miR-2116-5p functions as a tumor suppressor in lung adenocarcinoma by targeting ADAM12 and serves as a prognostic biomarker

Abstract

Background

MicroRNAs play key roles in tumor progression. miR-2116-5p is downregulated in lung adenocarcinoma (LUAD), and this study investigated its prognostic value and functional role in the disease.

Materials and methods

A total of 125 LUAD patients contributed tissue samples. miR-2116-5p and ADAM12 expression in tissues and cell lines were detected by RT‑qPCR. Clinicopathological correlations of miR-2116-5p were analyzed using the chi-square test. Kaplan‑Meier and Cox regression were employed to assess prognostic significance. CCK‑8, Transwell, and dual‑luciferase reporter assays were performed to investigate miR‑2116-5p function and its targeting of ADAM12. Rescue experiments validated the functional involvement of ADAM12.

Results

Significant downregulation of miR-2116-5p was observed in LUAD tissues and cell lines. Low expression was markedly linked to lymph node metastasis (P = 0.013) and advanced TNM stage (P = 0.002). Patients exhibiting reduced miR-2116-5p levels showed worse overall survival, and it was identified as an independent prognostic factor (HR = 2.521, 95% CI: 1.129–5.628, P = 0.020). Functional experiments showed that increasing miR-2116-5p expression suppressed LUAD cell proliferation, migration, and invasion, whereas its knockdown promoted these processes. ADAM12 was confirmed as a direct target, with expression inversely correlated in LUAD tissues (r = -0.749, P < 0.001). ADAM12 overexpression effectively counteracted the ability of miR-2116-5p to suppress proliferation, migration, and invasion.

Conclusion

miR‑2116-5p suppresses LUAD progression by targeting ADAM12, suggesting it may serve as a prognostic biomarker and therapeutic target.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13019-026-04473-3.

Introduction

Lung cancer contributes to the highest levels of both incidence and mortality in global oncology statistics. As the most frequent histological subtype, lung adenocarcinoma (LUAD) represents roughly 40% of total lung cancer cases [1]. Early-stage LUAD typically lacks distinct clinical signs, and the scarcity of reliable detection tools means that diagnosis often occurs at an advanced stage in most cases [2]. Patients with advanced LUAD face elevated risks of both recurrence and metastasis. Even when treatment includes surgery, radiotherapy, and systemic approaches, the five-year overall survival rate stays persistently poor [3]. Therefore, the identification and validation of reliable prognostic biomarkers are of significant clinical importance for improving risk stratification and personalized treatment for LUAD patients.

MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression post-transcriptionally by binding to target mRNA 3′UTRs, through gene expression regulation, these molecules contribute to proliferation, differentiation, apoptosis, and metabolic regulation [4]. Recent research has increasingly revealed that abnormal miRNA expression contributes significantly to the initiation and progression of malignant tumors. As an illustration, miR-3648 facilitates the proliferation, migration, and invasive capacity of LUAD cells by downregulating SOCS2 [5], miR-126-5p has been shown to increase the radiosensitivity of LUAD cells via regulation of the KLF2/EZH2/BIRC signaling axis [6]. In addition, miR-148b-3p expression correlates significantly with both tumor grade and size in LUAD patients, functioning as an independent predictor of overall survival [7]. miR-2116-5p regulates vascular endothelial function, inflammatory responses, proliferation, migration, and angiogenesis [8, 9]. Preliminary research indicates that miR-2116-5p expression is significantly downregulated in non-small cell lung cancer cells under high-glucose conditions [10]. Furthermore, miR-2116-5p expression is low in CD4 + CD69+ T cells derived from malignant pleural effusions of NSCLC patients [11]. However, the biological function, downstream network, prognostic potential, and mechanisms of miR-2116-5p in LUAD progression remain to be defined.

This study investigates how miR-2116-5p contributes to LUAD development, thereby providing a novel theoretical basis for prognosis evaluation and therapeutic targeting in LUAD patients.

