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

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.

5-Aza-2'-deoxycytidine, a DNA methylation inhibitor, attenuates hypoxic pulmonary hypertension via demethylation of the PTEN promoter.

Phosphatase and tensin homolog (PTEN) plays an important role in the pathogenesis of hypoxic pulmonary hypertension (HPH). A decrease in PTEN expression is associated with the hypermethylation of its promoter. However, whether the demethylation of the PTEN gene could attenuate HPH remains unknown. 5-Aza-2'-deoxycytidine (5-Aza-dC) is a DNA methyltransferase (DNMT) inhibitor. The present study was designed to investigate the effects and mechanisms of 5-Aza-dC on HPH. The proliferation, migration and apoptosis of rat pulmonary artery smooth muscle cells (PASMCs) induced by hypoxia and treated with 5-Aza-dC were detected. The expression of PTEN and DNMTs and the PTEN methylation status of PASMCs were detected. SD rats were randomly divided into normal group, hypoxia group and hypoxia + 5-Aza-dC group. The expression of PTEN was decreased, the expression of DNMTs was increased, and the methylation status of PTEN was increased in hypoxia-induced PASMCs. However, 5-Aza-dC can rescue the decreased PTEN, inhibit DNMT levels in a dose-dependent manner and suppress PTEN methylation. Furthermore, the demethylation of PTEN, which was induced by 5-Aza-dC, inhibited the proliferation, migration and promoted apoptosis in PASMCs. In vivo studies further demonstrated that the expression of PTEN, mean pulmonary artery pressure and right ventricular hypertrophy index in HPH rats was attenuated by 5-Aza-dC. 5-Aza-dC also suppressed the expression of DNMTs and PTEN methylation in the lungs of HPH rats. These results indicated that PTEN promoter methylation status is involved in HPH. 5-Aza-dC, as a DNMT inhibitor, has the potential to attenuate HPH via demethylation of the PTEN promoter.