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. 2023 Dec 1;76(2):179–190. doi: 10.1007/s10616-023-00607-w

MiR-4763-3p accelerates lipopolysaccharide-induced cardiomyocyte apoptosis and inflammatory response by targeting IL10RA

Lei Yang 1, Qian Dai 2, Xiaoming Bao 3, Wang Li 4, Jie Liu 5,✉
PMCID: PMC10940562  PMID: 38495290

Abstract

In order to investigate miR-4763-3p and associated genes’ roles in myocarditis, AC16 cell line was divided into LPS + miR-4763-3p inhibitor, LPS + NC inhibitor, LPS + miR-4763-3p inhibitor + si-IL10RA and NC groups, and Q-PCR was used to find out whether miR-4763-3p was expressed; Targetscan, Genecards, and MiRDB were used to estimate the miR-4763-3p target; Targetscan was used to display binding sites. Western blot assay was undertaken to detect Bax, Bcl-2, and IL10RA expression. Proliferation and apoptosis were processed using CCK8 and the flow cytometry assay, respectively. Migration and invasion were confirmed utilizing Transwell test. ELISA assay was processed to show the content of IL-6, IL-1ß, IL-10 and TGF-ß in the cell culture supernatant. After being exposed to LPS, cardiomyocyte cells expressed more miR-4763-3p. MiR-4763-3p inhibitor accelerated proliferation, migration and invasion behavior, while it also decreased apoptosis rate in LPS-treated cardiomyocyte cells. MiR-4763-3p inhibitor attenuated the inflammatory response by up-regulating Bax expression and down-regulating Bcl-2 level in LPS-treated cardiomyocyte cells. In cardiomyocyte cells treated with LPS, MiR-4763-3p expression was elevated. si-IL10RA The miR-4763-3p inhibitor restored its effects. MiR-4763-3p accelerates lipopolysaccharide-induced cardiomyocyte apoptosis and inflammatory response by targeting IL10RA, which might be a potential target for myocarditis.

Keywords: Myocarditis, miR-4763-3p, IL10RA, Lipopolysaccharide, Apoptosis, Inflammatory

Introduction

Myocarditis, commonly referred to as inflammatory cardiomyopathy, can exhibit itself in both children and adults, which was localized or diffuse (Mason 2003). In affluent nations, viral infection is the most typical cause of myocarditis (Blauwet and Cooper 2009). Bacterial infections, toxins, adverse drug reactions, and autoimmune conditions are additional reasons (Blauwet and Cooper 2009). Clinically, moderate individuals show no overt signs, whereas severe patients may pass away suddenly or have cardiac failure (Cobelli et al. 2006). Currently, various phases of myocarditis are often treated with medications, intravenous immune globulin, implantable cardiac defibrillators, and heart transplantation (Mcnamara et al. 1997). Numerous laboratory tests, such as brain natriuretic peptide, creatine kinase, lactate dehydrogenase, and troponin I or T, are employed to support a clinical diagnosis of cardiovascular disease (Pei et al. 2015). The levels of these biomarkers, however, alter significantly during myocardial damage and cardiac failure, making the diagnosis of fulminant myocarditis less certain. Therefore, novel biomarkers with increased sensitivity and specificity are urgently needed for the diagnosis of fulminant myocarditis. Numerous diseases are affected by miRNAs, which commonly regulate gene expression by attaching to the 3′UTR of their target mRNAs (Heggermont et al. 2013). MiRNAs may remain stable in plasma or serum for more than 10 years, according to earlier research (Tang et al. 2015). Heart failure (HF) is caused by a number of miRNAs, including miR-665, miR-320a, and miR-22. These miRNAs also act as biomarkers for the diagnosis of HF (Goren et al. 2012; Jiahui et al. 2018; Wang et al. 2021). MiR-208a in human plasma has also been verified to be a brand-new biomarker for the early detection of heart injury (Xu Ji 2009). Additionally, miR-122-5p was a consistent indicator of neurological recovery and survival after cardiac arrest, which aided in the prediction for cardiac arrest that occurred outside of a hospital (Yvan et al. 2017). Notably, miR-4763-3p expression in plasma was up-regulated during the onset of fulminant myocarditis and returned to normal with the improvement of clinical symptoms, according to real-time quantitative PCR and microarray analyses (Nie et al. 2020). But more research is required to determine the precise molecular mechanism of miR-4763-3p in myocarditis. The anti-inflammatory action of IL10 is mediated by IL10RA, which controls excessive tissue destruction brought on by inflammation (Wilson et al. 2007; Chakraborty et al. 2014). After attaching to IL10, it changes the conformation of IL10RB, causing IL10RB and IL10 to bond (Yoo et al. 2011). Additionally, JAK1 and TYK2 are activated by heterotetrameric assembly complexes made up of two each of the IL10RA and IL10RB subunits (Cabral-Marques et al. 2019). These kinases phosphorylate STAT3 after phosphorylating certain tyrosine residues in the intracellular domain of IL10RA (Isomki et al. 2014). The expression of anti-inflammatory genes is then activated as a result of STAT3 homodimerization, nuclear translocation, and gene activation (Zhang et al. 2018). Additionally, starvation-induced autophagy is inhibited by STAT3 activation via IL10RA (Beck et al. 2016). IL10RA is therefore essential for myocarditis. In this study, Both the role of miR-4763-3p and the molecular basis of its downstream gene (IL10RA) were examined.