Materials and methods

Study subjects

A total of 125 patients pathologically diagnosed with LUAD at the First Hospital of Putian City were enrolled in this study. None had undergone any form of antitumor therapy (radiotherapy, chemotherapy, or targeted therapy) prior to specimen collection. LUAD tissues and matched adjacent normal specimens were collected post-surgery, snap-frozen in liquid nitrogen, and stored at -80 °C until RNA extraction. Clinicopathological parameters, including age, sex, tumor size, lymph node metastasis, TNM stage, and smoking history, were retrieved from electronic medical records. All patients received standard postoperative treatment and completed a 5-year follow-up period. Overall survival information was used for prognostic analysis. The Ethics Committee of the First Hospital of Putian City approved this study, and all patients signed written informed consent.

Inclusion criteria were age ≥ 18 years and histologically confirmed LUAD. Exclusion criteria included receipt of any form of treatment (including chemotherapy) before surgical resection, presence of other malignant tumors, severe systemic diseases such as cardiovascular or renal disease, lung surgery or biological therapy within the previous three months, and pregnancy or lactation.

Cell culture and transfection

PC-9, A549, NCI-H23, H1975, and 16HBE cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 (Gibco, Gaithersburg, MD, USA) with 10% FBS (Gibco, USA) at 37 °C in 5% CO₂.

The miR-2116-5p mimic, inhibitor, negative controls, and oe-ADAM12 plasmid were sourced from GenePharma (Shanghai, China). Transfection was conducted using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the provided protocol, with cell collection occurring 48 h post-transfection.

Quantitative real-time PCR (RT-qPCR)-qPCR)

Total RNA was isolated from tissues and cell lines with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) per the manufacturer’s instructions. The PrimeScript RT reagent Kit (Takara, Shiga Prefecture, Japan) was used for cDNA synthesis. Real-time PCR was conducted on a 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using SYBR Green I Premix Ex Taq™ (Invitrogen, Carlsbad, USA) to assess miR-2116-5p and ADAM12 levels. The PCR conditions were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. U6 and GAPDH were used as reference genes for normalization, with relative expression determined by the 2^−ΔΔCt method. The primer sequences used are listed in Supplementary Table 1.

CCK-8 cell proliferation assay (CCK8)

Proliferation was evaluated using the CCK-8 assay (Beyotime, Shanghai, China). A549 and NCI-H23 cells were seeded in 96-well plates (3 × 10³ cells/well) after transfection. At 0, 24, 48, and 72 h, 10 µl of CCK-8 reagent was added per well, incubated for 2 h, and absorbance at 450 nm was recorded.

Transwell migration and invasion assays

Migration and invasion were evaluated using 24-well Transwell chambers (Corning, Lowell, MA, USA). Membranes were precoated with Matrigel for invasion assays or left uncoated for migration. Cells (2 × 10⁵/well) in serum-free medium were placed in the upper chambers, with 10% FBS medium below. After 24 h, non-migrated cells were wiped off, while invaded or migrated cells on the lower membrane were fixed, stained with 0.1% crystal violet, and counted in five random fields.

Bioinformatics analysis

Potential target genes of miR-2116-5p were predicted using four online databases. miRDB (http://mirdb.org/), TargetScan (https://www.targetscan.org/vert_72/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/), and miRTARGET (https://mirtarget.com/?). The overlapping genes from the predictions of all four databases were selected as candidate targets for subsequent validation.

Dual-luciferase reporter gene assay

Genomeditech (Shanghai, China) constructed luciferase reporter plasmids carrying either the wild-type (WT) or mutated (MT) version of the predicted miR-2116-5p binding site within the 3′UTR of ADAM12 mRNA. These reporters were introduced into cells together with miR-2116-5p mimic, inhibitor, or their respective negative controls using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). After 48 h, firefly and Renilla luciferase signals were measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA), and Renilla luciferase was employed as an internal reference for data normalization.

Statistical analysis

Statistical analyses were performed using SPSS 26.0 and GraphPad Prism 9.0. All experiments were conducted in triplicate, with data expressed as mean ± SD. Student’s t‑test was used for two-group comparisons, while one‑way ANOVA with Tukey’s post hoc test was applied for multiple groups. The chi‑square test assessed associations between miR-2116-5p expression and clinicopathological features, and Pearson’s correlation coefficient evaluated its relationship with ADAM12 expression. Survival curves were generated via the Kaplan‑Meier method. Univariate and multivariate Cox regression models were employed to identify independent prognostic factors. The multivariate model was adjusted for potential confounders selected based on univariate analysis, including gender, age, tumor size, smoking status, and tumor grade.

