LINC00941 affects the proliferation, apoptosis and differentiation of osteoblasts by regulating the miR-335-5p/KAT7 axis

Abstract

Background

Fractures are the prevalent traumatic conditions encountered in orthopedic practices. The rising incidence of fractures has emerged as a pressing global health concern. Although the majority of individuals with fractures experience complete recovery of bone structure and function, approximately 10% of those with fractures exhibit delayed fracture healing (DFH). The objective of this investigation was to explore the function and underlying mechanisms of LINC00941 in the advancement of DFH, as well as its involvement in the regulation of osteoblastic differentiation by regulating the miR-335-5p/KAT7 axis.

Methods

The expression levels of LINC00941, miR-335-5p, KAT7 and osteoblast differentiation-related markers were assessed using RT-qPCR. The proliferation of MC3T3-E1 cells was evaluated through the CCK-8 assay, and cell apoptosis was analyzed via flow cytometry. The targeted regulatory relationships between LINC00941 and miR-335-5p, as well as between miR-335-5p and KAT7 were verified by a dual-luciferase reporter gene assay.

Result

The expression of LINC00941 was significantly up regulated, while miR-335-5p exhibited a notable downregulation in DFH patients, both of LINC00941 and miR-335-5p have been identified as potential predicted markers for DFH. Furthermore, LINC00941 has been demonstrated to inhibit osteoblast proliferation, promote apoptosis, and suppress osteoblast differentiation through the regulation of the miR-335-5p/KAT7 axis.

Conclusion

LINC00941/ miR-335-5p/KAT7 axis may be a therapeutic target for DFH.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13018-025-05469-w.

Background

Fractures constitute a prevalent category of traumatic conditions encountered in orthopedics, and the escalating incidence of fractures has emerged as a significant global health concern. Although the majority of individuals with fractures achieve complete recovery in both bone structure and function, approximately 10% of those with fractures exhibit irregular healing processes, such as delayed fracture healing (DFH) [1]. At the same time, aspects including insufficient nutrition, blood circulation disruption, and invasion of pathogens become critical signs of DFH [2]. Studies have also reported a variety of treatments for fractures, including the administration of calcitonin [3], the application of intermittent pneumatic compression (IPC) [4], and the use of low-intensity pulsed ultrasound (LIPUS) [5]. Nonetheless, surgical intervention remains the treatment of choice for fractures, but it is more difficult to treat intra-articular fractures with surgery. Hence, ensuring optimal therapeutic outcomes continues to be a formidable challenge for surgeons specializing in trauma [6]. Accordingly, the quest for reliable biomarkers to investigate the mechanism of fracture repair is of considerable practical importance for the recovery of individuals experiencing fractures.

The expression profiles of long non-coding RNAs (LncRNAs) have been shown to undergo significant alterations throughout the process of bone tissue healing. These insights suggest that lncRNAs may hold promise as potential therapeutic biomarkers for addressing intricate bone fractures, congenital skeletal abnormalities, and related diseases [7, 8]. For instance, lncRNA ZFAS1 has been implicated in exacerbating the progression of osteosarcoma [9]. MALAT1 has been observed to enhance osteogenic differentiation and prevent apoptosis, positioning it as a promising target for osteoporosis treatment [10]. Additionally, Lei et al. found that lncRNAs mediate cell autophagy, which leads to bone loss in rheumatoid arthritis [11].

LINC00941 has been implicated in the progression of various malignancies, including pancreatic cancer [12], esophageal squamous cell carcinoma [13], thyroid cancers [14], and others. Moreover, it has the potential to serve as a biomarker for identifying conditions associated with hepatitis B virus infection [15]. Notably, one study observed that LINC00941 is downregulated during osteogenic differentiation induced by TiO2 nanotubes [16]. However, the specific role of LINC00941 in DFH remains to be elucidated.

