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Downregulation of long non?coding RNA ANRIL promotes proliferation and migration in hypoxic human pulmonary artery smooth muscle cells.

ABSTRACT: Pulmonary arterial hypertension (PAH) is a progressive syndrome. When PAH occurs, the circulatory resistance of the pulmonary vasculature will gradually increase, which may lead to right heart failure and death. Pathological features of PAH include abnormal proliferation of pulmonary vascular smooth muscle cells and pulmonary vascular remodeling. Hypoxia is the main cause of PAH, which directly induces the contraction and proliferation of pulmonary artery smooth muscle cells (PASMCs), and eventually leads to pulmonary vascular remodeling. Recent studies have shown that long non?coding RNAs (lncRNAs) play key roles in numerous biological processes, including cell proliferation and the occurrence and development of cardiovascular diseases. Studies have also shown that lncRNA antisense noncoding RNA in the INK4 locus (ANRIL) can promote the proliferation of vascular smooth muscle cells. Therefore, the hypothesis of the present study was that ANRIL may be expressed in PASMCs and play a regulatory role. In this study, the expression of ANRIL was analyzed by quantitative PCR. The effects of ANRIL on human pulmonary artery smooth muscle cells (HPASMCs) were assessed by MTT assay, flow cytometry, bromodeoxyuridine incorporation assay, Transwell assay, scratch?wound assay, immunofluorescence assay and western blotting. These experiments revealed that the expression of ANRIL was significantly downregulated in HPASMCs induced by hypoxia. The downregulation of ANRIL affected the cell cycle, making more HPASMCs move from the G0/G1 phase to the G2/M+S phase and strengthening the cell proliferation. Moreover, downregulated ANRIL increased the migration of HPASMCs under hypoxia. This study identified ANRIL as a critical regulator in HPASMCs induced by hypoxia and demonstrated the potential of gene therapy and drug development for treating PAH.

SUBMITTER: Wang S

PROVIDER: S-EPMC6947933 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Downregulation of long non‑coding RNA ANRIL promotes proliferation and migration in hypoxic human pulmonary artery smooth muscle cells.

Wang Siqi S Zhang Chen C Zhang Xiaodan X

Molecular medicine reports 20191217 2

Pulmonary arterial hypertension (PAH) is a progressive syndrome. When PAH occurs, the circulatory resistance of the pulmonary vasculature will gradually increase, which may lead to right heart failure and death. Pathological features of PAH include abnormal proliferation of pulmonary vascular smooth muscle cells and pulmonary vascular remodeling. Hypoxia is the main cause of PAH, which directly induces the contraction and proliferation of pulmonary artery smooth muscle cells (PASMCs), and eventu ...[more]

PMID: 31974617

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Project description:The chronic phase of pulmonary arterial hypertension (PAH) is associated with vascular remodeling, especially thickening of the smooth muscle layer of large pulmonary arteries and muscularization of small pulmonary vessels, which normally have no associated smooth muscle. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMC), and may be important for in vivo pulmonary vascular remodeling. Here, we show that 5-HT stimulates migration of pulmonary artery PASMC. Treatment with 5-HT for 16h increased migration of PASMC up to four-fold as monitored in a modified Boyden chamber assay. Increased migratory responses were associated with cellular morphological changes and reorganization of the actin cytoskeleton. 5-HT-induced alterations in morphology were previously shown in our laboratory to require cAMP [Lee SL, Fanburg BL. Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP. J Cell Phys 1992;150(2):396-405], and the 5-HT4 receptor was pharmacologically determined to be the primary activator of cAMP in bovine PASMC [Becker BN, Gettys TW, Middleton JP, Olsen CL, Albers FJ, Lee SL, et al. 8-Hydroxy-2-(di-n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells. Mol Pharmacol 1992;42(5):817-25]. We examined the role of the 5-HT4 receptor and cAMP in 5-HT-induced bovine PASMC migration. PASMC express 5-HT4 receptor mRNA, and a 5-HT4 receptor antagonist and a cAMP antagonist completely blocked 5-HT-induced cellular migration. Consistent with our previous report that a cAMP-dependent Cl(-) channel is required for 5-HT-induced morphological changes in PASMC, phenylanthranilic acid, a Cl(-) channel blocker, inhibited actin cytoskeletal reorganization and migration produced by 5-HT. We conclude that 5-HT stimulates PASMC migration and associated cytoskeletal reorganization through the 5-HT4 receptor and cAMP activation of a chloride channel.

Project description:Pulmonary arterial hypertension is a progressive and fatal disease. Recent studies suggest that circular RNA (circRNAs/circs) can regulate various biological processes, including cell proliferation. Therefore, it is possible that circRNA may have important roles in pulmonary artery smooth muscle cell proliferation in hypoxic pulmonary hypertension (HPH). The aim of the present study was to determine the role and mechanism of circRNA‑glutamate metabotropic receptor 1 (circ‑Grm1; mmu_circ_0001907) in pulmonary artery smooth muscle cell (PASMC) proliferation and migration in HPH. High‑throughput transcriptome sequencing was used to screen circRNAs and targeted genes involved in HPH. Cell Counting Kit‑8 (CCK‑8), 5‑ethynyl‑2‑deoxyuridine and wound healing assays were employed to assess cell viability and migration. Reverse transcription‑quantitative PCR and western blotting were used to detect target gene expression in different groups. Bioinformatical approaches were used to predict the interaction probabilities of circ‑Grm1 and Grm1 with FUS RNA binding protein (FUS). The interactions of circ‑Grm1, Grm1 and FUS were evaluated using RNA silencing and RNA immunoprecipitation assays. The results demonstrated that circ‑Grm1 was upregulated in hypoxic PASMCs. Further experiments revealed that the knockdown of circ‑Grm1 could suppress the proliferation and migration of hypoxic PASMCs. Transcriptome sequencing revealed that Grm1 could be the target gene of circ‑Grm1. It was found that circ‑Grm1 could competitively bind to FUS and consequently downregulate Grm1. Moreover, Grm1 could inhibit the function of circ‑Grm1 by promoting the proliferative and migratory abilities of hypoxic PASMCs. The results also demonstrated that circ‑Grm1 influenced the biological functions of PASMCs via the Rap1/ERK pathway by regulating Grm1. Overall, the current results suggested that circ‑Grm1 was associated with HPH and promoted the proliferation and migration of PASMCs via suppression of Grm1 expression through FUS.

Dataset Information

Downregulation of long non?coding RNA ANRIL promotes proliferation and migration in hypoxic human pulmonary artery smooth muscle cells.

Publications

Downregulation of long non‑coding RNA ANRIL promotes proliferation and migration in hypoxic human pulmonary artery smooth muscle cells.

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