Elevated expression of microRNA-378 in children with asthma aggravates airway remodeling by promoting the proliferation and apoptosis resistance of airway smooth muscle cells

Abstract

The present study determined the expression of microRNA (miR)-378 in the peripheral blood and lung tissues of children with asthma, and investigated its effect and mechanism of action on the biological functions of airway smooth muscle cells. A total of 23 asthmatic children and 15 healthy children were included in the study. Peripheral blood and tissues were obtained from asthmatic children. Healthy children provided peripheral blood. Quantitative real-time polymerase chain reaction was used to determine the expression of miR-378. Airway smooth muscle cells were isolated and cultured in vitro. The cells were transfected with miR-378 mimics or miR-378 inhibitor. Following transfection, proliferation of the cells was determined using the CCK-8 assay. In addition, flow cytometry was used to detect the cell cycles and apoptosis of smooth muscle cells. Western blotting was performed to determine the expression of extracellular matrix proteins in smooth muscle cells. Furthermore, bioinformatics was used to predict potential target genes of miR-378 and their downstream signaling pathways. Results indicated that the expression of miR-378 in peripheral blood and lung tissues from asthmatic children was increased compared with that in healthy children. Serum from asthmatic children promoted the proliferation of smooth muscle cells in vitro by affecting the cell cycle, and enhanced apoptotic resistance of smooth muscle cells. Notably, overexpression of miR-378 increased the proliferation of smooth muscle cells by affecting the cell cycle, and this upregulated apoptotic resistance of smooth muscle cells and enhanced the expression of extracellular matrix-related proteins in smooth muscle cells. However, downregulation of miR-378 expression reversed the promoting effect of serum from asthmatic children on the biological functions of smooth muscle cells. These findings suggested that miR-378 possibly affects the proliferation, apoptosis and motility of airway smooth muscle cells via downstream signaling pathways. To conclude, the present study demonstrated that miR-378 expression was elevated in the peripheral blood and lung tissues from children with asthma. Furthermore, miR-378 promoted the biological functions of extracellular matrix-related proteins of smooth muscle cells, and possibly exerts its effect via its target genes through downstream signaling pathways.

Keywords: airway smooth muscle cells; asthma; microRNA-378.

Figure 1.

Expression of miR-378 in blood, lung tissues or airway smooth muscle cells. (A and B) Expression of miR-378 in (A) peripheral blood or (B) lung tissues from children with asthma measured by quantitative real-time polymerase chain reaction. *P<0.05 compared with control group. (C and D) Expression of miR-378 in airway smooth muscle cells (C) transfected with miR-378 mimics or (D) treated with serum from asthmatic children for 7 days measured by quantitative real-time polymerase chain reaction. *P<0.05 compared with miR-NC group or NC group. Student's t-test was used to compare differences between groups (n=3–5). Control, samples from healthy children; Asthma, samples from asthmatic children; miR, microRNA; miR-NC, cells transfected with miR-negative control; miR-378 mimics, cells transfected with miR-378 mimics; NC, smooth muscle cells treated with serum from healthy children; Asthma serum, smooth muscle cells treated with serum from asthmatic children.

Figure 2.

Culture of primary airway smooth muscle cells and effect of serum from asthmatic children on their biological functions. (A) Identification of the expression of α-smooth muscle actin in smooth muscle cells by immunohistochemistry. Magnification, ×100. (B) Absorbance (490 nm) of airway smooth muscle cells at 24, 48 and 72 h. Proliferation was determined by CCK-8 assay. *P<0.05 compared with NC group. (C) Percentage of smooth muscle cells in G1, S and G2/M phases. Cell cycles were identified by flow cytometry. *P<0.05 compared with NC group. Student's t-test was used to compare differences between groups (n=3–5). NC, smooth muscle cells treated with serum from healthy children; Asthma serum, smooth muscle cells treated with serum from asthmatic children. PI-A-PI, phosphatidylserine-Annexin V-phosphatidylserine.

Figure 3.

Effect of serum from asthmatic children on the apoptosis of airway smooth muscle cells. Flow cytometry was performed to detect apoptosis. *P<0.05 compared with NC group. Student's t-test was used to compare differences between groups (n=3–5). NC, smooth muscle cells treated with serum from healthy children; Asthma serum, smooth muscle cells treated with serum from asthmatic children; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 4.

Effect of miR-378 on the proliferation and cell cycle of airway smooth muscle cells. (A) Determination of the proliferation of cells by CCK-8 assay. *P<0.05 compared with miR-NC group. (B) Detection of cell cycle of the cells by flow cytometry. *P<0.05 compared with miR-NC group. Student's t-test was used to compare differences between groups (n=3–5). miR, microRNA; miR-NC, cells transfected with miR-negative control; miR-378 mimics, cells transfected with miR-378 mimics.

Figure 5.

Effect of miR-378 on the apoptosis of airway smooth muscle cells. Flow cytometry was performed to detect apoptosis. *P<0.05 compared with miR-NC group. Student's t-test was used to compare differences between groups (n=3–5). miR, microRNA; miR-NC, cells transfected with miR-negative control; miR-378 mimics, cells transfected with miR-378 mimics; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 6.

Effect of miR-378 on the expression of extracellular matrix-related proteins in airway smooth muscle cells. Expression of COL-I and fibronectin was measured by western blotting. *P<0.05 compared with miR-NC group. Student's t-test was used to compare differences between groups (n=3–5). miR, microRNA; miR-NC, cells transfected with miR-negative control; miR-378 mimics, cells transfected with miR-378 mimics; COL-I, type I collagen.

Figure 7.

Effect of miR-378 inhibition on the biological functions of airway smooth muscle cells treated with serum from asthmatic children. (A) Proliferation of smooth muscle cells evaluated by CCK-8 assay. *P<0.05 compared with miR-NC+serum group. (B) Cell cycles of smooth muscle cells determined by flow cytometry. *P<0.05 compared with miR-NC+serum group. (C) Apoptosis of smooth muscle cells detected by flow cytometry. Multigroup measurement data were analyzed using one-way ANOVA. In case of homogeneity of variance, Least Significant Difference and Student-Newman-Keuls methods were used; in case of heterogeneity of variance, Tamhane's T2 or Dunnett's T3 method was used. *P<0.05 compared with miR-NC+serum group. Student's t-test was used to compare differences between groups (n=3–5). miR, microRNA; miR-NC, cells transfected with miR-negative control; miR-NC+serum, cells transfected with miR-NC and treated with serum from asthmatic children; miR-378 inhibitor+serum, cells transfected with miR-378 inhibitor and treated with serum from asthmatic children.