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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 induces miR-322 promoter activity in a HIF-1α-dependent manner.(a) Schematic diagram of cloned rat putative promoter region of miR-322, stretching from upstream 1000 bp. The position of the putative HRE matching the core sequence (A/G)CGTG is labeled in box, between two functional CACAG elements (P1k). A mutant promoter was constructed with the sequence as shown below (P1k-m). (b) Luciferase reporter assays of miR-322 promoter activity with both wild (pGL4-P1k) and mutant constructs (pGl4-P1k-m) in the presence or absence of CoCl2 (200μM) (left panel); Under these conditions, the protein levels of HIF-1α and HIF-2α were stabilized after CoCl2 treatment in the cells as determined by western blot analysis and normalized to β-actin levels (right panel). (c) miR-322 promoter activity assessed by luciferase reporter assay after CoCl2 treatment was diminished by HIF-1α knockdown. Cells were transduced with shRNA targeting HIF-1α (shHIF-1α) or HIF-2α (shHIF-2α) and miR-322 promoter activity was determined in the luciferase reporter assay after transfection of wild type and mutant constructs and CoCl2 treatment (left panel) as described under Methods; Control (sh-Con) or HIF-1α/-2α shRNA-transfected cells were harvested for protein analysis by western blot to determine knockdown specificity (right panel); *p < 0.05 compared with sh-Con. (d & e) A7r5 cells were transfected with recombinant adenoviruses expressing oxygen-dependent degradation domains (ODDD-wt) or mutated ODDD (ODDD-mut) under normoxic conditions. The influence on the miR-322 promoter reporter activity (d) and the endogenous expression levels (e) of miR-322 were determined by real-time PCR. All the bar plots represent means ± SD. *p < 0.05, **p < 0.01,compared with ODDD-mut. (f) Western blot for HIF-1α and HIF-2α in ODDD–transfected A7r5 cells after 24 hours. β-actin served as loading control. The full-length blots with these antibodies were presented in supplementary Figure S3. All gels have been run simultaneously under the same experimental conditions. (g) HIF-1α dynamic binding on the HRE site (−797 to −793) on the miR-322 promoter. ChIP assays were performed with indicated antibodies in the absence or presence of CoCl2 (left), or transfected with ODDD-wt or ODDD-mut (right) as described under Methods. Representative gel with input lanes showing products after PCR amplification and before immunoprecipitation.
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f3: Hypoxia induces miR-322 promoter activity in a HIF-1α-dependent manner.(a) Schematic diagram of cloned rat putative promoter region of miR-322, stretching from upstream 1000 bp. The position of the putative HRE matching the core sequence (A/G)CGTG is labeled in box, between two functional CACAG elements (P1k). A mutant promoter was constructed with the sequence as shown below (P1k-m). (b) Luciferase reporter assays of miR-322 promoter activity with both wild (pGL4-P1k) and mutant constructs (pGl4-P1k-m) in the presence or absence of CoCl2 (200μM) (left panel); Under these conditions, the protein levels of HIF-1α and HIF-2α were stabilized after CoCl2 treatment in the cells as determined by western blot analysis and normalized to β-actin levels (right panel). (c) miR-322 promoter activity assessed by luciferase reporter assay after CoCl2 treatment was diminished by HIF-1α knockdown. Cells were transduced with shRNA targeting HIF-1α (shHIF-1α) or HIF-2α (shHIF-2α) and miR-322 promoter activity was determined in the luciferase reporter assay after transfection of wild type and mutant constructs and CoCl2 treatment (left panel) as described under Methods; Control (sh-Con) or HIF-1α/-2α shRNA-transfected cells were harvested for protein analysis by western blot to determine knockdown specificity (right panel); *p < 0.05 compared with sh-Con. (d & e) A7r5 cells were transfected with recombinant adenoviruses expressing oxygen-dependent degradation domains (ODDD-wt) or mutated ODDD (ODDD-mut) under normoxic conditions. The influence on the miR-322 promoter reporter activity (d) and the endogenous expression levels (e) of miR-322 were determined by real-time PCR. All the bar plots represent means ± SD. *p < 0.05, **p < 0.01,compared with ODDD-mut. (f) Western blot for HIF-1α and HIF-2α in ODDD–transfected A7r5 cells after 24 hours. β-actin served as loading control. The full-length blots with these antibodies were presented in supplementary Figure S3. All gels have been run simultaneously under the same experimental conditions. (g) HIF-1α dynamic binding on the HRE site (−797 to −793) on the miR-322 promoter. ChIP assays were performed with indicated antibodies in the absence or presence of CoCl2 (left), or transfected with ODDD-wt or ODDD-mut (right) as described under Methods. Representative gel with input lanes showing products after PCR amplification and before immunoprecipitation.