Materials and methods

Cell grouping and treatment

Human myocardial AC16 cell line was purchased from ATCC. 10% FBS was used to cultivate the cells in a 37 °C incubator with 5% CO2. The cells were split into four groups according to the treatment: LPS + miR-4763 inhibitor, LPS + NC inhibitor, LPS + miR-4763-3p inhibitor + si-IL10RA, and NC groups. Cells were treated with 10 0 mg/L LPS for 24 h in the LPS + miR-4763 inhibitor, LPS + NC inhibitor, and LPS + miR-4763-3p inhibitor + si-IL10RA groups. Besides, miR-4763-3p mimic and NC mimic were also transfected. Besides, miR-4763-3p mimic and NC mimic were also transfected. MiR-4763 inhibitor, si-IL10RA, miR-4763-3p mimic and their controls were designed and synthesized by Genewiz Co., ltd. Moreover, they were transfected by liposome 2000 according to Instructions.

Q-PCR assay

After the total RNA was extracted using Trizol Reagent, it was reverse-transcribed into cDNA in accordance with the guidelines. MiR-4763-3p expression was determined by q-PCR, and U6 was used as an internal control. The amplification conditions included pre-denaturation at 95 °C for 15 s, denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 15 s, for a total of 40 cycles. The primer of miR-4763-3p was 5′-AGGCAGGGCUGGUGCUGGGCGGG-3′. The primers of IL10RA were as follows, (F) 5′-TTTGCCTTTGTCCTGCTG-3′, (R) 5′-CGCTGGCTGATGAAGATG-3′.

Bioinformatics tools and dual-luciferase reporter gene assay

Targetscan, genecards and miRDB online software were used to predict the target of miR-4763-3p. Targetscan was used to display binding sites. The wild-type (WT) and mutant (MUT) sequences of IL10RA were synthesized based on the predicted binding sites, and the above sequences were cloned into pmirGLO luciferase vector to construct WT-IL10RA and MUT-IL10RA. Dual-luciferase reporter kit was used to gauge the relative luciferase activity in cells of the WT-IL10RA or IL10RA group 48 h after miR-4763-3p mimic and NC mimic were transfected.

Western blot assay

Each group’s cardiomyocytes’ total protein content was extracted, and the BCA method was used to calculate each group’s protein concentration., and the sample solution was prepared. 20 µL of each group of samples and 5 µL of protein marker were added to the SDS-PAGE electrophoresis device for electrophoresis. After being transferred to the membrane, the cells were blocked with skim milk for 2 h before being incubated at 4 °C overnight with the matching primary antibody (Bax, Bcl-2, IL10RA, ß-actin, 1:1000, Abcam). After three TBST washes, The membrane was exposed to secondary anti-rabbit antibody (1:1 000, Abcam) and incubated for 2 h at room temperature. ECL imaging was carried out after three TBST membrane washes, darkroom exposure was undertaken, and the film was scanned and analyzed with a gel image processing system. ß-actin was used as internal reference.

