Open Veterinary Journal, (2026), Vol. 16(4): 2540-2551

Research Article

10.5455/OVJ.2026.v16.i4.54

Upregulation of miRNA-374: A novel diagnostic biomarker and insight into inflammatory pathways in idiopathic pulmonary fibrosis

Asseel Yassin^1* and Alaa Mohammad Hasson Al-Husseini²

¹Department of Anatomy and Histology, College of Veterinary Medicine, University of Al-Qadisiyah, Al Diwaniyah, Iraq

²Department of Biology, College of Science, University of Al-Qadisiyah, Al Diwaniyah, Iraq

*Corresponding Author: Asseel Yassin. Department of Anatomy and Histology, College of Veterinary Medicine, University of Al-Qadisiyah, Al Diwaniyah, Iraq. Email: asseel.yassin [at] qu.edu.iq

Submitted: 22/11/2025 Revised: 03/03/2026 Accepted: 17/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by declining lung function and severe, irreversible scarring. The early diagnosis of IPF is particularly challenging due to the presence of nonspecific symptoms and a shortage of accurate, noninvasive biomarkers. Recently, interest in microRNAs (miRNAs) as IPF diagnostic biomarkers has emerged due to their stability and involvement in inflammation and fibrosis regulation.

Aim: This study aimed to measure the level of circulating miRNA-374 in patients with IPF, assess the level of diagnostic accuracy of miRNA-374, and determine the relationship between miRNA-374 and lung function and various inflammatory indices in the blood.

Methods: A case-control design was applied where 30 patients with IPF and 30 age-, sex-, and body mass index-matched healthy controls were included. Lung function was assessed using forced vital capacity (FVC%) and FEV1/FVC%. Hematological indices were measured with a focus on white blood cells (WBC), neutrophils, lymphocytes, and neutrophil percentage. The expression level of miRNA-374 was quantified using stem-loop Reverse Transcription Quantitative Polymerase Chain Reaction and Glyceraldehyde-3-Phosphate Dehydrogenase as the internal control. Data were reported as means, and the comparison of the means was achieved using t-tests, and correlation was done using Pearson’s coefficient, with receiver operating characteristic curve analysis used to determine diagnostic accuracy.

Results: FVC% in IPF patients was much lower than in controls, showing clear restrictive physiology (41.81 ± 6.11 vs. 97.00 ± 4.36; p < 0.001). Furthermore, patients with IPF had significantly higher levels of FEV1/FVC (91.64 ± 10.1 vs. 80.45 ± 1.41; p=0.017). Furthermore, the WBC, neutrophils, neutrophil% levels, and lymphocytes showed statistically significant changes (all p < 0.05). Patients with IPF had significantly elevated levels of miRNA-374 expression (2.51 ± 0.58) compared with controls (0.41 ± 0.10; p < 0.001). In the ROC analysis, the diagnostic accuracy was perfect (AUC=1.000; sensitivity=specificity=100%). miRNA-374 was positively correlated with neutrophil% (r=0.500, p=0.001), further supporting the association between miRNA-374 and IPF inflammation.

Conclusion: IPF is associated with elevated levels of circulating miRNA-374, which is likely to be of diagnostic significance as it relates to neutrophilic inflammation. Therefore, circulating miRNA-374 may serve as an important non-invasive biomarker and provide an opportunity as a target for therapeutic intervention.

Keywords: Biomarker, Idiopathic pulmonary fibrosis, Inflammation, miRNA-374.

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Introduction

Idiopathic pulmonary fibrosis (IPF) is one of the most challenging interstitial lung diseases to treat due to the irreversible changes in lung architecture, the continued decline in lung function, and high mortality that occur despite existing treatments. In the early stages of the disease, the slow and insidious onset and nonspecific symptoms camouflage the pathological changes of the disease and contribute to the significant delays in diagnosis (Raghu et al., 2018; Raghu et al., 2022). The associated pathological processes are complex and poorly understood, but recent literature suggests that fibrotic remodeling is primarily due to repetitive injury of the pulmonary epithelium, dysregulated aberrant repair processes, activation of fibroblasts and myofibroblasts, and excessive synthesis of the extracellular matrix (Martinez et al., 2021). The need to better understand the disease on a molecular level, diagnose it earlier, and identify indicators that do not require the use of invasive procedures to detect disease before irreversible damage is done to lung tissue is greater than the need for existing antifibrotic agents like nintedanib and pirfenidone, as they slow the disease progression but do not reverse the fibrosis (Maher et al., 2021).

