MiRNA-21 Regulates Bronchial Epithelial Cell Proliferation by Activating Tgfβ1/Smad Signaling Pathway and Its Correlation with Asthma Severity in Children

Abstract

Background: This research was designed to probe into the role of miRNA-21 in the pathogenesis of childhood asthma and its correlation with the severity.

Methods: Fifty-four children with bronchial asthma admitted to the Third Affiliated Hospital of Qiqihar Medical University from Jun 2018 to Dec 2019 were included. Forty nine healthy children underwent physical examination at this time period were also enrolled. The miR-21 expression in peripheral blood serum was analyzed by qRT-PCR. The relationship between the expression and severity of asthma in children was explored by Spearman correlation analysis and ROC curve. Bronchial epithelial cell lines were cultured in vitro and divided into blank control group, negative control group and miR-21 inhibition and activation group. The changes of cell proliferation after treatment were detected by CCK-8 test in different groups. The expression of TGF-β1/Smad signaling pathway protein in cells was assessed by Western blot (WB).

Results: Compared with that of healthy children, the miR-21 expression in peripheral blood serum of asthmatic children was higher (P<0.001). MiR-21 expression was positively correlated with the severity of illness (r=0.853, P<0.001). The results of cell experiments in vitro signified that miR-21 can promote the proliferation of bronchial epithelial cells, and may be involved in regulating the expression of TGF-β1/Smad3 signaling pathway, thus affecting cell proliferation.

Conclusion: miRNA-21 regulates the proliferation of bronchial epithelial cells by activating TGFβ1/Smad signaling pathway. And it is positively correlated with the severity of asthma in children.

Keywords: Childhood asthma; MicroRNA; Proliferation; Transforming growth factor β1.

Fig. 1:

Expression of miR-21 in peripheral blood serum of children with asthma increases Note: A: expression of miR-21 in peripheral blood serum of healthy and asthmatic children; B: expression of miR-21 in peripheral blood of children with different asthma grades. P<0.01 means **, and P<0.001 means ***

Fig. 2:

Relationship between miR-21 expression in peripheral blood serum and severity of asthma Note: A: the relationship between miR-21 expression in peripheral blood serum of healthy and asthma children and disease classification is assessed by Spearman correlation analysis; B: the specificity and sensitivity of miR-21 in diagnosing childhood asthma are investigated by ROC curve

Fig. 3:

Effect of miR-21 over-expression and knockout on proliferation in bronchial epithelial cells Note: the effect of miR-21 inhibition and over-expression on proliferation of bronchial epithelial cells 16HBE is assessed by CCK-8 proliferation test

Fig. 4:

miR-21 can directly regulate expression of Smad signal pathway inhibitor Smad7 Note: A: the binding sites of miR-21 and Smad7 are analyzed by using Targetscan software; B: Dual luciferase reporter gene experiment verifies that there is a regulatory relationship between miR-21 and Smad7; C: the effect of miR-21 inhibition or over-expression on Smad7 protein expression is explored by WB experiment

Fig. 5:

miR-21 can activate Smad signaling pathway and promote cell proliferation by affecting Smad7 expression Note: A: CCK8 experiment explores the effects of miR-21 over-expression and Smad7 agonist on bronchial epithelial cell proliferation; B: WB experiment verifies the over-expression of miR-21 and the activation of Smad signaling pathway by Smad7 agonist