Mentions: Since HIF-1 and -2 mediate most of the cellular responses to hypoxia, we hypothesized that the hypoxia-induced increase in expression of miR-322 is HIF-related. We searched for hypoxia-responsive elements (HRE) in the putative promoter sequence, 1000 bp upstream from the rat pre-miR-322. One potential HIF-binding site was identified within this region, containing a HRE core sequence (A/G)CGTG stretching from −797 bp to −793 bp. Two functional HRE elements (CACAG) are located 138 bp upstream and 55 bp downstream, respectively (Fig. 3a). We constructed both wild and HRE-mutant promoter-driven luciferase reporter plasmids (pGL4-P1k and pGL4-P1km, respectively) and transfected A7r5 cells to determine whether HIF-1/2α influences the miR-322 promoter activity. Cells were treated with cobalt chloride (CoCl2, 200 μM, 24 h), a hypoxia-mimetic compound that has been shown to stabilize HIF protein in PASMC21. The results show that the native promoter but not the mutant promoter of miR-322 was activated by CoCl2 (Fig. 3b). Moreover, CoCl2-induced expression of HIF-1α and -2α in A7r5 cells was confirmed by western blotting (Fig. 3b). We further tested the roles of HIF-1α and -2α in regulation of miR-322 promoter activity using a gene knockdown approach. As shown in Fig. 3c, shRNA targeting of HIF-1α almost completely abolished the activation of miR-322 promoter reporter induced by CoCl2, but HIF-2α silencing had no effect. And both shRNAs had no effect on the mutant promoter. Western blot analysis confirmed decreased expression of HIF-1α or HIF-2α after CoCl2-treatment with specific shRNA (Fig. 3c). This indicates that the HRE site within the miR-322 promoter is required for HIF-1α-induced upregulation in hypoxia.


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 induces miR-322 promoter activity in a HIF-1α-dependent manner.(a) Schematic diagram of cloned rat putative promoter region of miR-322, stretching from upstream 1000 bp. The position of the putative HRE matching the core sequence (A/G)CGTG is labeled in box, between two functional CACAG elements (P1k). A mutant promoter was constructed with the sequence as shown below (P1k-m). (b) Luciferase reporter assays of miR-322 promoter activity with both wild (pGL4-P1k) and mutant constructs (pGl4-P1k-m) in the presence or absence of CoCl2 (200μM) (left panel); Under these conditions, the protein levels of HIF-1α and HIF-2α were stabilized after CoCl2 treatment in the cells as determined by western blot analysis and normalized to β-actin levels (right panel). (c) miR-322 promoter activity assessed by luciferase reporter assay after CoCl2 treatment was diminished by HIF-1α knockdown. Cells were transduced with shRNA targeting HIF-1α (shHIF-1α) or HIF-2α (shHIF-2α) and miR-322 promoter activity was determined in the luciferase reporter assay after transfection of wild type and mutant constructs and CoCl2 treatment (left panel) as described under Methods; Control (sh-Con) or HIF-1α/-2α shRNA-transfected cells were harvested for protein analysis by western blot to determine knockdown specificity (right panel); *p < 0.05 compared with sh-Con. (d & e) A7r5 cells were transfected with recombinant adenoviruses expressing oxygen-dependent degradation domains (ODDD-wt) or mutated ODDD (ODDD-mut) under normoxic conditions. The influence on the miR-322 promoter reporter activity (d) and the endogenous expression levels (e) of miR-322 were determined by real-time PCR. All the bar plots represent means ± SD. *p < 0.05, **p < 0.01,compared with ODDD-mut. (f) Western blot for HIF-1α and HIF-2α in ODDD–transfected A7r5 cells after 24 hours. β-actin served as loading control. The full-length blots with these antibodies were presented in supplementary Figure S3. All gels have been run simultaneously under the same experimental conditions. (g) HIF-1α dynamic binding on the HRE site (−797 to −793) on the miR-322 promoter. ChIP assays were performed with indicated antibodies in the absence or presence of CoCl2 (left), or transfected with ODDD-wt or ODDD-mut (right) as described under Methods. Representative gel with input lanes showing products after PCR amplification and before immunoprecipitation.
