T Dong, X Huang, Y Han, J Zhu

Abstract

Kawasaki disease (KD) is an acute, self-limiting vascular inflammatory disease that primarily affects the small and medium arteries of the whole body, particularly the coronary arteries. About 25% of untreated KD children have coronary artery injury, which is mainly manifested as coronary artery dilatation (CAD), coronary artery aneurysm (CAA) and myocardial infarction. KD is now the leading cause of acquired heart disease in the developed world. Recent studies have demonstrated that non-coding RNA (ncRNA), especially microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), are intimately associated with the onset and progression of numerous diseases. These studies have also indicated that ncRNAs may play an indispensable role in the pathogenesis of KD via differential expression and participation in the central pathogenesis of KD, which comprises the modulation of immunity, inflammatory response and vascular dysregulation. Despite the increasing number of studies examining the expression profile and functional mechanism of ncRNA in KD, the exact role of these molecules in the disease remains unclear. This review aims to synthesise the current opinion of the potential functions and potential mechanisms of miRNAs, lncRNAs, and circRNAs in KD based on existing studies.

Introduction

Kawasaki disease (KD), also known as mucocutaneous lymph node syndrome, is an acute, self-limited medium -vessel vasculitis that has a predilection for the coronary arteries and is most common in children under 5 years of age.¹ Non-coding RNAs (ncRNAs) could accomplish a remarkable variety of biological functions, including regulating gene expression at the levels of transcription, RNA processing, and translation.² Since the first ncRNA- Alanine tRNA was reported in 1965 by Holly, other ncRNAs have been discovered in subsequent years, such as ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), circular RNA (circRNA), long non-coding RNA (lncRNA) etc.³ Previous studies have demonstrated that miRNAs, lncRNAs, circRNAs can be considered as potential biomarkers for detecting various diseases, including cancer,⁴ cardiovascular disease,⁵ and autoimmune diseases.⁶ Moreover, numerous studies have investigated the potential correlation between ncRNAs and the risk or pathogenesis of KD. Although a large number of studies have been conducted, they have also revealed that certain ncRNAs may serve as diagnostic markers for KD, which may help clinicians improve the diagnostic accuracy of KD.

Micro RNAs

miRNAs are short RNA molecules, 19 to 25 nucleotides in size, which affect the expression levels of proteins by binding to the untranslated regions of messenger RNA (mRNA) or by participating in functional interactive pathway.⁷ Furthermore, emerging studies have revealed that miRNAs play a vital role in the pathogenesis of various diseases, including cancer, autoimmune disease, allergic disease, cardiovascular disease and genetic disease progression and prognosis.⁸ In 2013, Shimizu et al⁹ initially identified significant differential expression of miRNA-145, miRNA-199b-5p, miRNA-618, miRNA-145, and miRNA-145* (complementary strand) in the acute phase of KD. The researchers then further discovered that miRNA-145 may act in conjunction with other differentially expressed miRNAs to affect the transforming growth factor-β (TGF-β) pathway in the acute phase of KD, which then influences gene expression. However, previous studies have implicated that the TGF-β pathway is associated with disease pathogenesis and arterial wall myofibroblast generation, and have also found that the formation of coronary aneurysm (CAA) in KD is related to altered TGF-β pathway.^10,11 As increasing numbers of scholars have begun to study the relationship between miRNAs and KD in recent years, it has gradually become one of the hotspots in the study of the pathogenesis of KD. These miRNAs have been studied from a wide range of sources, mainly from serum, platelets and exosomes. A study conducted by He et al¹² demonstrated that sera from healthy children and those with KD were incubated with human umbilical vein endothelial cells (HUVECs), and the expression level of miRNA-483 in KD was significantly lower than that healthy controls, as determined by quantitative reverse transcription-polymerase chain (qRT-PCR). They also investigated how KD serum could inhibit the KLF4-miR-483 axis in endothelial cells, resulting in increased expression of connective tissue growth factor (CTGF) and induction of endothelial-to-mesenchymal transition (EndoMT). This detrimental process in the endothelium may contribute to coronary artery abnormalities in KD patients. In addition, the use of statins may restore the KLF4-miRNA-483 axis and reduce the incidence of coronary lesions (CALs) in patients with acute KD.¹² miRNAs always play an important role in KD with CALs; for example, Li et al¹³ first demonstrated that miR‐182‐5p and miR‐183‐5p exhibited higher levels in KD patients with CALs compared to those without CALs (p<0.05). Furthermore, machine learning alignment confirmed that the formation of CALs could be predicted, with an area under the receiver operating characteristic curve (auROC) value of 0.86. Subsequently, they further treated neutrophil cells with miR‐182‐5p mimic, followed by an in vitro transendothelial migration assay. As a consequence, the overexpression of miR-182-5p was found to significantly enhance the infiltration of neutrophil cells into the endothelial layer, which was composed of human coronary artery endothelium cells (p<0.05).