Results

miR-2116-5p is downregulated in LUAD tissues and correlates with poor patient prognosis

Compared with adjacent normal tissues, LUAD tissues displayed significantly lower miR-2116-5p levels based on RT-qPCR measurements (Fig. 1A). Based on median expression, 125 patients were separated into a high-expression group (n = 60) and a low-expression group (n = 65). Chi-square test analysis revealed that low miR-2116-5p expression was significantly correlated with positive lymph node metastasis (P = 0.013) and advanced TNM stage (P = 0.002), but showed no association with age, sex, smoking history, tumor size, or histological subtype (Table 1). Kaplan-Meier analysis indicated that reduced miR-2116-5p expression was associated with poorer overall survival outcomes (P = 0.002, Fig. 1B). Univariate Cox analysis showed that low miR-2116-5p expression (HR = 3.112, 95% CI: 1.438–6.737, P = 0.004), TNM stage (HR = 2.513, 95% CI: 1.224–5.159, P = 0.012), and lymph node metastasis (HR = 2.394, 95% CI: 1.191–4.813, P = 0.014) correlated with worse prognosis. After adjusting for potential confounders including gender, age, tumor size, smoking status, and tumor grade, only variables that were significant in univariate analysis were entered into the multivariate Cox model, which further established low miR-2116-5p expression (HR = 2.521, 95% CI: 1.129–5.628, P = 0.020) and TNM stage (HR = 2.373, 95% CI: 1.146–4.913, P = 0.024) as independent predictors of overall survival (Table 2).

Fig. 1.

Expression of miR-2116-5p in LUAD tissues and its prognostic significance. (A). Relative expression levels of miR-2116-5p in LUAD tissues and paired adjacent normal tissues detected by RT-qPCR. (B). Kaplan-Meier survival curves analysis of overall survival in LUAD patients stratified by miR-2116-5p expression level. All the experiments were independently repeated three times (n = 3). ^***P < 0.001

Table 1.

Association between miR-2116-5p level and pathologic characteristics in patients with lung adenocarcinoma

Parameters		miR-2116-5p expression			P-value
		Total	High	Low
		125	60	65
Gender	Male	68	33	35	0.897
	Female	57	27	30
Age (years)	<60	64	28	36	0.330
	≥60	61	32	29
Tumor size (cm)	<3	69	34	35	0.124
	≥3	56	26	30
Smoking history	No	48	24	24	0.724
	Yes	77	36	41
Grade	1-2	78	41	37	0.082
	3	47	19	28
TNM stage	Ⅰ-Ⅱ	81	47	34	0.002
	Ⅲ	44	13	31
LNM	Negative	69	40	29	0.013
	Positive	56	20	36

TNM: tumor node metastasis; LNM: lymph node metastasis

Table 2.

Univariate and multivariate analyses of clinical characteristics associated with overall survival of lung adenocarcinoma patients

Variable	Univariate analysis			Multivariate analysis
	HR	95% CI	P-value	HR	95% CI	P-value
miR-2116-5p	3.112	1.438–6.737	0.004	2.521	1.129–5.628	0.020
Gender	1.215	0.613–2.871	0.320
Age	1.299	0.632–2.668	0.477
Tumor size	1.760	0.833–3.717	0.138
Smoking	1.322	0.624–2.799	0.466
Grade	1.967	0.981–3.943	0.057
TNM stage	2.513	1.224–5.159	0.012	2.373	1.146–4.913	0.024
LNM	2.394	1.191–4.813	0.014	1.761	0.853–3.639	0.126

TNM: tumor node metastasis; LNM: lymph node metastasis

miR-2116-5p overexpression inhibits LUAD cell proliferation, migration, and invasion

Compared with normal bronchial epithelial cells (16HBE), miR-2116-5p expression was downregulated in LUAD cell lines (PC-9, A549, NCI-H23, H1975), the most pronounced decreases were observed in A549 and NCI-H23 cells, which were thus chosen for subsequent functional assays (Fig. 2A). miR-2116-5p mimic transfection markedly increased its expression, whereas the inhibitor effectively reduced it (Fig. 2B). miR-2116-5p overexpression suppressed the proliferation of A549 and NCI-H23 cells, while its knockdown promoted it, as determined by CCK-8 assays (Fig. 2C, D). Transwell assays revealed that increased miR-2116-5p expression suppressed cell migration and invasion, whereas its suppression enhanced these abilities (Fig. 2E, F).