MiRNAs, which are non-coding Ribonucleic Acids approximately 22 nucleotides in length, have shown remarkable stability over time, express disease-specific patterns, and modulate differentiation, proliferation, apoptosis, and immune responses (Colin Waldo et al., 2024). Because of these traits, they have been labeled as ideal candidates for protein biomarker discovery (Waldo et al., 2024). Negative regulatory uotomes in miRNA pathways are directly involved in mod altering IPF pathobiology via transforming growth factor Beta signaling, epithelial-mesenchymal transition, fibroblast activation, and matrix remodeling (Pandit et al., 2021; Xie, 2023). These have sparked mild interest as non-invasive molecular signatures to fibrotic lung diseases, with growing interest in mechanistic interpretations. Even so, miRNAs are seemingly left unexplored in IPF research.

One such candidate is miRNA-374. Although its roles in inflammation, cell proliferation, and immunity are well defined, its contribution to IPF has not been specifically explored. miRNA-374 has been shown to modulate the PI3K/Akt and Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NF-κB) pathways, which are integral in oncology and inflammatory disorders, and are critical in the activation of fibroblasts and amplification of inflammation (Jiang et al., 2020; Wang et al., 2022), both of which are prominent in the pathophysiology of IPF. Neutrophilic inflammation is an important driver of disease progression and acute exacerbation in IPF (Achaiah et al., 2022; Scott et al., 2024). Thus, miRNA-374 expression may illuminate the fibrotic remodeling phenotypic inflammatory microenvironment. Therefore, miRNA-374 as a potential diagnostic biomarker and an isolated and characterized regulatory factor of the inflammatory and fibrotic pathways would be an important contribution to IPF research, paving the way for early diagnosis and targeted therapies.

Evidence has shown how miRNA-374 participates in some critical pathways of the inflammatory response, immune cell activation, and tissue remodeling. Studies have shown that in addition to these, miRNA-374 has effects on the pathways of fibrosis and the tissues of the body that become chronically inflamed, along with the pathways of the NF-κB and others that incite the production of cytokines. This is true even though there have been studies on many areas of inflammation and cancer where this miRNA is analyzed; studies on idiopathic pulmonary fibrosis are lacking, which is why we are conducting this study (Shang et al., 2023; Waldo et al., 2024).

This study aimed to measure the level of circulating miRNA-374 in patients with IPF, assess the level of diagnostic accuracy of miRNA-374, and determine the relationship of miRNA-374 with lung function and various inflammatory indices in the blood.

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Materials and Methods

Study design and participants

Considering the requirements of the institutional ethics committee, we conducted a case–control study. The total number of study participants included 120, of which 60 were patients diagnosed with IPF per the latest criteria from the American Thoracic Society/European Thoracic Society/Japanese Thoracic Society/American Association of Thoracic Surgeons guidelines, and the other 60 were healthy age, sex, and body mass index (BMI) matched controls. All participants signed informed consent forms before study participation. Clinically diagnosed patients were seen and assessed after a diagnosis was made and before the patients started any antifibrotic medication. Controls were selected from a cohort of healthy individuals with no prior history of any pulmonary, autoimmune, or other chronic inflammatory diseases.

Sample collection and demographic data

Peripheral venous blood samples were drawn from each participant under aseptic conditions using two types of collection tubes: Ethylenediaminetetraacetic Acid (EDTA) tubes for complete blood count (CBC) and separation for RNA and stabilization tubes (RNAlater, Thermo Fisher Scientific, USA) for sparing microRNA.

Age, sex, BMI, smoking, and family history were collected from participants using a structured questionnaire, and these data were corroborated with clinical registration files for added accuracy.

Assessment of lung function

A pilot test was completed in accordance with the ATS/ERS 2019 guidelines. The forces of vital capacity (FVC%) and the ratio of 1 second to total capacity (FEV1/FVC%) controlling the spirometer calibration. On average, each participant completed a minimum of 3 acceptable trials, with the highest repeatable spirometry values retained and analyzed.

Hematological CBC analysis

Blood in EDTA tubes was analyzed on an automated hematology analyzer (Sysmex XN-Series, Sysmex Corporation, Kobe, Japan) for total white blood cells (WBC), absolute neutrophil count, absolute lymphocyte count, and neutrophil percentage. These biomarkers helped us understand the systemic inflammatory state of individuals with IPF and helped us understand how heterogeneous these patients are from the control patients.

Hematological parameters were analyzed using an automated hematology analyzer (Sysmex XN-1000, Sysmex Corporation, Kobe, Japan).

