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Abstract

Background

Osteoporosis (OP) is a progressive metabolic disorder resulting from an uncoupling of bone formation and breakdown processes.

Objectives

This study mainly explored the effects of lncRNA DUXAP8 on the biological functions and osteogenic potential of hBMSCs (human bone marrow mesenchymal stem cells).

Methods

This study enrolled 35 OP patients and 35 healthy individuals. The expression profiles of lncRNA DUXAP8 and miR-24-3p in serum and cells were analyzed using RT-qPCR. Additionally, osteogenic marker expression at mRNA and protein levels was assessed. The proliferation ability of hBMSCs was evaluated by CCK-8 assay, with cell motility and invasiveness assessed by Transwell assay. The regulatory relationship between lncRNA DUXAP8 and miR-24-3p was verified by Dual-Luciferase.

Results

Serum lncRNA DUXAP8 levels were reduced in OP patients, with an AUC of 0.967 (95% CI: 0.931-1.000), sensitivity 0.914, and specificity 0.943. Inhibition of lncRNA DUXAP8 decreased osteogenic markers (mRNA/protein) and hBMSCs proliferation, migration, invasion, while overexpression had opposite effects. LncRNA DUXAP8 targeted miR-24-3p, and miR-24-3p overexpression reversed the promoting effects of oe-DUXAP8 on hBMSCs functions.

Conclusion

LncRNA DUXAP8 in the serum of OP patients is decreased. LncRNA DUXAP8 has a high diagnostic value for patients with OP. Overexpression of lncRNA DUXAP8 may enhance hBMSCs’ proliferative and osteogenic potential by targeting miR-24-3p.

Keywords

osteoporosis hBMSCs lncRNA DUXAP8 miR-24-3p diagnosis proliferation migration invasion

Introduction

Osteoporosis (OP) is a metabolic disease caused by multiple factors, primarily characterized by a higher rate of bone resorption than bone formation. This imbalance leads to disrupted bone microstructure, reduced bone mineral density and quality, increased bone fragility, and elevated fracture risk.¹ Human bone marrow mesenchymal stem cells (hBMSCs) are pluripotent stem cells widely present in cancellous bone and bone marrow cavities. They exhibit strong proliferative capacity and multi-tissue differentiation potential, capable of differentiating into chondrocytes, osteoblasts, adipocytes, and other cell types under specific microenvironmental induction.² Studies have shown that hBMSCs play a critical role in maintaining the balance between bone formation and resorption. Exploring the molecular mechanisms influencing hBMSCs proliferation and differentiation can provide potential molecular targets for OP treatment.

LncRNAs and miRNAs, as key regulatory non-coding RNAs, modulate critical cellular activities including proliferation, apoptosis, and differentiation, playing significant roles in pathological processes.^3,4 Research has demonstrated that lncRNAs play significant roles in OP pathogenesis.^5–8 Chen et al. reported that overexpression of lncRNA BMNCR significantly inhibits osteoclastogenesis and bone resorption capacity in RANKL-induced osteoclast differentiation in vitro, and slows down the progression of osteoporosis.⁹ Lnc-ob1 exerts an osteo-promotive effect during osteogenic differentiation.¹⁰ MIR31HG can directly bind to IκBα and activate the NF-κB pathway, thereby inhibiting osteogenic differentiation.¹¹ A recent study reported that lncRNA-DUXAP8 (double homeobox A pseudogene 8), a 2107-bp lncRNA located at chromosome 22q11, exhibits abnormal expression in various tumor tissues.^12,13 In NSCLC, lncRNA DUXAP8 regulates miR-498 through the AKT/mTOR pathway to inhibit proliferation and metastasis.¹⁴ LncRNA DUXAP8 can promote the proliferation, and invasion of pancreatic cancer cells.¹⁵ Reduced lncRNA DUXAP8 expression has been observed in bone marrow tissues of AML patients, suggesting its involvement in AML development.¹⁶ However, the role of lncRNA DUXAP8 in osteoporosis and its effects on hBMSCs proliferation and differentiation remain unclear.

