The diagnostic and short-term prognostic significance of miR-183-5p in pulmonary embolism and its regulation of disease progression through PDCD4

Abstract

The incidence of pulmonary thromboembolism (PTE) has increased year on year, with high rates of misdiagnosis and recurrence, as well as significant heterogeneity in prognosis. This study investigated the clinical value of miR-183-5p in PTE, as well as its mechanism of regulating disease progression through PDCD4. This study enrolled 126 PTE patients and 103 non-PTE patients. Serum miR-183-5p was detected using qPCR, and its diagnostic and prognostic significance were analyzed using ROC and logistic analyses. A PTE cell model was established using 150 µg/mL ox-LDL. The effects of inhibiting only miR-183-5p, and co-inhibiting with PDCD4, on the HPMECs functions, inflammation/oxidative stress, and adhesion molecules were analyzed. Serum miR-183-5p in PTE patients increased gradually with risk stratification. The diagnostic efficacy of miR-183-5p for PTE was superior to that of D-dimer, and combining the two provides even better diagnostic results. miR-183-5p was an independent risk factor for prognosis in PTE patients. In a PTE cell model, inhibiting miR-183-5p restored HPMECs proliferation and suppressed apoptosis and autophagy. It also reduced the inflammatory factors release, alleviated oxidative stress damage and down-regulated the adhesion molecules expression. There was a direct target relationship between miR-183-5p and PDCD4. Co-suppression both miR-183-5p and PDCD4 reversed the hindering effect of inhibiting only miR-183-5p on PTE progression. miR-183-5p is a novel biomarker for the supplementary diagnosis and 30-day short-term prognosis assessment of PTE, and it participates in PTE progression by targeting PDCD4 to regulate vascular endothelial cell function.

Introduction

Pulmonary thromboembolism (PTE), is a severe pulmonary vascular disease with a sudden onset and rapid progression [1]. It poses a significant threat to patients’ health and well-being. The high misdiagnosis and recurrence rates of PTE, as well as its heterogeneous prognosis, remain major challenges in clinical diagnosis and treatment [2]. Current clinical diagnosis primarily relies on D-dimer testing and CTPA; however, the former lacks specificity and the latter is limited by equipment availability and patient tolerance [3]. Therefore, there is an urgent need to explore more precise biomarkers and investigate their regulatory mechanisms to optimize PTE diagnosis, assessment and treatment strategies.

MicroRNA hold promise as markers for various diseases [4]. Recent studies have revealed that several specific miRNAs play important roles in the pathogenesis of vascular diseases [5]. Among these, research on miR-183-5p, has expanded from pulmonary diseases to various vascular-related disease fields. In patients with lung cancer and chronic obstructive pulmonary disease, abnormally expressed miR-183-5p serves as a potential biomarker and participates in regulating pathogenesis [6, 7]. Elevated expression of miR-183-5p has been observed in the serum of patients with coronary artery disease (CAD) and atherosclerosis (AS). It is positively correlated with disease severity [8] and regulates the growth of VSMCs [9]. In acute coronary syndrome (ACS), miR-183-5p demonstrates superior diagnostic efficacy for ACS subtypes compared to traditional markers [10]. Elevated expression of miR-183-5p in the plasma of patients with acute venous thromboembolism (AVTE) may be involved in the thrombosis process [11]. The aforementioned cardiovascular diseases share similar pathological mechanisms with PTE, including vascular endothelial injury, haemodynamic abnormalities, and so on. Given the association between miR-183-5p and disease severity in related conditions, as well as its regulatory role in endothelial cell function, we hypothesize that miR-183-5p may play a part in the PTE development. However, the clinical significance of miR-183-5p in PTE and its role in disease progression remain to be elucidated.

As a tumor suppressor gene and inflammatory regulatory factor, PDCD4 plays a significant role in maintaining vascular homeostasis [12, 13]. Studies have shown that PDCD4 expression is down-regulated in AS patients and is positively correlated with plaque stability [14]. Its absence relieves the inhibition of vascular smooth muscle cell proliferation and exacerbates inflammatory responses, which can lead to thickening of the vascular wall and narrowing of the lumen. This suggests that PDCD4 may participate in vascular lesions by regulating cell proliferation and inflammatory responses [15]. However, the changes in PDCD4 expression in PTE and whether its interaction with miR-183-5p mediates PTE progression remain to be explored.

This study therefore analyzed the expression characteristics of miR-183-5p in the serum of PTE patients, exploring its potential as a diagnostic tool and short-term prognostic indicator for PTE. Additionally, a cell model was employed to ascertain whether miR-183-5p influences the function of endothelial cells by targeting PDCD4. This provides experimental evidence to elucidate the pathogenesis of PTE and develop new biomarkers.