Fig. 2.

Effects of miR-2116-5p on proliferation, migration, and invasion of LUAD cells. (A). Relative expression levels of miR-2116 in normal bronchial epithelial cell line 16HBE and LUAD cell lines (A549, PC-9, NCI-H23, H1975) detected by RT-qPCR. (B). Transfection efficiency of miR-2116-5p mimic or inhibitor in A549 and NCI-H23 cells detected by RT-qPCR. (C-D). Proliferation capacity of A549 (C) and NCI-H23 (D) cells after transfection assessed by CCK-8 assay. (E-F). Migration (E) and invasion (F) capacities of A549 and NCI-H23 cells after transfection evaluated by Transwell assay. All the experiments were independently repeated three times (n = 3). ^**P < 0.01, ^***P < 0.001

miR-2116-5p negatively regulates ADAM12 expression by direct targeting

Overlapping analysis of predictions from TargetScan, miRDB, miRWalk, and miRTARGET databases identified six candidate target genes: ADAM12, LPGAT1, PDCD6IP, POLR1B, HLCS, and PTPN3 (Fig. 3A). Following miR-2116-5p overexpression in A549 cells, only ADAM12 mRNA levels showed a consistent and significant downregulation (Fig. 3B), and similar results were observed in NCI-H23 cells (Fig. 3C). Therefore, ADAM12 was selected for further investigation. A potential miR-2116-5p recognition sequence was identified within the ADAM12 3′-UTR via bioinformatic prediction (Fig. 3D). In dual-luciferase assays, miR-2116-5p mimic suppressed luciferase activity of the wild-type ADAM12 3′-UTR reporter in A549 cells and NCI-H23 cells, while miR-2116-5p inhibitor enhanced its activity; however, no significant effects were observed on the mutated binding site reporter (ADAM12-MT) (Fig. 3E, F). Compared with adjacent normal tissues, LUAD tissues exhibited markedly higher ADAM12 mRNA expression, according to clinical sample analysis (Fig. 3G) and was negatively correlated with miR-2116-5p expression (r = -0.749, P < 0.001, Fig. 3H). Furthermore, LUAD cell lines showed substantially increased ADAM12 expression relative to 16HBE cells (Fig. 3I).

Fig. 3.

miR-2116-5p targets and regulates ADAM12 expression. (A). Venn diagram showing the intersection of miR-2116-5p target genes predicted by four databases. (B-C). mRNA expression changes of candidate genes in A549 (B) and NCI-H23 (C) cells following miR-2116-5p overexpression detected by RT-qPCR. (D). Predicted binding site of miR-2116-5p within the ADAM12 3’UTR. (E-F). Validation of the targeting relationship between miR-2116-5p and ADAM12 by dual-luciferase reporter assay in A549 (E) and NCI-H23 (F) cells. (G). Expression of ADAM12 mRNA in LUAD tissues and paired adjacent normal tissues detected by RT-qPCR. (H) Correlation analysis between miR-2116-5p and ADAM12 expression in LUAD tissues. (I) Relative expression levels of ADAM12 in normal bronchial epithelial cell line 16HBE and LUAD cell lines (A549, PC-9, NCI-H23, H1975) detected by RT-qPCR. All the experiments were independently repeated three times (n = 3). ^***P < 0.001

miR-2116-5p exerts tumor-suppressive functions by downregulating ADAM12

To determine whether miR-2116-5p influences LUAD malignant phenotypes through ADAM12 regulation, rescue experiments were performed. RT-qPCR analysis showed that ADAM12 mRNA levels were markedly reduced by miR-2116-5p mimic treatment, with co-transfection of an ADAM12 overexpression plasmid abrogating this reduction (Fig. 4A). CCK-8 proliferation assays demonstrated that ADAM12 overexpression reversed the inhibitory effect on cell proliferation induced by miR-2116-5p mimic (Fig. 4B, C). Transwell assays further confirmed that ADAM12 overexpression partially restored the migration and invasion capabilities suppressed by miR-2116-5p overexpression (Fig. 4D, E). These findings indicate that miR-2116-5p inhibits the malignant biological behavior of LUAD cells, at least in part, through negative regulation of ADAM12 expression.

