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Hypoxia inducible factor-1 mediates expression of miR-322: potential role in proliferation and migration of pulmonary arterial smooth muscle cells.

Zeng Y, Liu H, Kang K, Wang Z, Hui G, Zhang X, Zhong J, Peng W, Ramchandran R, Raj JU, Gou D - Sci Rep (2015)

Bottom Line: We demonstrated that HIF-1α, but not HIF-2α, transcriptionally upregulates the expression of miR-322 in hypoxia.Furthermore, miR-322 facilitated the accumulation of HIF-1α in the nucleus and promoted hypoxia-induced cell proliferation and migration.Direct targeting BMPR1a and smad5 by miR-322 was demonstrated in PASMCs suggesting that downregulation of BMP-Smad signaling pathway may be mediating the hypoxia-induced PASMC proliferation and migration.

View Article: PubMed Central - PubMed

Affiliation: 1] Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresourse and Eco-environmental Science, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China [2] Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong, 518060, China.

ABSTRACT
There is growing evidence that microRNAs play important roles in cellular responses to hypoxia and in pulmonary hypertensive vascular remodeling, but the exact molecular mechanisms involved are not fully elucidated. In this study, we identified miR-322 as one of the microRNAs induced in lungs of chronically hypoxic mice and rats. The expression of miR-322 was also upregulated in primary cultured rat pulmonary arterial smooth muscle cells (PASMC) in response to hypoxia. We demonstrated that HIF-1α, but not HIF-2α, transcriptionally upregulates the expression of miR-322 in hypoxia. Furthermore, miR-322 facilitated the accumulation of HIF-1α in the nucleus and promoted hypoxia-induced cell proliferation and migration. Direct targeting BMPR1a and smad5 by miR-322 was demonstrated in PASMCs suggesting that downregulation of BMP-Smad signaling pathway may be mediating the hypoxia-induced PASMC proliferation and migration. Our study implicates miR-322 in the hypoxic proliferative response of PASMCs suggesting that it may be playing a role in pulmonary vascular remodeling associated with pulmonary hypertension.

No MeSH data available.


Related in: MedlinePlus

miR-322 is induced by hypoxia in mouse lung.(a) Differential expression of 1040 miRNAs in lung tissue from mice exposed to normoxia (21% O2)(Con) or hypoxia (10% O2) for 2 days (H-2D), 1 week (H-1W), 2 weeks (H-2W) and 3 weeks (H-3W) (n = 3 or 4 for each group). Cluster analysis of miRNAs expression from individual specimens were assessed by microarray analysis. (p < 0.05 compared with normoxic controls). (b) Validation of miRNAs upregulated by hypoxia by real-time PCR assay. Bar charts showing the relative expression level by normalizing to the respective normoxia control (Con). Data are shown as means ± SD, *p < 0.05, **p < 0.01. (c) Hypoxia results in stabilization of HIF-1α and HIF-2α proteins in the lung. Representative immunoblots showing the lung HIF-1α and HIF-2α levels in normoxia and hypoxia-exposed mice. β-actin levels served as loading control. The full-length blots with these antibodies were presented in supplementary Figure S1. All gels have been run simultaneously under the same experimental conditions.
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f1: miR-322 is induced by hypoxia in mouse lung.(a) Differential expression of 1040 miRNAs in lung tissue from mice exposed to normoxia (21% O2)(Con) or hypoxia (10% O2) for 2 days (H-2D), 1 week (H-1W), 2 weeks (H-2W) and 3 weeks (H-3W) (n = 3 or 4 for each group). Cluster analysis of miRNAs expression from individual specimens were assessed by microarray analysis. (p < 0.05 compared with normoxic controls). (b) Validation of miRNAs upregulated by hypoxia by real-time PCR assay. Bar charts showing the relative expression level by normalizing to the respective normoxia control (Con). Data are shown as means ± SD, *p < 0.05, **p < 0.01. (c) Hypoxia results in stabilization of HIF-1α and HIF-2α proteins in the lung. Representative immunoblots showing the lung HIF-1α and HIF-2α levels in normoxia and hypoxia-exposed mice. β-actin levels served as loading control. The full-length blots with these antibodies were presented in supplementary Figure S1. All gels have been run simultaneously under the same experimental conditions.

Mentions: To determine the lung miRNA profile in chronic hypoxia (10% O2)-induced PH in mice, we performed a microarray analysis. The microarray profile revealed that several miRNAs, including miR-466i-5p, −199a-3p, −322, −351 and −379, were significantly upregulated after 3-weeks of hypoxic exposure (Fig. 1a). Then, an independent quantitative real-time PCR (qRT-PCR) assay was carried out to confirm the expression pattern of these miRNAs, normalized to sno202. The results showed that the expression of miR-322 and miR-351 was increased significantly with the duration of hypoxia exposure (Fig. 1b). Western blot analysis showed increased expression of HIF-1α and HIF-2α in lung tissue extracts in response to hypoxia, with β-actin serving as an internal control (Fig. 1c).


Hypoxia inducible factor-1 mediates expression of miR-322: potential role in proliferation and migration of pulmonary arterial smooth muscle cells.