Studies have shown that lncRNAs can regulate miRNA expression, and the two jointly participate in disease development.¹⁷ Bioinformatics software predicts that miR-24-3p may be a target gene of lncRNA DUXAP8. Previous studies have demonstrated that miR-24-3p exerts a critical regulatory function in OP^18,19

Therefore, this study first detected the lncRNA DUXAP8 and miR-24-3p in the serum of OP patients, and explored whether lncRNA DUXAP8 can regulate miR-24-3p expression to influence hBMSCs proliferation and differentiation, aiming to provide new insights for OP treatment.

Materials and methods

Study subjects

From January 2023 to December 2024, our study enrolled 35 OP patients (10 male, 25 female; mean age 64.71 ± 5.38 years; range 54-76) from hospital admissions. The inclusion criteria were: (1) positive characteristics in bone mineral density examination; (2) X-ray examination showing generalized bone rarefaction, increased bone translucency, with the most specific spinal changes including widened intervertebral space, wedge-shaped deformation of the thoracic vertebrae, and scattered/multiple involved vertebral bodies; (3) low back and limb pain, muscle cramps, chills and cold limbs, mostly with obvious lumbosacral pain. In this study, patients had not used any medications that could potentially interfere with bone metabolism prior to enrollment. Moreover, blood samples from all patients were collected prior to the initiation of treatment. Consequently, the medications used during the treatment process (such as hormone drugs that may affect bone metabolism, antiepileptic drugs, and anticonvulsant drugs) will not have an impact on the data of this study. This research design ensures the independence and reliability of the study results, accurately reflecting the physiological state of patients prior to treatment. Additionally, the control group comprised 35 age-matched healthy examinees (11 male, 24 female; mean age 64.65 ± 6.22 years, range 45-79). Fasting venous blood samples were collected from OP patients and healthy controls in the morning, centrifuged at 3500 r/min for 5 min, and the supernatants were taken for standby.

The study was conducted in accordance with the Declaration of Helsinki. The research protocol was approved by the Ethics Committee of Shanghai Zhongye Hospital, and all of the participants provided signed informed consent.

Cell culture

hBMSCs were sourced from Cyagen Biosciences Inc. (Guangzhou, China). After thawing, hBMSCs were cultured in complete growth medium at 37°C in a humidified 5% CO₂ incubator. Upon reaching 80–90% confluence, the medium was aspirated and cells were washed with ice-cold PBS. Following PBS aspiration, 0.25% trypsin solution was added for digestion, and cells were subcultured.

Osteogenic induction

hBMSCs (2.5 × 10⁴ cells/well) in logarithmic phase were cultured in osteogenic differentiation medium (24-well plate), with medium refreshed every 48 h. Cells were collected after 0, 3, 7, and 14 days of culture for subsequent analysis.

Cell transfection

hBMSCs (1 × 10⁵ cells/well) in logarithmic growth phase were seeded into 6-well plates. When cell confluence reached 60%, si-NC, si-lncRNA DUXAP8, oe-NC, oe-lncRNA DUXAP8, oe-lncRNA DUXAP8 + miR-24-3p-NC, or oe-lncRNA DUXAP8 + miR-24-3p-mimic were transfected into hBMSCs using Lipofectamine™ 2000 (Invitrogen, USA) according to the instruction manual. After 12 h of transfection, the cells were cultured in conventional medium for another 48 h and collected for subsequent experiments.

CCK-8 assay

Transfected cells were plated in 96-well plates (5 × 10³ cells/well). After 24 h of culture in conventional medium, 10 μL of CCK-8 solution (Shanghai LiJi Biotechnology Co., Ltd) was added to each well, and plates were incubated for an additional 4 h. OD450 was measured using a microplate reader.

Transwell assay

Migration: Following 24 h post-transfection, cells were resuspended in serum-free medium and plated in Transwell inserts (8-μm pores; 4 × 10⁵ cells/well). After 24 h incubation, non-migrated cells were scraped from the upper membrane surface. Migrated cells were fixed (4% PFA), stained (0.1% crystal violet), imaged, and quantified (ImageJ). Invasion: Cells from each group were cultured for 24 h, resuspended in serum-free medium, and seeded into the upper chamber of a Matrigel-coated Transwell insert (8-μm pore size; BD Biosciences, USA) at 8 × 10⁵ cells per well. The lower chamber contained complete medium. After a 24-h incubation, non-invaded cells were discarded, and invaded cells were fixed, stained, imaged, and quantified using the same protocol as the migration assay.