microRNAs (miRNAs) are crucial regulators of gene expression, capable of either degrading mRNA or inhibiting its translation [17]. The therapeutic potential of miRNAs for tendon injury has been reported [18]and can be used in the diagnosis and management of osteoarthritis [19]. Several studies have identified specific miRNAs, including miR-214-3p [20], miR-92b [21], and miR-98-5p [22], as significant contributors to the onset of DFH. Notably, miR-335-5p has emerged as a pivotal player in the modulation of bone growth, demonstrating its influential role in fostering bone development and repair processes [23]. miR-335-5p has been found to enhance the process of bone formation and regeneration in murine models [24]. miR-335-5p is involved in the regulation of bone formation and in mesenchymal stem cells [25]. Moreover, studies have shown that miR-335-5p promotes chondrogenesis of mouse mesenchymal stem cells [26]. These studies suggest that miR-335-5p potentially plays a modulatory function in bone homeostasis. However, the clinical significance of miR-335-5p in the context of DFH, as well as the interplay between LINC00941 and miR-335-5p in fractures are still unknown.

The primary objective of this investigation was to elucidate the function and underlying mechanisms of LINC00941 in the progression of DFH, as well as to examine its involvement in the modulation of osteoblastic differentiation.

Methods

Inclusion of patients

In this study, a cohort of 182 adults over the age of 18 who were receiving treatment at Zhongxian People’s Hospital of Chongqing for unilateral tibial fractures were enrolled. Following observation of fracture healing four months after surgery, patients were divided into normal fracture healing (NFH) (n = 89) group and DFH (n = 93) group. All individuals in the NFH group exhibited the formation of bony calluses at fracture site, experiencing pain-free normal function. In comparison, the DFH group demonstrated a discernible space between the fractured ends on X-ray images, with no dense sclerotic bone and continuous callus. Individuals with a history of osteoporosis, fractures, or other fractures were excluded from the study. After allowing the blood samples from the participants to stand for 10 min, they were centrifuged at 2000 rpm for 15 min. Thereafter, the serum was separated and preserved cryogenically. This study was reviewed and approved by the Ethics Committee of Zhongxian People’s Hospital of Chongqing, and all participants provided informed consent.

Cell culture and osteoblast differentiation

The pre-osteoblastic cell line MC3T3-E1 was procured from the ATCC cell repository (Manassas, USA) and cultured in α-MEM with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS).The cells were cultured in a humidified environment with 5% CO₂ at 37 °C. Upon reaching a confluence of 80%, the differentiation of the cells was induced. After being inoculated in 6-well plates, the cells were incubated for 14 days using osteogenic induction media that was made with ascorbic acid (50 mg/L), dexamethasone (100 nmol/L), and L-glycerophosphate (5 mmol/L).

Cell transfection

In order to overexpress LINC00941, a full-length cDNA of LINC00941 was constructed and inserted into the pcDNA3.1 expression vector. Subsequently, a pcDNA3.1 vector without any insert was used as the negative control (NC). Furthermore, LINC00941-specific small interfering RNAs (si-LINC00941) and corresponding negative control siRNA (si-NC) were acquired. Additionally, miR-335-5p mimics, miR-335-5p inhibitors, as well as mimic negative controls (mimic NC) and inhibitor negative controls (inhibitor NC) were synthesized by Shanghai GenePharm. The cells were transfected utilizing Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

RT-qPCR

Total RNA was isolated using Trizol agent (Invitrogen, Carlsbad, CA, USA). The process of reverse transcription was carried out utilizing the HiFiScript cDNA Synthesis Kit (Cwbiotech, China). Subsequently, once cDNA was produced, a quantitative PCR analysis was performed on an MX3000p Real-time PCR instrument using the SYBR premix Ex Taq II Kit (Takara Bio, Japan). The relative expression levels were assessed using the 2^−ΔΔCT method, normalizing the data to the levels of GAPDH and U6 snRNA, respectively. The sequences of the primers used were listed in Table S1

Cell counting-8 kit (CCK-8) assay

The CCK-8 (Dojindo Laboratories, Japan) was utilized to evaluate cell proliferation. Following transfection, cells were plated in 96-well plates at a density of 5000 cells per well. A mixture of 100 µL α-MEM medium and 10 µL of CCK-8 reagent was added to the cells at 0, 24, 48, and 72 h. The cells were then subjected to an additional incubation period of 2.5 h. Subsequently, the optical density (OD) was recorded at a wavelength of 450 nm.