Patients and Methods

Research subject selection and grouping

This single-center retrospective study enrolled 229 patients who visited Zibo Central Hospital between January 2022 and October 2024. Of these, 126 were in the PTE group and were all diagnosed using CTPA or pulmonary angiography and met the diagnostic criteria [16]. The non-PTE group included 103 cases. Patients in this group exhibited clinical manifestations highly similar to PTE, including sudden breathing difficulty, chest pain and lower limb swelling. However, after PTE was excluded via CTPA, pulmonary angiography and other examinations, they were ultimately diagnosed with pneumonia (n = 42), heart failure (n = 35) or isolated deep vein thrombosis (n = 26). The three disease groups were distributed in a balanced manner, enabling robust representation of the target population for differential diagnosis of non-PTE.

All study participants had complete clinical records, including medical history, laboratory test results, and imaging data. Patients with concomitant malignant tumors, severe liver or kidney failure, or autoimmune diseases were excluded. Those with a history of major surgery, trauma, or blood transfusion within the past three months, or with mental disorders or cognitive impairments, were also excluded. This study was approved by the Medical Ethics Committee of Zibo Central Hospital. All participants provided written informed consent prior to enrollment.

PTE patients were stratified into different risk groups: (1) Low-risk group: Hemodynamically stable patients without right heart failure (32 cases); (2) Low-intermediate-risk group: Patients with either right heart failure or abnormal laboratory findings (41 cases); (3) High-intermediate-risk group: Patients with both right heart failure and abnormal laboratory findings (33 cases); (4) High-risk group: Patients with hemodynamic instability or shock (20 cases).

Following admission, PTE patients were monitored for 30 days. Based on complications occurring during the follow-up period, such as death, PTE recurrence or pulmonary hypertension, patients were divided into a good (79 cases) and a poor (47 cases) prognosis group. While this 30-day window is clinically relevant for assessing acute-phase outcomes and early complications, it does not capture long-term prognostic events such as chronic thromboembolic pulmonary hypertension (CTEPH), recurrent VTE beyond one month, or late mortality. Future studies should incorporate extended follow-up periods to evaluate the long-term prognostic value of miR-183-5p.

Serum specimen collection

All patients had 8 mL of venous blood collected while fasting the morning after enrollment. The blood was left to stand until stratification and centrifuged at 3000 rpm (4 °C,15 min). The upper serum was then carefully aspirated and divided into RNase-free centrifuge tubes.

PTE cell model construction

The human pulmonary microvascular endothelial cells (HPMECs) were purchased from ScienCell Research Laboratories (USA). These cells were cultured in a specialized endothelial cell culture medium containing 10% fetal bovine serum (FBS), 1% endothelial cell growth supplement and 1% penicillin-streptomycin mixture. The cells were cultured and passaged in a cell culture incubator at 37 °C with 5% CO₂ and 95% humidity. Cells from passages 3 to 5 were selected for the experiment.

The HPMECs were seeded at a density of 5 × 10⁵ cells per well in a 6-well plate and cultured for 24 h until cell confluence reached 70–80%. The medium was then replaced with medium (serum-free) containing 150 µg/mL ox-LDL (Thermo, USA) and cultured them for a further 24 h to establish the PTE cell model. Control cells were treated with serum-free medium only, with all other conditions remaining consistent.

Cell transfection

Seeded ox-LDL-treated HPMECs in a 6-well plate, ensuring that cell confluence reaches 50–60% at the time of transfection. The following constructs were provided by Hanbio Company in China: inhibitor-miR-183-5p, mimic-miR-183-5p, negative controls, along with small interfering RNA (si-PDCD4) targeting PDCD4. Transfection was performed strictly according to the Lipofectamine 3000 protocol. Six hours after transfection, replaced the medium and cultured them for an additional 48 h.

Gene expression

Total RNA (including miRNA) was extracted from serum or cells using the Quick-RNA Miniprep Kit (Zymo Research, USA), with the procedure strictly following the manufacturer’s instruction. The concentration and quality of the RNA were quantified using the Qubit™ RNA Quantification Kit on the Qubit Fluorescence Meter (Thermo Fisher Scientific, USA). Took 1 µg of total RNA and used PrimeScript RT Master Mix (Takara, Japan) for reverse transcription to synthesize cDNA.

The qPCR amplification was performed using Hieff UNICON^® Universal Blue qPCR SYBR Green Master Mix (Yeasen, China) on a StepOnePlus real-time fluorescent quantitative PCR instrument (Thermo Fisher, USA). Each 20 µL reaction mixture contained 10 µL of 2× SYBR Green Master Mix, 0.8 µL of forward primer (10 µM), 0.8 µL of reverse primer (10 µM), 2 µL of cDNA template, and 6.4 µL of nuclease-free water. The thermal cycling protocol consisted of an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 30 s. A melting curve analysis was performed at the end of each run (95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s) to verify the specificity of amplification. Either miR-183-5p with U6 or PDCD4, ICAM-1 and VCAM-1 with GAPDH as the internal control. All primers were designed and synthesized by Sangon Biotech Co., Ltd. in China. Relative expression levels of the target genes were calculated using the 2^(−ΔΔ)Ct method, with each sample replicated 3 times.