DLR (Dual-Luciferase reporter)

Wild-type (DUXAP8-WT) and mutant (DUXAP8-MUT) luciferase reporter vectors for lncRNA DUXAP8 were constructed. DUXAP8-WT or DUXAP8-MUT was co-transfected with miR-24-3p mimic, miR-24-3p inhibitor, or miR-24-3p-NC into hBMSCs. Post-12 h transfection, medium was refreshed followed by 48 h culture. Luciferase activity was measured using a commercial DLR kit (Beyotime).

RT-PCR

Total RNA was extracted using Trizol reagent (Invitrogen, USA), and RNA purity and concentration were measured by ultraviolet spectrophotometry. cDNA was synthesized using a reverse transcription kit (Jingmei Biotechnology, Shenzhen) according to the protocol. cDNA served as the template for PCR amplification. ALP (alkaline phosphatase), RUNX2 (Runt-related transcription factor 2), OCN (osteocalcin), OPN (osteopontin), and lncRNA DUXAP8 were normalized to β-actin, while miR-24-3p was normalized to U6. Gene expression was quantified via 2^−ΔΔCt analysis.

Western blot analysis of osteogenic-related proteins

The Western blot analysis was used to confirm the protein expression levels of the osteogenic-related markers ALP, RUNX2, OCN, and OPN. At ∼90% confluence, cells were PBS-washed, mechanically scraped into 15 mL tubes, and centrifuged (10,000 rpm, 10 min, 4°C). Pellets were lysed in ice-cold RIPA buffer for 20–30 min, followed by centrifugation at 13,500 rpm for 15 min at 4°C. Following BCA protein quantification, the supernatants were stored at −80°C.

Statistical methods

Statistical analyses used SPSS 25.0 and GraphPad Prism 9. Normally distributed measurement data are presented as mean ± standard deviation (x ± s). Comparisons between two groups were performed using independent samples t-tests, while comparisons among multiple groups were conducted using analysis of variance (one-way ANOVA and two-way ANOVA). Enumeration data are presented as counts, and intergroup comparisons were performed using chi-square tests. Statistical significance was set at *P < 0.05. All experiments in this study were conducted with three independent biological replicates and three technical replicates to ensure the reliability of the results.

Results

Comparison of clinical data between OP patients and healthy controls

There were no significant differences in age, gender, BMI, smoking history, or drinking history between the two groups (P > 0.05). Significant BMD reductions were observed at all measured sites (L1-L4/FN/TH) in the OP cohort (***P < 0.001, Table 1).

Table 1.

Clinical data of the study subjects.

Indicators	Healthy control (n = 35)	OP patients (n = 35)	P-value
Age	64.65 ± 6 .22	64.71 ± 5.38	0.967
Gender			0.794
Male	11	10
Female	24	25
BMI	22.79 ± 1.15	22.90 ± 1.30	0.721
Smoking history			0.799
No	23	24
Yes	12	11
Drinking history			0.803
No	22	13
Yes	13	12
L1-L4 BMD/(g/cm²)	1.075 ± 0.14	0.69 ± 0.07	<0.001
FN BMD/(g/cm²)	0.89 ± 0.12	0.6 ± 0.10	<0.001
TH BMD/(g/cm²)	0.98 ± 0.17	0.63 ± 0.11	<0.001

Note: OP, osteoporosis. BMI,Body Mass Index. L1-L4 BMD, Bone Mineral Density of Lumbar Vertebrae L1 to L4. FN, Femoral Neck. TH, Total Hip. The data are presented as the number of cases or the mean ± standard deviation, and n represents the sample size.

Expression level and diagnostic efficacy of lncRNA DUXAP8

LncRNA DUXAP8 in the serum of OP patients was significantly lower than that in healthy controls (***P < 0.001, Figure 1(a)). ROC curve analysis showed that lncRNA DUXAP8 had an AUC of 0.967, a 95% CI of 0.931–1.000, a sensitivity of 0.914, and a specificity of 0.943 (***P < 0.001, Figure 1(b)).

Figure 1.

Expression of lncRNA DUXAP8 in serum. (a) Expression of lncRNA DUXAP8 in serum from osteoporosis (OP) patients and healthy controls. (b) ROC curve evaluating the predictive efficacy of lncRNA DUXAP8 for clinical diagnosis of OP patients. The data are presented as mean ± standard deviation. ***P < 0.001.