RNA extraction and stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis

Total RNA was extracted from isolated whole blood using the TRIzol reagent kit (Bioneer, Korea) according to the manufacturer’s protocol. RNA was quantified and spectrophotometrically assessed for purity and concentration using a Nanodrop (Thermo Fisher Scientific, USA).

For miRNA quantification, the stem-loop reverse transcription method was implemented on the reverse transcribed 10 ng of the RNA sample into cDNA, which was performed using the Reverse Transcription Kit (Applied Biosystems, USA) with the specific GoI stem-loop primers for hsa-miR-374-5p, alongside the housekeeping gene, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). The amplification was monitored in real time using a Bio-Rad real-time polymerase chain reaction system (USA) with Promega Sybr Green Master Mix (USA). Non-template controls were included in the analysis.

GAPDH is considered a reference gene because of its consistent expression in both sample types (inflammatory lung disease and peripheral blood). Initial analysis has shown that there is a low cycle threshold (Ct) value variation in both patients with IPF and controls, further proving the adequacy of this gene for normalization.

Total RNA concentration and purity were evaluated using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, USA). The RNA concentration was between 30 and 65 ng/µl, and the A260/280 ratios fell between 1.8 and 2.0. These results indicate a high purity, which is satisfactory for subsequent RT-qPCR analysis.

The purity of RNA was evaluated using spectrophotometric absorbance ratios (A260/280 and A260/230). No gel electrophoresis was used to evaluate the RNA integrity. All RNA samples met the quality criteria for RT-qPCR analysis.

Primers and probes used

The qPCR commercial TaqMan^® Assays were purchased from Applied Biosystems (USA). For this study, specific qPCR primers for mature HSA-miR-374 were designed based on the selected miRNA sequence using an online tool for miRNA primer design. Conversely, the qPCR primers for the housekeeping gene GAPDH (accession number NM_001256799.3) were designed in this study using the sequence from the National Center for Biotechnology Information database and the online software, Primer3Plus. All primers were synthesized by Macrogen (Korea). The details of the primers are provided in Table 1.

Table 1. Primers used in this study.

Calculations of Gene expression

Quantifying miRNA-374 expression levels relatively was accomplished via the 2^–ΔΔCt method (Livak and Schmittgen 2001). U6 snRNA was used as the endogenous reference, and one of the control samples was used as the calibrator to facilitate the standardization of fold-change calculations.

RT-qPCR specificity was confirmed by amplification and melting curve analyses, which demonstrated a single sharp melting peak for miRNA-374, indicating specific amplification.

SYBR Green chemistry was employed for miRNA-374 quantification; therefore, no probe sequences were used. Only gene-specific primers were applied and are listed in Table 1.

Data analysis

SPSS version 26 (IBM Corp, USA) and Microsoft Excel 2010 were used for data analysis. Normality was examined using the Kolmogorov–Smirnov test. Continuous variables are presented as mean ± standard deviation (SD) and categorical variables as counts and proportions.

Ethical approval

This study was approved by the Committee for Research Ethics at the College of Biology, University of Al-Qadisiyah (263-Oct-27-2025).

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Results

Demographic characteristics of patients with IPF and controls

A total of 60 patients with IPF and 60 matched healthy controls were included in the study, and no imbalances in the baseline characteristics were observed. The mean age of patients with IPF was 58.23 ± 11.9 years and 53.95 ± 11.6 years for controls (p=0.113), with no age disparity. The age group distribution was also similar, as no differences were detected (p=0.651). No statistically significant difference was found in the gender distribution, with 65.0% of the IPF group and 75.0% of the control group being men (p=0.232). There was no difference in the mean BMI of patients with IPF (23.1 ± 3.96 kg/m²) and controls (22.90 ± 3.69 kg/m²) (p=0.808). The classification of participants into the obesity categories of normal, overweight, and obese also did not significantly differ (p=0.893). Therefore, all these demographic characteristics, both from the patients and controls, confirmed that both study groups were well matched, and thus, the differences observed in the study must reflect the disease status of the individuals and not the imbalances in the populations (Table 2).

Table 2. Demographic characteristics of patients with IPF and healthy control subjects.

Family history and disease severity in patients with IPF

A family history is considered a possible predisposing risk factor. Of the 60 patients with IPF, only 9 (15%) recounted a family history. This suggests that the majority of cases were sporadic, as 85% had no family history. Disease severity evaluation showed that 42 (70%) patients had severe IPF, and 18 (30%) patients had moderate disease. This pattern is consistent with the clinical natural history, as patients are often seen after a considerable functional decline. The findings underscore the non-familial predominance of IPF and the advanced stage of the disease at diagnosis.