Transwell assay

The 24-well plate’s upper chamber of the Transwell chamber received a total of 100 mL of Matrigel, which was then placed in the incubator for 6 h to form a gel. The basement membrane was moistened for 0.5 h while culture media devoid of serum was placed in the lower chamber. The cell suspension (5 × 103) from each group was prepared in serum-free media and added to the upper chamber. 500 L of full culture media were poured into the lower chamber. After an 8-h incubation period, samples were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min. Five randomly chosen fields of view were taken photos of, watched over, and counted.The migration experiment steps are the same as the invasion experiment but without Matrigel. Cell migration and invasion abilities were expressed as the number of transmembrane cells.

CCK8 assay

Cell proliferation was assessed using the CCK-8 method. Cells from each group were seeded into 96-well plates at a density of 5 × 103 cells per well. Each well received 10 L of CCK-8 reagent after being cultured for 24, 48, and 72 h. After that, the wells were incubated for 2.5 h at 37 °C. The optical density value (OD) at 450 nm wavelength was calculated using a microplate reader.

Flow cytometry assay

The cardiomyocytes in each group were digested with trypsin for 20–25 min, and calf serum was added to terminate the digestion. Larger clumps were removed by sieving through a 300-mesh nylon mesh. The samples were centrifuged for five minutes at 500 rpm before the supernatant was discarded. The pellet was mixed with physiological saline, centrifuged for five minutes, and the supernatant was discarded. 5 L of AnnexinV-FITC was added after the single cell solution was adjusted to include 1 105 cells. 5 L of PI was added and thoroughly mixed before the reaction was carried out at room temperature for 15 min in the dark. FSC was used as a threshold, the debris in the sample and small particles in the sheath fluid were excluded, and the data was acquired and analyzed by the CELQuest software.

ELISA assay

The amount of pro-inflammatory and anti-inflammatory substances in the cell culture supernatant was measured using an enzyme-linked immunosorbent assay. ELISA kits were purchased from SYT ImmuneScience Inc (college park, MD). The cell culture supernatant from each well was collected after the cells in each group had been cultivated in a 37 °C, 5% CO2 incubator for 48 h. According to the ELISA kit’s instructions for use, the microplate reader was used to establish a standard curve, and the content of IL-6, IL-1ß, IL-10 and TGF-ß in the cell culture supernatant was detected.

Statistical analysis

The experimental results were completely statistically assessed using the SPSS 21.0 program, and the measurement data were expressed as mean ± SD. To compare two groups of data, compare differences between several groups, and compare between groups, the T-test, one-way variance, and SNK-q tests were utilized, respectively. Statistics were considered different if P < 0.05.

Results

MiR-4763-3p expressed higher in LPS-induced cardiomyocytes

LPS, also known as endotoxin, is the main pathogenic component of Gram-negative bacteria. LPS activates a variety of cells to release inflammatory mediators, causing a systemic inflammatory response. This study used q-PCR to identify the expression of miR-4763-3p in control and LPS-induced cardiomyocytes. In LPS-treated AC16 cell line, miR-4763-3p was highly expressed (Fig. 1A). Thereby, miR-4763-3p expression was up-regulated in LPS-treated cardiomyocyte cells.

Fig. 1.

Fig. 1

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MiR-4763-3p expressed higher in LPS-induced cardiomyocytes. Vs control group, ***P < 0.01

MiR-4763-3p inhibitor accelerated proliferation, migration and invasion behavior of LPS-treated cardiomyocyte cells

In order to research the function of miR-4763-3p in LPS-treated cardiomyocyte cells, miR-4763-3p inhibitor was transfected. And the cells were divided into 3 groups, including LPS + NC inhibitor, LPS + miR-4763-3p inhibitor and control groups. After treatment, miR-4763-3p inhibitor obviously reduced miR-4763-3p expression in LPS-treated cardiomyocyte cells, indicating that the transfection was successful (Fig. 2A). Interestingly, LPS dramatically reduced the capacity of AC16 cells to proliferate, migrate, and invade while it increased apoptosis. However, miR-4763-3p inhibitor was with positive effect compared with LPS, which accelerated proliferation, migration and invasion behavior of LPS-treated cardiomyocyte cells (Fig. 2B, C). MiR-4763-3p inhibitor also decreased apoptosis rate in LPS-treated cardiomyocyte cells (Fig. 2D). Moreover, apoptosis related proteins, including Bax and Bcl-2 was detected. The results were shown in Fig. 2E. Bax was up-regulated by LPS treatment, while it down-regulated by miR-4763-3p inhibitor. Bcl-2 was increased in LPS group, and miR-4763-3p inhibitor partly rescued the effect of LPS. The results reconfirmed the effect of miR-4763-3p inhibitor on apoptosis.