Expression levels of ALP, RUNX2, OCN, and OPN after osteogenic induction

At osteogenic days 3, 7, 14, mRNA levels of ALP (Figure 2(a)), RUNX2 (Figure 2(b)), OCN (Figure 2(c)), and OPN (Figure 2(d)) significantly increased during osteogenic induction (***P < 0.001), confirming successful osteogenic differentiation of hBMSCs.

Figure 2.

Expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN). (a) ALP, (b) RUNX2, (c) OCN, (d) OPN. The data are presented as mean ± standard deviation. ***P < 0.001.

Effects of oe-lncRNA DUXAP8 and si-lncRNA DUXAP8 on the biological functions of hBMSCs

The results showed that after knocking down lncRNA DUXAP8, lncRNA DUXAP8 was significantly reduced, and after overexpressing lncRNA DUXAP8, lncRNA DUXAP8 was significantly increased, indicating successful transfection (***P < 0.001, Figure S1). Inhibition of lncRNA DUXAP8 significantly decreased the mRNA levels of ALP, RUNX2, OCN, and OPN, while overexpression of lncRNA DUXAP8 significantly increased the mRNA levels of osteogenesis-related markers (***P < 0.001, Figure 3(a)). After inhibiting lncRNA DUXAP8, the protein levels of ALP, RUNX2, OCN, and OPN significantly decreased, while overexpression of lncRNA DUXAP8 significantly increased the protein levels of osteogenesis-related markers (***P < 0.001, Figure 3(b)).

Figure 3.

Effects of oe-DUXAP8 and si-DUXAP8 on osteogenesis-related genes and proteins. (a) mRNA levels, (b) Protein levels. The data are presented as mean ± standard deviation. ***P < 0.001.

Knockdown of lncRNA DUXAP8 significantly decreased the proliferation (Figure 4(a)), migration (Figure 4(b)), and invasion (Figure 4(c)) abilities of hBMSCs (****P < 0.0001). In contrast, overexpression of lncRNA DUXAP8 significantly increased the proliferation, migration, and invasion abilities of hBMSCs (**P < 0.01, Figure 4).

Figure 4.

Effects of oe-DUXAP8 and si-DUXAP8 on biological functions of hBMSCs. (a) Proliferation, (b) Migration, (c) Invasion. The data are presented as mean ± standard deviation. **P < 0.01, ***P < 0.001.

LncRNA DUXAP8 targets and regulates miR-24-3p

Bioinformatics website prediction identified the binding sites between lncRNA DUXAP8 and miR-24-3p (Figure 5(a)). DLR showed that overexpression of miR-24-3p markedly inhibited the activity of DUXAP8-WT, while knockdown of miR-24-3p markedly enhanced the activity of DUXAP8-WT (***P < 0.001, Figure 5(b)). Overexpression or inhibition of miR-24-3p did not affect the activity of DUXAP8-MUT (P > 0.05). In cells transfected with miR-24-3p mimics or inhibitors, the expression levels of miR-24-3p were significantly upregulated or downregulated, respectively, confirming successful transfection (***P < 0.001, Figure 5(c)). Overexpression of lncRNA DUXAP8 in hBMSCs significantly increased lncRNA DUXAP8. However, compared with oe-lncRNA DUXAP8 + miR-24-3p-NC, overexpression of miR-24-3p did not significantly change lncRNA DUXAP8 (P > 0.05, Figure 5(d)). In contrast, overexpression of miR-24-3p reversed the inhibitory effect of oe-lncRNA DUXAP8 on miR-24-3p (***P < 0.001, Figure 5(e)), indicating that lncRNA DUXAP8 targets and regulates miR-24-3p.

Figure 5.

LncRNA DUXAP8 targets and regulates miR-24-3p. (a) Binding sites between lncRNA DUXAP8 and miR-24-3p, (b) Luciferase reporter gene assay, (c) The expression of miR-24-3p in cells, (d) Expression level of lncRNA DUXAP8 after transfection. (e) Expression level of miR-24-3p after transfection. The data are presented as mean ± standard deviation. ***P < 0.001.

