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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

Hypoxia upregulates expression of miR-322 in rat PASMC and A7r5 cells.(a) Quantitative RT-PCR assay of miR-322 in rat lungs exposure to hypoxia (n = 3 for each group). Data were shown as mean ± SD, **p < 0.01 compared to Con. (b) Purity of rat PASMCs in primary culture. Smooth muscle cells were isolated from rat pulmonary artery and immunostained with smooth muscle α-actin (α-SMA, green) antibody and DAPI (blue) to assess purity. Scale bar, 40 μm. (c) Rat PASMCs and A7r5 cells were exposed to normoxia (21% O2) (N-Con) or hypoxia (3% O2) for 24 h (H-24 h). The relative levels of miR-322 was estimated by real-time PCR. Data are shown as means ± SD relative to respective normoxia controls, **p < 0.01. (d) Representative western blot showing HIF-1α, HIF-2α and β-actin (as loading control) protein levels in rat PASMCs and A7r5 cells exposed to either normoxia or hypoxia for 24 h. The full-length blots with these antibodies were presented in supplementary Figure S2. All gels have been run simultaneously under the same experimental conditions.
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f2: Hypoxia upregulates expression of miR-322 in rat PASMC and A7r5 cells.(a) Quantitative RT-PCR assay of miR-322 in rat lungs exposure to hypoxia (n = 3 for each group). Data were shown as mean ± SD, **p < 0.01 compared to Con. (b) Purity of rat PASMCs in primary culture. Smooth muscle cells were isolated from rat pulmonary artery and immunostained with smooth muscle α-actin (α-SMA, green) antibody and DAPI (blue) to assess purity. Scale bar, 40 μm. (c) Rat PASMCs and A7r5 cells were exposed to normoxia (21% O2) (N-Con) or hypoxia (3% O2) for 24 h (H-24 h). The relative levels of miR-322 was estimated by real-time PCR. Data are shown as means ± SD relative to respective normoxia controls, **p < 0.01. (d) Representative western blot showing HIF-1α, HIF-2α and β-actin (as loading control) protein levels in rat PASMCs and A7r5 cells exposed to either normoxia or hypoxia for 24 h. The full-length blots with these antibodies were presented in supplementary Figure S2. All gels have been run simultaneously under the same experimental conditions.

Mentions: Next we determined whether hypoxia-induced expression of miR-322 in rat lung and in vitro cultured PASMCs parallels the mouse lung miRNA profile. As shown in Fig. 2a, miR-322 level in rat lungs was increased about 2-fold after 3-weeks hypoxic treatment. PASMCs isolated from Sprague-Dawley rats were determined to be 98% pure by staining with smooth muscle-specific α-actin (α-SMA) (Fig. 2b). PASMCs were grown either in normoxia (21% O2) or hypoxia (3% O2) for 24 h, and miRNA levels were measured by qRT-PCR. As shown in Fig. 2c, the expression of miR-322 was upregulated 1.6-fold in hypoxia compared to normoxia. A similar study was carried out in A7r5 cells a smooth muscle cell line derived from rat thoracic aorta. In these cells miR-322 expression was increased about 2.2-fold in hypoxia. The increase in HIF-1α and HIF-2α levels in hypoxia was also confirmed in PASMCs and A7r5 cells (Fig. 2d).


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)

Hypoxia upregulates expression of miR-322 in rat PASMC and A7r5 cells.(a) Quantitative RT-PCR assay of miR-322 in rat lungs exposure to hypoxia (n = 3 for each group). Data were shown as mean ± SD, **p < 0.01 compared to Con. (b) Purity of rat PASMCs in primary culture. Smooth muscle cells were isolated from rat pulmonary artery and immunostained with smooth muscle α-actin (α-SMA, green) antibody and DAPI (blue) to assess purity. Scale bar, 40 μm. (c) Rat PASMCs and A7r5 cells were exposed to normoxia (21% O2) (N-Con) or hypoxia (3% O2) for 24 h (H-24 h). The relative levels of miR-322 was estimated by real-time PCR. Data are shown as means ± SD relative to respective normoxia controls, **p < 0.01. (d) Representative western blot showing HIF-1α, HIF-2α and β-actin (as loading control) protein levels in rat PASMCs and A7r5 cells exposed to either normoxia or hypoxia for 24 h. The full-length blots with these antibodies were presented in supplementary Figure S2. All gels have been run simultaneously under the same experimental conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Hypoxia upregulates expression of miR-322 in rat PASMC and A7r5 cells.(a) Quantitative RT-PCR assay of miR-322 in rat lungs exposure to hypoxia (n = 3 for each group). Data were shown as mean ± SD, **p < 0.01 compared to Con. (b) Purity of rat PASMCs in primary culture. Smooth muscle cells were isolated from rat pulmonary artery and immunostained with smooth muscle α-actin (α-SMA, green) antibody and DAPI (blue) to assess purity. Scale bar, 40 μm. (c) Rat PASMCs and A7r5 cells were exposed to normoxia (21% O2) (N-Con) or hypoxia (3% O2) for 24 h (H-24 h). The relative levels of miR-322 was estimated by real-time PCR. Data are shown as means ± SD relative to respective normoxia controls, **p < 0.01. (d) Representative western blot showing HIF-1α, HIF-2α and β-actin (as loading control) protein levels in rat PASMCs and A7r5 cells exposed to either normoxia or hypoxia for 24 h. The full-length blots with these antibodies were presented in supplementary Figure S2. All gels have been run simultaneously under the same experimental conditions.
Mentions: Next we determined whether hypoxia-induced expression of miR-322 in rat lung and in vitro cultured PASMCs parallels the mouse lung miRNA profile. As shown in Fig. 2a, miR-322 level in rat lungs was increased about 2-fold after 3-weeks hypoxic treatment. PASMCs isolated from Sprague-Dawley rats were determined to be 98% pure by staining with smooth muscle-specific α-actin (α-SMA) (Fig. 2b). PASMCs were grown either in normoxia (21% O2) or hypoxia (3% O2) for 24 h, and miRNA levels were measured by qRT-PCR. As shown in Fig. 2c, the expression of miR-322 was upregulated 1.6-fold in hypoxia compared to normoxia. A similar study was carried out in A7r5 cells a smooth muscle cell line derived from rat thoracic aorta. In these cells miR-322 expression was increased about 2.2-fold in hypoxia. The increase in HIF-1α and HIF-2α levels in hypoxia was also confirmed in PASMCs and A7r5 cells (Fig. 2d).

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