Long non‐coding RNA colorectal neoplasia differentially expressed correlates negatively with miR‐33a and miR‐495 and positively with inflammatory cytokines in asthmatic children

1 INTRODUCTION

Asthma is a chronic respiratory disease commonly observed in children under 16 years, whose hallmarks are inflammation of the airway, increased mucus secretion, bronchoconstriction as well as hypersensitivity to the external stimuli.1, 2 The propriate management (including pharmacological therapy, ventilation and allergenic-specific immunotherapy) may prevent most paediatric patients with asthma from developing further chronic lung diseases, thereby impairing their long-term pulmonary function.1, 3 However, some paediatric patients with asthma may experience acute asthmatic exacerbation which requires immediate medical attention.4 Therefore, identifying reliable biomarkers to prevent asthmatic exacerbation and monitor disease progression in paediatric patients with asthma is necessary.

Long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) located on human chromosome 16, is highly involved in cancer biology, which has been reported to be involved in promotion of cell proliferation, inhibition of cell apoptosis and assistance of epithelial-to-mesenchymal transition.5 Regarding its role in inflammation diseases, overexpression of lncRNA CRNDE activates toll-like receptor (TLR)-4/nuclear factor (NF)-κB pathway and further leads to production of inflammatory cytokines (such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6) in lipopolysaccharide (LPS) treated HK-2 cells.6 Meanwhile, upregulated lncRNA CRNDE promotes inflammatory cytokines production through the activation of NF-κB and Janus kinase/signal transduction and activator of transcription (JAK–STAT) signalling pathways in LPS treated WI-38 cells.7 As for its clinical application, lncRNA CRNDE serves as a prognostic factor for increased mortality rate in sepsis patients.8 From the previous data, lncRNA CRNDE targets several microRNAs (miRNA) (miR-33a, miR-181a and miR-495) to promote inflammation in several diseases. For instance, lncRNA CRNDE downregulates miR-181a and miR-495 to produce TNF-α, IL-1β and IL-6 in the cell model of sepsis and inflammatory bowel disease, respectively.9, 10 Meanwhile, lncRNA CRNDE also downregulates miR-33a.11, 12 Although lncRNA CRNDE and other above-mentioned miRNAs are heavily involved in the inflammation process of several diseases, fewer studies report their interaction and its clinical application for paediatric asthma management.

Hence, we performed this study and aimed to explore the interaction of lncRNA CRNDE with miR-33a, miR181a and miR-495, also to investigate their correlation with inflammatory cytokines and exacerbation severity in paediatric patients with asthma.

2 MATERIALS AND METHODS 2.1 Subjects

This study was carried out with the permission by Ethics Committee of Xingtai People's Hospital. From October 2019 to June 2020, this study consecutively recruited 65 asthmatic exacerbation children, 65 asthmatic controlled children, and 65 healthy subjects as controls. All asthma children were required to meet the diagnosis criteria of Global Initiative for Asthma (available on www.ginasthma.org.) and have age within 1 to 16 years. All asthmatic exacerbations were diagnosed by physicians. Besides, all asthmatic controlled children had a previous physician diagnosed asthma. The asthma children were excluded from study if they had other allergic diseases, respiratory diseases, infections, autoimmune disorders, or had history of haematological system diseases or malignancies. The controls were screened out from the healthy children who underwent health examination in Xingtai People's Hospital during the same period. The recruited controls were required to have age between 1 and 16 years, without obvious abnormality in medical examination, and have no history of asthma, infection, allergic diseases, respiratory diseases, autoimmune diseases, inflammatory diseases, or malignancies. The guardians of recruited children signed the informed consents; meanwhile, the recruited children with age ≥10 years also signed the informed consents.

2.2 Definition

The asthmatic exacerbation was defined as an acute or subacute episode of progressive increase in asthma symptoms, associated with airflow obstruction (Exacerbations of asthma were episodes characterised by a progressive increase in symptoms of shortness of breath, cough, wheezing or chest tightness and progressive decrease in lung function, that is, they represented a change from the patient's usual status that was sufficient to require a change in treatment.). Moreover, the severity of asthmatic exacerbation was assessed according to The Global Strategy for Asthma Management and Prevention. The detailed category of mild, moderate and severe asthmatic exacerbation was listed in Table S1. The asthmatic controlled was defined as symptoms and signs disappeared after treatment or without treatment, and the pulmonary function recovered and maintained for at least 3 months.