Apoptosis assay

Apoptosis in cells was quantified by flow cytometry utilizing the FITC Annexin-V Apoptosis Detection Kit (BD, Franklin Lakes, NJ, USA). Following two washes with PBS, the cells were suspended in flow cytometry binding buffer and then incubated with Annexin V-FITC and propidium iodide (PI) for a duration of 15 min at room temperature in the dark. The analysis of apoptotic cells was then performed using the FACSCalibur flow cytometer (Becton-Dickinson, CA, USA).

Dual-luciferase reporter assay

The binding sites of LINC00941 and miR-335-5p, as well as the interaction between KAT7 and miR-335-5p were predicted using the lncRNASNP3 and miRBD online database. Subsequently, the interaction between LINC00941 and miR-335-5p, miR-335-5p and KAT7 was validated using a dual-luciferase assay. The sequences of LINC00941 and KAT7 that containing the wild-type or altered binding site for miR-335-5p were inserted into the pmirGLO vectors, resulting in the production of recombinant plasmids containing the wild-type sequences WT-LINC00941, WT-KAT7, and mutated sequences MUT-LINC00941 and MUT-KAT7. MC3T3-E1 cells were cultured in 6-well plates and subjected to transfection at the exponential growth phase. The transfection reagent Lipofectamine 2000 was mixed with the previously described transfection vector and either miR-335-5p mimic, miR-335-5p inhibitor, or miR NC. Following a 48-hour incubation period, luciferase activity was measured utilizing a luciferase reporting system.

Statistical analysis

Statistical analysis was performed utilizing SPSS 26.0 and GraphPad prism 9.0. Quantitative data were expressed as mean ± SD. Receiver operating characteristic (ROC) curve analysis was used to evaluate the predictive efficacy of LINC00941 and miR-335-5p for DFH. One-way or two-way ANOVA was used to examine differences between several groups, and the Student’s T-test was used to compare data between two groups. A difference that was statistically significant was indicated by a p-value of less than 0.05.

Results

Clinicopathological data of subjects

No significant differences were observed between individuals with NFH and those with DFH regarding various demographic and clinical variables, including age, GENDER, BMI, smoking, alcohol intake, diabetes mellitus, method of osteosynthesis, and fracture severity (p > 0.05, Table 1). This result implies that these patient cohorts are somewhat comparable. In addition, the flow chart of this study is presented in figure S1.

Table 1.

Comparison of the baseline data of study objects

Parameters	NFH	DFH	p value
	(n = 89)	(n = 93)
Age (years)	39.37 ± 10.31	40.99 ± 9.07	0.262
BMI (kg/m2)	23.04 ± 3.04	22.81 ± 3.14	0.618
Gender			0.674
male	47	52
female	42	41
Smoking			0.540
yes	25	30
no	64	63
Alcohol intake			0.431
yes	36	43
no	53	50
Diabetes			0.191
yes	19	24
no	70	69
Osteosynthesis method			0.360
open reduction	38	46
closed reduction	51	47
Severity of fracture			0.441
complete	42	45
incomplete	47	48

NFH, Normal fracture healing; DFH, Delayed fracture healing; BMI, body mass index. Data were expressed as mean ± standard deviation or n

Expression and predictive value of LINC00941 and mir-335-5p in DFH

The expression of LINC00941 in serum was found to be significantly elevated in patients with DFH (Fig. 1a), whereas the expression of miR-335-5p in serum exhibited a notable decrease among these patients (Fig. 1b). Furthermore, a significant negative correlation was observed between the expression levels of LINC00941 and miR-335-5p in DFH (Fig. 1c).

Fig. 1.

Expression and predictive value of LINC00941 and miR-335-5p in DFH. Expression of LINC00941 in patients with NFH (n = 89) and DFH (n = 93) (a). Expression of miR-335-5p in patients with NFH (n = 89) and DFH (n = 93) (b). Correlation between expression levels of LINC00941 and miR-335-5p in patients with DFH (c). ROC curves of LINC00941 and miR-335-5p for predicting delayed healing in fracture patients (d)

Fig. 2.

Changes of LINC00941, miR-335-5p and osteoblast differentiation-related markers at different induction times. Expression of LINC00941 (a), miR-335-5p (b) and osteoblast differentiation-related markers (c) in osteoblasts at different differentiation times

Furthermore, both LINC00941 and miR-335-5p exhibited remarkable predictive significance for the development of DFH. The AUC of the ROC curve for LINC00941 in predicting DFH was 0.882, whereas for miR-335-5p, it was 0.863. Notably, when employed in conjunction, the AUC was found to be higher (Fig. 1d).