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f3: Hypoxia induces miR-322 promoter activity in a HIF-1α-dependent manner.(a) Schematic diagram of cloned rat putative promoter region of miR-322, stretching from upstream 1000 bp. The position of the putative HRE matching the core sequence (A/G)CGTG is labeled in box, between two functional CACAG elements (P1k). A mutant promoter was constructed with the sequence as shown below (P1k-m). (b) Luciferase reporter assays of miR-322 promoter activity with both wild (pGL4-P1k) and mutant constructs (pGl4-P1k-m) in the presence or absence of CoCl2 (200μM) (left panel); Under these conditions, the protein levels of HIF-1α and HIF-2α were stabilized after CoCl2 treatment in the cells as determined by western blot analysis and normalized to β-actin levels (right panel). (c) miR-322 promoter activity assessed by luciferase reporter assay after CoCl2 treatment was diminished by HIF-1α knockdown. Cells were transduced with shRNA targeting HIF-1α (shHIF-1α) or HIF-2α (shHIF-2α) and miR-322 promoter activity was determined in the luciferase reporter assay after transfection of wild type and mutant constructs and CoCl2 treatment (left panel) as described under Methods; Control (sh-Con) or HIF-1α/-2α shRNA-transfected cells were harvested for protein analysis by western blot to determine knockdown specificity (right panel); *p < 0.05 compared with sh-Con. (d & e) A7r5 cells were transfected with recombinant adenoviruses expressing oxygen-dependent degradation domains (ODDD-wt) or mutated ODDD (ODDD-mut) under normoxic conditions. The influence on the miR-322 promoter reporter activity (d) and the endogenous expression levels (e) of miR-322 were determined by real-time PCR. All the bar plots represent means ± SD. *p < 0.05, **p < 0.01,compared with ODDD-mut. (f) Western blot for HIF-1α and HIF-2α in ODDD–transfected A7r5 cells after 24 hours. β-actin served as loading control. The full-length blots with these antibodies were presented in supplementary Figure S3. All gels have been run simultaneously under the same experimental conditions. (g) HIF-1α dynamic binding on the HRE site (−797 to −793) on the miR-322 promoter. ChIP assays were performed with indicated antibodies in the absence or presence of CoCl2 (left), or transfected with ODDD-wt or ODDD-mut (right) as described under Methods. Representative gel with input lanes showing products after PCR amplification and before immunoprecipitation.
Mentions: Since HIF-1 and -2 mediate most of the cellular responses to hypoxia, we hypothesized that the hypoxia-induced increase in expression of miR-322 is HIF-related. We searched for hypoxia-responsive elements (HRE) in the putative promoter sequence, 1000 bp upstream from the rat pre-miR-322. One potential HIF-binding site was identified within this region, containing a HRE core sequence (A/G)CGTG stretching from −797 bp to −793 bp. Two functional HRE elements (CACAG) are located 138 bp upstream and 55 bp downstream, respectively (Fig. 3a). We constructed both wild and HRE-mutant promoter-driven luciferase reporter plasmids (pGL4-P1k and pGL4-P1km, respectively) and transfected A7r5 cells to determine whether HIF-1/2α influences the miR-322 promoter activity. Cells were treated with cobalt chloride (CoCl2, 200 μM, 24 h), a hypoxia-mimetic compound that has been shown to stabilize HIF protein in PASMC21. The results show that the native promoter but not the mutant promoter of miR-322 was activated by CoCl2 (Fig. 3b). Moreover, CoCl2-induced expression of HIF-1α and -2α in A7r5 cells was confirmed by western blotting (Fig. 3b). We further tested the roles of HIF-1α and -2α in regulation of miR-322 promoter activity using a gene knockdown approach. As shown in Fig. 3c, shRNA targeting of HIF-1α almost completely abolished the activation of miR-322 promoter reporter induced by CoCl2, but HIF-2α silencing had no effect. And both shRNAs had no effect on the mutant promoter. Western blot analysis confirmed decreased expression of HIF-1α or HIF-2α after CoCl2-treatment with specific shRNA (Fig. 3c). This indicates that the HRE site within the miR-322 promoter is required for HIF-1α-induced upregulation in hypoxia.

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