Fig. 4.

miR-2116-5p affects proliferation, migration, and invasion of LUAD cells by regulating ADAM12. (A). ADAM12 mRNA expression levels in A549 and NCI-H23 cells under different treatment conditions detected by RT-qPCR. (B-C) Proliferation capacity of cells in each group assessed by CCK-8 assay in A549 (B) and NCI-H23 (C) cells. (D-E). Migration (D) and invasion (E) capacities of cells in each group evaluated by Transwell assay. All the experiments were independently repeated three times (n = 3). ^**P < 0.01, ^***P < 0.001

Discussion

As the chief histological variant of non-small cell lung cancer, LUAD has exhibited a sustained upward trend in both incidence and prevalence in recent years. This shift may be closely associated with multiple contributing factors, including worsening environmental pollution and changing lifestyles [12]. Currently, clinical treatment for LUAD primarily relies on surgical resection, supplemented by radiotherapy, chemotherapy, and targeted therapy. However, these therapeutic strategies have limited efficacy in improving long-term patient survival, and the overall prognosis remains unsatisfactory [13]. Accordingly, deeper insight into the mechanisms underlying LUAD pathogenesis and the search for novel targeted biomarkers are needed, holds significant clinical importance for optimizing early diagnosis, precision treatment, and prognostic assessment.

The regulatory importance of miRNAs in LUAD progression has become a key area of focus in tumor biology in recent years [14, 15]. This study explored the expression profile, prognostic value, and biological functions of miR-2116-5p in LUAD. In line with findings from hepatocellular carcinoma, LUAD tissues and cell lines exhibited significant downregulation of miR-2116-5p [16], suggesting that its low expression may have universal significance across tumor types. In the context of LUAD, various miRNAs have been identified as prognostic markers; for instance, miR-21 functions as an oncogenic miRNA whose overexpression predicts poor survival [17]. A significant correlation was observed between decreased miR-2116-5p expression and the presence of lymph node metastasis as well as advanced TNM stage, as revealed by clinicopathological evaluation. Similarly, LUAD tissues exhibited decreased miR-198-5p expression, which was markedly linked to lymph node metastasis, advanced TNM stage, and worse prognosis [18]. A systematic meta‑analysis has identified that high expression of miR-21 and miR-155 in NSCLC tissues and blood samples is consistently associated with poor overall survival, while low expression of miR‑148a, miR‑148b, and let‑7 also correlates with worse prognosis, underscoring the complexity and diversity of miRNA prognostic signatures in LUAD [19]. Additionally, elevated expression of miR-200c and miR-141 has been linked to shorter overall survival in lung adenocarcinoma, functioning through mesenchymal‑epithelial transition and angiogenesis pathways [20]. Conversely, low expression of miR-637 in NSCLC patients has been shown to be associated with significantly lower survival rates and poorer prognosis, consistent with a tumor‑suppressive role [21]. The data suggest a tumor-suppressive role for miR-2116-5p in LUAD, with low expression correlating significantly with reduced overall survival, and multivariate Cox analysis validated its independence as a prognostic marker. Together, these results provide clinical validation for miR-2116-5p as a prognostic biomarker in LUAD.

The unlimited proliferative capacity of tumor cells forms the basis for malignant expansion, while migration and invasion are key processes in distant metastasis, the latter being the leading cause of lung cancer mortality [22]. miR-2116-5p was found to be significantly downregulated in both hepatocellular carcinoma and its adjacent non‑cancerous tissues relative to normal liver tissues, as shown in earlier reports, suggesting its involvement as a suppressor in liver cancer development [16]. This study showed that overexpression of miR-2116-5p significantly inhibited LUAD cell proliferation, migration, and invasion, while its knockdown had the contrary effect. These observations support a role for miR-2116-5p as a tumor suppressor that governs malignant phenotypes in this cancer type. Combined with its clinical value as an independent prognostic factor, miR-2116-5p may hold potential as a molecular indicator for prognostic assessment and therapeutic targeting in lung adenocarcinoma, a role that parallels the previously reported tumor-suppressive activity of miR-374a-5p in non-small cell lung cancer [23].