Endothelial microparticles (EMPs) may be involved in vasculitis in acute KD, which considered to be both biomarkers and effectors of cell signalling that maintain and/or initiate cell dysfunction.¹⁴ In the study that measured EMPs using flow cytometry and performed miRNA expression profiling by miRNA arrays, it was found that the percentage of EMPs in patients with acute KD was significantly higher than that in the control group (P<0.0001).¹⁵ Furthermore, Nakaoka et al identified two specific miRNAs wrapped in EMPs: hsa-miR-145-5p and hsa-miR-320a, and confirmed that hsa-miR-145-5p was preferentially expressed in CALs. The involvement of hsa-miR-145-5p and hsa-miR-320a in the regulation of inflammatory cytokines and the pathogenesis of acute KD vasculitis is a potential avenue for further investigation. Finally, they suggested that hsa-miR-145-5p and hsa-miR-320a may be involved in the regulation of inflammatory cytokines and the pathogenesis of acute KD vasculitis.

A recent study showed that platelet-derived miRNA-223 was significantly down-regulated in KD patients compared with healthy controls.¹⁶ In a previous study, platelet-derived miRNA-223 was found to promote vascular smooth quiescence and resolution of wound healing after vessel injury.¹⁷ It is believed that the down-regulation of miRNA-223 may be related to the formation of CALs in KD, which has been verified in a mouse experiment. Administration of miRNA-223 mimics or the platelet-derived growth factor receptor β (PDGFRβ) inhibitor, imatinib mesylate, may alleviate the severity of CALs in KD.¹⁶

Here, many scholars have screened out differentially expressed miRNAs through gene chip technology or high-throughput sequencing, and have verified these differentially expressed miRNAs by qRT-PCR testing. The results showed that miRNA-455-5p¹⁸ was significantly down-regulated, miRNA-937,¹⁹ miRNA-210-3p,²⁰ miRNA-184,²⁰ miRNA-19a-3p,²⁰ miRNA-222-3p²¹ were significantly up-regulated in KD patients (p<0.05). In addition, miRNA-122,²² miRNA-21,²³ miRNA-186,²⁴ miRNA-197-3p²⁵ and miRNA-27a-3p²⁶ in KD serum were significantly up-regulated and can be used as diagnostic markers of KD. We have summarised the clinically valuable miRNAs in Table 1.

While numerous studies have been published on the potential involvement of miRNAs in the pathogenesis of KD and their utility as diagnostic markers, researchers have also suggested their potential as promising therapeutic targets for KD. Nevertheless, the lack of sufficiently large sample sizes and multi-centre study data has precluded the formulation of a consensus conclusion.

Exosomes miRNA

Exosomes are 40-100 nm nano-sized vesicles that contain a variety of proteins and nucleic acids, including mRNAs, miRNAs, and other ncRNAs.^27,28 They are released from most cell types into the extracellular space after fusion with the plasma membrane. In terms of function, exosomal miRNAs play an important role in disease progression, and can stimulate angiogenesis and facilitate metastasis in cancers.²⁷ In 2017, it was first reported that the serum exosomal miRNAs (namely, CT (mirna-1246) - CT (mirna-4436b-5p) and CT (mirna-197-3p) - CT (mirna-671-5p)) can act as candidate diagnostic biomarkers for KD.²⁹ Later, a study by Zhang et al³⁰ demonstrated that serum exosomal miR-328, miR-575, miR-134 and miR-671-5p may be used as potential biomarkers for the diagnosis of KD and the prediction of outcomes of the intravenous immunoglobulin (IVIG) therapy. Recently, it was also found that serum exosomal miRNAs, such as miRNA-7i-3P, identified by the qRT-PCR, may act as biomarker candidates for KD patients with CAA.³¹ In summary, exosomal miRNAs might serve as both prognostic biomarkers and the therapeutic targets for KD or KD with CAA.

