Aberrant expression of serum circANRIL and hsa_circ_0123996 in children with Kawasaki disease

Background Kawasaki disease is a childhood systemic vasculitis that causes coronary artery abnormalities. The etiology remains unknown and there are no specific diagnostic tests. Circular non‐coding RNAs are a special class of endogenous RNAs that display some characteristics of an ideal biomarker. However, few studies have examined the expression of circRNAs in the serum of Kawasaki disease (KD) patients. The aim of this study was to identify circRNAs in the serum that can serve as potential biomarkers for KD diagnosis. Methods The cases were children diagnosed with KD (n = 56). The controls comprised healthy children (n = 56). Blood was collected from the patients before and after intravenous immunoglobulin therapy, and from the healthy controls. Levels of circANRIL and hsa_circ_0123996 in the serum were measured by quantitative reverse transcription PCR. Then, the potential relationship between serum circRNA levels and patients’ biochemical parameter levels was investigated. Receiver operating characteristic curves were constructed for evaluating the diagnostic value of these circRNAs. Results The serum levels of circANRIL were lower in patients with KD before therapy than in the controls, but became higher in the patients after therapy than before therapy. The serum levels of hsa_circ_0123996 were higher in patients with KD before therapy than in healthy controls. Conclusion Our study indicated that the circANRIL and hsa_circ_0123996 levels in the serum of patients with KD were significantly different from those in healthy individuals. circANRIL and hsa_circ_0123996 may become potential biomarkers for early KD diagnosis.

important way, a delay in accurate diagnosis may lead to increased mortality and morbidity from complications of KD. 3 Thus, improving the diagnosis and treatment of KD is necessary for reducing mortality and morbidity associated with KD.
Circular non-coding RNAs (circRNAs) are a special class of endogenous RNAs characterized by jointed 3′ and 5′ ends via exon or intron circularization and are broadly expressed in eukaryotic cells, but their involvement in human disease remains to be elucidated. 4,5 Previously, circRNAs were thought to be expressed at such low levels that they were generally regarded as byproducts of splicing errors and were not considered biologically active. However, the use of next generation sequencing has shown that circRNAs are expressed abundantly and are ubiquitous among eukaryotes. 6,7 Several circRNAs have been reported to have physiological functions. 8,9 Additionally, circRNAs have been suggested to be potential novel biomarkers of disease, including cancer, 10 heart failure, 11 and Parkinson's disease. 12 However, there has been no report on the serum circRNAs levels in patients with KD.
Here, we studied circANRIL and hsa_circ_0123996 in KD because they are circRNAs that may be associated with coronary artery and heart failure according to a bioinformatics analysis in the CircBase database.

| Subject recruitment
In total, 56 patients diagnosed with Kawasaki disease in the Ningbo Women and Children's Hospital from January 2017 to December 2017 were recruited, including 36 males and 20 females. The minimum age of KD onset was 10 months, and the maximum age of onset was 7 years. The average age was 2.58 ± 1.73 years. Additionally, we collected 56 age-and gender-matched children who were physically healthy, without any clinical signs of infection or inflammation as controls. All patients diagnosed with KD had fever for at least five days and met at least four of the five clinical criteria for KD (lips and oral cavity, bilateral conjunctival congestion, acute non-purulent cervical lymphadenopathy, polymorphous exanthema and changes in the extremities) or three of the five criteria plus coronary artery abnormalities documented by echocardiogram. 13 The follow-up of all patients was record clinical and laboratory information, whether there is any abnormal coronary artery by cardiac color ultrasound. The demographic and clinical characteristics of the study subjects are summarized in Table 1. Serum samples were obtained from KD subjects at two points: within 24 hours before intravenous immunoglobulin (IVIG) therapy, followed by at 3 or 4 days after IVIG therapy. Serum samples were obtained from the controls only once. All patients with KD received 2 g/kg IVIG and were administered aspirin orally (Table 2).

| Collection of human blood samples and RNA extraction
Peripheral blood (3 mL) was collected from a direct venous puncture into a tube containing sodium citrate. Then, whole blood was centrifuged to obtain serum within 48 hours and stored at −80°C until use. RNA was isolated using Trizol-ls (Qiagen, Hilden, Germany), followed by phenol and chloroform.

| Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
RNAs were reverse transcribed with the HiFi-MMLV cDNA first strand synthesis kit (CWBIO). qRT-PCRs were performed on a 96-well format Roche LightCycler 480 real-time PCR machine to compare the levels of the two circRNAs between the case and control groups. The relative fold changes were calculated by the comparative threshold cycle method, and GAPDH was used as the internal normalization control. Primers are listed in Table 3. The dissociation curve of each sample was then assessed. CircRNA levels were calculated by the ΔCt method with GAPDH as the control. Larger ΔCt values indicate lower expression. All data in this study were expressed as the mean ± standard deviation (SD) of two independent experiments.

| Statistical analyses
The data were analyzed using SPSS version 24.0 (SPSS, Chicago, IL, USA). Categorical data were presented as count and percentile. were found to have coronary artery abnormalities (Table 1).
All patients with KD received 2 g/kg IVIG and were administered aspirin orally. As shown in Table 2

| Expression of circANRIL in KD patients serum
Serum circANRIL level was significantly lower in patients during the acute phase of KD than in controls (P = 0.035; Figure 1A). The serum circANRIL level in patients with KD was significantly increased following the IVIG therapy (P = 0.016, Figure 1A), to a level that was comparable to the acute phase.

| Expression of hsa_circ_0123996 in the serum of patients with KD
The serum level of hsa_circ_0123996 was measured in 45 KD patients during the acute phase and in 45 healthy individuals. The serum hsa_circ_0123996 level was significantly higher in patients during the acute phase of KD than in the controls (P < 0.001; Figure 1B).
The serum hsa_circ_0123996 level did not change in patients with KD following the IVIG therapy.

| Potential diagnostic values of circANRIL and hsa_circ_0123996 for KD
To estimate the diagnostic valued of circANRIL and hsa_circ_0123996 for KD, ROC curves were used. The larger the area under the ROC curve (AUC), the higher the diagnostic value. The AUC of circANRIL was 0.624 (95% confidence interval (CI) = 0.520-0.727; Figure 2).

| Relationship between circANRIL levels and clinic laboratory factors
To further evaluate the usefulness of circANRIL as KD biomarkers, we tested whether its level correlated with laboratory parameters.
As shown in Table 4, serum levels of circANRIL were negatively

| Association between hsa_circ_0123996 expression and clinical laboratory factors in KD
Laboratory parameters of 45 serum samples from KD patients and 56 control serum samples were analyzed. The data summarized in Table 4 show that hsa_circ_0123996 level was positively correlated with TP By using multivariable linear regression, pre-albumin, MCHC, UA, PDW and Ibil had influence on hsa_circ_0123996 levels ( Table 6).

| D ISCUSS I ON
Kawasaki disease is an acute vasculitis syndrome in infants and young children that affects small-and medium-sized arteries, particularly the coronary arteries. 14   In conclusion, our study showed that the serum circANRIL level was lower in serum patients during the acute phase of KD than in controls and was increased in the patients following IVIG. The serum hsa_circ_0123996 level was higher in KD patients than in healthy controls. Our results suggest that circANRIL and hsa_circ_0123996 may serve as novel biomarkers for the diagnosis of KD.