miRNAs participate in cancer development and progression by targeting and regulating tumor-associated gene expression [24]. A disintegrin and metalloproteinases (ADAMs) family consists of transmembrane proteins with multiple structural domains. Members of this family are critically involved in tumor invasion and metastasis through their roles in extracellular matrix degradation, cell adhesion, and signal transduction [25, 26]. ADAM12, as a member of the ADAM family, exists in both transmembrane and secreted forms, is highly expressed in skeletal, cartilage, and muscle tissues, and participates in cell fusion, growth, and repair processes [26]. Emerging evidence indicates that ADAM12 is aberrantly expressed across diverse malignant tumors and drives their progression [27, 28]. In lung cancer, ADAM12 is also highly expressed and is considered to have diagnostic and prognostic value [29, 30]. ADAM12 was confirmed in this study to be directly targeted by miR-2116-5p. n LUAD tissues, ADAM12 mRNA showed pronounced upregulation, along with a negative correlation to miR-2116-5p expression. Functional rescue experiments showed that the reduced proliferation, migration, and invasion caused by miR-2116-5p overexpression were partially restored by ADAM12 co‑overexpression. These findings identify ADAM12 as an important downstream effector responsible for the tumor-suppressive function of miR-2116-5p in LUAD. The miR-2116-5p/ADAM12 regulatory pathway may offer a novel therapeutic approach for LUAD.

The present study has limitations. First, the sample size is relatively small and derived from a single center, and no external validation cohort (e.g., from TCGA or GEO) was included to verify the association between miR‑2116‑5p and ADAM12 expression or prognosis. Larger, multi‑center cohorts are needed to confirm our findings. Second, Zhao et al. provided in vivo evidence that miR-651-5p antagomir increases CD8⁺ T cell infiltration and enhances PD-1 inhibitor efficacy in an EGFR-mutant lung adenocarcinoma mouse model, highlighting the importance of in vivo validation for miRNA function [31]. However, due to technical difficulties in establishing animal models, our study was conducted solely in vitro. Consequently, the complex tumor microenvironment, including immune cells, cancer‑associated fibroblasts, and the vascular system, which may critically modulate miR‑2116‑5p function, could not be recapitulated. The potential effects of miR‑2116‑5p on tumor growth, immune infiltration, and therapeutic response in a physiologically relevant setting thus remain unexplored. Further investigations using animal models are required to fully clarify its in vivo biological functions and therapeutic potential. Third, although our findings suggest that miR‑2116‑5p may serve as a valuable biomarker and therapeutic target, several challenges must be addressed before clinical translation. These include the lack of efficient and safe delivery systems for miRNA‑based therapeutics (e.g., lipid nanoparticles or viral vectors), potential off‑target effects, and the need for validation in larger, more diverse patient cohorts. Overcoming these hurdles is essential for realizing the clinical potential of miR‑2116‑5p‑based strategies.

Collectively, these findings indicate that miR-2116-5p is expressed at reduced levels in LUAD, with diminished expression correlating significantly with the presence of lymph node metastasis, higher TNM stage, and worse prognosis, functioning as an independent prognostic indicator. Experimental functional analyses demonstrated that elevated miR-2116-5p expression reduces LUAD cell proliferation, migration, and invasion, with the reverse observed upon its knockdown. Mechanistically, miR-2116-5p exerts tumor-suppressive effects through targeted negative regulation of ADAM12. In LUAD, miR-2116-5p shows promise as a valuable biomarker for prognosis and a viable target for therapy.

Author contributions

HXH and NW conceived and designed the experiments. LHX and MX performed the experiments. KYC and SMC contributed sample collection and statistical analysis. HXH and NW wrote the manuscript. All authors revised it critically for important intellectual content. All authors read and approved the final manuscript.

Funding

No funding was received to assist with the preparation of this work.

Data availability

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

Declarations

Ethics approval and consent to participate

The study protocol was approved by The Ethics Committee of The First Hospital of Putian City and followed the principles outlined in the Declaration of Helsinki. In addition, informed consent has been obtained from the participants involved.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.