2.3 Data collection

The characteristics of the enrolled children were documented after necessary examinations and tests, which covered the age, gender, height, weight, family history of asthma, forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), FEV1 (%predicted), eosinophil count and immune globulin E (IgE) level. With regard to asthmatic exacerbation children, the exacerbation severity was evaluated on the admission depending on the clinical manifestation and necessary tests in accordance with the guidelines of International consensus on (ICON) paediatric asthma.13

2.4 Sample collection and determination

Collection of peripheral blood samples was carried out for the children after enrolment. Following that, the centrifugal separation and the Ficoll density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania, USA) were performed to isolate the serum samples and the peripheral blood mononuclear cells (PBMCs). The PBMCs were used for the quantitative analysis of lncRNA CRNDE, miR-33a, miR-181a and miR-495 by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) assay. The serum samples were used for the determination of the inflammatory cytokines including tumour necrosis factor alpha (TNF-α), interleukin (IL)-1 beta (IL-1β), IL-6 and IL-17 by enzyme-linked immunosorbent assay (ELISA) using Human ELISA Kits (Shanghai Enzyme-linked Biotechnology Co., Ltd, Shanghai, China).

2.5 RT-qPCR assay

LncRNA CRNDE, miR-33a, miR-181a and mi-495 expressions were detected by RT-qPCR. In brief, total RNA from PMBCs were extracted by PureZOL™ RNA isolation reagent (Bio-Rad, Hercules, California, USA). Then reverse transcription was applied using iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, California, USA). After that, qPCR reaction was completed by SYBR® Green Realtime PCR Master Mix (Toyobo, Osaka, Kansai, Japan). The relative expression was calculated by 2−ΔΔCt method using GAPDH as the internal reference for lncRNA CRNDE and U6 as the internal reference for miRNA. The primer sequences were listed in the Table S2.

2.6 Statistical analysis

Data were analysed by SPSS 21.0 software (IBM Corp., Armonk, New York, USA), and diagrams were formed using GraphPad Prism 8.01 software (GraphPad Software Inc., San Diego, CA, USA). Mean with standard deviation (SD), median with interquartile range (IQR), and count with percentage were used for descriptive analysis. One-way analysis of variance (ANOVA) Kruskal–Wallis test and Chi-square test were used for comparison among groups. Student t test, Wilcoxon rank sum test and Chi-square test were applied for comparison between two groups. Analysis of covariance (ANCOVA) was performed to detect difference of slope in different groups. Spearman's rank correlation test was carried out for correlation analysis. P value < 0.05 was considered as statistical significance.

3 RESULTS 3.1 Patients' clinical characteristics

The mean age of asthmatic exacerbation children, asthmatic controlled children and controls was 6.3 ± 2.7, 6.5 ± 2.6 and 6.6 ± 2.8 years, respectively (Table 1). There were 32 (49.2%) females and 33 (50.8%) in asthmatic exacerbation children, then 30 (46.2%) females and 35 (53.8%) males in asthmatic controlled children, and 27 (41.5%) females and 38 (58.5%) males in controls. The comparison of biochemical indexes, lung function indexes and inflammatory cytokines among three groups as well as intergroup were listed shown in Table 1.

