Circ_0070934 promotes MGAT3 expression and inhibits epithelial-mesenchymal transition in bronchial epithelial cells by sponging miR-199a-5p

Abstract

Background: Circular RNA (circRNA) has the potential to serve as a crucial regulator in the progression of bronchial asthma. The objective of this investigation was to elucidate the functional dynamics of the circ_0070934/miR-199a-5p/Mannoside acetylglucosaminyltransferase 3 (MGAT3) axis in the development of asthma.

Methods: Circ_0070934, miR-199a-5p and MGAT3 in peripheral venous blood of 38 asthmatic patients and 43 healthy controls were detected by qRT-PCR, and the expression of MGAT3 protein was examined by ELISA. The GSE148000 dataset was analyzed for differences in MGAT3. The BEAS-2B cells were transfected with circ_0070934 plasmid and small interfering RNA, miR-199a-5p mimics and inhibitors. The apoptosis level was detected by flow cytometry and MGAT3 was detected by qRT-PCR and Western blot. The expression of E-cadherin, N-cadherin, Vimentin was examined by Western blot. Interleukin-4 (IL-4) and IL-13 were used to co-stimulate BEAS-2B cells as an asthmatic airway epithelial cell model. BEAS-2B cells exposed to type 2 cytokines (IL-4 and IL-13) were treated with circ_0070934 plasmid, and the expression of E-cadherin, N-cadherin, and Vimentin was detected by Western blot. The binding relationships were verified using dual-luciferase reporter assay and miRNA pull-down assay.

Results: The expression of circ_0070934 and MGAT3 in peripheral venous blood of asthmatic patients was down-regulated, and the expression of miR-199a-5p was up-regulated. And the expression of MGAT3 was reduced in sputum of asthma patients. Down-regulating the expression of circ_0070934 could promote apoptosis of BEAS-2B cells and increase epithelial-mesenchymal transition (EMT), and this effect can be partially reversed by down-regulating miR-199a-5p. Circ_0070934 could inhibit the process of epithelial mesenchymal transition induced by IL-4 and IL-13 in BEAS-2B cells. In addition, miR-199a-5p could respectively bind to circ_0070934 and MGAT3.

Conclusion: The findings of this study indicate that circ_0070934 may function as a competitive endogenous RNA (ceRNA) of miR-199a-5p, thereby modulating the expression of MGAT3 and impacting the process of EMT in bronchial epithelial cells. These results contribute to the establishment of a theoretical framework for advancing the prevention and treatment strategies for asthma.

Keywords: Asthma; Epithelial-mesenchymal transition; Mannoside acetylglucosaminyltransferase 3; circ_0070934; miR-199a-5p.

Fig. 1

circ_0070934, miR-199a-5p and MGAT3 were differentially expressed in asthma patients and healthy controls. A–C The expression of circ_0070934, miR-199a-5p and MGAT3 in peripheral venous blood of healthy controls (N = 43) and asthmatic patients (N = 38) was determined by qRT-PCR. D ELISA was used to detect the expression level of MGAT3 protein in peripheral plasma of healthy controls (N = 43) and asthmatic patients (N = 38). E The expression level of MGAT3 in sputum of healthy controls (N = 7) and asthmatic patients (N = 9) was presented by analyzing the GSE148000 gene expression profiling dataset. The data were presented as mean ± SEM. qRT-PCR and ELISA assays were repeated three times. ns, no significant, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Fig. 2

Regulation of the expression of circ_0070934 and miR-199a-5p could affect the expression of MGAT3. A qRT-PCR was used to detect the expression of circ_0070934 after transfection of vector or circ_0070934 plasmid, and si-NC or si-circ_0070934 in BEAS-2B cells. B The expressions of miR-199a-5p in BEAS-2B cells after overexpression and knockdown miR-199a-5p was measured by qRT-PCR. C and D mRNA levels of MGAT3 in BEAS-2B cells after regulating circ_0070934 and miR-199a-5p was detected by qRT-PCR. E and F The expression level of MGAT3 protein in BEAS-2B cells was determined by Western blot. The data were presented as mean ± SEM. Our experiments were repeated three times. ns, no significant; P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 3

