Effect of miR-23a on anoxia-induced phenotypic transformation of smooth muscle cells of rat pulmonary arteries and regulatory mechanism

Abstract

We investigated the possible implication of miR-23a in anoxia-induced phenotypic transformation of the pulmonary arterial smooth muscle and studied the mechanism of upregulation of miR-23a expression in anoxia. The collagenase digestion method was used for preparing rat primary pulmonary artery smooth muscle cell (PASMC) culture. SM-MHC, SM-α-actin, calponin-1 and SM22α protein expression levels were evaluated using western blot analysis after the ASMCs were subjected to anoxia treatment (3% O2). Transfection with miR-23a mimics were conducted when PASMCs were under normoxia and anoxia conditions. EdU staining was used to detect the proliferative activity of PASMCs. Cells were transfected with HIF-1α specific siRNA under anoxia condition. RT-qPCR was used to detect miR-23a expression in PASMCs. Chromatin immunoprecipitation method was employed to verify the binding sites of HIF-1α. The dual-luciferase reporter gene was used to study the role of HIF-1 and its binding sites. Rat hypoxic pulmonary hypertension models were established to study the expression of miR-23a using RT-qPCR method and to verify the expression of miR-23a in the arteriole of the rat pulmonary. Our results showed that compared with normoxia condition, under anoxia condition (3% O2), the expression levels of the contractile phenotype marker proteins decreased significantly after 24 and 48 h. The positive rate of the EdU staining increased significantly and the expression of miR-23a increased. Transfection with miR-23a-mimic downregulated the expression of contractile marker proteins and improved the positive rate of the EdU staining under normoxia. Anoxia and transfection with HIF-1α enhanced the activity of the wild-type Luc-miR-23a-1 (WT) reporter gene. We concluded that miR-23a participated in the anoxia-induced phenotypic transformation of PASMCs. Increased expression of miR-23a under anoxia may primarily be due to miR-23a-1 and miR-23a-3 upregulation. The anoxia-induced upregulation of miR-23a was regulated by HIF-1.

Construction and analysis of the abnormal lncRNA–miRNA–mRNA network in hypoxic pulmonary hypertension

“…MiR-210-5p promotes epithelial–mesenchymal transformation, an important contributor to the development of HPH, by inhibiting the expression of PIK3R5 [ 33 ]. Yan et al reported that miR-23a is highly expressed in hypoxia-treated pulmonary artery smooth muscle cells (PASMCs) and plays an important role in hypoxia-induced phenotypic transformation of PASMCs [ 34 ]. In the past 5 years, miR-23a has been found to be a biomarker for idiopathic PH [ 35 ] and a possible therapeutic target for pulmonary arterial hypertension because it promotes cell proliferation and migration by targeting the BMPR2/Smad1 signal in hypoxia-induced PASMCs [ 36 ].…”

An Overview of miRNAs Involved in PASMC Phenotypic Switching in Pulmonary Hypertension

“…The mechanism by which hypoxia leads to changes in miRNA expression can be mediated by hypoxia inducible factor-1- (HIF-1-) dependent and HIF-1-independent pathways. Although hypoxia reportedly induces PASMCs phenotypic switching, the expression levels of miR-23a [ 92 ], miR-9 [ 93 ], miR-214 [ 36 ], and miR-20a [ 94 ] are increased, while those of miR-449 [ 95 ], miR-206 [ 96 ], miR-124 [ 97 ], miR-30c [ 98 ], and miR-140 [ 99 ] are decreased.…”

“…miR-23a: miR-23a is involved in the development of various cancers, promotes cardiac hypertrophy, and antagonizes muscle atrophy [ 102 , 103 ]. Yan et al showed that under hypoxic conditions the expression of miR-23a was upregulated in primary rat PASMCs through a mechanism involving HIF-1a [ 92 ]. In addition, the expression of contractile protein markers was significantly downregulated, suggesting that miR-23a regulates the dedifferentiation of PASMCs.…”

“…Many studies have confirmed that HIF-1 is upregulated and plays an important role in various types of PH, especially in hypoxic PH (HPH) [101]. In the early stage of hypoxia, HIF can activate the transcription of more than 100 genes, including miRNAs [92,93], which affect and regulate various pulmonary vascular functions, such as reactive oxygen species generation, angiogenesis, and vascular homeostasis, including PASMCs phenotypic switching. miR-23a: miR-23a is involved in the development of various cancers, promotes cardiac hypertrophy, and antagonizes muscle atrophy [102,103].…”