TABLE 1. Clinical characteristics of participants Items Controls (N = 65) Asthmatic controlled children (N = 65) Asthmatic exacerbation children (N = 65) P valuea P valueb P valuec P valued Age (years), mean ± SD 6.6 ± 2.8 6.5 ± 2.6 6.3 ± 2.7 0.848 0.675 0.584 0.897 Gender, No. (%) 0.675 0.725 0.378 0.596 Female 27 (41.5) 30 (46.2) 32 (49.2) Male 38 (58.5) 35 (53.8) 33 (50.8) Height (cm), median (IQR) 121.6 ± 18.6 117.0 ± 16.0 116.7 ± 16.6 0.184 0.914 0.100 0.124 Weight (kg), median (IQR) 24.9 ± 9.6 23.1 ± 7.6 22.9 ± 7.5 0.301 0.901 0.162 0.202 Family history of asthma, No. (%) 0.265 0.212 0.140 0.818 No 54 (83.1) 53 (81.5) 47 (72.3) Yes 11 (16.9) 12 (18.5) 18 (27.7) Biochemical index, median (IQR) Eosinophil count (X109/L) 0.1 (0.1–0.1) 0.2 (0.1–0.3) 0.4 (0.4–0.7) <0.001 <0.001 <0.001 <0.001 IgE (IU/ml) 34.3 (20.0–51.8) 78.5 (56.6–121.1) 219.8 (149.2–365.9) <0.001 <0.001 <0.001 <0.001 Lung function index, median (IQR) FEV1/FVC (%) 82.8 (81.6–85.4) 78.3 (75.5–80.7) 65.0 (60.2–69.6) <0.001 <0.001 <0.001 <0.001 FEV1 (% predicted) 98.6 (94.6–104.4) 82.4 (79.9–84.8) 74.4 (70.6–78.7) <0.001 <0.001 <0.001 <0.001 Inflammatory cytokine, median (IQR) TNF-α (pg/ml) 19.3 (16.5–29.2) 29.5 (24.7–41.2) 63.8 (46.8–99.1) <0.001 <0.001 <0.001 <0.001 IL-1β (pg/ml) 1.4 (1.0–1.9) 2.5 (1.7–3.2) 5.6 (4.2–8.1) <0.001 <0.001 <0.001 <0.001 IL-6 (pg/ml) 12.8 (9.1–16.9) 20.5 (15.6–27.2) 65.2 (37.5–89.6) <0.001 <0.001 <0.001 <0.001 IL-17 (pg/ml) 21.4 (15.5–24.9) 29.3 (23.8–36.8) 83.3 (47.4–139.4) <0.001 <0.001 <0.001 0.001 a Comparison among three groups. b Comparison between asthmatic exacerbation children and asthmatic controlled children. c Comparison between asthmatic exacerbation children and controls. d Comparison between asthmatic controlled children and controls; SD, standard deviation; IQR, interquartile range; IgE, immunoglobulin E; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; FEV1 (%predicted), the percentage of the tested FEV1 value against the predicted normal FEV1 value; TNF-α, tumour necrosis factor-α; IL-1β, interleukin-1 β; IL-6, interleukin- 6; IL-17, interleukin-17. 3.2 Expressions of lncRNA CRNDE and miRNAs

LncRNA CRNDE (P < 0.001) (Figure 1A), miR-33a (P < 0.001) (Figure 1B) and miR-495 (P = 0.003) (Figure 1D) expressions were differed, while miR-181a expression (P = 0.064) (Figure 1C) was similar among asthmatic exacerbation children, asthmatic controlled children and controls.

image

Expressions of long non-coding RNA colorectal neoplasia differentially expressed (lncRNA CRNDE), miR-33a, miR-181a and miR-495. Comparison of lncRNA CRNDE (a), miR-33a (B), miR-181a (C) and miR-495 (D) in controls, asthmatic exacerbation children and asthmatic controlled children. LncRNA CRNDE: Long non-coding RNA colorectal neoplasia differently expressed, miRNA: microRNA

Subgroup analysis exhibited that lncRNA CRNDE expression (P < 0.001) (Figure S1A) was reduced, while miR-181a (P = 0.036) (Figure S1C) and miR-495 (P = 0.003) (Figure S1D) expressions were elevated in asthmatic controlled children with treatment compared to those without treatment. Furthermore, miR-33a expression was similar between asthmatic controlled children with treatment and asthmatic controlled children without treatment (P = 0.054) (Figure S1D).

3.3 Correlation of lncRNA CRNDE expression with miR-33a, miR-181a and miR-495 expressions

In asthmatic exacerbation children, lncRNA CRNDE was negatively correlated with miR-33a (P = 0.004) (Figure 2A) and miR-495 (P = 0.008) (Figure 2C). In asthmatic controlled children, lncRNA CRNDE also negatively associated with miR-33a (P = 0.005) (Figure 2D) and miR-495 (P = 0.018) (Figure 2F). In controls, lncRNA CRNDE was inversely correlated with miR-495 (P = 0.007) (Figure 2I) as well.

image

Association of Long non-coding RNA colorectal neoplasia differentially expressed (lncRNA CRNDE) with miR-33a, miR-181a and miR-495. Association of lncRNA CRNDE with miR-33a (a), miR-181a (B) and miR-495 (C) in asthmatic exacerbation children. Association of lncRNA CRNDE with miR-33a (D), miR-181a (E) and miR-495 (F) in asthmatic controlled children. Association of lncRNA CRNDE with miR-33a (G), miR-181a (H) and miR-495 (I) in controls. LncRNA CRNDE: Long non-coding RNA colorectal neoplasia differently expressed, miRNA: microRNA

3.4 Correlation of lncRNA CRNDE, miR-33a, miR-181a and miR-495 expressions with biochemical indexes and respiratory function indexes

LncRNA CRNDE was negatively correlated with FEV1/FVC in asthmatic exacerbation children (P = 0.015), while it was positively correlated with eosinophil count in controls (P = 0.033) (Table 2). While these findings were weak and inconsistent among different groups of paediatric patients.