Cell proliferation

Transfected HPMECs were seeded at a density of 1 × 10⁴ cells per well in a 96-well plate. Once the cells had stabilized, proliferation activity was assessed using CCK-8 assay. Based on preliminary experiments that established the growth kinetics of HPMECs under the described culture conditions, a 4-day observation window was selected to encompass the lag phase, logarithmic growth phase, and early plateau phase of the cell growth curve. Day 0 was designated as the baseline time point (approximately 6 h after seeding) to normalize the initial cell adhesion and eliminate plating efficiency variations among groups. On days 0, 1, 2, 3 and 4 after culturing, 10 µL of CCK-8 solution (Dojindo, Japan) was added and the plate was incubated for 2 h. After zeroing the blank wells, the OD value (450 nm) was measured using a microplate reader (Bio-Tek, USA).

Cell apoptosis

Collected the cells 48 h after transfection. Re-suspended them in 500 µL of Binding Buffer. Added 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI, Yeasen, China) and incubated the cells at room temperature in the dark for 15 min. Then, added 400 µL binding buffer and analyzed immediately using a CytoFLEX flow cytometer (excitation wavelengths: 488 nm for Annexin V-FITC and 535 nm for PI), collecting 10,000 cells per sample. Analyzed and calculated the apoptosis rate using FlowJo software.

Western Blotting (WB)

The transfected cells were collected and lysed in RIPA lysis buffer (Beyotime, China) supplemented with 1% protease inhibitor cocktail (PMSF, Beyotime, China) and 1% phosphatase inhibitor cocktail (Beyotime, China) to prevent protein degradation and preserve phosphorylation status. The lysis buffer was prepared immediately prior to use, following the specified composition, and vortexed thoroughly before applying it to cell lysis. The cells were then centrifuged at 12,000 rpm (4 °C, 15 min) and the supernatant was collected.

The 12% SDS-PAGE was performed (80 V constant voltage for 30 min and then 120 V constant voltage for 90 min). For protein transfer, a wet transfer system (Bio-Rad, USA) was used. Proteins were transferred onto a 0.45 μm PVDF membrane (Millipore, USA) at 100 V constant voltage for 90 min at 4 °C in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3). The PVDF membrane was pre-activated with methanol for 15 s and equilibrated in transfer buffer for 5 min prior to transfer. The gel-membrane sandwich was assembled with filter papers and sponges soaked in transfer buffer, ensuring the removal of air bubbles. Primary antibodies (p62, LC3-I/II, GAPDH, 1:1000, 1:1000, 1:5000 respectively, Sigma-Aldrich, USA) were added at 4 °C overnight. HRP-labelled secondary antibody (1:5000, Abcam, UK) was added at room temperature for 1 h. The image was developed using an ECL chemiluminescence kit (Thermo Fisher, USA). The grey values of the bands were analyzed using ImageJ software.

Inflammatory factors and oxidative stress indicators

The cell culture supernatant was collected after transfection and centrifuged at 12,000 rpm (4 °C, 10 min). The supernatant was then collected. The ELISA kits for the inflammatory factors were purchased from R&D Systems in the United States. The ELISA kits for the oxidative stress markers were purchased from Beyotime and Nanjing Jiancheng Company, in China. Antibody incubation and chromogenic reaction were performed according to the instructions. The changes in ROS were monitored under FITC fluorescence detection conditions. The OD value was measured using a fluorescence microplate reader.

Dual luciferase reporter gene assay

The binding sites of miR-183-5p and PDCD4 were predicted using the ENCORI database and constructed PDCD4 wild-type (PDCD4-wt, 5’-GCUACCUUUUGUAAGUGCCAUG-3’) and mutant (PDCD4-mut, with a mutated binding site, 5’-GCUACCUUUUGUAUCACGGUAG-3’) luciferase reporter vectors (vector backbone: pmirGLO, Promega, USA). Seeded HPMECs in a 24-well plate. Untreated as the control group. PDCD4-wt/PDCD4-mut and mimic-miR-183-5p/inhibitor-miR-183-5p were then co-transfected using Lipofectamine 3000 as the transfection reagent. After 48 h, the cells were lysed using dual luciferase reporter gene assay kit (Promega, USA), firefly and renilla luciferase substrates were added sequentially, and the activity of both luciferases was detected using a luminometer (Berthold, Germany).

Statistical analysis

Comparisons of quantitative data between 2 groups were performed using an independent samples t-test; comparisons between multiple groups, using ANOVA; and pairwise comparisons, using the LSD-t test. Comparisons of count data between groups were performed using the chi-squared test. The diagnostic value of miR-183-5p and D-dimer for PTE was analyzed using receiver operating characteristic (ROC) curves. Binary logistic regression analysis was used to identify independent risk factors for a poor prognosis in PTE patients. Correlation was performed using Pearson method.