Down-regulation of circ_0070934 can promote apoptosis and affect the process of EMT in BEAS-2B cells. A BEAS-2B cells were divided into four groups: si-NC, si-circ_0070934, inhibitor NC and miR-199a-5p inhibitor. The level of cell apoptosis after transfection was quantified by Flow cytometry. B and C Western blot was used to detect the expression levels of epithelial-mesenchymal transition related molecular markers, including E-cadherin, N-cadherin and vimentin after transfection of si-NC or si-circ_0070934, and vector or circ_0070934 plasmid in BEAS-2B cells. The data were presented as mean ± SEM. Our experiments were repeated three times. ns, no significant; P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 4

MiR-199a-5p could reverse the effect of circ_0070934 on apoptosis and EMT of BEAS-2B cells. A BEAS-2B cells were divided into four groups: si-NC + inhibitor NC, si-circ_0070934 + inhibitor NC, si-circ_0070934 + miR-199a-5p inhibitor, si-NC + miR-199a-5p inhibitor. Flow cytometry was used to measure the level of cell apoptosis. B BEAS-2B cells were divided into three groups: si-NC + inhibitor NC, si-circ_0070934 + inhibitor NC, and si-circ_0070934 + miR-199a-5p inhibitor. The expression levels of proteins associated with EMT was detected by Western blot. C BEAS-2B cells were divided into three groups: vector + miR-NC, circ_0070934 + miR-NC, and circ_0070934 + miR-199a-5p mimics. Western blot was used to determine the expression levels of proteins associated with EMT. The data were presented as mean ± SEM. Our experiments were repeated three times. ns, no significant; P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 5

Circ_0070934 suppressed the EMT process induced by BEAS-2B cells exposed to IL-4 and IL-13. BEAS-2B cells were divided into three groups: vector, vector + IL4/13, and circ_0070934 + IL-4/13. Western blot was used to detect expression levels of proteins associated with EMT in BEAS-2B cells. The data were presented as mean ± SEM. Our experiments were repeated three times. ns, no significant; P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 6

Circ_0070934 had a targeted binding relationship with miR-199a-5p. A There are potential base binding sites between circ_0070934 and miR-199a-5p. B Dual-luciferase reporter assay was conducted to explicit the binding relationship between circ_0070934 and miR-199a-5p. C MiR-199a-5p-pull-down and NC-pull-down were the two groups of cells transfected with miR-199a-5p and NC probes, respectively. The input was the sample control, equivalent to total RNA, and the pulldown was the RNA after magnetic bead pull-down. There are four groups of RNA. The expression of hsa circ 0070934 and GAPDH were detected by qPCR. The data were presented as mean ± SEM. Our experiments were repeated three times. ns, no significant; P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 7

MGAT3 was a downstream target of miR-199a-5p. A There are potential base binding sites between MGAT3 and miR-199a-5p. B Dual-luciferase reporter assay was used to verify the binding relationship between MGAT3 and miR-199a-5p. C BEAS-2B cells were divided into four groups: vector, circ_0070934, si-NC and si-circ_0070934. D BEAS-2B cells were divided into four groups: miR-NC, miR-199a-5p mimics, inhibitor NC and miR-199a-5p inhibitor. C and D mRNA expression level of MGAT3 was determined by qRT-PCR. E and F The protein expression level of MGAT3 was determined by Western blot. The data were presented as mean ± SEM. Our experiments were repeated three times. ns, no significant; P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig.8

Schematic diagram demonstrating mechanisms by which circ_0070934 promotes MGAT3 expression and inhibits EMT progress in bronchial epithelial cells by sponging miR-199a-5p