Fig. 2.

Fig. 2

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MiR-4763-3p inhibitor accelerated proliferation, migration and invasion behavior of LPS-treated cardiomyocyte cells. A MiR-4763-3p inhibitor reduced miR-4763-3p expression in LPS-treated cardiomyocyte cells. B CCK8 assay for proliferation. C Migration and invasion ability detected by transwell assay. D Apoptosis behavior. E The expression of apoptosis related proteins (Bax and Bcl-2). Vs control group, ***P < 0.001, vs. LPS + NC inhibitor group, ##P < 0.01, ###P < 0.001

MiR-4763-3p inhibitor attenuated the inflammatory response

To investigate the function of miR-4763-3p in the inflammatory response, pro-inflammatory factors (IL-6 and IL-1ß) and anti-inflammatory factor (IL-10 and TGF-ß) expressions were detected by ELISA assay. The results were shown in Fig. 3A–D. IL-6 and IL-1ß were increased in LPS + NC inhibitor group, while they were partly down-regulated after miR-4763-3p inhibitor transfection (Fig. 3A and B). Interestingly, the levels of anti-inflammatory factor (IL-10 and TGF-ß) were lowest in LPS + NC inhibitor group, while miR-4763-3p inhibitor up-regulated the expressions of IL-10 and TGF-ß (Fig. 3C and D). Based on these results, miR-4763-3p inhibitor significantly attenuated the inflammatory response.

Fig. 3.

Fig. 3

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MiR-4763-3p inhibitor attenuated the inflammatory response. ELISA assay for expressions of IL-6 (A), IL-1ß (B), IL-10 (C) and TGF-ß (D). Vs control group, ***P < 0.001, vs. LPS + NC inhibitor group, #P < 0.05, ##P < 0.01

MiR-4763-3p targeted IL10RA

According to the online databases of targetscan, genecards and miRDB, total 4 targets were obtained and shown as Venn diagram (Fig. 4A). Then, targetscan database showed the binding of miR-4763-3p and IL10RA (Fig. 4B). Then the target relationship was verified by luciferase reporter gene assay. In IL10RA wt group, the expression of IL10RA was dramatically reduced by the miR-4763-3p mimic. However, no difference was observed in IL10RA Mut group (Fig. 4C). The outcomes demonstrated that miR-4763-3p targeted and inhibited the expression of IL10RA. After that, the regulated relationship was also confirmed by western blot assay. LPS treatment decreased IL10RA expression, while miR-4763-3p inhibitor increased IL10RA level in LPS-treated AC16 cells (Fig. 4D). Above evidences verified that miR-4763-3p targeted and negatively regulated IL10RA expression.

Fig. 4.

Fig. 4

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MiR-4763-3p targeted IL10RA. A The target of miR-4763-3p was predicted by targetscan, genecards and miRDB, and shown as Venn diagram. B The binding of miR-4763-3p and IL10RA. C The target relationship was verified by luciferase reporter gene assay. D The regulated relationship between miR-4763-3p and IL10RA showed by western blot. Vs control group, ***P < 0.001, vs. LPS + NC inhibitor group, ###P < 0.001

Si-IL10RA rescued the effect of mir-4763-3p inhibitor

Rescue assay was undertaken to further explore the roles of IL10RA and miR-4763-3p in LPS-treated cardiomyocyte cells. Si-IL10RA significantly reduced IL10RA expression in LPS-treated AC16 cells, indicating the si-IL10RA transfection was successful (Fig. 5A). The rescue assay was set with 4 groups, including LPS + NC inhibitor, LPS + miR-4763-3p inhibitor, LPS + miR-4763-3p inhibitor + si-IL10RA and control groups. Notably, si-IL10RA rescued the effect of miR-4763-3p inhibitor on cell proliferation, migration and invasion ability (Fig. 5B, C). Furthermore, miR-4763-3p inhibitor decreased apoptosis ability induced by LPS. However, the apoptotic cell rate was higher in LPS + miR-4763-3p inhibitor + si-IL10RA group than that in LPS + miR-4763-3p inhibitor group (Fig. 5D). Similarly results were also obtained by western blot. Bax and Bcl-2, two proteins associated with apoptosis, were detected. Compared with LPS + NC inhibitor group, Bax-2 was down-regulated, while Bcl-2 was up-regulated in LPS + miR-4763-3p inhibitor group, Moreover, Bax was up-regulated, while Bcl-2 was down-regulated by si-IL10RA compared with LPS + miR-4763-3p inhibitor group (Fig. 5E). According to these results, si-IL10RA rescued the effect of miR-4763-3p inhibitor on proliferation, migration, invasion and apoptosis ability.

