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Up-Regulation of miR-9-5p Inhibits Hypoxia-Ischemia Brain Damage Through the DDIT4-Mediated Autophagy Pathways in Neonatal Mice

Gai, Chengcheng, Xing, Xiaohui, Song, Yan, Zhao, Yijing, Jiang, Zige, Cheng, Yahong, Xiao, Yilei, Wang, Zhen·2023·Drug Design, Development and Therapy
DOI 10.2147/dddt.s393362PMC PMC10128084
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Figure 1 MiR-9-5p expression was decreased post HI and overexpression of miR-9-5p could ameliorate brain injury. (A) The expression levels of miR-9-5p mRNA were detected by qRT-PCR at 0 to 7 days after HI injury. (B) The expression levels of miR-9-5p mRNA were measured after HI with miR-9-5p mimics treatment. (C and D) Representative pictures of the brain and TTC staining of the brain. (E) Quantification of brain infarct area in each group. The data were presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 according to One-way ANOVA with Dunnett corrections in (A); ***p < 0.001 according to t-test in (B); **p < 0.01, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (E).
Figure 1 MiR-9-5p expression was decreased post HI and overexpression of miR-9-5p could ameliorate brain injury. (A) The expression levels of miR-9-5p mRNA were detected by qRT-PCR at 0 to 7 days after HI injury. (B) The expression levels of miR-9-5p mRNA were measured after HI with miR-9-5p mimics treatment. (C and D) Representative pictures of the brain and TTC staining of the brain. (E) Quantification of brain infarct area in each group. The data were presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 according to One-way ANOVA with Dunnett corrections in (A); ***p < 0.001 according to t-test in (B); **p < 0.01, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (E).CC BY-NC · PubMed Central
Figure 2 MiR-9-5p alleviated apoptosis after HI injury. (A) TUNEL assay to detect apoptotic in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group. Scale bar: 400 μm. Magnified views of boxed regions scale bar: 50 μm. (B and C) The expression levels of cleaved caspase-3 and caspase-3 protein were analyzed by Western blotting. The data were presented as mean ± SD, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (C).
Figure 2 MiR-9-5p alleviated apoptosis after HI injury. (A) TUNEL assay to detect apoptotic in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group. Scale bar: 400 μm. Magnified views of boxed regions scale bar: 50 μm. (B and C) The expression levels of cleaved caspase-3 and caspase-3 protein were analyzed by Western blotting. The data were presented as mean ± SD, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (C).CC BY-NC · PubMed Central
Figure 3 MiR-9-5p inhibited inflammatory activation after HI injury. (A) Representative images of Iba-1 immunohistochemical staining at 2 days following HI insult. Scale bar: 50 μm. Red arrow: microglia/macrophages. Magnified views of black box regions in A shows morphology of microglia/macrophages indicated by red arrow. (B) Quantification of Iba-1+ cells in the Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group. (C) Quantitative analysis of the effect of miR-9-5p on microglia/macrophage activation scores. (D and E) Western blotting was detected protein expression levels of iNOS, IL-1β and Arg-1 at 2 days post HI. The data were presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (B, C and E).
Figure 3 MiR-9-5p inhibited inflammatory activation after HI injury. (A) Representative images of Iba-1 immunohistochemical staining at 2 days following HI insult. Scale bar: 50 μm. Red arrow: microglia/macrophages. Magnified views of black box regions in A shows morphology of microglia/macrophages indicated by red arrow. (B) Quantification of Iba-1+ cells in the Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group. (C) Quantitative analysis of the effect of miR-9-5p on microglia/macrophage activation scores. (D and E) Western blotting was detected protein expression levels of iNOS, IL-1β and Arg-1 at 2 days post HI. The data were presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (B, C and E).CC BY-NC · PubMed Central
Figure 4 The effect of miR-9-5p on brain atrophy at 28 days after HI. (A) Representative images of brain atrophy at 28 days after HI insults. (B) Quantitative analysis of brain atrophy area in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group. The data were presented as mean ± SD, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (B).
