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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 regulates BMP mediated signaling.(a) Expression of BMP signaling related components in rat PASMCs. Left: representative western blot for hypoxia-dependent decrease of BMP signaling factors; Middle and right: Overexpression/ knockdown of miR-322 altered the protein level of factors in BMP signaling. The full-length blots with these antibodies were presented in supplementary Figure S5. All gels have been run simultaneously under the same experimental conditions. (b) Reporter gene studies on the interaction between miR-322 and 3′-UTR of the predicted targets in 293A cells. Luciferase activities were measured at 48 h after cotransfecting with the 3′-UTR constructs and the miR-322 overexpressing vectors or its vector control. (c) Validation of BMPR1a and Smad5 as direct targets of miR-322. Upper panel: conserved miR-322 binding sites in the 3′-UTR of Smad5 and BMPR1a along with the mutation site; Bottom panel: 3′-UTR luciferase reporter assay with targets and their mutant along with miR-322/miR-Con overexpressing vectors. Bar charts of luciferase reporter analysis represent means ± SD (n = 3), *p < 0.05, **p < 0.01 compared with miR-Con/3′UTR of targets. (d) Rescue experiments on EdU incorporation assay and wound-healing assay. Cells are transfected with miRNA inhibitor or/and siRNAs against BMPR1a or Smad5. Data are shown as means ± SD. *p < 0.05, **p < 0.01 compared to anti-Con + si-Con.
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f7: miR-322 regulates BMP mediated signaling.(a) Expression of BMP signaling related components in rat PASMCs. Left: representative western blot for hypoxia-dependent decrease of BMP signaling factors; Middle and right: Overexpression/ knockdown of miR-322 altered the protein level of factors in BMP signaling. The full-length blots with these antibodies were presented in supplementary Figure S5. All gels have been run simultaneously under the same experimental conditions. (b) Reporter gene studies on the interaction between miR-322 and 3′-UTR of the predicted targets in 293A cells. Luciferase activities were measured at 48 h after cotransfecting with the 3′-UTR constructs and the miR-322 overexpressing vectors or its vector control. (c) Validation of BMPR1a and Smad5 as direct targets of miR-322. Upper panel: conserved miR-322 binding sites in the 3′-UTR of Smad5 and BMPR1a along with the mutation site; Bottom panel: 3′-UTR luciferase reporter assay with targets and their mutant along with miR-322/miR-Con overexpressing vectors. Bar charts of luciferase reporter analysis represent means ± SD (n = 3), *p < 0.05, **p < 0.01 compared with miR-Con/3′UTR of targets. (d) Rescue experiments on EdU incorporation assay and wound-healing assay. Cells are transfected with miRNA inhibitor or/and siRNAs against BMPR1a or Smad5. Data are shown as means ± SD. *p < 0.05, **p < 0.01 compared to anti-Con + si-Con.

Mentions: Dysregulated BMP signaling in PASMC and pulmonary artery endothelial cells (PAECs) is thought to have a pro-proliferative and pro-migratory effect through the Smad-dependent signaling pathway, a mechanism that is involved in hypoxia-induced PAH2425. Therefore, we measured the expression of specific components of the BMP signaling pathway in rat PASMCs. Western blot analysis showed that the expression of BMPR2, BMPR1a (BMP type IA receptor), Smad4, Smad5, ID1 and ID2 (Inhibitors of DNA binding) in PASMCs was decreased in response to hypoxia (Fig. 7a, left panel). We also measured protein levels of these molecules involved in BMP signaling after overexpressing or silencing miR-322. Notably, the protein levels of BMPR1a, BMPR2, Smad5 and ID2 were decreased in miR-322 overexpressing cells under normoxia (Fig. 7a, middle panel). And they were all increased by miR-322 knockdown (Fig. 7a, right panel). These results suggest that hypoxia-induced miR-322 may play a regulatory role in PASMC proliferation and migration via the BMP signaling pathway.


