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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 promotes proliferation of PASMCs.(a, d) PASMCs were transduced with miR-Con, miR-322 or anti-Con, anti-322 and exposed to hypoxia (3% O2) or normoxia for 24h before measuring proliferative index as described under methods. Bar chart represents fold change in OD 490nm readings relative to miR-Con or anti-Con in both normoxic and hypoxic conditions.** p < 0.01 compared to miR-Con or anti-Con. (b, e) EdU incorporation assay showing proliferation activity of miR-322-overexpressed or inhibited cells and each control cells under normoxia and hypoxia. Data are shown as means ± SD, **p < 0.01 compared to miR-Con or anti-Con. (c, f) Staining of transfected cells with propidium iodide (PI) to assess cell cycle phase distribution by DNA content as described under Methods. Data are shown as means ± SD, **p < 0.01 compared to miR-Con or anti-Con.
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f5: miR-322 promotes proliferation of PASMCs.(a, d) PASMCs were transduced with miR-Con, miR-322 or anti-Con, anti-322 and exposed to hypoxia (3% O2) or normoxia for 24h before measuring proliferative index as described under methods. Bar chart represents fold change in OD 490nm readings relative to miR-Con or anti-Con in both normoxic and hypoxic conditions.** p < 0.01 compared to miR-Con or anti-Con. (b, e) EdU incorporation assay showing proliferation activity of miR-322-overexpressed or inhibited cells and each control cells under normoxia and hypoxia. Data are shown as means ± SD, **p < 0.01 compared to miR-Con or anti-Con. (c, f) Staining of transfected cells with propidium iodide (PI) to assess cell cycle phase distribution by DNA content as described under Methods. Data are shown as means ± SD, **p < 0.01 compared to miR-Con or anti-Con.

Mentions: To understand the consequences of increased miR-322 in hypoxia, we investigated the effects of miR-322 overexpression and knockdown on the proliferation and migration in rat PASMCs. As shown in Fig. 5a, stable overexpression of miR-322 significantly accelerated the proliferation rate of PASMCs compared to its control cells both under normoxia and hypoxia by MTS assay. EdU incorporation assay, a specific assay that labels replicating cells, also showed higher fraction of proliferating cells in miR-322-expressing cells compared with control cells (Fig. 5b). FACS analysis of PI-stained cells indicated that the proportion of cells in S and G2/M phase was significantly increased, and cells in G1 phase decreased by a comparable degree (Fig. 5c). To further confirm these results, we performed the proliferation assay in PASMCs after miRNA knockdown. In contrast with miRNA overexpression studies, proliferation was significantly reduced in anti-322 transfected cells compared to that of control cells, both when exposed to normoxia or hypoxia (Fig. 5d,e,f). These data indicate that miR-322 can significantly accelerate the proliferation of PASMCs.


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 promotes proliferation of PASMCs.(a, d) PASMCs were transduced with miR-Con, miR-322 or anti-Con, anti-322 and exposed to hypoxia (3% O2) or normoxia for 24h before measuring proliferative index as described under methods. Bar chart represents fold change in OD 490nm readings relative to miR-Con or anti-Con in both normoxic and hypoxic conditions.** p < 0.01 compared to miR-Con or anti-Con. (b, e) EdU incorporation assay showing proliferation activity of miR-322-overexpressed or inhibited cells and each control cells under normoxia and hypoxia. Data are shown as means ± SD, **p < 0.01 compared to miR-Con or anti-Con. (c, f) Staining of transfected cells with propidium iodide (PI) to assess cell cycle phase distribution by DNA content as described under Methods. Data are shown as means ± SD, **p < 0.01 compared to miR-Con or anti-Con.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: miR-322 promotes proliferation of PASMCs.(a, d) PASMCs were transduced with miR-Con, miR-322 or anti-Con, anti-322 and exposed to hypoxia (3% O2) or normoxia for 24h before measuring proliferative index as described under methods. Bar chart represents fold change in OD 490nm readings relative to miR-Con or anti-Con in both normoxic and hypoxic conditions.** p < 0.01 compared to miR-Con or anti-Con. (b, e) EdU incorporation assay showing proliferation activity of miR-322-overexpressed or inhibited cells and each control cells under normoxia and hypoxia. Data are shown as means ± SD, **p < 0.01 compared to miR-Con or anti-Con. (c, f) Staining of transfected cells with propidium iodide (PI) to assess cell cycle phase distribution by DNA content as described under Methods. Data are shown as means ± SD, **p < 0.01 compared to miR-Con or anti-Con.
Mentions: To understand the consequences of increased miR-322 in hypoxia, we investigated the effects of miR-322 overexpression and knockdown on the proliferation and migration in rat PASMCs. As shown in Fig. 5a, stable overexpression of miR-322 significantly accelerated the proliferation rate of PASMCs compared to its control cells both under normoxia and hypoxia by MTS assay. EdU incorporation assay, a specific assay that labels replicating cells, also showed higher fraction of proliferating cells in miR-322-expressing cells compared with control cells (Fig. 5b). FACS analysis of PI-stained cells indicated that the proportion of cells in S and G2/M phase was significantly increased, and cells in G1 phase decreased by a comparable degree (Fig. 5c). To further confirm these results, we performed the proliferation assay in PASMCs after miRNA knockdown. In contrast with miRNA overexpression studies, proliferation was significantly reduced in anti-322 transfected cells compared to that of control cells, both when exposed to normoxia or hypoxia (Fig. 5d,e,f). These data indicate that miR-322 can significantly accelerate the proliferation of PASMCs.

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