Figure 4 The effect of miR-9-5p on brain atrophy at 28 days after HI. (A) Representative images of brain atrophy at 28 days after HI insults. (B) Quantitative analysis of brain atrophy area in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group. The data were presented as mean ± SD, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (B).CC BY-NC · PubMed Central
Figure 5 MiR-9-5p up-regulated HI injury-induced down-regulation of synapse-associated protein expression. (A and B) Western blotting analysis and quantitative measurement were used to monitor protein expression levels of PSD95 and NLG-1 at 2 days post HI. (C and D) Western blotting analysis and quantitative measurement were used to examine protein expression levels of PSD95 and NLG-1 at 28 days post HI. (E) Double immunofluorescence analysis was performed with PSD95 and NeuN in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group at 28 days post HI. Scale bar: 50 μm. The data were presented as mean ± SD, *p < 0.05, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in B, D and E.
Figure 5 MiR-9-5p up-regulated HI injury-induced down-regulation of synapse-associated protein expression. (A and B) Western blotting analysis and quantitative measurement were used to monitor protein expression levels of PSD95 and NLG-1 at 2 days post HI. (C and D) Western blotting analysis and quantitative measurement were used to examine protein expression levels of PSD95 and NLG-1 at 28 days post HI. (E) Double immunofluorescence analysis was performed with PSD95 and NeuN in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group at 28 days post HI. Scale bar: 50 μm. The data were presented as mean ± SD, *p < 0.05, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in B, D and E.CC BY-NC · PubMed Central
Figure 6 Effects of miR-9-5p t on long-term neurological function post HI. (A) The number of crossing, feces, glooming and rearing in the open field test at 28 days post HI. (B) Representative occupancy plots shown the activity track of mice during the Y maze test and the frequency of entries into the novel arm of the Y maze. The data were presented as mean ± SD, *p < 0.05, **p < 0.01 according to One-way ANOVA with Bonferroni corrections in A and B.
Figure 6 Effects of miR-9-5p t on long-term neurological function post HI. (A) The number of crossing, feces, glooming and rearing in the open field test at 28 days post HI. (B) Representative occupancy plots shown the activity track of mice during the Y maze test and the frequency of entries into the novel arm of the Y maze. The data were presented as mean ± SD, *p < 0.05, **p < 0.01 according to One-way ANOVA with Bonferroni corrections in A and B.CC BY-NC · PubMed Central
Figure 7 Downregulation of DDIT4 expression alleviates brain injury. (A) Different downstream targets (BTG2, FURIN, CXCL12, CXCR4 and DDIT4) mRNA levels of miR-9-5p were detected by qRT-PCR. (B and C) Luciferase activity was measured in HEK293T cells transfected with DDIT4-Mut, DDIT4-WT, miR-9-5p NC and miR-9-5p mimics. (D) The DDIT4 mRNA level was detected after transfection with si-DDIT4, si-NC, vector, or DDIT4 overexpression (OE). (E) Representative brain pictures at 2 days following HI in each group treated with si-NC, si-DDIT4, miR-9-5p mimics+vector and miR-9-5p mimics+DDIT4 OE, miR-9-5p NC+vector and miR-9-5p NC+DDIT4 OE. (F) Example of images of TTC staining at 2 days following HI treated with si-NC, si-DDIT4, miR-9-5p mimics+vector and miR-9-5p mimics+DDIT4 OE, miR-9-5p NC+vector and miR-9-5p NC+DDIT4 OE. (G) Quantification of brain infarct area at 2 days following HI treated with si-NC, si-DDIT4, miR-9-5p mimics+vector and miR-9-5p mimics+DDIT4 OE, miR-9-5p NC+DDIT4 vector and miR-9-5p NC+DDIT4 OE. The data were presented as mean ± SD, **p < 0.01, *p < 0.05, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (A, C and G); *p < 0.05, **p < 0.01 according to t-test in (D).
