans or human tissues were performed for the present study.
The authors declared that written informed consent was obtained from all participants before enrollment into this study.
The Research Ethics Committee of the University of Baghdad’s Institute of Genetic Engineering and Biotechnology reviewed the protocol of this study and granted an approval.
The authors declared that no experiments on animals were performed for the present study.
The authors declared that no commercially available immortalized human and animal cell lines were used in the present study.

Conflict of interest

The authors have declared that no competing interests exist.

Artificial Intelligence (AI) use

The authors accept full responsibility for the content of the manuscript, including the disclosure of any use of AI. No AI tools were used in the preparation of this manuscript.

Funding

No funding was reported.

Author contributions

All authors have contributed equally.

Author ORCIDs

Hamsa Ahmed Jasim 0009-0005-6421-669X

Data availability

All of the data that support the findings of this study are available in the main text.