MiR-221-3p improves the prognostic value of lung ultrasound score in acute respiratory distress syndrome and modulates the inflammatory response

Introduction

Acute respiratory distress syndrome (ARDS) is defined as a sudden and severe lung injury caused by a variety of different factors, both within the lungs and outside the lungs, other than those related to heart problems. It manifests as progressive respiratory distress and persistent hypoxemia [1]. The pathogenesis of ARDS is complex, and the mortality among patients is extremely high. The causes of ARDS include direct and indirect lung injuries such as pneumonia, sepsis, noncardiac shock, aspiration, trauma, contusions, and massive blood transfusions [2]. Among these, sepsis is the main etiological cause of ARDS [3]. Despite significant progress in the study of the pathogenesis, pathophysiology, and respiratory support therapy of ARDS, the clinical mortality rate remains high [4].

Lung ultrasound (LUS) is an invaluable diagnostic tool for identifying and ruling out various pulmonary conditions. Its advantages are numerous: it is rapid, reproducible, radiation-free, and non-invasive [5]. The utilization of LUS in the ICU not only permits the monitoring of a patient’s lung disease progression but also enables the timely detection of secondary lung disease resulting from ventilator use [6]. It has been demonstrated that standardized LUS features can diagnose and quantify pleural effusion, alveolar syndrome, interstitial syndrome, and pneumothorax [7]. The LUS score is a semi-quantitative method of assessing lung ventilation based on ultrasound signs. Extensive research has highlighted the clinical utility of LUS scoring in diagnosing and assessing ARDS [5], 8]. However, as a semi-quantitative diagnostic tool, LUS exhibits several limitations, including susceptibility to extrapulmonary confounding factors, operator-dependent variability in technique, moderate precision, and restricted diagnostic specificity [9]. These shortcomings highlight the need for complementary biomarkers that could enhance prognostic accuracy and facilitate tailored treatment strategies.

The clinical significance of microRNAs (miRNAs) in disease pathology has garnered increasing attention, with numerous studies highlighting their potential as diagnostic biomarkers in ARDS [10]. For instance, miR-155 was found to exhibit a positive correlation with lung function in sepsis patients complicated by ARDS, which is important for the early diagnosis of ARDS [11]. Moreover, miR-141-3p was demonstrated to be remarkably correlated with the severity of ARDS and to act as a risk factor for pulmonary fibrosis [12]. Previous investigations have also revealed that miR-221-3p is dysregulated in ARDS patients, suggesting its potential as a diagnostic biomarker for the disease [13]. Furthermore, miR-221-3p has been linked to lung function in individuals who have survived ARDS caused by Corona Virus Disease 2019 [14]. Therefore, miR-221-3p may be a valuable tool in improving the prognostic assessment of ARDS.

This investigation systematically evaluated the clinical relevance and prognostic value of miR-221-3p in ARDS through comprehensive data analysis. Furthermore, the study employed in vitro experimental approaches to elucidate the potential mechanistic role of miR-221-3p in pulmonary injury pathogenesis.

Materials and methods

Results

Serum miR-221-3p was upregulated in dead ARDS patients

This study enrolled 156 patients diagnosed with sepsis-induced ARDS, with a 28-day mortality rate of 41.03 % (n=64). Comparative analysis of baseline characteristics discovered statistically significant elevations in SOFA scores, APACHE II scores, procalcitonin levels, and LUS scores among non-survivors compared to survivors. No other clinically relevant differences were observed in demographic or laboratory parameters between the two groups (Table 1). Comparative analysis indicated markedly elevated miR-221-3p levels in non-survivors compared to survivors (Figure 1).

Table 1:

Comparison of clinical features between the survival group and death group.