Comparison of the lung function of patients with IPF and healthy controls

Patients with IPF demonstrated a greater decline in lung function than healthy controls. The percentage of forced vital capacity (FVC%) was lowest in the IPF group, with an average of 41.81 ± 6.11 compared to 97.00 ± 4.36 in the control group (p < 0.001), corroborating the extreme restrictive ventilatory defect of IPF. On the other hand, the FEV1/FVC ratio was greater in the patients’ group (91.64 ± 10.1) than in the control group (80.45 ± 1.41; 0.017), a standard observation in patients with lung disease with a restrictive ventilatory pattern. Logistic regression analysis revealed an inverse association between FVC% and FEV1/FVC%, indicating that the extent of volume loss in IPF intensifies the restrictive ventilatory pattern. These functional variances offer convincing physiological evidence of significant parenchymal fibrosis in the advanced stage in these patients (Table 3).

Table 3. Comparison of lung function test results (FVC% and FEV1/FVC%1) between patients and healthy controls.

Lung function according to family history and disease severity

Analysis based on family history showed no statistically significant difference in lung function between patients with and without family history. FVC% and FEV1/FVC were also comparable (38.34 ± 8.7 vs. 42.42 ± 7.9; p=0.313; 90.92 ± 4.76 vs. 91.66 ± 5.43; p=0.703). Disease severity was strongly associated with lung function. Patients with moderate disease had a significantly higher FVC% (55.14 ± 4.44) than those with severe disease (36.11 ± 6.9; p < 0.001). Similarly, FEV1/FVC% also showed a significant difference between moderate and severe (87.63 ± 4.57 vs. 93.23 ± 4.71; p < 0.001). Thus, family history did not affect physiological impairment; however, functional lung decline correlated with the higher order of clinical severity classification (Figs. 1 and 2).

Fig. 1. Frequency distribution of lung function test (FVC% and FEV1/FVC%1) according to the patient’s family history.

Fig. 2. Frequency distribution of lung function test (FVC% and FEV1/FVC%1) according to disease severity. Different uppercase letters mean significant (p < 0.05).

Inflammatory blood profile parameters in patients with IPF and controls

Comparison of blood parameters showed that patients with IPF had an inflammatory profile that was unique compared to the control group. Patients had an increased total white blood cell count (8.89 ± 1.8) compared with the controls (7.82 ± 1.3; p=0.015). When considering controls and then the patients and controls (5.23 ± 1.1 and 75.11 ± 10.1, respectively; p=0.001 and p=0.002, respectively). In contrast, lymphocyte counts were significantly lower in patients with IPF (1.66 ± 0.41) than in controls (2.19 ± 0.32; p=0.001). Overall, these data suggest an overall shift toward neutrophil-dominant inflammation, which has been documented as a hallmark of the immunological responses to IPF.

Blood parameters by family history and illness severity

In all family history strata, blood parameters did not exhibit any statistically significant differences. Patients with a positive family history did not show any differences in WBC, neutrophil, lymphocyte, and neutrophil percentage from those with a negative family history (all p > 0.05). However, the severity of the disease was the same as that for inflammatory markers. Patients with severe IPF had elevated neutrophil levels (7.03 ± 1.69 vs. 5.90 ± 1.20; p=0.032), lower lymphocyte levels (1.54 ± 0.41 vs. 1.93 ± 0.61; p=0.029), and higher neutrophil percentages (77.78 ± 8.6 vs. 68.91 ± 5.61; p=0.001) than those with moderate disease. Thus, there is an obvious increase in systemic inflammation with regard to the severity of fibrosis (Tables 4–6).

Table 4. Comparison of some blood parameters (WBC, neutrophils, lymphocytes, and neutrophils%) between patients with IPF and healthy controls.

Table 5. The frequency distribution of blood parameters (WBC, neutrophils, lymphocytes, and neutrophils%) according to family history was calculated.

Table 6. Frequency distribution of blood parameters (WBC, neutrophils, lymphocytes, and neutrophils%) according to disease severity.