Results

Comparison of the basic characteristics of the research subjects

There were no statistically significant differences in general patient characteristics between the PTE group and the non-PTE group (P > 0.05), indicating that the baseline characteristics of the 2 groups were matched well. However, the prevalence of hyperlipoidemia and diabetes was significantly higher in the PTE group (P < 0.05).

Regarding laboratory test indicators, no significant differences in red blood cell (RBC) and blood platelet counts (BPC) (P > 0.05). Compared with the non-PTE group, PTE patients had significantly higher white blood cell counts (WBC) and D-dimer levels (P < 0.05). Meanwhile, blood gas analysis indicators were significantly lower (P < 0.01, Table 1).

Table 1.

Comparison of the basic characteristics of the research subjects in each group

Indicators	Non-PTE (N = 103)	PTE (N = 126)	P value
Age (years)	58.05 ± 13.30	60.47 ± 11.37	0.139
Gender (male, %)	56 (54.37)	80 (63.49)	0.162
BMI (kg/m2)	25.92 ± 4.13	26.77 ± 3.82	0.107
Smoking History (yes, %)	28 (27.18)	45 (35.71)	0.168
Drinking History (yes, %)	36 (34.95)	54 (42.86)	0.223
Hypertension History (yes, %)	52 (50.49)	79 (62.70)	0.063
Hyperlipoidemia History (yes, %)	27 (26.21)	56 (44.44)	0.004
Diabetes History (yes, %)	20 (19.42)	39 (30.95)	0.047
CHD History (yes, %)	18 (17.48)	26 (20.63)	0.546
WBC (109/L)	9.58 ± 3.07	10.42 ± 3.06	0.040
RBC (1012/L)	4.51 ± 0.65	4.46 ± 0.61	0.562
BPC (109/L)	231.60 ± 60.85	243.20 ± 59.24	0.148
PaO2 (mmHg)	70.31 ± 11.35	65.59 ± 12.41	0.003
PaCO2 (mmHg)	35.12 ± 4.47	33.40 ± 5.40	0.010
OI (mmHg)	325.90 ± 84.21	289.70 ± 79.58	0.001
D-Dimer(ng/mL, FEU)	1009.21 ± 525.44	2586.48 ± 1976.51	<0.001
sPESI score	-	1.91 ± 1.31	-

Notes: PTE, pulmonary thromboembolism; BMI, body mass index; CHD, coronary heart disease; WBC, white blood cell count; RBC, red blood cell count; BPC, blood platelet count; PaO2, partial pressure of oxygen; PaCO2, pressure of carbon dioxide; OI, oxygenation index; FEU, fibrinogen equivalent units; sPESI, simplified pulmonary embolism severity index; P < 0.05 means a significant difference

The expression and diagnostic value of miR-183-5p in PTE patients

The expression of serum miR-183-5p were significantly higher in the PTE group (P < 0.001, Fig. 1A) and increased progressively with risk stratification (P < 0.05, Fig. 1B).

Fig. 1.

Serum miR-183-5p levels in non-PTE and PTE patients (A). miR-183-5p expression in different risk groups of PTE patients (B). Diagnostic performance of D-Dimer (C), miR-183-5p (D), and their combination (E) for PTE. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Diagnostic performance analysis showed that the AUC for diagnosing PTE using D-dimer alone was 0.783 (P < 0.001, Fig. 1C). By contrast, the AUC for serum miR-183-5p for independently diagnosing PTE was higher (0.846, 73.81% sensitivity, 82.52% specificity) (P < 0.001, Fig. 1D). This indicates superior classification performance and predictive accuracy of serum miR-183-5p. Using the two markers in combination for diagnosis further improved the AUC to 0.882 (75.40% sensitivity, 93.20% specificity) (P < 0.001, Fig. 1E), significantly enhancing diagnostic efficacy.

The relationship between miR-183-5p and the short-term prognosis of PTE patients

Patients in the poor prognosis group had significantly higher proportions of hypertension, hyperlipoidemia and diabetes (P < 0.05). Their WBC, D-dimer levels and sPESI scores were also significantly higher (P < 0.001). Furthermore, blood gas analysis parameters (PaO2, PaCO2 and OI) were significantly lower (P < 0.01, Table 2).

Table 2.

