YKL-40 Promotes the Expression of Inflammatory Factors in Type Ⅱ Alveolar Epithelial Cell Model of A549 Cell Line

MIAO Zhiyong, RAN Jingjing, MOU Dachao, WU Shasha, CHEN Yanqiong, LI Cong, CHEN Yuehong, YANG Min, XI

Abstract

YKL-40, also known as chitinase-3-like-1 (CHI3L1), is a human cartilage glycoprotein-39, with its N-terminus consisting of tyrosine (Y), lysine (K), and leucine (L), hence the name YKL-40. In this study, we explored whether YKL-40 could promote the expression of inflammatory factors in type Ⅱ alveolar epithelial cells.  Methods  A549 cells were cultured in vitro with interleukin (IL)-1β (20 ng/mL), IL-6 (20 ng/mL), tumor necrosis factor-alpha (TNF-α) (20 ng/mL), and interferon-gamma (IFN-γ) (20 ng/mL). The expression of YKL-40 transcription was determined by RT-qPCR. A549 cells were cultured with IL-1β at 5, 10, and 20 ng/mL and the expression of YKL-40 protein was determined by Western blot. A549 cells were cultured with recombinant YKL-40 protein at 0, 100, 500, and 1000 ng/mL and the expression levels of IL-6 and IL-8 were measured by RT-qPCR. Three pairs of small interfering RNAs targeting YKL-40 (si-YKL-40-1/2/3) and the negative control (NC) were designed and used to transfect A549 cells, respectively, and the expression of YKL-40 was determined by RT-qPCR and Western blot. si-YKL-40-3 was screened out for subsequent experiments. In A549 cells, si-YKL-40-3 and si-NC were transfected and, then, IL-1β (20 ng/mL) was added in for culturing. The expression of YKL-40, IL-6, and IL-8 was determined by RT-qPCR and the expression of multiple factors in the supernatant was measured with the QAH-INF-1 kit.   Results  RT-qPCR results showed that IL-1β could up-regulate YKL-40 protein transcription level compared with that of the control group and the difference was statistically significant (P<0.01), but IL-6, TNF-α, and IFN-γ could not up-regulate YKL-40 protein transcription level. Western blot results showed that IL-1β (20 ng/mL) could significantly promote the expression of YKL-40 and, compared with that of the control group, the differences showed by groups treated with different concentrations of IL-1β were all statistical significant (P<0.01). After adding human recombinant YKL-40 protein to A549 cells, the results showed that the expression of inflammatory factors IL-6 and IL-8 was significantly increased and the difference was statistically significant compared with that of the control group (P<0.05). After the expression of YKL-40 was decreased by si-YKL-40-3 transfection, the expression of IL-6 (P<0.05), IL-8 (P<0.05), and other inflammatory factors was inhibited compared with that of the control group.   Conclusion  YKL-40 can promote the expression and secretion of IL-6, IL-8, and other acute inflammatory factors in A549 cell line, a type Ⅱ alveolar epithelial cell model, thus aggravating the inflammatory response. Targeted inhibition of YKL-40 expression may effectively inhibit inflammatory response.


Keywords:YKL-40, Type Ⅱ alveolar epithelial cell, Small interfering RNA, Inflammatory response


 

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