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 regulates BMP mediated signaling.(a) Expression of BMP signaling related components in rat PASMCs. Left: representative western blot for hypoxia-dependent decrease of BMP signaling factors; Middle and right: Overexpression/ knockdown of miR-322 altered the protein level of factors in BMP signaling. The full-length blots with these antibodies were presented in supplementary Figure S5. All gels have been run simultaneously under the same experimental conditions. (b) Reporter gene studies on the interaction between miR-322 and 3′-UTR of the predicted targets in 293A cells. Luciferase activities were measured at 48 h after cotransfecting with the 3′-UTR constructs and the miR-322 overexpressing vectors or its vector control. (c) Validation of BMPR1a and Smad5 as direct targets of miR-322. Upper panel: conserved miR-322 binding sites in the 3′-UTR of Smad5 and BMPR1a along with the mutation site; Bottom panel: 3′-UTR luciferase reporter assay with targets and their mutant along with miR-322/miR-Con overexpressing vectors. Bar charts of luciferase reporter analysis represent means ± SD (n = 3), *p < 0.05, **p < 0.01 compared with miR-Con/3′UTR of targets. (d) Rescue experiments on EdU incorporation assay and wound-healing assay. Cells are transfected with miRNA inhibitor or/and siRNAs against BMPR1a or Smad5. Data are shown as means ± SD. *p < 0.05, **p < 0.01 compared to anti-Con + si-Con.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4499844&req=5

f7: miR-322 regulates BMP mediated signaling.(a) Expression of BMP signaling related components in rat PASMCs. Left: representative western blot for hypoxia-dependent decrease of BMP signaling factors; Middle and right: Overexpression/ knockdown of miR-322 altered the protein level of factors in BMP signaling. The full-length blots with these antibodies were presented in supplementary Figure S5. All gels have been run simultaneously under the same experimental conditions. (b) Reporter gene studies on the interaction between miR-322 and 3′-UTR of the predicted targets in 293A cells. Luciferase activities were measured at 48 h after cotransfecting with the 3′-UTR constructs and the miR-322 overexpressing vectors or its vector control. (c) Validation of BMPR1a and Smad5 as direct targets of miR-322. Upper panel: conserved miR-322 binding sites in the 3′-UTR of Smad5 and BMPR1a along with the mutation site; Bottom panel: 3′-UTR luciferase reporter assay with targets and their mutant along with miR-322/miR-Con overexpressing vectors. Bar charts of luciferase reporter analysis represent means ± SD (n = 3), *p < 0.05, **p < 0.01 compared with miR-Con/3′UTR of targets. (d) Rescue experiments on EdU incorporation assay and wound-healing assay. Cells are transfected with miRNA inhibitor or/and siRNAs against BMPR1a or Smad5. Data are shown as means ± SD. *p < 0.05, **p < 0.01 compared to anti-Con + si-Con.
Mentions: Dysregulated BMP signaling in PASMC and pulmonary artery endothelial cells (PAECs) is thought to have a pro-proliferative and pro-migratory effect through the Smad-dependent signaling pathway, a mechanism that is involved in hypoxia-induced PAH2425. Therefore, we measured the expression of specific components of the BMP signaling pathway in rat PASMCs. Western blot analysis showed that the expression of BMPR2, BMPR1a (BMP type IA receptor), Smad4, Smad5, ID1 and ID2 (Inhibitors of DNA binding) in PASMCs was decreased in response to hypoxia (Fig. 7a, left panel). We also measured protein levels of these molecules involved in BMP signaling after overexpressing or silencing miR-322. Notably, the protein levels of BMPR1a, BMPR2, Smad5 and ID2 were decreased in miR-322 overexpressing cells under normoxia (Fig. 7a, middle panel). And they were all increased by miR-322 knockdown (Fig. 7a, right panel). These results suggest that hypoxia-induced miR-322 may play a regulatory role in PASMC proliferation and migration via the BMP signaling pathway.

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