Blood parameters and lung function

Some hematological parameters exhibited significant correlations with each other in patients with IPS. White blood cells showed positive correlations with neutrophil (r=0.527, p=0.001) and lymphocyte (r=0.302, p=0.019) counts, whereas neutrophils had a strong correlation with neutrophil percentage (r=0.499, p=0.001). There was a strong negative correlation between the lymphocyte count and neutrophil cell percentage (r=0.813, p=0.001), suggesting a shift in the neutrophil balance in the immune system. Notably, blood parameters did not exhibit significant correlations with FVC% and FEV1/FVC%, indicating that the systemic inflammation might reflect the disease biology but not to what extent the disease is physiologically severe (Fig. 3 and Table 7).

Fig. 3. Correlation between blood parameters (WBC, neutrophils, lymphocytes, and neutrophils%) and lung function test parameters in patients with IPF.

Table 7. Correlation between blood parameters (WBC, neutrophils, lymphocytes, and neutrophils%) and lung function test parameters in patients with IPF.

Gene expression of miRNA 374 in the IPF and control groups

MiRNA374 expression was higher in patients with IPF than in controls. In IPF, the miRNA 374 mean Ct was 24.45, whereas it was 26.64 in the controls, suggesting the presence of more target transcripts in IPF. In accordance with the 2^–ΔCt method, the control samples had a mean expression of 0.41, whereas in IPF, it was 2.51, which suggests a significant difference (p < 0.001). This expression value indicates that miRNA 374 is possibly involved in the disease molecular pathways of IPF and thus is a sensitive molecular marker (Table 8 and Fig. 4).

Fig. 4. Relative expression of circulating miRNA-374 in patients with idiopathic pulmonary fibrosis and healthy controls.

Table 8. Comparison of (Ct, ^2‾∆ct and folding) between the patients and healthy controls.

Implications of findings on miRNA-374 expression

The Receiver Operating Characteristic (curve) (ROC) curves demonstrated the efficiency of miRNA-374 alone and showed an optimal cutoff of >0.93 with an AUC of (1.000; 95% CI: 1.000–1.000; p=0.0001). The sensitivity, specificity, and positive and negative predictive values were all equal to 100%. The high diagnostic accuracy of miRNA-374 identifies it as one of the best potential non-invasive diagnostic biomarkers for IPF (Table 9).

Table 9. Roc curve of miRNA-374 expression.

Implications of miRNA-374 and its relationship with disease and family history

Further analysis of the clinical subgroup data suggested that there were no statistically significant differences in miRNA-374 expression between the clinical subgroups of family history and disease severity. Family history patients had a miRNA-374 expression that was statistically the same as those without a family history of 2.57 ± 0.54 versus 2.38 ± 0.46; p=0.516. There was also no statistically significant difference in the expression levels between the cases of moderate and severe IPF. (2.31 ± 0.36 vs. 2.44 ± 0.64; p=0.566). Therefore, as the expression of miRNA-374 was significantly above the control level, IPF was already present rather than a familial predisposition or a more advanced clinical stage (Fig. 5).

Fig. 5. Expression of miRNA-374 according to family history and disease severity in patients with IPF.

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Discussion

The extraordinary upregulation of circulating miRNA-374 observed in this study intersects with the correlation of miRNA dysregulation with pathobiological processes of IPF. miR-374 has not been studied in the context of IPF; however, recent studies in other pathologies have suggested its involvement. Jiang et al. (2020) reported that miR-374b inhibits cellular overgrowth by targeting CCND1, implying that members of the miR-374 family may regulate IPF pathways that involve fibrogenic myofibroblast overgrowth, extracellular matrix production, and apoptotic resistance. Conversely, Zhang et al. (2022) reported that miRNA-374 may enhance oncogenic activity by the Wnt/β-catenin and the PI3K/Akt pathways, which are well-known drivers of fibroblast differentiation and apoptotic resistance. Therefore, the increased level of miRNA-374 in this study is in alignment with the recent literature, providing additional evidence that this entity has the potential to interface with the core profibrotic pathways, much as it does in malignancies and other inflammatory conditions. The large magnitude of upregulation observed in our cohort supports the potential of miRNA-374 as a candidate diagnostic biomarker, particularly due to its mechanistic links to signaling pathways that have already been recognized in fibrotic diseases.

