| Backgrounds: The pathologic process of hypoxic pulmonary hypertension(HPH) plays an important role in the development of chronic obstructive pulmonary disease and high altitude heart disease. Chronic hypoxia will ultimately lead to pulmonary vascular remodeling. During this process, the pulmonary arterial smooth muscle cells(PASMCs) convent from contractile, differentiated phenotype to synthetic, undifferentiated phenotype, accompany with the abnormal proliferation, migration, and dedifferentiation. Recently, several studies have demonstrated that micro RNAs(mi RNAs)have effects on secretion, proliferation, migration, and apoptosis of PASMCs, as well as the pulmonary vascular remodeling. However, the understanding of how the mi RNAs regulate the molecular mechanism of hypoxia-induced PASMCs is very limited. Objective: To investigate the effect and mechanism of mi R-20 a in phenotype transition of hypoxia-induced PASMCs. Methods and Results: First, in chronic hypoxia-induced PAH mouse, the increased expression of mi R-20 a was detected by q RT-PCR both in lung tissue and isolated pulmonary arteries, which was also observed in pulmonary arterial smooth muscle cells cultured in vitro under oxygen deprivation. Through Ed U incorporation assay, wound-healing assay and western blot method, we had investigated the function of mi R-20 a on HPASMCs by overexpression or knockdown.We found that mi R-20 a could significantly promote proliferation, migration and down-regulating the expression of differentiation phenotype protein including calponin、 α-SMA 、Smoothelin、MHC and SM-22 in HPASMC. These results revealed that chronic hypoxia could up-regulate the expression of mi R-20 a and then promot cells convert to dedifferentiated phenotype in HPASMCs. Second, by q RT-PCR and western blot, we verified that the expression of both m RNA and protein of PKG1 significantly decreased in chronic hypoxia-treated primary cultured HPASMCs, which played a key role in maintaining cells phenotypic differentiation. Using western blot, we investigated that overexpression and knockdown of the mi R-20 a could up or down-regulating the expression of PKG1 respectively in HPASMC. The results presumed that PKG1 may be the target genes of mi R-20 a. By using Dual luciferase reporter gene assay, we found that there were no interactions between mi R-20 a with the PRKG1 promoter and 3’-UTR. Through further analysis of PKG1 coding sequence, we found mi R-20 a suppressed the expression of PKG1 protein in post-transcription level by interacting the two highly conserved binding sites in its coding region. The cell function experimental results show that PKG1 could up-regulate the expression of differentiation phenotype protein suppress proliferation, migration by overexpression or knockdown of PKG1 in HPASMC respectively, which was contrary to the function of mi R-20 a. Besides, the expression of mi R-20 a in serum also increased in patients with congenital heart disease associated pulmonary arterialhy pertention. Conclusion: In this study, it showed that mi R-20 a was up-regulated in hypoxia-induced human PASMC and inhibited PKG1 expression by targeting its coding region, and then the proliferation and migration was promoted, the cell converted to differentiated phenotype in PASMC. In summary, we provided a possible mechanism, that is, hypoxia resulted in decreased PKG expression and the phenotypic switching of PASMC, also provided a new way of the clinical diagnosis and treatment of pulmonary hypertension... |
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