Expression of LINC00941, mir-335-5p and osteogenic markers in osteoblast differentiation

To gain a deeper understanding of the underlying mechanisms by which LINC00941 influences the fracture healing process, the osteogenic differentiation of MC3T3-E1 was induced. As the duration of osteogenic differentiation prolonged, the levels of LINC00941 were reduced (Fig. 2a), while the levels of miR-335-5p were elevated (Fig. 2b). Furthermore, the expression of osteogenic marker genes, including RUNX2, ALP, OCN, and OPN, exhibited a significant upregulation consistent with the prolongation of the osteogenic induction period (Fig. 2c).

Fig. 3.

Effects of LINC00941 and miR-335-5p on proliferation, apoptosis and differentiation of osteoblasts. Detection of transfection efficiency of si-LINC00941 and oe-LINC00941 (a). Effect of inhibiting or overexpressing LINC00941 on cell proliferation (b), apoptosis (c) and osteoblast differentiation-related markers (d). Detection of transfection efficiency of miR-335-5p inhibitor and miR-335-5p mimic (e). Effect of inhibiting or overexpressing miR-335-5p on cell proliferation (f), apoptosis (g) and osteoblast differentiation-related markers (h)

Effects of LINC00941 and mir-335-5p on proliferation, apoptosis and differentiation of osteoblasts

When the expression of LINC00941 was either inhibited or increased, the proliferation ability of MC3T3-E1 cells was enhanced and weakened, respectively (Fig. 3a and b). Following the downregulation of LINC00941 expression, a corresponding decline in the apoptosis rate of MC3T3-E1 cells was noted. Conversely, the upregulation of LINC00941 led to a significant increase in the apoptosis rate of MC3T3-E1 cells (Fig. 3c). Moreover, the suppression of LINC00941 correlated with an enhancement in the expression of osteoblast differentiation markers, whereas overexpression of LINC00941 resulted in a reduction of these markers (Fig. 3d).

Following transfection with miR-335-5p inhibitor, a notable downregulation of miR-335-5p expression was observed. while transfection with miR-335-5p mimic resulted in a significant increase in miR-335-5p levels (Fig. 3e). When the miR-335-5p expression was suppressed, a decrease in cell proliferation was observed (Fig. 3f), as well as an elevation in the apoptosis ratio (Fig. 3g) and a reduction in the expression levels of osteoblast markers (Fig. 3h). In contrast, the promotion of miR-335-5p expression led to an improvement in cell proliferation, a reduction of the apoptosis rate, and an increase in the levels of osteoblast markers.

Mir-335-5p may be a potential target of LINC00941

The online website displayed the binding sites of miR-335-5p and LINC00941 (Fig. 4a). The results indicated that the miR-335-5p inhibitor enhanced the luciferase activity of WT-LINC00941, whereas it significantly suppressed the luciferase activity in WT-LINC00941 but had no effect on MUT-LINC00941 (Fig. 4b). Furthermore, the downregulation of LINC00941 was associated with an increase in miR-335-5p expression, which was reversed by the miR-335-5p inhibitor. Nevertheless, the miR-335-5p inhibitor failed to counteract the reduction of LINC00941 expression (Fig. 4c and d), suggesting that miR-335-5p may be a potential target of LINC00941.

Fig. 4.

miR-335-5p may be a potential target of LINC00941. The binding site of LINC00941 and miR-335-5p (a). Dual-luciferase reporter gene assay verified the relationship between LINC00941 and miR-335-5p (b). LINC00941 negatively regulated the expression of miR-335-5p (c-da)