TABLE 2. Correlation of lncRNA CRNDE, miR-33a, miR-181a and miR-495 expressions with biochemical indexes and respiratory function indexes Items LncRNA CRNDE miR-33a miR-181a miR-495 r P value r P value r P value r P value Asthmatic exacerbation children Eosinophil count 0.165 0.188 −0.216 0.084 −0.094 0.455 −0.118 0.349 IgE 0.148 0.240 −0.199 0.112 <0.001 0.999 −0.095 0.454 FEV1/FVC −0.300 0.015 0.240 0.055 0.211 0.091 0.236 0.059 FEV1 (% predicted) −0.223 0.074 0.212 0.090 0.086 0.495 0.166 0.185 Asthmatic controlled children Eosinophil count 0.137 0.275 −0.022 0.863 0.129 0.306 −0.082 0.514 IgE 0.198 0.115 −0.073 0.565 0.158 0.209 −0.172 0.171 FEV1/FVC 0.123 0.329 −0.102 0.420 −0.064 0.614 −0.140 0.268 FEV1 (% predicted) 0.182 0.147 −0.122 0.331 0.017 0.895 −0.194 0.122 Controls Eosinophil count 0.265 0.033 −0.092 0.467 −0.068 0.588 0.009 0.942 IgE 0.172 0.170 −0.097 0.440 −0.005 0.968 0.062 0.626 FEV1/FVC −0.131 0.298 −0.162 0.198 −0.184 0.142 0.007 0.956 FEV1 (% predicted) −0.096 0.447 0.086 0.497 −0.017 0.896 0.095 0.450 Note: LncRNA, long non-coding RNA; CRNDE, colorectal neoplasia differentially expressed; miR, microRNA; IgE, immunoglobulin E; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; FEV1 (%predicted): the percentage of the tested FEV1 value against the predicted normal FEV1 value. 3.5 Correlation of lncRNA CRNDE, miR-33a, miR-181a and miR-495 expressions with inflammatory cytokines

In asthmatic exacerbation children, lncRNA CRNDE positively correlated with all tested inflammatory cytokines (all P < 0.05) (Table 3), while miR-33a, miR-181a and miR-495 inversely correlated with inflammatory cytokines except for miR-181a and IL-17. In asthmatic controlled children, lncRNA CRNDE positively correlated with majority of tested inflammatory cytokines. While miR-33a, miR-181a and miR-495 negatively correlated with some inflammatory cytokines. In controls, only miR-181a was negatively correlated with TNF-α (P = 0.039). Furthermore, the detailed results were displayed in Table 3.

TABLE 3. Correlation of lncRNA CRNDE, miR-33a, miR-181a and miR-495 expressions with inflammatory cytokines Items LncRNA CRNDE miR-33a miR-181a miR-495 r P value r P value r P value r P value Asthmatic exacerbation children TNF-α 0.302 0.014 −0.331 0.007 −0.260 0.037 −0.319 0.010 IL-1β 0.297 0.016 −0.256 0.039 −0.268 0.031 −0.247 0.047 IL-6 0.214 0.088 −0.306 0.013 −0.245 0.049 −0.300 0.015 IL-17 0.276 0.026 −0.375 0.002 −0.095 0.453 −0.285 0.021 Asthmatic controlled children TNF-α 0.384 0.002 −0.324 0.008 −0.266 0.033 −0.262 0.035 IL-1β 0.297 0.016 −0.267 0.032 −0.288 0.020 −0.245 0.049 IL-6 0.187 0.137 −0.275 0.027 −0.138 0.273 −0.223 0.074 IL-17 0.263 0.034 −0.226 0.070 −0.149 0.235 −0.189 0.132 Controls TNF-α 0.150 0.234 −0.171 0.173 −0.257 0.039 −0.137 0.277 IL-1β 0.170 0.176 −0.119 0.345 −0.167 0.183 −0.220 0.078 IL-6 0.066 0.602 −0.178 0.155 −0.177 0.158 −0.243 0.051 IL-17 0.142

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