MiR-24-3p-mimic reverses the effect of lncRNA DUXAP8 overexpression on the biological functions of hBMSCs. Overexpression of lncRNA DUXAP8 significantly increased the mRNA (***P < 0.001, Figure 6(a)) and protein (***P < 0.001, Figure 6(b)) levels of ALP, RUNX2, OCN, and OPN in hBMSCs. However, overexpression of miR-24-3p reversed the promoting effect of oe-lncRNA DUXAP8 on the mRNA and protein levels of ALP, RUNX2, OCN, and OPN (***P < 0.001, Figure 6).

Figure 6.

Effects of oe-DUXAP8 and miR-24-3p-mimic on the osteogenic differentiation of hBMSCs (a) mRNA levels, (b) Protein levels. The data are presented as mean ± standard deviation. ***P < 0.001.

Overexpression of lncRNA DUXAP8 significantly enhanced the proliferation (Figure 7(a)), migration (Figure 7(b)), and invasion (Figure 7(c)) abilities of hBMSCs (**P < 0.01). However, overexpression of miR-24-3p reversed the promoting effect of oe-lncRNA DUXAP8 on the proliferation, migration, and invasion of hBMSCs.

Figure 7.

Effects of oe-DUXAP8 and miR-24-3p-mimic on the biological functions of hBMSCs (a) Proliferation, (b) Migration, (c) Invasion. The data are presented as mean ± standard deviation. **P < 0.01, ***P < 0.001.

Discussion

Osteoporosis is a prevalent metabolic bone disorder.²⁰ It is estimated that there are 200 million osteoporosis patients worldwide.²¹ Osteoporosis and osteoporosis-related fractures are more prevalent in women over 55 years old and men over 65 years old.^22,23 Exploring specific genes causing osteoporosis and identifying new therapeutic targets are particularly important. With the recent development of high-throughput technologies, lncRNAs have been discovered, and their roles and associations with various cellular functions and pathological conditions have been rapidly identified. Although the mechanistic details of lncRNA functions have not been fully elucidated, reports still provide strong evidence for their involvement in osteogenic gene regulation and pathogenesis.²⁴

Early diagnosis and treatment of osteoporosis can help relieve pain and symptoms and facilitate timely medical intervention.²⁵ This study found that lncRNA DUXAP8 was significantly downregulated in the serum of osteoporosis patients. Through ROC curve analysis, lncRNA DUXAP8 was found to have good predictive value in diagnosing OP patients, suggesting it may be a potential biomarker for clinical adjunct diagnostic tool of OP.

The pathogenesis of OP is closely associated with abnormal osteogenic differentiation of BMSCs.²⁶ Meanwhile, studies have revealed that abnormal proliferation, migration, and invasion of BMSCs can also disrupt bone tissue homeostasis and contribute to the pathological process of OP.²⁷ Multiple markers play critical roles in osteogenic differentiation and bone metabolism regulation. ALP is an important substance involved in the formation, metabolism, and regeneration of mineralized tissues such as bone and teeth. Its expression level reflects the trend of osteoblastic transformation and bone formation, serving as a key marker of osteogenic differentiation.²⁸ RUNX2, a member of the RUNX family, promotes osteogenic differentiation of hBMSCs.²⁹ OCN is the most abundant non-collagen protein in bone tissue, participating in regulating mineralization and bone turnover.³⁰ OPN serves as an extracellular matrix protein that participates in bone metabolism and osteoclast activation. It plays an important role in both bone metabolism and osteoclast activation, while also being associated with cell migration and invasion processes.³¹ After inducing hBMSCs with osteogenic induction medium, the gene levels of ALP, RUNX2, OCN, and OPN increased with prolonged induction time, indicating successful osteogenic differentiation of hBMSCs. This study also showed that low expression of lncRNA DUXAP8 reduced the mRNA and protein levels of ALP, RUNX2, OCN, and OPN, as well as the proliferation, migration, and invasion abilities of hBMSCs, while overexpression of lncRNA DUXAP8 produced opposite effects. Previous studies have shown that abnormal proliferation, migration and invasion of cells can disrupt the normal repair and reconstruction balance of bone tissue, thereby promoting the occurrence and development of OP.³² Therefore, the results suggest that low lncRNA DUXAP8 expression decreases hBMSC proliferation and differentiation, disrupts normal bone metabolism and repair, and may contribute to OP pathogenesis, making it a potential therapeutic target.