Comparison of basic characteristics of the research subjects between the good prognosis group and the poor prognosis group

Indicators	Good Prognosis (n = 79)	Poor Prognosis (n = 47)	P value
Age (years)	59.75 ± 11.62	61.68 ± 10.95	0.358
Gender (male, %)	48 (60.76)	32 (68.09)	0.409
BMI (kg/m2)	26.36 ± 3.81	27.46 ± 3.79	0.116
Smoking History (yes, %)	25 (31.65)	20 (42.55)	0.217
Drinking History (yes, %)	32 (40.51)	22 (46.81)	0.489
Hypertension History (yes, %)	44 (55.70)	35 (74.47)	0.035
Hyperlipoidemia History (yes, %)	25 (31.65)	31 (65.96)	0.001
Diabetes History (yes, %)	18 (22.78)	21 (44.68)	0.010
CHD History (yes, %)	14 (17.72)	12 (25.53)	0.295
WBC (109/L)	9.42 ± 2.59	12.11 ± 3.08	<0.001
RBC (1012/L)	4.52 ± 0.65	4.37 ± 0.54	0.190
BPC (109/L)	248.30 ± 61.01	234.50 ± 55.70	0.208
PaO2 (mmHg)	68.41 ± 11.98	60.85 ± 11.76	0.001
PaCO2 (mmHg)	34.35 ± 5.48	31.79 ± 4.93	0.009
OI (mmHg)	307.70 ± 79.97	259.40 ± 69.81	<0.001
D-Dimer(ng/mL, FEU)	1655.73 ± 1061.10	4150.94 ± 2172.12	<0.001
sPESI score	1.35 ± 0.83	2.85 ± 1.44	<0.001

Notes: PTE, pulmonary thromboembolism; BMI, body mass index; CHD, coronary heart disease; WBC, white blood cell count; RBC, red blood cell count; BPC, blood platelet count; PaO2, partial pressure of oxygen; PaCO2, pressure of carbon dioxide; OI, oxygenation index; FEU, fibrinogen equivalent units; sPESI, simplified pulmonary embolism severity index; P < 0.05 means a significant difference

Analysis of serum miR-183-5p expression showed that it was significantly up-regulated in the poor prognosis group (P < 0.001, Fig. 2A).

Fig. 2.

Serum miR-183-5p levels in PTE patients with good and poor prognosis (A). Logistic regression analysis of independent prognostic risk factors for PTE (B). ***, P < 0.001

Binary logistic regression analysis revealed that a history of hyperlipoidemia [odds ratio (OR) = 3.597], increased WBC (OR = 3.303), elevated D-dimer levels (OR = 7.542), an increased sPESI score (OR = 9.315), and high miR-183-5p expression (OR = 3.886) were independent risk factors for a poor prognosis (P < 0.05). Of these factors, D-dimer, sPESI score and serum miR-183-5p demonstrated the greatest predictive value for patient prognosis (P < 0.001). Notably, PaO2 (OR = 0.351) and PaCO2 (OR = 0.445) were protective factors for short-term prognosis in PTE patients (P < 0.05, Fig. 2B).

The potential role of miR-183-5p in the PTE cell functions

Ox-LDL treatment resulted in significantly increased expression of miR-183-5p (P < 0.01, Fig. 3A) and significantly reduced proliferation capacity (P < 0.001, Fig. 3B) in HPMECs. The apoptosis rate was also significantly increased (P < 0.001, Fig. 3C). Concurrently, the level of the autophagy-related protein p62 decreased (P < 0.01, Fig. 3D) and the LC3-II/LC3-I ratio increased (P < 0.01, Fig. 3E). Further experiments showed that inhibiting the expression of miR-183-5p significantly reversed its high expression level in HPMECs (P < 0.01, Fig. 3A), restored their proliferation capacity (P < 0.01, Figs. 3B), inhibited apoptosis (P < 0.01, Figs. 3C) and markedly weakened autophagy function (P < 0.05, Figs. 3D–E).

Fig. 3.

miR-183-5p levels in the ox-LDL induction and miR-183-5p inhibition groups (A). The proliferation activity (B) and apoptosis rate (C) of HPMECs in different treatment groups. Relative level of the autophagy-related protein p62 (D) and the LC3-II/LC3-I ratio (E) in different treatment groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Analysis of inflammatory factors, oxidative stress markers and adhesion molecules revealed that ox-LDL treatment significantly increased the release of the inflammatory factors in HPMECs (P < 0.001, Fig. 4A), resulting in a significant rise in oxidative stress markers ROS and MDA levels and a fall in SOD activity (P < 0.01, Fig. 4B), as well as up-regulating the mRNA expression of the adhesion molecules ICAM-1 and VCAM-1 (P < 0.001, Fig. 4C). However, inhibiting the expression of miR-183-5p effectively reversed all of these changes. Specifically, the release of inflammatory factors was significantly reduced (P < 0.001, Fig. 4A), oxidative stress levels were restored (ROS and MDA decreased significantly, and SOD increased significantly, P < 0.05, Fig. 4B), and adhesion molecule mRNA expression was also inhibited (P < 0.01, Fig. 4C).

Fig. 4.

