MicroRNA-214-3p Regulates Hypoxia-Mediated Pulmonary Artery Smooth Muscle Cell Proliferation and Migration by Targeting ARHGEF12

Abstract

BACKGROUND miR-214-3p has been found to inhibit proliferation and migration in cancer cells. The objective of this study was to determine whether ARHGEF12 is involved in miR-214-3p-mediated suppression of proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). MATERIAL AND METHODS PASMCs were cultured under normoxia or hypoxia. miR-214-3p mimics or inhibitors were transiently transfected into PASMCs. Proliferation, apoptosis, and migration of PASMCs were evaluated using MTT assay, flow cytometry, and Boyden chamber apparatus. Western blot analysis was used to examine expression of Rho guanine nucleotide exchange factor 12 (ARHGEF12), c-fos, c-jun, and caspase-3. Luciferase reporter assay was used to test the direct regulation of miR-214-3p on the 3'-untranslated region (UTR) of ARHGEF12. RESULTS miR-214-3p was significantly upregulated in hypoxia-treated PASMCs. Knockdown of miR-214-3p significantly attenuated hypoxia-induced proliferation and migration in PASMCs and promoted apoptosis, whereas this effect was aggravated by overexpression of miR-214-3p. In addition, dual-luciferase reporter assay demonstrated that ARHGEF12 is a direct target gene of miR-214-3p. The protein levels of ARHGEF12 were downregulated after knockdown of miR-214-3p in PASMCs. Rescue experiment results indicated that decreased proliferation of PASMCs resulted from knockdown of miR-214-3p were partially reversed by silencing of ARHGEF12 by siRNA. Furthermore, knockdown of miR-214-3p reduced expression of c-jun and c-fos, but increased expression of caspase-3 in PASMCs under hypoxia. CONCLUSIONS In conclusion, these results indicate that miR-214-3p acts as a novel regulator of hypoxia-induced proliferation and migration by directly targeting ARHGEF12 and dysregulating c-jun and c-fos in PASMCs, and may be a potential therapeutic target for treating pulmonary hypertension.

Figure 1

The expression of miR-214-3p in rat PASMCs. QRT-PCR results showing (A) The expression of miR-214-3p was increased in rat PASMCs following hypoxia treatment. n=3; * P<0.05 vs. N 0 h, ^# P<0.05 vs. H 6 h and H 12 h; (B) QRT-PCR results showing the expression of miR-214-3p in rat PASMCs treated with miR-214-3p mimics or inhibitors following normoxia or hypoxia treatment. n=3; ^# P<0.05 vs. control, * P<0.05 vs. control.

Figure 2

Knockdown of miR-214-3p inhibited proliferation and migration and promoted apoptosis of PASMCs. PASMCs treated with miR-214-3p control, negative control, mimics, or inhibitors were cultured in normoxia or hypoxia for 24 h. (A) The proliferation was assessed using MTT assay; (B, C) The apoptosis of PASMCs assessed by flow cytometry; (D, E) Quantification of the migration of PASMCs by Transwell. n=3; ^# P<0.05 vs. control, * P<0.05 vs. control.

Figure 3

miR-214-3p targeted the 3′-UTR of ARHGEF12. (A, B) Luciferase reporter assay was performed in HEK293 cells transfected with wild-type ARHGEF12-3′UTR or mutant ARHGEF12-3′UTR and miR-214-3p mimics or negative control. * P<0.05 vs. NC. NC – negative control; WT – wild-type; MUT – mutated; (C, D) The effect of miR-214-3p on the expression of ARHGEF12 in PASMCs; ^# P<0.05 vs. control, * P<0.05 vs. control. (E) The expression of ARHGEF12 mRNA was specifically decreased by si-ARHGEF12; ^# P<0.05 vs. control; (F) Specific silencing of ARHGEF12 promoted proliferation of PASMCs. ^# P<0.05 vs. miR-214-3p inhibitors group.

Figure 4

The effect of miR-214-3p on the expression of c-jun, c-fos, and caspase-3 in PASMCs. (A) Representative Western blot showing the expression of c-jun, c-fos, and caspase-3 in PASMCs treated with miR-214-3p control, negative control, mimics, or inhibitors following normoxia or hypoxia exposure for 24 h. GAPDH was used as a loading control; Quantification of the expression of c-jun (B), c-fos (C) and caspase-3 (D). n=3; ^# P<0.05 vs. control, * P<0.05 vs. control.