https://orcid.org/0000-0001-6699-0463
Figures & data
Figure 1. MiR-30b-5p is highly expressed in human ASM cells treated with PDGF
a. The volcano plot was used to show the expression changes of miRNAs in asthmatic bronchial epithelial cells. Horizontal axis: log2 (fold change); vertical axis: -log10 (P-value). The vertical line corresponds to 1.0 times up and down, and the horizontal line represents a P-value of 0.05. High expression is represented by ‘red’, low expression is represented by ‘blue’, and ‘black’ represents miRNAs with no significant differences. As shown, miR-30b-5p was among the up-regulated miRNAs in the bronchial epithelial cells obtained from asthma patients.b. The heat map was used to show 31 miRNAs, including miR-30b-5p, which were up-regulated in asthmatic bronchial epithelial cells.c. qRT-PCR was used to detect miR-30b-5p expression in human ASM cells with or without 25 ng/mL PDGF treatment. It showed that miR-30b-5p was markedly up-regulated in ASM cells after PDGF treatment.***P < 0.001.
Figure 2. MiR-30b-5p promotes PDGF-induced human ASM cell proliferation, migration and cell cycle progression
Human ASM cells were transfected with miR-30b-5p mimics or inhibitors, and then treated with 25 ng/mL PDGF for 24 h.a.MiR-30b-5p expression in ASM cells was detected by qRT-PCR, and it showed that the transfection was successful.b&c. ASM cells’ proliferation was detected by MTT and BrdU assays, and the results showed that miR-30b-5p positively regulated the proliferation of ASM cells.d.ASM cells’ migration was evaluated by Transwell assay, and the results showed that miR-30b-5p positively regulated the migration of ASM cells.e.ASM cells’ cell cycle was analyzed by flow cytometry, and the results showed that miR-30b-5p promoted the cell cell progression of ASM cells.**P < 0.01, and ***P < 0.001.
Figure 3. PTEN is a downstream target of miR-30b-5p
a. The binding sequences between miR-30b-5p and PTEN mRNA 3ʹ-UTR, which are predicted by bioinformatics analysis. b.The targeted relationship between miR-30b-5p and PTEN was verified by dual-luciferase reporter gene assay.c&d. qRT-PCR and Western blot were used to detect the expression of PTEN mRNA and protein in human ASM cells transfected with miR-30b-5p mimics and then treated with 25 ng/mL PDGF. The results showed that PDGF induced the down-regulation of PTEN expression in ASM cells, and miR-30b-5p negatively regulated PTEN expression.**P < 0.01, and ***P < 0.001.
Figure 4. MiR-30b-5p participates in PDGF-mediated ASM cell proliferation and migration by regulating the PTEN/PI3K/AKT axis
MiR-30b-5p mimics and PTEN overexpression plasmids were transfected into human ASM cells, or the cells were co-transfected with miR-30b-5p mimics and PTEN overexpression plasmids, and then the cells were stimulated with 25 ng/mL PDGF for 24 h.a&b. The relative expression of PTEN mRNA and protein in ASM cells were detected by qRT-PCR and Western blot.c&d. ASM cells’ proliferation was detected by MTT and BrdU assays. The results showed that PTEN restoration reversed the promoting effects of miR-30b-5p on ASM cells’ proliferation.e. Transwell assay was used to evaluate the migration of ASM cells. The results showed that PTEN restoration reversed the promoting effects of miR-30b-5p on ASM cells’ migration.f. Flow cytometry was used to analyze the cell cycle progression of ASM cells. The results showed that PTEN restoration reversed the promoting effects of miR-30b-5p on ASM cells’ cell cycle progression.g. Western blot was used to detect the expression of p-PI3K, p-AKT, PI3K and AKT proteins in ASM cells. The results showed that PDGF activated PI3K/AKT pathway, and miR-30b-5p further promoted the activation of PI3K/AKT pathway, and PTEN overexpression counteracted the effects of miR-30b-5p.**P < 0.01, and ***P < 0.001.
Data Availability Statement
The data used to support the findings of this study are available from the corresponding author upon request.