Feature	Survival group (n=92)	Death group (n=64)	p-Value
Age, years, mean ± SD	65.13 ± 9.84	66.09 ± 9.43	0.542
Male, n, %	49 (53.26)	36 (56.25)	0.712
BMI (kg/m2), mean ± SD	23.93 ± 3.71	24.09 ± 3.60	0.787
Hypertension, n, %	38 (41.30)	28 (43.75)	0.761
Diabetes, n, %	30 (32.60)	24 (37.50)	0.528
APACHE II score, median (IQR)	17 (14, 19)	21 (18, 24)	<0.001
SOFA score, median (IQR)	9 (8, 10)	11 (10, 12)	<0.001
CRP (mg/L), median (IQR)	87.60 (78.30, 107.70)	91.55 (74.35, 117.30)	0.757
Albumin (g/L), mean ± SD	29.97 ± 5.31	28.43 ± 4.52	0.060
Procalcitonin (ng/mL), median (IQR)	3.10 (1.50, 6.08)	5.55 (2.20, 9.80)	0.001
Blood lactate (mmol/L), median (IQR)	2.10 (1.53, 2.48)	2.20 (1.70, 2.80)	0.105
LUS score, median (IQR)	21 (17, 25)	26 (24, 28)	<0.001

1. SD, standard deviation; BMI, body mass index; APACHE II, Acute Physiology and Chronic Health Evaluation II; IQR, interquartile range; SOFA, Sequential Organ Failure Assessment; CRP, C‐reactive protein; LUS, lung ultrasound. Differences were significant at p<0.05 with Student’s t-test, Chi-square test, or Wilcoxon test.

Figure 1:

Serum levels of miR-221-3p in ARDS patients in the death and survival groups. ***p<0.001.

MiR-221-3p predicted the prognosis of ARDS

Logistic analysis identified miR-221-3p (OR=12.303, 95 % CI: 4.513–33.542, p<0.001) and LUS score (OR=4.655, 95 % CI: 1.663–13.031, p=0.003) as risk factors for mortality in ARDS patients (Table 3). The analysis also revealed significant associations between ARDS mortality and three key indicators: SOFA score (OR=4.727, 95 % CI: 1.700–13.147, p=0.003), APACHE II score (OR=3.278, 95 % CI: 1.113–9.654, p=0.031), and procalcitonin (OR=2.752, 95 % CI: 1.027–7.373, p=0.044), as detailed in Table 3.

Table 3:

Logistic regression analysis of clinical characteristics associated with ARDS prognosis.

Variables	OR	95 % CI	p-Value
Age	0.896	0.333–2.413	0.829
Sex	1.095	0.417–2.880	0.853
BMI	2.368	0.848–6.612	0.100
Hypertension	0.635	0.238–1.696	0.365
Diabetes	1.105	0.408–2.992	0.844
APACHE II score	3.278	1.113–9.654	0.031
SOFA score	4.727	1.700–13.147	0.003
CRP	1.197	0.445–3.222	0.722
Albumin	0.490	0.182–1.315	0.157
Procalcitonin	2.752	1.027–7.373	0.044
Blood lactate	1.667	0.596–4.661	0.330
LUS score	4.655	1.663–13.031	0.003
MiR-221-3p	12.303	4.513–33.542	<0.001

1. ARDS, acute respiratory distress syndrome OR, odds ratio; CI, confidence interval; BMI, body mass index; APACHE II, Acute Physiology and Chronic Health Evaluation II; SOFA, Sequential Organ Failure Assessment; CRP, C‐reactive protein; LUS, lung ultrasound.

The AUC values for miR-221-3p (Figure 2A) and LUS score (Figure 2B) in predicting 28-day death in ARDS patients were 0.838 and 0.770. The AUC value for miR-221-3p combined with LUS score to predict death in ARDS patients was 0.903 (sensitivity: 0.797, specificity: 0.870) (Figure 2C).

Figure 2:

The ROC curve for miR-221-3p (A), LUS score (B), and the combination of them (C) for predicting the prognosis of ARDS.

