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Silencing lncRNA CDKN2B-AS1 Alleviates Childhood Asthma Progression Through Inhibiting ZFP36 Promoter Methylation and Promoting NR4A1 Expression

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Abstract

LncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) was found to be upregulated in plasma of patients with bronchial asthma. This study aimed to explore the roles and mechanisms of CDKN2B-AS1 in childhood asthma. We found that CDKN2B-AS1 was upregulated and zinc finger protein 36 (ZFP36) mRNA was downregulated in blood samples of children with asthma compared with healthy controls as measured by RT-qPCR. Human bronchial epithelial cell line BEAS-2B was treated with LPS to induce inflammation model. Small interfering RNA against CDKN2B-AS1 (si-CDKN2B-AS1) was transfected into LPS-treated BEAS-2B cells, and we observed that CDKN2B-AS1 silencing increased cell viability and inhibited apoptosis and inflammation cytokine levels in LPS-treated BEAS-2B cells. Methylation‐specific PCR, ChIP, and RIP assays indicated that CDKN2B-AS1 inhibited ZFP36 expression by recruiting DNMT1 to promote ZFP36 promoter methylation. Co-immunoprecipitation (Co-IP) assay verified the interaction between ZFP36 and nuclear receptor subfamily 4 group A member 1 (NR4A1) proteins. Then rescue experiments revealed that ZFP36 knockdown reversed the effects of CDKN2B-AS1 silencing on BEAS-2B cell functions. ZFP36 overexpression facilitated apoptosis, inflammation, and p-p65 expression in BEAS-2B cells, while NR4A1 knockdown reversed these effects. Additionally, CDKN2B-AS1 silencing alleviated airway hyperresponsiveness and inflammation in ovalbumin (OVA)-induced asthma mice. In conclusion, silencing lncRNA CDKN2B-AS1 enhances BEAS-2B cell viability, reduces apoptosis and inflammation in vitro, and alleviated asthma symptoms in OVA-induced asthma mice in vivo through inhibiting ZFP36 promoter methylation and NR4A1-mediated NF-κB signaling pathway.

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Availability of Data and Materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

lncRNAs:

Long non-coding RNAs

lnc-BAZ2B:

LncRNA bromodomain adjacent to zinc finger domain 2B

RMRP:

RNA component of mitochondrial RNAase P

CDKN2B-AS1:

Cyclin-dependent kinase inhibitor 2B antisense RNA 1

ANRIL:

Antisense non-coding RNA in the INK4 locus

ZFP36:

Zinc finger protein 36

NR4A1:

Nuclear receptor subfamily 4 group A member 1

ELISA:

Enzyme-linked immunosorbent assay

TNF-α:

Tumor necrosis factor-α

IL-1β:

Interleukin-1β

IL-6:

Interleukin-6

MSP:

Methylation‐specific PCR

ChIP:

Chromatin immunoprecipitation

RIP:

RNA immunoprecipitation

Co-IP:

Co-immunoprecipitation

OVA:

Ovalbumin

BALF:

Bronchoalveolar lavage fluid

HNECs:

Human nasal epithelial cells

ADAM10:

A disintegrin and metalloprotease 10

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Authors and Affiliations

  1. Department of Pediatrics, Nanyang Central Hospital, No. 312, Gongnong Road, Henan Province, 473000, China

    Zhixin Chen, Guangsheng Shen & Jing Yang

  2. Department of Pathology, Nanyang Traditional Chinese Medicine Hospital, Henan Province, 473000, China

    Nuandong Fan

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  1. Zhixin Chen
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Contributions

Zhixin Chen designed the experiments. Zhixin Chen, Nuandong Fan, and Guangsheng Shen performed the experimental work. Jing Yang provided statistical analysis as well as figures for the manuscript. Zhixin Chen wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Zhixin Chen.

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This study was approved by the Ethics Committee of Nanyang Central Hospital, and all subjects had read and signed the informed consent. All animal care and experimental procedures in this study were approved by Animal Care and Use Committee of Nanyang Central Hospital.

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The authors declare no competing interests.

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Chen, Z., Fan, N., Shen, G. et al. Silencing lncRNA CDKN2B-AS1 Alleviates Childhood Asthma Progression Through Inhibiting ZFP36 Promoter Methylation and Promoting NR4A1 Expression. Inflammation 46, 700–717 (2023). https://doi.org/10.1007/s10753-022-01766-2

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