Zeng Y, Liu H, Kang K, Wang Z, Hui G, Zhang X, Zhong J, Peng W, Ramchandran R, Raj JU, Gou D - Sci Rep (2015)

miR-322 is induced by hypoxia in mouse lung.(a) Differential expression of 1040 miRNAs in lung tissue from mice exposed to normoxia (21% O2)(Con) or hypoxia (10% O2) for 2 days (H-2D), 1 week (H-1W), 2 weeks (H-2W) and 3 weeks (H-3W) (n = 3 or 4 for each group). Cluster analysis of miRNAs expression from individual specimens were assessed by microarray analysis. (p < 0.05 compared with normoxic controls). (b) Validation of miRNAs upregulated by hypoxia by real-time PCR assay. Bar charts showing the relative expression level by normalizing to the respective normoxia control (Con). Data are shown as means ± SD, *p < 0.05, **p < 0.01. (c) Hypoxia results in stabilization of HIF-1α and HIF-2α proteins in the lung. Representative immunoblots showing the lung HIF-1α and HIF-2α levels in normoxia and hypoxia-exposed mice. β-actin levels served as loading control. The full-length blots with these antibodies were presented in supplementary Figure S1. All gels have been run simultaneously under the same experimental conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499844&req=5

f1: miR-322 is induced by hypoxia in mouse lung.(a) Differential expression of 1040 miRNAs in lung tissue from mice exposed to normoxia (21% O2)(Con) or hypoxia (10% O2) for 2 days (H-2D), 1 week (H-1W), 2 weeks (H-2W) and 3 weeks (H-3W) (n = 3 or 4 for each group). Cluster analysis of miRNAs expression from individual specimens were assessed by microarray analysis. (p < 0.05 compared with normoxic controls). (b) Validation of miRNAs upregulated by hypoxia by real-time PCR assay. Bar charts showing the relative expression level by normalizing to the respective normoxia control (Con). Data are shown as means ± SD, *p < 0.05, **p < 0.01. (c) Hypoxia results in stabilization of HIF-1α and HIF-2α proteins in the lung. Representative immunoblots showing the lung HIF-1α and HIF-2α levels in normoxia and hypoxia-exposed mice. β-actin levels served as loading control. The full-length blots with these antibodies were presented in supplementary Figure S1. All gels have been run simultaneously under the same experimental conditions.
Mentions: To determine the lung miRNA profile in chronic hypoxia (10% O2)-induced PH in mice, we performed a microarray analysis. The microarray profile revealed that several miRNAs, including miR-466i-5p, −199a-3p, −322, −351 and −379, were significantly upregulated after 3-weeks of hypoxic exposure (Fig. 1a). Then, an independent quantitative real-time PCR (qRT-PCR) assay was carried out to confirm the expression pattern of these miRNAs, normalized to sno202. The results showed that the expression of miR-322 and miR-351 was increased significantly with the duration of hypoxia exposure (Fig. 1b). Western blot analysis showed increased expression of HIF-1α and HIF-2α in lung tissue extracts in response to hypoxia, with β-actin serving as an internal control (Fig. 1c).

Bottom Line: We demonstrated that HIF-1α, but not HIF-2α, transcriptionally upregulates the expression of miR-322 in hypoxia.Furthermore, miR-322 facilitated the accumulation of HIF-1α in the nucleus and promoted hypoxia-induced cell proliferation and migration.Direct targeting BMPR1a and smad5 by miR-322 was demonstrated in PASMCs suggesting that downregulation of BMP-Smad signaling pathway may be mediating the hypoxia-induced PASMC proliferation and migration.

View Article: PubMed Central - PubMed

Affiliation: 1] Shenzhen Key Laboratory of Microbial Genetic Engineering, Shenzhen Key Laboratory of Marine Bioresourse and Eco-environmental Science, College of Life Sciences, Shenzhen University, Shenzhen, Guangdong, 518060, China [2] Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong, 518060, China.

ABSTRACT
There is growing evidence that microRNAs play important roles in cellular responses to hypoxia and in pulmonary hypertensive vascular remodeling, but the exact molecular mechanisms involved are not fully elucidated. In this study, we identified miR-322 as one of the microRNAs induced in lungs of chronically hypoxic mice and rats. The expression of miR-322 was also upregulated in primary cultured rat pulmonary arterial smooth muscle cells (PASMC) in response to hypoxia. We demonstrated that HIF-1α, but not HIF-2α, transcriptionally upregulates the expression of miR-322 in hypoxia. Furthermore, miR-322 facilitated the accumulation of HIF-1α in the nucleus and promoted hypoxia-induced cell proliferation and migration. Direct targeting BMPR1a and smad5 by miR-322 was demonstrated in PASMCs suggesting that downregulation of BMP-Smad signaling pathway may be mediating the hypoxia-induced PASMC proliferation and migration. Our study implicates miR-322 in the hypoxic proliferative response of PASMCs suggesting that it may be playing a role in pulmonary vascular remodeling associated with pulmonary hypertension.

No MeSH data available.


Related in: MedlinePlus