KAT7 may be a potential target of miR-335-5p

The downstream target genes of miR-335-5p were predicted from miRDB (target score > 80, n = 106), ENCORI (TDMD score > 0.9, n = 627), TargetScan (total context +++ score < -0.3, n = 55), and miRWalk (binding score > 0.9, n = 357) databases. By drawing the venn diagram, six overlapping target genes were obtained from the four databases. We randomly selected 10 patients from the NFH group and DFH group to detect the mRNA expression of 6 overlapping genes in their serum and found that only KAT7 expression was significantly different between the two groups of patients (figure S2). We then measured KAT7expression levels in all patients, the expression of KAT7 was increased in DFH patients (Fig. 5a) and was positively associated with the levels of LINC00941 (Fig. 5b) and inversely related to the levels of miR-335-5p (Fig. 5c). Furthermore, as osteoblast differentiation progressed, the expression of KAT7 in osteoblasts was observed to decline (Fig. 5d). Figure 5e showed the binding sites of KAT7 and miR-335-3p. miR-335-5p inhibitor enhanced the luciferase activity of WT-KAT7, while miR-335-5p mimic reduced the luciferase activity of WT-KAT7 (Fig. 5f). In addition, the inhibition of miR-335-5p increased KAT7 expression, and si-KAT7 reversed the increase in KAT7 expression induced by miR-335-5p inhibitor (Fig. 5g).

Fig. 5.

KAT7 may be a target of miR-335-5p. Expression of KAT7 in patients with NFH (n = 89) and DFH (n = 93) (a). Correlation between expression levels of LINC00941 and KAT7 in patients with DFH (b). Correlation between expression levels of miR-335-5p and KAT7 in patients with DFH (c). Expression of KAT7 in osteoblasts at different differentiation times (d). The binding site of KAT7 and miR-335-5p (e). Dual-luciferase reporter gene assay verified the relationship between KAT7 and miR-335-5p (f). miR-335-5p negatively regulated the expression of KAT7 (g)

LINC00941 affected the proliferation, apoptosis and differentiation of osteoblasts by regulating the miR-335-5p/KAT7 axis

The inhibitor of miR-335-5p effectively counteracted the promotion of cell proliferation caused by si-LINC00941, however, this effect was subsequently negated following si-KAT7 transfection (Fig. 6a). Additionally, the miR-335-5p inhibitor reversed the reduction in apoptosis rate and the elevation of osteogenic differentiation marker levels caused by si-LINC00941, with this trend being attenuated upon the inhibition of KAT7 (Fig. 6b and c).

Fig. 6.

LINC00941 affected the proliferation, apoptosis and differentiation of osteoblasts

LINC00941 affected the proliferation (a), apoptosis (b), and level of differentiation-related markers (c) of osteoblasts by regulating the miR-335-5p/KAT7 axis

Discussion

The healing of fracture is a complex process that relies on the cooperation of various osteoblasts, cytokines, and genetic elements. However, the multifaceted nature of the causes and the diversity of fracture patterns often result in many patients experiencing incomplete healing. This impediment triggers a delay in the healing process, consequently impacting both the physical and mental well-being of the patients [27]. Significantly, the physiological role of osteoblasts in fracture healing has been identified, with their capacity for replication and the transformation of osteoblasts being indispensable for the creation of new bones [28]. Additionally, the levels of osteogenic markers increase as osteoblasts become more metabolically active; these markers act as indirect indicators to track the healing process of fractures [29]. Numerous investigations have highlighted the critical importance of lncRNAs in the fracture healing process. Further investigations have demonstrated that lncRNAs can modulate osteogenic differentiation, indicating their possible roles in the advancement of the fracture healing process. For instance, the expression level of KCNQ1OT1 is markedly reduced in the bones of individuals experiencing DFH, and it promotes cell proliferation and prevents cell apoptosis [30]. SNHG1 is up regulated in serum of patients with DFH and inhibits tibial fracture healing [31]. The elevation of MAGI2-AS3 could expedite the mending process in fracture patients, thus offering a novel biomarker for identifying individuals with DFH [32].

In this study, we have observed that LINC00941 is upregulated in the serum of individuals suffering from DFH, and it serves as an effective predictor of healing delays in patients with fractures. Previous studies have reported that the metabolic process of osteoporosis is regulated by many genes and cytokines targeted by small interfering RNAs (siRNA) [33]. SiRNA influence gene dysregulation of growth factors and tendon structural proteins [34] and can be used in the treatment of rheumatoid arthritis [35]. In this study, we inhibited the expression of LINC00941 by transfecting siRNA targeting LINC00941, and found that si-LINC00941 promoted the proliferation and differentiation of osteoblasts, and inhibited the apoptosis of osteoblasts. Consequently, we propose that LINC00941 may play a crucial role in the process of fracture healing and could potentially act as a biomarker for the DFH. However, further experimental validation is necessary to substantiate these findings.