Figure 7 Downregulation of DDIT4 expression alleviates brain injury. (A) Different downstream targets (BTG2, FURIN, CXCL12, CXCR4 and DDIT4) mRNA levels of miR-9-5p were detected by qRT-PCR. (B and C) Luciferase activity was measured in HEK293T cells transfected with DDIT4-Mut, DDIT4-WT, miR-9-5p NC and miR-9-5p mimics. (D) The DDIT4 mRNA level was detected after transfection with si-DDIT4, si-NC, vector, or DDIT4 overexpression (OE). (E) Representative brain pictures at 2 days following HI in each group treated with si-NC, si-DDIT4, miR-9-5p mimics+vector and miR-9-5p mimics+DDIT4 OE, miR-9-5p NC+vector and miR-9-5p NC+DDIT4 OE. (F) Example of images of TTC staining at 2 days following HI treated with si-NC, si-DDIT4, miR-9-5p mimics+vector and miR-9-5p mimics+DDIT4 OE, miR-9-5p NC+vector and miR-9-5p NC+DDIT4 OE. (G) Quantification of brain infarct area at 2 days following HI treated with si-NC, si-DDIT4, miR-9-5p mimics+vector and miR-9-5p mimics+DDIT4 OE, miR-9-5p NC+DDIT4 vector and miR-9-5p NC+DDIT4 OE. The data were presented as mean ± SD, **p < 0.01, *p < 0.05, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (A, C and G); *p < 0.05, **p < 0.01 according to t-test in (D).CC BY-NC · PubMed Central
Figure 8 Excessive autophagy after HI injury was reversed by overexpression of miR-9-5p. (A and B) The protein expression levels of LC3 II/LC3 I and Beclin-1 in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group were determined at 2 days post HI by Western blotting. (C) Immunofluorescence staining of LC3B in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group was determined at 2 days post HI. Scale bar: 50 μm. (D and E) The protein expression levels of LC3 II/LC3 I and Beclin-1 in ipsilateral cortex following treated with si-NC, si-DDIT4, miR-9-5p mimics+vector and miR-9-5p mimics+DDIT4 OE, miR-9-5p NC+DDIT4 and miR-9-5p NC+DDIT4 OE were detected at 2 days post-HI. The data were presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (B and E).
Figure 8 Excessive autophagy after HI injury was reversed by overexpression of miR-9-5p. (A and B) The protein expression levels of LC3 II/LC3 I and Beclin-1 in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group were determined at 2 days post HI by Western blotting. (C) Immunofluorescence staining of LC3B in Sham group, HI group, HI+miR-9-5p NC group and HI+miR-9-5p mimics group was determined at 2 days post HI. Scale bar: 50 μm. (D and E) The protein expression levels of LC3 II/LC3 I and Beclin-1 in ipsilateral cortex following treated with si-NC, si-DDIT4, miR-9-5p mimics+vector and miR-9-5p mimics+DDIT4 OE, miR-9-5p NC+DDIT4 and miR-9-5p NC+DDIT4 OE were detected at 2 days post-HI. The data were presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 according to One-way ANOVA with Bonferroni corrections in (B and E).CC BY-NC · PubMed Central

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Metadata

Authors
Gai, C., Xing, X., Song, Y., Zhao, Y., Jiang, Z., Cheng, Y., Xiao, Y., & Wang, Z.
ORCID
Venue
Drug Design, Development and Therapy
Publisher
Informa UK Limited
Published
2023-04
Volume
Volume 17
Pages
1175-1189
ISSN
1177-8881
DOI
10.2147/dddt.s393362
PMC
PMC10128084
Cited by
14
References
3
Language
en
Licence
creativecommons.org/licenses/by-nc/3.0/ (unspecified)

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