Relative change level of inflammatory factors (A), oxidative stress markers (B), as well as adhesion molecules expression (C) in ox-LDL induction and miR-183-5p inhibition groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001

miR-183-5p can target and regulate PDCD4

Serum PDCD4 expression was significantly lower in the PTE group (P < 0.001, Fig. 5A), and was negatively correlated with serum miR-183-5p (r = − 0.757, P < 0.001, Fig. 5B).

Fig. 5.

Serum PDCD4 mRNA levels in non-PTE and PTE patients (A). The correlation between miR-183-5p and PDCD4 expression in PTE patients (B). The binding sites between miR-183-5p and the 3’-UTR of PDCD4 (C). Validation of the targeting relationship between miR-183-5p and PDCD4 (D-E). **, P < 0.01; ***, P < 0.001

Prediction results from the ENCORI database indicated that there was a binding site for miR-183-5p with PDCD4 (Fig. 5C). Overexpression of miR-183-5p significantly reduced the luciferase activity of PDCD4-wt, whereas inhibition of miR-183-5p expression increased the luciferase activity (P < 0.01, Fig. 5D). However, there was no effect on PDCD4-mut (P > 0.05, Fig. 5E), which confirmed their direct binding.

The effects of co-inhibiting miR-183-5p and PDCD4 on the PTE progression

Compared with the control group, PDCD4 expression in HPMECs was significantly reduced following ox-LDL treatment (P < 0.01). Further inhibition of miR-183-5p effectively restored PDCD4 expression (P < 0.05), whereas co-inhibition of miR-183-5p and PDCD4 again significantly inhibited PDCD4 expression (P < 0.01, Fig. 6A).

Fig. 6.

PDCD4 mRNA levels in the ox-LDL induction, miR-183-5p inhibition, and miR-183-5p and PDCD4 co-inhibition groups (A). The proliferation activity (B) and apoptosis rate (C) of HPMECs in miR-183-5p and PDCD4 co-inhibition group. Relative level of the autophagy-related protein p62 (D) and the LC3-II/LC3-I ratio (E) in miR-183-5p and PDCD4 co-inhibition group. *, P < 0.05; **, P < 0.01; ***, P < 0.001

In the PTE cell model, inhibition of only miR-183-5p expression significantly increased the proliferative activity of HPMECs, inhibited apoptosis and autophagy, reduced the release of inflammatory factors, alleviated oxidative stress damage, and decreased the expression of adhesion molecule mRNA, thereby inhibiting PTE progression (Figs. 3 and 4). Further co-inhibition of miR-183-5p and PDCD4 significantly reduced the proliferative activity of HPMECs (P < 0.001, Fig. 6B) while promoting apoptosis (P < 0.01, Fig. 6C) and autophagy (P < 0.05, Figs. 6D–E). Concurrently, the release of inflammatory factors increased again (P < 0.001, Fig. 7A), ROS and MDA levels increased significantly, SOD activity decreased significantly, oxidative stress damage worsened (P < 0.01, Fig. 7B) and the mRNA expression of the adhesion molecules increased significantly (P < 0.01, Fig. 7C).

Fig. 7.

Relative change level of inflammatory factors (A), oxidative stress markers (B), as well as adhesion molecules expression (C) in miR-183-5p and PDCD4 co-inhibition group. **, P < 0.01; ***, P < 0.001

Discussion

PTE is a common critical condition that arises suddenly in clinical practice. Due to its non-specific clinical manifestations, it is often misdiagnosed, which poses a threat to patients’ lives. Although tests such as D-dimer and CTPA have improved diagnostic accuracy, they fail to meet the demands of precision diagnosis and treatment [17]. Furthermore, while the overall prognosis for PTE patients has improved considerably, high-risk patients still face high mortality rates and risks of long-term complications [18]. Therefore, developing specific biomarkers and gaining a deep understanding of their regulatory mechanisms is crucial for optimizing PTE diagnostic and therapeutic strategies.