Studies have elucidated that lncRNAs act as competitive endogenous RNAs to negatively modulate miRNAs, thereby contributing to the modulation of various cellular processes [36]. We retrieved miRNAs regulated by lncRNAs from online databases, with miR-335-5p standing out as a notable candidate. Wang et al. revealed that LINC00941 governs the advancement of pancreatic cancer through a competitive binding interaction with miR-335-5p [37]. Our investigations further confirmed that LINC00941 is capable of regulating the expression levels of miR-335-5p. Notably, the study highlighted that miR-335-5p is expressed at diminished levels in the bone tissue of patients and rats with osteoporosis, indicating a potential novel approach for the prevention and management of osteoporosis [38]. Moreover, melatonin has been shown to upregulate the expression of miR-335-5p, which enhances bone growth in bone defect healing [39]. Our findings revealed that miR-335-5p expression was down-regulated in DFH patients, and the low expression of miR-335-5p predicted delayed healing in fracture patients, indicating a beneficial role for miR-335-5p in fracture recovery. To further investigate the roles of LINC00941 and miR-335-5p in fracture healing, we conducted a series of in vitro experiments focusing on osteoblasts. Our results demonstrated significant alterations in the levels of LINC00941 and miR-335-5p correlating with the prolongation of osteogenic differentiation time. The levels of proteins linked to cellular differentiation, including ALP, OCN, Runx2, and OPN, as well as the vitality of cells, serve as markers for the activity of osteoblasts and the formation of new bone [32]. The inhibition and overexpression of LINC00941 and miR-335-5p had significant effects on the proliferation, apoptosis and differentiation of osteoblasts. Collectively, these findings provide more substantial to support our conclusions and validate previous studies [16, 24, 38, 39].

In addition, this study found that miR-335-5p negatively regulated KAT7 expression. KAT7 functions as a histone acetyl transferase, pivotal in regulating gene transcription and immune control. The study discovered that KAT7 is markedly elevated in human osteosarcoma at both the mRNA and protein levels [40]. In this study, KAT7 was up-regulated in DFH patients, and KAT7 could reverse the effects of miR-335-5p on osteoblast proliferation, apoptosis, and differentiation. The current investigation revealed through a responsive experiment that LINC00941 modulates the process of fracture healing by controlling the miR-335-5p/KAT7 axis.

However, this study still has some limitations. First, we only detected the mRNA expression of osteoblast differentiation markers, without detecting their protein expression levels, which we need to strengthen in future experiments. In addition, LINC00941 may also play a role in fracture healing by regulating other miRNAs, which needs more experiments to prove, which will provide more evidence for the clinical application of LINC00941. Additionally, we confined our analysis to the detection of LINC00941, miR-335-5p, and KAT7 expression levels in the serum of patients and in osteoblasts, without examining these in the bone tissues of the patients. To delve into this aspect, subsequent studies will involve an additional cohort of patients, as well as animal studies, to generate further data that corroborates the findings of the current experiment.

Conclusions

Collectively, the study indicates that LINC00941 and miR-335-5p exhibit altered expression in DFH and serve as indicators for forecasting delayed healing in individuals with fractures. Furthermore, LINC00941 exerts its influence on osteoblast proliferation, apoptosis, and differentiation by modulating the miR-335-5p/KAT7 axis.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

CCK-8

Cell Counting-8 Kit

DFH

Delayed fracture healing

FBS

Fetal bovine serum

LncRNAs

Long non-coding RNAs

miRNAs

microRNAs

NC

Negative control

PI

Propidium iodide

ROC

Receiver operating characteristic

Author contributions

All authors designed this study. LJ L, Y Y and PX S conducted the experiment and analyzed the data. LJ L, Y Y wrote the manuscript. PX S revised the manuscript. All authors reviewed and approved for publication.

Funding

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

Data availability

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

Declarations

Ethics approval and consent to participate

This study was reviewed and approved by the Ethics Committee of Zhongxian People’s Hospital of Chongqing, and all participants provided informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.