Fig. 5.

Fig. 5

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Si-IL10RA rescued the effect of miR-4763-3p inhibitor. A Si-IL10RA significantly reduced IL10RA expression in LPS-treated AC16 cells. B The proliferation ability was decreased by LPS and si-IL10RA, while it increased by miR-4763-3p inhibitor. C Transwell assay for migration and invasion ability in LPS + NC inhibitor, LPS + miR-4763-3p inhibitor, LPS + miR-4763-3p inhibitor + si-IL10RA and control groups. D Si-IL10RA accelerated apoptosis ability induced by LPS. E. Si-IL10RA up-regulated Bax expression, while it decreased Bcl-2 level in LPS + miR-4763-3p inhibitor treated cell. Vs control group, ***P < 0.001, vs. LPS + NC inhibitor group, #P < 0.05, ###P < 0.001.Vs LPS + miR-4763-3p inhibitor group, $$P < 0.01, $$$P < 0.001

Si-IL10RA rescued the effect of mir-4763-3p inhibitor on inflammation

The effect of si-IL10RA and miR-4763-3p inhibitor on apoptosis was researched. The rescue assay was also set with 4 groups, including LPS + NC inhibitor, LPS + miR-4763-3p inhibitor, LPS + miR-4763-3p inhibitor + si-IL10RA and control groups. Compared with LPS + NC inhibitor group, the levels of IL-6 and IL-1ß were decreased, while IL-10 and TGF-ß expression were up-regulated in LPS + miR-4763-3p inhibitor group. However, compared with LPS + miR-4763-3p inhibitor group, IL-6 and IL-1ß were up-regulated, while IL-10 and TGF-ß was down regulated in LPS + miR-4763-3p inhibitor + si-IL10RA group (Fig. 6A–D). Thereby, si-IL10RA rescued the effect of miR-4763-3p inhibitor on inflammation.

Fig. 6.

Fig. 6

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Si-IL10RA rescued the effect of miR-4763-3p inhibitor on inflammation. ELISA assay for expressions of IL-6 (A), IL-1ß (B), IL-10 (C) and TGF-ß (D). Vs control group, ***P < 0.001, vs. LPS + NC inhibitor group, ##P < 0.01, ###P < 0.001. Vs LPS + miR-4763-3p inhibitor group, $P < 0.05, $$P < 0.01

Discussion

Lipopolysaccharide is one of the main mediators of inflammation and shock, which can induce the expression of inflammatory factors in cardiomyocytes, trigger cardiomyocyte hypertrophy and apoptosis, and lead to congestive heart failure (Wagner et al. 1998). miRNAs play a key regulatory role in a variety of cellular metabolic pathways (Wang et al. 2013). Relevant studies have shown that miRNA was involved in the pathophysiological process of lipopolysaccharide, and lipopolysaccharide regulated apoptosis and inflammatory response by affecting miRNA expression (Deng et al. 2019). In this study, miR-4763-3p was verified to express higher in LPS-induced cardiomyocytes.

In previous study, miR-4763-3p has been confirmed to be potential biomarkers of fulminant myocarditis based on microarray analysis (Nie et al. 2019). Moreover, miR-4763-3p was overexpressed in plasma sample of fulminant myocarditis (Nie et al. 2020). Interestingly, miR-4763-3p inhibitor accelerated proliferation, migration and invasion behavior of LPS-treated cardiomyocyte cells in this study. Adult cardiomyocytes greatly lose their ability to divide and proliferate after a narrow proliferation window during the neonatal period (Shen et al. 2019). As a representative organ, with poor regenerative capacity, the adult heart is particularly vulnerable to damage responses (Carson et al. 2014). However, increased cardiomyocyte proliferation has been shown in animal experiments to indirectly induce cardiac repair in the adult heart.