miRNAs are easily accessible and highly stable in bodily fluids, have shown potential as novel biomarkers in recent years [19]. Previous studies found that low miR-21 expression is linked to higher recurrence and post-thrombotic syndrome risk in VTE patients, making it a potential predictive factor [20]. Coronary artery thrombus miR-34a-5p up-regulation is significantly associated with endothelial cell inflammatory damage and macrophage polarization [21]. miR-150-5p and miR-221-3p are effective in identifying acute PE patients and predicting pulmonary hypertension [22, 23]. Studies on miR-183-5p highlight its value in thrombotic diseases. miR-183-5p has good diagnostic efficacy and participates in the thrombosis process of AVTE patients [11]. Furthermore, miR-183-5p plays a significant role in the regulation of vascular pathology, serving as a diagnostic and prognostic biomarker for CAD and ACS [8, 10] and influencing the growth of VSMCs in AS patients [9]. Under the influence of ox-LDL, it targets FOXO1, causing an imbalance in VSMC proliferation/apoptosis [24]. This study found that serum miR-183-5p increased with risk stratification in PTE patients, suggesting that it may reflect disease severity. The diagnostic performance of miR-183-5p was superior to that of D-dimer for the PTE diagnosis, which is consistent with clinical practice where D-dimer is primarily used to rule out PTE due to its low specificity and high false-positive rate (which can lead to excessive testing or misdiagnosis). Combined detection was more effective, suggesting that it can help reduce missed diagnoses. Furthermore, elevated miR-183-5p was a risk factor for a poor prognosis in PTE patients, highlighting its importance as a parameter for assessing prognosis. Notably, the current study design cannot determine whether elevated miR-183-5p is a cause or consequence of thrombus formation. It remains unclear whether miR-183-5p upregulation precedes thrombus formation (suggesting a pathogenic role), parallels the inflammatory response during active thrombosis (indicating an ongoing process), or reflects post-thrombotic tissue injury (serving as a damage marker). Serial sampling from symptom onset, stratified by time to diagnosis, would help distinguish these possibilities and establish the clinical utility of miR-183-5p for early detection versus disease monitoring.

Hyperlipoidemia induces atherosclerosis and raises blood viscosity, while diabetes damages vascular endothelium and boosts coagulation; both are key thrombotic risk factors [25]. Elevated WBCs relate to pathological mechanisms like inflammatory activation and vascular endothelial damage [26]. Elevated D-dimer levels often indicate thrombus formation and increased fibrinolysis in the body [27]. Reduced blood gas parameters reflect impaired pulmonary circulation function, which can further induce vascular endothelial damage, coagulation dysfunction and inflammatory responses [28]. Notably, the severity of these abnormalities was more pronounced in the poor prognosis group, aligning with the trend of elevated miR-183-5p expression. This suggests that miR-183-5p may contribute to the pathological progression of PTE by participating in inflammatory responses, thrombosis and pulmonary circulation regulation. High miR-183-5p expression was not only a signal of disease severity, but might also promote disease progression, further highlighting its importance as a clinical assessment indicator.

HPMECs are the main cells responsible for maintaining the anticoagulant-antithrombotic homeostasis of pulmonary vessels. Damage to these cells or abnormalities in their function can activate the coagulation system, promote platelet aggregation and enhance inflammatory responses, ultimately inducing thrombosis [29]. Ox-LDL is a key driver of AS and thrombotic diseases and can contribute to thrombosis formation by damaging the vascular endothelium and accelerating inflammatory responses. This is highly consistent with the pathological mechanisms of PTE [30]. This study established a PTE model by treating HPMECs with ox-LDL, which caused its functions damage. Inhibiting miR-183-5p in the model significantly reversed functions abnormalities, suggesting that miR-183-5p may disrupt vascular endothelial homeostasis (e.g., regulating cell survival, inflammation, and oxidative stress), disturb the anticoagulant-coagulation balance, accelerate thrombus formation to promote PTE progression.