LncRNAs can regulate miRNA expression, thereby influencing disease development.³³ Bioinformatics software predictions showed that miR-24-3p might be a target gene of lncRNA DUXAP8, which was confirmed by this study to be negatively regulated by lncRNA DUXAP8 in hBMSCs. MiR-24 is a miRNA associated with bone remodeling.³⁴ Studies have found that miR-24 is an effective inhibitor of bone formation in premenopausal osteoporosis patients.³⁵ Moreover, miR-24-3p can significantly inhibit the osteogenic differentiation of BMSCs.³⁶ Research has found that microRNA-24 can inhibit the protein expression of T-cell factor-1 by targeting its 3′-untranslated region, thereby exerting a negative regulatory role in osteoblast differentiation and maturation.³⁷ In addition, miR-24-2-5p reduces the risk of early bone metastasis by inhibiting the malignant behavior of breast cancer cells and protecting the bone microenvironment.³⁸ This is similar to the results of this study. We found that overexpression of miR-24-3p not only reversed the promoting effects of lncRNA DUXAP8 overexpression on the mRNA and protein levels of ALP, RUNX2, OCN, and OPN but also reversed its promoting effects on hBMSCs proliferation, migration, and invasion. This indicates that lncRNA DUXAP8 may regulate hBMSCs proliferation and differentiation through miR-24-3p.

This study thoroughly investigated the impact of long non-coding RNA DUXAP8 on the biological functions and osteogenic potential of hBMSCs, yet certain limitations remain. First, the small sample size and single-center cohort study may limit the generalizability and accuracy of the results. Future research should conduct larger-scale multicenter validation to confirm the diagnostic value of lncRNA DUXAP8, and its correlation with clinical indicators such as BMD, T-scores, and fracture history was analyzed to more accurately assess its potential as a biomarker for osteoporosis. Secondly, although this study attempted in vivo animal experiments, due to experimental constraints and operational difficulties, it failed to successfully establish a stable osteoporosis animal model, resulting in challenging and non-reproducible data acquisition. During the experiment, some mice developed health issues unrelated to the disease, which increased the complexity of the experiment and raised ethical concerns. Future plans include collaborating with a professional animal experimentation center to optimize experimental conditions and procedures, ensuring the successful establishment of animal models and the reliability of data, while strictly adhering to ethical guidelines and prioritizing animal welfare. Finally, the use of commercialized hBMSCs may not fully represent the actual conditions within patients, as these cells are cultured under specific conditions and may exhibit biological differences compared to the cells of actual patients. In future studies, the use of patient-derived BMSCs or the conduct of in vivo experiments should be considered to enhance the translational value and clinical relevance of the research findings.

In conclusion, lncRNA DUXAP8 is significantly downregulated in the serum of OP patients, demonstrating high diagnostic value for OP. Additionally, lncRNA DUXAP8 may promote hBMSCs proliferation and osteogenic differentiation via miR-24-3p, providing a novel therapeutic target for OP treatment.

Supplemental Material

Supplemental Material - A study on the diagnostic significance and biological roles of lncRNA DUXAP8 in osteoporosis

Supplemental Material for A study on the diagnostic significance and biological roles of lncRNA DUXAP8 in osteoporosis by Huiang Chen, Rui Wang, Yue Du, Zhongxiang Liu, Xin Zhang, Qi Zhan, Xianmin Wu in Journal of Orthopaedic Surgery

Footnotes

ORCID iD

Xianmin Wu

Ethical considerations

The study was conducted in accordance with the Declaration of Helsinki. The research protocol was approved by the Ethics Committee of Shanghai Zhongye Hospital.

Consent to participate

All of the participants provided signed informed consent.

Author contributions

Huiang Chen and Rui Wang made substantial contributions to conception and design, performed all the experiment, and was a major contributor in writing the manuscript. Yue Du, Zhongxiang Liu and Xin Zhang contributed to acquisition of patients and tissues specimens, analysis and interpretation of data. Qi Zhan and Xianmin Wu contributed with interpretation of the results and critically revised the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by Science and Technology Innovation Special Fund Project of Baoshan District Science and Technology Commission, Shanghai (Grant numbers: 2024-E-67).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.*

Supplemental Material

Supplemental material for this article is available online.

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