The heightened inflammatory profile present in our patients with IPF, specifically the surge in neutrophils, increase in neutrophil percentage, and decrease in lymphocyte count, supports the findings of a previous study demonstrating that immune dysregulation is a major contributor to the IPF disease trajectory. Neutrophils, as described in Achaiah et al. (2022), gain aberrant degranulation and oxidative burst that chronically injure tissues. This supports the correlation between neutrophil percentage and miRNA-374, as miRNA-374 is likely that miRNA-374 is modified in response to or is modified by neutrophils. The fibroproliferative sequelae of repetitive epithelial barrier injury and persistent stimulation of fibroblasts, described by Bringardner et al. (2023) inflates the basic immune response. The substantial and uniform increase in miRNA-374 across subjects in this study, irrespective of family history and disease severity, is also indicative of this molecule functioning as a primary regulator or an early inflammatory biomarker. The outstanding diagnostic capability of our ROC analysis underscores the increasing recognition that miRNA profiles, unlike conventional inflammatory indices, are consistent and not confounded by comorbidities.

Recent advances in fibrosis research complement the work of Jenkins et al. (2023). For example, Jenkins et al. (2023) described the multifaceted nature of fibrotic remodeling. Jenkins et al. (2023) described the multifaceted nature of fibrotic remodeling, noting that multiple molecular drivers coalesce to form the unique progression of IPF. Jenkins et al. (2023) emphasized the importance of determining accurate, stable biomarkers to identify early molecular dysfunction of Jenkins et al. (2023) emphasized the importance of identifying stable biomarkers to capture early molecular dysfunction, particularly those relating to fibroblast diversity, heterogeneous fibroblast activation, and aberrant tissue repair. Xie et al. (2023) focused on single-cell analysis. Xie et al. (2023) described distinct subpopulations of fibroblasts that possessed diverse transcriptional programs, many of which were regulated by miRNA. Xie et al. (2023) did not characterize miRNA-374, and their study design suggests the fibroblast activation of diverse microRNA networks. Therefore, it is most likely that Xie et al. (2023) and Jenkins et al. (2023) described regulatory networks in which miRNA-374 is not expressed.

The clinical significance of circulating miRNA-374 hinges on its usefulness for the identification of possible early-stage IPF, considering that current diagnostic methods are deficient in sensitivity. miRNA-based biomarkers are most likely to detect nascent inflammatory and fibrotic responses and, therefore, make it possible to diagnose patients in a more non-invasive manner.

Idiopathic pulmonary fibrosis is more commonly found in males than in females. Observations of male predominance in IPF have been documented in numerous registry-based studies and large population studies. Some of the reasons for this include cumulative smoking exposure, inhalational exposure in the workplace, some unknown biological factors based on sex, and flavors of bias in the diagnosis that favor men over women (Sesé et al., 2021; Podolanczuk et al., 2023; Sia et al., 2025).

Study limitations

This research has several shortcomings. Because of the small sample size and the study’s single-center design, negative consequences may occur due to a lack of generalizability. There was also no longitudinal study; therefore, miRNA-374 cannot be assessed, which means it cannot be considered a prognostic marker. Future multicenter studies with larger sample sizes are needed, and longitudinal tracking has to be conducted.

In previous studies, miRNA-374 has been associated with the modulation of NF-κB signaling, but this study did not directly assess NF-κB activation. However, NF-κB activity along with miRNA profiling would be of considerable value in future studies to help clarify the role of miRNA-374 in IPF-related inflammation.

Age-stratified graphical analysis could not be performed because individual-level miRNA-374 expression data linked to patient age were not available; this limitation has been acknowledged, and future studies with subject-level datasets are warranted.

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Conclusion

Circulating miRNA-374 is significantly upregulated in the samples, indicating case versus control in IPF patients, according to our study’s results. This biomarker can differentiate patients with IPF from healthy controls with 100% sensitivity and 100% specificity. The miRNA-374 of the biomarker likely resides in the case of neutrophil-rich inflammatory disorders and is likely transcriptionally controlled in relation to an IPF dysregulated step of the immune cascade. Nevertheless, the diseased miRNA-374 seems to be an unqualified, independent marker of related family history and of the degree of disease, as the miRNA-374 is likely to be a stable representation of disease. We believe that this study will define miRNA-374 as a non-invasive biomarker that will aid in providing etiological and clinical hypotheses in the case of inflammation and fibrotic pathways in IPF. We believe that the biomarker will provide multiple clinical hypotheses in the case of dysregulated inflammatory pathways in IPF. In this case, IPF can be postulated with high inflammation levels. Additional multicenter studies with larger sample sizes will be invaluable in this clinical study.

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Acknowledgment

The authors thank the College of Veterinary Medicine, University of Al-Qadisiyah, for their support in this study.

Conflict of interest

The authors have no conflicts of interest to declare.

Funding

The authors have self-funded the study. No external funding source is available.

Authors’ contributions

All authors have participated in the study.

Data availability

Data are available when requested by the corresponding author.

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