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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 stimulates the accumulation of HIF-1α.(a) Western blotting analysis of HIF-1α subcellular expression in rat PASMCs exposed to hypoxia for 24h. TBP (TATA box Binding Protein) and α-tubulin were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (b) Quantitative real-time PCR quantification of miR-322 in stably transfected rat PASMCs. Cells were transfected with recombinant lentiviral particles expressing miR-322 (miR-322) or control lentiviral vector (miR-Con), or with lentivirus of dTud construct against mature miR-322 (anti-322) or scramble control vector (anti-Con). Data are shown as means ± SD relative to respective controls, *p < 0.05 compared with controls. (c) Left: Immunoblotting to determine HIF-1α expression in cells transfected with lentivirus expressing miR-322 or control lentivirus under normoxia. Right: Immunoblotting to determine HIF-1α expression in PASMCs transfected with anti-322 construct or scramble control and exposed to hypoxia. The full-length blots with these antibodies were presented in supplementary Figure S4. All gels have been run simultaneously under the same experimental conditions.
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f4: miR-322 stimulates the accumulation of HIF-1α.(a) Western blotting analysis of HIF-1α subcellular expression in rat PASMCs exposed to hypoxia for 24h. TBP (TATA box Binding Protein) and α-tubulin were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (b) Quantitative real-time PCR quantification of miR-322 in stably transfected rat PASMCs. Cells were transfected with recombinant lentiviral particles expressing miR-322 (miR-322) or control lentiviral vector (miR-Con), or with lentivirus of dTud construct against mature miR-322 (anti-322) or scramble control vector (anti-Con). Data are shown as means ± SD relative to respective controls, *p < 0.05 compared with controls. (c) Left: Immunoblotting to determine HIF-1α expression in cells transfected with lentivirus expressing miR-322 or control lentivirus under normoxia. Right: Immunoblotting to determine HIF-1α expression in PASMCs transfected with anti-322 construct or scramble control and exposed to hypoxia. The full-length blots with these antibodies were presented in supplementary Figure S4. All gels have been run simultaneously under the same experimental conditions.

Mentions: Ghosh et al. have reported that human miR-424, the homolog of rodent miR-322, stabilizes HIF-1α leading to its accumulation in the nucleus in human umbilical vein endothelial cells (HUVEC)23. Therefore, we determined whether manipulating miR-322 expression could affect the levels of HIF-1α in rat PASMCs. Cells were transfected with recombinant lentivirus expressing miR-322 (miR-322), or no miRNA sequence as a negative control (miR-Con). Stably transfected cells were generated by puromycin selection. As shown in Fig. 4a, exposure to hypoxia induced HIF-1α accumulation in the nucleus of PASMCs. qRT-PCR revealed an over 12-fold increase in miR-322 in the cells overexpressing miR-322 compared with control cells (Fig. 4b). Western blot showed that the accumulation of HIF-1α increased in the nucleus when overexpressing miR-322 in normoxia (Fig. 4c). Cells were also transfected with dTud constructs for miR-322 knockdown studies. As shown in Fig. 4b, cells with miR-322 inhibition (anti-322) exhibited significantly reduced expression in miR-322 compared to its control cells (anti-Con). Knockdown of miR-322 abolished the stabilization of HIF-1α in PASMCs under hypoxic condition (Fig. 4c). Taken together these results indicate that hypoxia-induced miR-322 is involved in regulating the stability of HIF-1α in 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 stimulates the accumulation of HIF-1α.(a) Western blotting analysis of HIF-1α subcellular expression in rat PASMCs exposed to hypoxia for 24h. TBP (TATA box Binding Protein) and α-tubulin were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (b) Quantitative real-time PCR quantification of miR-322 in stably transfected rat PASMCs. Cells were transfected with recombinant lentiviral particles expressing miR-322 (miR-322) or control lentiviral vector (miR-Con), or with lentivirus of dTud construct against mature miR-322 (anti-322) or scramble control vector (anti-Con). Data are shown as means ± SD relative to respective controls, *p < 0.05 compared with controls. (c) Left: Immunoblotting to determine HIF-1α expression in cells transfected with lentivirus expressing miR-322 or control lentivirus under normoxia. Right: Immunoblotting to determine HIF-1α expression in PASMCs transfected with anti-322 construct or scramble control and exposed to hypoxia. The full-length blots with these antibodies were presented in supplementary Figure S4. All gels have been run simultaneously under the same experimental conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: miR-322 stimulates the accumulation of HIF-1α.(a) Western blotting analysis of HIF-1α subcellular expression in rat PASMCs exposed to hypoxia for 24h. TBP (TATA box Binding Protein) and α-tubulin were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (b) Quantitative real-time PCR quantification of miR-322 in stably transfected rat PASMCs. Cells were transfected with recombinant lentiviral particles expressing miR-322 (miR-322) or control lentiviral vector (miR-Con), or with lentivirus of dTud construct against mature miR-322 (anti-322) or scramble control vector (anti-Con). Data are shown as means ± SD relative to respective controls, *p < 0.05 compared with controls. (c) Left: Immunoblotting to determine HIF-1α expression in cells transfected with lentivirus expressing miR-322 or control lentivirus under normoxia. Right: Immunoblotting to determine HIF-1α expression in PASMCs transfected with anti-322 construct or scramble control and exposed to hypoxia. The full-length blots with these antibodies were presented in supplementary Figure S4. All gels have been run simultaneously under the same experimental conditions.
Mentions: Ghosh et al. have reported that human miR-424, the homolog of rodent miR-322, stabilizes HIF-1α leading to its accumulation in the nucleus in human umbilical vein endothelial cells (HUVEC)23. Therefore, we determined whether manipulating miR-322 expression could affect the levels of HIF-1α in rat PASMCs. Cells were transfected with recombinant lentivirus expressing miR-322 (miR-322), or no miRNA sequence as a negative control (miR-Con). Stably transfected cells were generated by puromycin selection. As shown in Fig. 4a, exposure to hypoxia induced HIF-1α accumulation in the nucleus of PASMCs. qRT-PCR revealed an over 12-fold increase in miR-322 in the cells overexpressing miR-322 compared with control cells (Fig. 4b). Western blot showed that the accumulation of HIF-1α increased in the nucleus when overexpressing miR-322 in normoxia (Fig. 4c). Cells were also transfected with dTud constructs for miR-322 knockdown studies. As shown in Fig. 4b, cells with miR-322 inhibition (anti-322) exhibited significantly reduced expression in miR-322 compared to its control cells (anti-Con). Knockdown of miR-322 abolished the stabilization of HIF-1α in PASMCs under hypoxic condition (Fig. 4c). Taken together these results indicate that hypoxia-induced miR-322 is involved in regulating the stability of HIF-1α in 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