Besides, we also confirmed that miR-4763-3p inhibitor suppressed apoptosis of AC16 in this study. Bax was up-regulated by LPS treatment, while it down-regulated by miR-4763-3p inhibitor. Bcl-2 was increased in LPS group, and miR-4763-3p inhibitor partly rescued the effect of LPS. Bcl-2’s primary biological role is to prolong cell life and increase a cell’s resistance to apoptotic triggers, therefore the protein prevented apoptosis brought on by a variety of circumstances (Raffo et al. 1995). Bax overexpression may reduce Bcl-2’s effectiveness and increase apoptosis (Cheng et al. 2001). The ratio between Bcl-2 and Bax determines whether cells receive apoptosis-inducing signals (Shinoura et al. 2000). In recent years, scholars have confirmed that apoptosis was an important mechanism of myocardial injury in viral myocarditis through myocardial biopsies in patients with myocarditis and animal experiments (Schulz et al. 2002). Sarasle et al. (Sarasle et al. 2005) infected mice with two variants of cVB3 to induce mild and severe myocarditis, respectively, and confirmed that mice infected with two types of cVB3 had significantly more apoptotic cardiomyocytes than uninfected mice. Kyto et al. (Kyt et al. 2009) also referred that in viral myocarditis, the virus causes pathogenic damage to cardiomyocytes through apoptosis and necrosis. Thereby, miR-4763-3p accelerated lipopolysaccharide-induced cardiomyocyte apoptosis and inflammatory response.

It worth noting that miR-4763-3p inhibitor attenuated the inflammatory response. It decreased pro-inflammatory factors (IL-6 and IL-1ß) and up-regulated anti-inflammatory factor (IL-10 and TGF-ß) expressions in this study. Recent studies have verified that cardiomyocytes synthesized and released inflammatory factors in addition to their traditional functions (Yoshida et al. 2023). For example, tumor necrosis factor alpha, one of the important early mediators of endotoxin-induced septic shock (Hinshaw et al. 1990). The traditional opinion was that it was derived from activated macrophages, but cardiomyocytes could also secrete TNFα during sepsis (Shen et al. 2007). TNFα increased the synthesis of heavy chains of actin and myosin (Dogan et al. 2017). TNFα causes cardiomyocyte hypertrophy under concentration-dependent conditions (Higuchi et al. 2002). At the same time, it could also act together with IL-1 and IL-6 to cause cardiomyocyte hypertrophy and induce cardiomyocyte apoptosis. In this study, miR-4763-3p inhibitor attenuated the inflammatory response.

Furthermore, miR-4763-3p targeted IL10RA in this study. IL-10 binds to its receptors in the form of homodimers and activates Jak1 and Tyk2 (Finbloom and Winestock 1995). Together, they initiate signaling mediated by transcription factors STAT1 and STAT3 (Finbloom and Winestock 1995). Among them, the tyrosine kinase Jak1 binds to the cytoplasmic side of IL10RA (Pereira et al. 1998). If the expression regulation or signal transduction function of IL-10RA is abnormal, the biological activity of IL-10 will be changed. Therefore, its anti-inflammatory effect is weakened, and the inhibition of monocytes, macrophages and neutrophils mediated by the STAT3 pathway is lost, which further promotes the occurrence of inflammation (Jacoby et al. 2003). In this investigation, similar results were also attained. Si-IL10RA rescued the effect of miR-4763-3p inhibitor. It weakens proliferation, migration, invasion ability, while promoted apoptosis ability and inflammatory response. However, there are limitations in this study. The function of miR-4763-3p/IL10RA in myocarditis disease in vivo has not been researched so far. In the future, more potential miRNAs and targets will be researched in vivo and vitro.

In conclusion, miR-4763-3p accelerates lipopolysaccharide-induced cardiomyocyte apoptosis and inflammatory response by targeting IL10RA, which might be a potential target for myocarditis.

Author contributions

LY and JL designed the experiments and QD, XMB, WL carried them out. JL prepared the manuscript with contributions from all co-authors. The authors applied the SDC approach for the sequence of authors.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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