Long Non-coding RNA

lncRNAs that are greater than 200 bp in size, play a diverse range of regulatory roles in gene expression, such as epigenetic regulation via molecular scaffolding, regulation of mRNA processing, molecular decoying and lncRNA-derived peptides.³² In general, lncRNAs have been detected and analysed in a wide range of biological samples, including tissues, organs, pathological specimens, cultured cells, and biological fluids.³³ Because different lncRNAs act as key regulatory molecules involved in various cellular processes and are dysregulated in various human diseases, they have broad application prospects in the clinic and can be used as diagnostic markers and therapeutic targets for clinical applications.³²

The earliest study of lncRNA and KD was conducted by Jiang et al,³⁴ who found that the expression of long-stranded non-coding RNA-PINC (Pregnancy induced noncoding RNA, PINC) was significantly increased in the serum of KD patients. At the same time, they found that PINC is involved in the process of TNF-α-induced vascular endothelial cell apoptosis, which might suggest a therapeutic target for KD. In addition, lnRNC may be involved in the progression of CAA in KD. A study published in 2019 was the first to perform a comprehensive analysis of lncRNAs in the blood of patients with KD, IVIG-resistant KD, or CAA, and found that XLOC_006277 expression in patients at the acute stage was 3.3-fold higher relative to patients with convalescent KD (P<0.05). Moreover, XLOC_006277 abundance increased significantly in patients with CAA, which might be associated with suppressed matrix metalloproteinase-8 (MMP-8) and MMP-9 expression and might contribute to further understanding of CAA pathogenesis in KD.³⁵ In conclusion, lncRNAs might play a role in the development and progression of KD or CAA, and could represent a potential diagnostic and therapeutic target in KD.

Circular RNAs

circRNAs are produced by a back-splicing mechanism, with a covalently closed loop structure without 5' caps and 3' poly-A tails. In function, they not only serve as a sponge for miRNAs and proteins but also regulate gene expression and epigenetic modification, translate into peptides, and generate pseudogenes.³⁶ Moreover, circRNAs are abundantly expressed in a tissue-specific manner, and natural circRNAs have been proved to be an abundant, stable, diverse and conserved class of RNA molecules.³⁷ It is becoming clear from several studies that dysregulation of circRNAs expression is associated with the development of several human diseases. Pathogenic roles of circRNAs are implicated in cancer,³⁸ cardiovascular diseases,³⁹ neurological diseases,⁴⁰ ocular diseases⁴¹ and rheumatoid arthritis⁴² progression.

In a recent study, the expression profiles of circRNAs from coronary artery tissue of patients with KD were analysed using the Gene Expression Omnibus (GEO) dataset. The researchers identified five circRNAs in coronary tissue but not in the blood of KD patients, and observed that these circRNAs returned to normal levels following treatment for the disease. They concluded that circRNAs may serve as promising biomarkers for KD.⁴³ In the same year, Wu et al⁴⁴ used qRT-PCR to detect the expression of circRNAs in the serum of KD patients and healthy children group. The results showed that circANRIL was significantly down-regulated in the KD group and the healthy control group, and hsa_circ_0123996 was significantly up-regulated in the KD group before treatment. However, the expression level of hsa_circ_0123996 showed no correlation with whether KD had been treated or not. In summary, although there are fewer studies on circRNAs in KD, circRNAs still have a broad value in the study of KD and can help us to understand the pathogenesis of KD more deeply.

Conclusion

Although KD is a self-limiting vasculitis disease, it can involve multiple systems and organs throughout the body, and the most important complication is coronary artery disease. Besides, a variety of ncRNAs are involved in the development of KD, which are related to the progression of KD or CAA through the regulation of target genes. Therefore, early diagnosis of KD is the key to preventing complications, and further study of the therapeutic methods of KD is of great significance for its future treatment.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Authors' Contributions

Dong Tong and Huang Xianmei designed the study. Dong Tong completed the review work and wrote the paper. Han Yong was responsible for the literature search. Huang Xianmei and Zhu Jiajun reviewed and edited the manuscript.

Funding/Support

This study was funded by Science and technology development fund of Nanjing Medical University Key Project (grant number: 2017NJMUZD08).

Data Availability Statement

Not applicable

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