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MicroRNA-107 contributes to post-stroke angiogenesis by targeting Dicer-1.

Li Y, Mao L, Gao Y, Baral S, Zhou Y, Hu B - Sci Rep (2015)

Bottom Line: We found that miR-107 was strongly expressed in ischemic boundary zone (IBZ) after permanent middle cerebral artery occlusion (pMCAO) in rats and inhibition of miR-107 could reduce capillary density in the IBZ after stroke.This resulted in translational desupression of VEGF (vascular endothelial growth factor) mRNA, thereby increasing expression of endothelial cell-derived VEGF (VEGF165/VEGF164), leading to angiogenesis after stroke.This process might be a protective mechanism for ischemia-induced cerebral injury and miR-107 might be used as a novel tool in stroke treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.

ABSTRACT
Previous studies have suggested that microRNA-107 (miR-107) regulates cell migration in tumor and promotes Hypoxia Inducible Factor 1α (HIF1α) regulated angiogenesis under hypoxia. We found that miR-107 was strongly expressed in ischemic boundary zone (IBZ) after permanent middle cerebral artery occlusion (pMCAO) in rats and inhibition of miR-107 could reduce capillary density in the IBZ after stroke. Such finding led us to hypothesize that miR-107 might regulate post-stroke angiogenesis and therefore serve as a therapeutic target. We also found that antagomir-107, a synthetic miR-107 inhibitor, decreased the number of capillaries in IBZ and increased overall infarct volume after pMCAO in rats. We demonstrated that miR-107 could directly down-regulate Dicer-1, a gene that encodes an enzyme essential for processing microRNA (miRNA) precursors. This resulted in translational desupression of VEGF (vascular endothelial growth factor) mRNA, thereby increasing expression of endothelial cell-derived VEGF (VEGF165/VEGF164), leading to angiogenesis after stroke. This process might be a protective mechanism for ischemia-induced cerebral injury and miR-107 might be used as a novel tool in stroke treatment.

No MeSH data available.


Related in: MedlinePlus

Dicer-1 is direct target of miR-107.(A) Heat-map of miRNAs that were differentially expressed at least 1.5-fold between RBMECs transfected with miR-107 and RBMECs transfected with scramble probe. (B)The putative targets genes of miR-107. (C) The expression of Dicer-1 in HUVECs, RBMECs and astrocytes under OGD for 12 h. *P < 0.05, vs. control. (D) The mRNA levels of Dicer-1 in rat IBZ on 3rd and 7th days after pMCAO were detected by qRT-PCR. *P < 0.05, vs. control. (E) Representative picture and analysis diagram show the protein levels of Dicer-1 in HUVECs as determined by Western blotting. *P < 0.05, vs. Control, ΔP < 0.05, vs. sham group. (F,G) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. (H,I) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with anti-miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. *P < 0.05, vs. control. (J) After pMCAO, rats were injected with antagomir control (antagomir-ctl) or antagomir-107 and divided into 3 group: pMCAO (control), negative control (antagomir-ctl) and antagomir-107. The mRNA level of Dicer-1 in the three groups were detected by using qRT-PCR on 7th day after pMCAO. *P < 0.05, vs. control, #P < 0.05, vs. antagomir-ctl group. (K) Luciferase activity of reporter constructs carrying luciferase cDNA and parts of the 3′UTR of target mRNAs. Left, Localization of binding sites for human miR-107 (hsa-miR-107) in the 3′UTR of target mRNA and their evolutionary conservation. Right, Quantitative analysis of 3′UTR luciferase activities. HUVECs were transfected with miR-107 or anti-miR-107 (in parallel with control molecules miR-ctl and anti-miR-ctl). Data are from 3 independent experiments performed in triplicate. Data are presented as mean ± SD. *P < 0.05, vs. control.
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f4: Dicer-1 is direct target of miR-107.(A) Heat-map of miRNAs that were differentially expressed at least 1.5-fold between RBMECs transfected with miR-107 and RBMECs transfected with scramble probe. (B)The putative targets genes of miR-107. (C) The expression of Dicer-1 in HUVECs, RBMECs and astrocytes under OGD for 12 h. *P < 0.05, vs. control. (D) The mRNA levels of Dicer-1 in rat IBZ on 3rd and 7th days after pMCAO were detected by qRT-PCR. *P < 0.05, vs. control. (E) Representative picture and analysis diagram show the protein levels of Dicer-1 in HUVECs as determined by Western blotting. *P < 0.05, vs. Control, ΔP < 0.05, vs. sham group. (F,G) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. (H,I) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with anti-miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. *P < 0.05, vs. control. (J) After pMCAO, rats were injected with antagomir control (antagomir-ctl) or antagomir-107 and divided into 3 group: pMCAO (control), negative control (antagomir-ctl) and antagomir-107. The mRNA level of Dicer-1 in the three groups were detected by using qRT-PCR on 7th day after pMCAO. *P < 0.05, vs. control, #P < 0.05, vs. antagomir-ctl group. (K) Luciferase activity of reporter constructs carrying luciferase cDNA and parts of the 3′UTR of target mRNAs. Left, Localization of binding sites for human miR-107 (hsa-miR-107) in the 3′UTR of target mRNA and their evolutionary conservation. Right, Quantitative analysis of 3′UTR luciferase activities. HUVECs were transfected with miR-107 or anti-miR-107 (in parallel with control molecules miR-ctl and anti-miR-ctl). Data are from 3 independent experiments performed in triplicate. Data are presented as mean ± SD. *P < 0.05, vs. control.