MiR-221-3p regulated LPS-induced cellular damage in HPMECs

LPS stimulation (0.5–10 μg/mL) markedly enhanced miR-221-3p expression in HPMECs in a concentration-dependent manner (Figure 3A). Further experiments were conducted using 1 μg/mL LPS as the optimal concentration. Transfection with specific inhibitors effectively blocked the LPS-induced upregulation of miR-221-3p (Figure 3B). The exposure to LPS substantially reduced cellular viability (Figure 3C) and promoted apoptosis (Figure 3D), effects that were significantly reversed by miR-221-3p inhibition. Furthermore, LPS-mediated dysregulation of apoptotic markers (increased Bax and decreased Bcl-2) was ameliorated following miR-221-3p silencing (Figure 3E).

Figure 3:

The effect of miR-221-3p on the cellular behaviors of LPS-induced HPMECs. (A) LPS stimulation (0.5–10 μg/mL) upregulated miR-221-3p expression in HPMECs, as detected by RT-qPCR at 24 h post-stimulation. (B) miR-221-3p expression was elevated in LPS (1 μg/mL) treated HPMECs, and the inhibitor restored miR-221-3p levels, as detected by RT-qPCR at 48 h post-transfection. MiR-221-3p attenuated the effects of LPS on cell viability (C) and apoptosis rate (D) of HPMECs, as detected by CCK-8 at 0, 24, 48, and 72 h post-transfection and by flow cytometry at 48 h post-transfection. (E) miR-221-3p reversed the effect of LPS on the expression of Bax and Bcl-2 in HPMECs, as detected by ELISA at 48 h post-transfection. *p<0.05, **p<0.01, ***p<0.001.

LPS treatment markedly enhanced the production of pro-inflammatory mediators (IL-1β, IL-6, and TNF-α) in HPMECs, while miR-221-3p knockdown significantly attenuated this inflammatory response (Figure 4A). In addition, experimental results demonstrated that LPS treatment markedly upregulated both ICAM-1 and E-selectin in HPMECs, an effect that was remarkably attenuated by miR-221-3p knockdown (Figure 4B).

Figure 4:

Effect of miR-221-3p on the levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) (A) and adhesion molecules (ICAM-1 and E-selectin) (B) in LPS-treated HPMECs, as detected by ELISA at 48 h post-transfection. ***p<0.001.

Discussion

ARDS is defined by an acute onset, rapid progression, and a high mortality rate. Although lung-protective ventilation strategies are effective in improving patients’ conditions, the overall prognosis remains unsatisfactory [19]. Therefore, early evaluation is of paramount importance for the treatment of ARDS patients. The study highlighted the utility of miR-221-3p, coupled with the LUS score, as a promising approach for the early prognostic evaluation of ARDS.

LUS score can be used as an important screening tool for assessing pulmonary ventilation and the degree of lung injury [5]. In this study, clinical data from patients were collected and analyzed. In sepsis patients with ARDS, the LUS score was markedly higher in patients who died, indicating more severe lung injury and loss of pulmonary ventilation. Herein, miR-221-3p expression was increased in deceased ARDS patients compared to survivors. Furthermore, miR-221-3p was correlated with APACHE II scores, SOFA scores, procalcitonin levels, and LUS score, indicating that miR-221-3p is linked to pulmonary dysfunction in patients. Recently, there has been increasing evidence implicating miR-221-3p in the development and progression of lung diseases, including ARDS, lung cancer, and chronic obstructive pulmonary disease [14], 20], 21]. Notably, miR-221-3p was reported to modulate lipopolysaccharide-induced lung injury in ARDS mice [22]. Therefore, miR-221-3p appears to be critically involved in the progression of ARDS, warranting further exploration to understand its possible role as a biomarker or treatment target in this context.