miRNAs typically regulate gene expression and downstream pathophysiological processes by binding specifically to the 3’UTR of target mRNAs. For instance, increased levels of miR-34a-3p can hinder the proliferation of pulmonary arterial smooth muscle cells in acute PE by targeting DUSP1 [31]. Similarly, miR-124 influences pulmonary artery endothelial metabolism in PTE by regulating the expression of PTBP1 and PKM, thereby participating in endothelial remodeling [32]. PDCD4 is an important functional molecule that has been shown to play a role in vascular diseases. As previously mentioned, downregulation of PDCD4 is closely associated with plaque stability in AS, and its absence leads to thickening of the vascular wall and narrowing of the lumen [14, 15]. Previous studies have confirmed that miR-183-5p can regulate inflammatory responses, the balance of oxidative stress, and cell proliferation and apoptosis by targeting PDCD4 [33–35], which provides a basis for exploring its role in PTE. This study measured serum PDCD4 mRNA by qPCR and found a significant negative correlation with miR-183-5p. However, several important caveats must be considered when interpreting serum PDCD4 mRNA levels as a surrogate for pulmonary vascular endothelial PDCD4 expression. First, PTE primarily causes pathological changes in pulmonary vascular endothelium, but there is currently no direct evidence that serum PDCD4 mRNA accurately reflects PDCD4 expression specifically in HPMECs. PDCD4 is a predominantly cytoplasmic protein, so its free mRNA in serum likely originates from cell lysis (e.g., leukocytes, platelets), endothelial injury, or extracellular vesicles (EVs)—which protect RNA from degradation and are major carriers of stable serum RNA. Since this study did not isolate EVs or separate blood cell fractions, we cannot distinguish between free and EV-encapsulated PDCD4 mRNA. Circulating PDCD4 mRNA likely originates mainly from leukocytes and platelets rather than specifically from pulmonary endothelium, so its changes may indicate systemic inflammation or generalized endothelial injury rather than local pulmonary vascular pathology. Serum mRNA stability also depends on preanalytical factors (processing time, anticoagulant, centrifugation, storage temperature). Although we used standardized handling (prompt centrifugation, − 80 °C storage), cell lysis during processing remains a possible confounder. Therefore, while the observed negative correlation between serum miR-183-5p and PDCD4 mRNA supports the existence of a regulatory relationship, serum PDCD4 mRNA may have lower tissue specificity than expected as a biomarker for pulmonary endothelial injury in PTE and should be interpreted alongside other endothelial-specific markers (e.g., vWF, ICAM-1). To clarify PDCD4 mRNA origin, future studies should isolate EVs (e.g., via density-gradient centrifugation) and quantify PDCD4 mRNA separately in vesicular and free fractions, as well as in specific blood cell populations. Complementary PDCD4 protein measurement (e.g., by ELISA or Western blot) and immunohistochemical staining of lung tissue would further support interpretation of local versus systemic PDCD4 expression. Although the dual-luciferase assay confirmed direct binding of miR-183-5p to the PDCD4 3′-UTR, serum PDCD4 mRNA levels may not reliably reflect PDCD4 expression in pulmonary vascular endothelial cells. Circulating PDCD4 mRNA likely originates mainly from leukocytes—not pulmonary endothelium—so its changes may indicate systemic inflammation rather than local endothelial injury. As a result, serum PDCD4 mRNA may have lower specificity than expected as a PTE biomarker and should be interpreted alongside other endothelial injury markers. In the PTE cell model, the simultaneous inhibition of both molecules reversed the protective effect of miR-183-5p silencing on HPMECs. This suggests that, miR-183-5p may exacerbate endothelial injury and thrombosis by targeting and inhibiting PDCD4. This deregulates cellular functions and promotes PTE progression. Unfortunately, this study was unable to establish a PTE animal model successfully. Further exploration is required into the mechanism of action of the miR-183-5p/PDCD4 axis in vivo and its downstream signaling pathways.

This study has several limitations. The study was conducted at a single center, which may introduce selection bias and limit the generalizability of the findings to broader populations with different demographic characteristics, genetic backgrounds, and clinical presentations. The relatively small sample size may also affect the statistical power and the robustness of the conclusions. Furthermore, this study only measured miR-183-5p expression at a single time point upon clinical diagnosis and did not capture its dynamic changes during the thrombus formation process. The timing of miR-183-5p upregulation—whether it occurs at the initiation of thrombosis, during propagation, or at maturation—remains unknown. Future studies should record the time interval from symptom onset to clinical diagnosis and perform serial measurements of miR-183-5p to classify thrombus formation stages and determine the optimal time window for biomarker detection. Moreover, the follow-up period was limited to 30 days, which precludes assessment of the long-term prognostic value of miR-183-5p for outcomes such as CTEPH, recurrent VTE, and late mortality. This short follow-up duration may underestimate the prognostic utility of miR-183-5p, as biomarkers with sustained elevation might better predict chronic complications. Therefore, future multicenter prospective studies with extended follow-up durations (e.g., 6–12 months or longer), larger and more diverse cohorts are warranted to validate the diagnostic and short-term and long-term prognostic value of miR-183-5p in PTE across different ethnicities and geographic regions. Serum PDCD4 mRNA detection in this study has methodological limitations. First, we did not trace its source—so we cannot determine whether it originated from circulating cell lysis, extracellular vesicles, or tissue injury. Second, as an intracellular protein, PDCD4 mRNA’s stability and degradation kinetics in serum are unknown; single-time-point measurements may miss its dynamic changes. Future studies should isolate extracellular vesicles and integrate proteomic and single-cell sequencing to clarify PDCD4’s circulating forms and biological relevance in PTE.

Conclusion

This study found that the serum miR-183-5p expression in PTE patients increases gradually with risk stratification, and serve as a novel biomarker for supplementary diagnosis and short-term prognosis assessment of PTE within a 30-day follow-up period. In vitro experiments demonstrated that miR-183-5p regulates HPMECs function by targeting PDCD4 and participates in thrombus formation, thereby influencing the PTE progression. Future investigations with prolonged follow-up are needed to determine whether miR-183-5p also predicts long-term outcomes such as CTEPH and recurrent VTE. This provides a new molecular perspective on the PTE pathogenesis.

Author contributions

SW, and YW carried out the concepts, design and definition of intellectual content, SW, QW, and WFX provided assistance for data acquisition, data analysis and statistical analysis. All authors performed the experiment, and draft of the manuscript. YW revised the manuscript critically for important intellectual content. All authors have read and approved the content of the manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

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

Declarations

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki. This study was approved by the Medical Ethics Committee of Zibo Central Hospital. All participants provided written informed consent prior to enrollment

Consent for publication

Not applicable.