Mentions: To explore the molecular mechanisms involved in regulation of VEGF165/VEGF164 expression by miR-107 in endothelial cells, we examined the potential target of miR-107 using gene-chip assay (Fig. 4A), and found 96 target genes (Fig. 4B). Notably, we found that transcription factor Dicer-1 possesses a specific binding site for miR-107 by using miRanda12, RNAhybrid13 and TargetScan14. When subjected to OGD for 12 h, which results in upregulation of miR-107, Dicer-1 dropped in both HUVECs and RBMECs whereas there was no effect in astrocytes (Fig. 4C, P < 0.05). Similarly, the mRNA level of Dicer-1 was significantly reduced in IBZ on day 3 and day 7 after pMCAO (Fig. 4D, P < 0.05). To validate that Dicer-1 was the target of miR-107, HUVECs and RBMECs were transfected with miR-107. Following transfection, Dicer-1 protein expression was decreased under normoxia and its expression at mRNA level was also inhibited (Fig. 4E–I, P < 0.05). On the other hand, transfection with anti-miR-107 in HUVECs and RBMECs increased Dicer-1 expression (Fig. 4E–I, P < 0.05) under OGD. In vivo, after miR-107 expression was down-regulated by lateral ventricular injection of antagomir-107 in rats brain, Dicer-1 expression was increased on day 7 after pMCAO (Fig. 4J, P < 0.05). Then, we integrated the respective 3′UTR regions of Dicer-1 into a luciferase reporter gene and determined the luciferase activity in HUVECs transfected with synthetic miR-107 precursors. We found that miR-107 significantly inhibited luciferase activity which is a measure of transcriptional activity (Fig. 4K, P < 0.05). These findings indicate that Dicer-1 was a direct target of miR-107.


MicroRNA-107 contributes to post-stroke angiogenesis by targeting Dicer-1.

Li Y, Mao L, Gao Y, Baral S, Zhou Y, Hu B - Sci Rep (2015)