Accurate clinical assessment of ARDS patients is essential for implementing prompt therapeutic interventions and enhancing survival rates. It has been demonstrated that the dysregulation of specific miRNA is related to the severity and prognosis of ARDS. For instance, miR-125b has been linked to the risk of developing ARDS in sepsis patients and is a predictor of 28-day mortality [23]. Previous studies have highlighted a correlation between LUS score and chest CT in assessing pulmonary ventilation in ARDS patients [24]. In this study, further analysis showed that miR-221-3p and LUS score were risk factors for patient mortality. MiR-221-3p was reported to exhibit aberrant expression in different diseases and is considered a promising biomarker. For example, the upregulation of miR-221-3p in early Parkinson’s disease makes it potentially useful as a noninvasive biomarker for diagnosing this disease [25]. Rincón et al. highlighted miR-221-3p as a key predictor of heart failure following acute myocardial infarction, underlining its prognostic relevance [26]. MiR-221-3p has demonstrated prognostic potential in various cancers, including gastric, breast, and prostate cancers [27], [28], [29]. Herein, miR-221-3p showed prognostic value in ARDS and was a risk factor for the patient’s death. LUS score also showed value in predicting the prognosis of ARDS. What’s more, the combination of them had better efficacy in prognostic prediction. These results indicate that combining miR-221-3p analysis with LUS scoring may serve as a promising tool for patient outcomes.

Current evidence suggests that miR-221-3p exerts regulatory effects on disease progression by facilitating cell injury through multiple interrelated processes, including apoptotic regulation, inflammatory mediation, oxidative stress modulation, and autophagy control [30], [31], [32]. Experimental evidence demonstrates that transfection with miR-221-3p mimics remarkably enhances the production of pro-inflammatory cytokines IL-6 and IL-8 in M2 macrophages [33]. Furthermore, research has demonstrated that miR-221-3p exacerbates inflammatory responses in BV2 microglial cells stimulated with LPS [34]. It has been established that HPMECs play a pivotal role in preserving the normal physiological functions of the lungs and regulating the development of lung diseases [35]. In conditions such as lung injury and pneumonia, dysfunction of HPMECs, including abnormalities in cell proliferation, migration, and apoptosis, affects vascular structure and function, further contributing to disease onset and progression [36], 37]. Research has demonstrated that HPMECs contribute to vascular barrier dysfunction and inflammatory responses, playing a pivotal role in the pathogenesis of ARDS [38], 39]. The present investigation employed LPS to establish an HPMEC injury model, which exhibited upregulated miR-221-3p expression. Functional analyses revealed that miR-221-3p knockdown enhanced HPMEC proliferation while suppressing apoptotic processes. Importantly, miR-221-3p downregulation attenuated LPS-mediated induction of inflammatory cytokines and adhesion molecules. Inflammatory mediators like TNF-α potently stimulate E-selectin upregulation in vascular endothelial cells, where it mediates key pathological events during acute inflammation [40]. Similarly, ICAM-1 serves critical functions in cell-cell adhesion, inflammatory signaling, and leukocyte recruitment, making it a pivotal player in numerous inflammatory disorders [41], 42]. Both molecules, recognized as biomarkers of endothelial dysfunction, demonstrate significant overexpression in ARDS and correlate strongly with disease severity [43]. The data demonstrate that increased miR-221-3p levels may participate in ARDS development by exacerbating endothelial cell damage and inflammatory activation. Nevertheless, the precise molecular mechanisms underlying the action of miR-221-3p require further investigation to fully understand its regulatory network.

Some limitations should be acknowledged. The clinical cohort in this study was from a single institution with a moderate sample size, which may limit generalizability. Future multi-center studies with larger populations are needed for validation. Although miR-221-3p was found to be a regulator of LPS-induced inflammation in endothelial cells, its precise molecular targets and signaling pathways require further validation. In vitro models (e.g., LPS-stimulated HPMECs) may not fully replicate the complexity of human ARDS. Complementary animal studies will reinforce mechanistic conclusions.

In summary, this research demonstrates that integrating serum miR-221-3p levels with LUS scoring enhances prognostic evaluation accuracy for ARDS patients, potentially guiding clinical decision-making. Furthermore, in vitro studies indicate the potential role of miR-221-3p in mediating inflammatory processes and cellular injury associated with ARDS.