Dicer-1 is direct target of miR-107.(A) Heat-map of miRNAs that were differentially expressed at least 1.5-fold between RBMECs transfected with miR-107 and RBMECs transfected with scramble probe. (B)The putative targets genes of miR-107. (C) The expression of Dicer-1 in HUVECs, RBMECs and astrocytes under OGD for 12 h. *P < 0.05, vs. control. (D) The mRNA levels of Dicer-1 in rat IBZ on 3rd and 7th days after pMCAO were detected by qRT-PCR. *P < 0.05, vs. control. (E) Representative picture and analysis diagram show the protein levels of Dicer-1 in HUVECs as determined by Western blotting. *P < 0.05, vs. Control, ΔP < 0.05, vs. sham group. (F,G) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. (H,I) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with anti-miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. *P < 0.05, vs. control. (J) After pMCAO, rats were injected with antagomir control (antagomir-ctl) or antagomir-107 and divided into 3 group: pMCAO (control), negative control (antagomir-ctl) and antagomir-107. The mRNA level of Dicer-1 in the three groups were detected by using qRT-PCR on 7th day after pMCAO. *P < 0.05, vs. control, #P < 0.05, vs. antagomir-ctl group. (K) Luciferase activity of reporter constructs carrying luciferase cDNA and parts of the 3′UTR of target mRNAs. Left, Localization of binding sites for human miR-107 (hsa-miR-107) in the 3′UTR of target mRNA and their evolutionary conservation. Right, Quantitative analysis of 3′UTR luciferase activities. HUVECs were transfected with miR-107 or anti-miR-107 (in parallel with control molecules miR-ctl and anti-miR-ctl). Data are from 3 independent experiments performed in triplicate. Data are presented as mean ± SD. *P < 0.05, vs. control.
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f4: Dicer-1 is direct target of miR-107.(A) Heat-map of miRNAs that were differentially expressed at least 1.5-fold between RBMECs transfected with miR-107 and RBMECs transfected with scramble probe. (B)The putative targets genes of miR-107. (C) The expression of Dicer-1 in HUVECs, RBMECs and astrocytes under OGD for 12 h. *P < 0.05, vs. control. (D) The mRNA levels of Dicer-1 in rat IBZ on 3rd and 7th days after pMCAO were detected by qRT-PCR. *P < 0.05, vs. control. (E) Representative picture and analysis diagram show the protein levels of Dicer-1 in HUVECs as determined by Western blotting. *P < 0.05, vs. Control, ΔP < 0.05, vs. sham group. (F,G) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. (H,I) miRNA levels of miR-107 and Dicer-1 were detected in HUVECs or RBMECs transfected with anti-miR-107 or scr-miR and in un-transfected HUVECs (control) by using qRT-PCR. *P < 0.05, vs. control. (J) After pMCAO, rats were injected with antagomir control (antagomir-ctl) or antagomir-107 and divided into 3 group: pMCAO (control), negative control (antagomir-ctl) and antagomir-107. The mRNA level of Dicer-1 in the three groups were detected by using qRT-PCR on 7th day after pMCAO. *P < 0.05, vs. control, #P < 0.05, vs. antagomir-ctl group. (K) Luciferase activity of reporter constructs carrying luciferase cDNA and parts of the 3′UTR of target mRNAs. Left, Localization of binding sites for human miR-107 (hsa-miR-107) in the 3′UTR of target mRNA and their evolutionary conservation. Right, Quantitative analysis of 3′UTR luciferase activities. HUVECs were transfected with miR-107 or anti-miR-107 (in parallel with control molecules miR-ctl and anti-miR-ctl). Data are from 3 independent experiments performed in triplicate. Data are presented as mean ± SD. *P < 0.05, vs. control.
Mentions: To explore the molecular mechanisms involved in regulation of VEGF165/VEGF164 expression by miR-107 in endothelial cells, we examined the potential target of miR-107 using gene-chip assay (Fig. 4A), and found 96 target genes (Fig. 4B). Notably, we found that transcription factor Dicer-1 possesses a specific binding site for miR-107 by using miRanda12, RNAhybrid13 and TargetScan14. When subjected to OGD for 12 h, which results in upregulation of miR-107, Dicer-1 dropped in both HUVECs and RBMECs whereas there was no effect in astrocytes (Fig. 4C, P < 0.05). Similarly, the mRNA level of Dicer-1 was significantly reduced in IBZ on day 3 and day 7 after pMCAO (Fig. 4D, P < 0.05). To validate that Dicer-1 was the target of miR-107, HUVECs and RBMECs were transfected with miR-107. Following transfection, Dicer-1 protein expression was decreased under normoxia and its expression at mRNA level was also inhibited (Fig. 4E–I, P < 0.05). On the other hand, transfection with anti-miR-107 in HUVECs and RBMECs increased Dicer-1 expression (Fig. 4E–I, P < 0.05) under OGD. In vivo, after miR-107 expression was down-regulated by lateral ventricular injection of antagomir-107 in rats brain, Dicer-1 expression was increased on day 7 after pMCAO (Fig. 4J, P < 0.05). Then, we integrated the respective 3′UTR regions of Dicer-1 into a luciferase reporter gene and determined the luciferase activity in HUVECs transfected with synthetic miR-107 precursors. We found that miR-107 significantly inhibited luciferase activity which is a measure of transcriptional activity (Fig. 4K, P < 0.05). These findings indicate that Dicer-1 was a direct target of miR-107.

Bottom Line: We found that miR-107 was strongly expressed in ischemic boundary zone (IBZ) after permanent middle cerebral artery occlusion (pMCAO) in rats and inhibition of miR-107 could reduce capillary density in the IBZ after stroke.This resulted in translational desupression of VEGF (vascular endothelial growth factor) mRNA, thereby increasing expression of endothelial cell-derived VEGF (VEGF165/VEGF164), leading to angiogenesis after stroke.This process might be a protective mechanism for ischemia-induced cerebral injury and miR-107 might be used as a novel tool in stroke treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.

ABSTRACT
Previous studies have suggested that microRNA-107 (miR-107) regulates cell migration in tumor and promotes Hypoxia Inducible Factor 1α (HIF1α) regulated angiogenesis under hypoxia. We found that miR-107 was strongly expressed in ischemic boundary zone (IBZ) after permanent middle cerebral artery occlusion (pMCAO) in rats and inhibition of miR-107 could reduce capillary density in the IBZ after stroke. Such finding led us to hypothesize that miR-107 might regulate post-stroke angiogenesis and therefore serve as a therapeutic target. We also found that antagomir-107, a synthetic miR-107 inhibitor, decreased the number of capillaries in IBZ and increased overall infarct volume after pMCAO in rats. We demonstrated that miR-107 could directly down-regulate Dicer-1, a gene that encodes an enzyme essential for processing microRNA (miRNA) precursors. This resulted in translational desupression of VEGF (vascular endothelial growth factor) mRNA, thereby increasing expression of endothelial cell-derived VEGF (VEGF165/VEGF164), leading to angiogenesis after stroke. This process might be a protective mechanism for ischemia-induced cerebral injury and miR-107 might be used as a novel tool in stroke treatment.

No MeSH data available.


Related in: MedlinePlus