MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis

Abstract

Expression of apoptotic protease activating factor-1 (Apaf-1) gradually decreases during brain development, and this decrease is likely responsible for the decreased sensitivity of brain tissue to apoptosis. However, the mechanism by which Apaf-1 expression is decreased remains elusive. In the present study, we found that four microRNAs (miR-23a/b and miR-27a/b) of miR-23a-27a-24 and miR-23b-27b-24 clusters play key roles in modulating the expression of Apaf-1. First, we found that miR-23a/b and miR-27a/b suppressed the expression of Apaf-1 in vitro. Interestingly, the expression of the miR-23-27-24 clusters in the mouse cortex gradually increased in a manner that was inversely correlated with the pattern of Apaf-1 expression. Second, hypoxic injuries during fetal distress caused reduced expression of the miR-23b and miR-27b that was inversely correlated with an elevation of Apaf-1 expression during neuronal apoptosis. Third, we made neuronal-specific transgenic mice and found that overexpressing the miR-23b and miR-27b in mouse neurons inhibited the neuronal apoptosis induced by intrauterine hypoxia. In conclusion, our results demonstrate, in central neural system, that miR-23a/b and miR-27a/b are endogenous inhibitory factors of Apaf-1 expression and regulate the sensitivity of neurons to apoptosis. Our findings may also have implications for the potential target role of microRNAs in the treatment of neuronal apoptosis-related diseases.

Figure 1

Decreased Apaf-1 gene expression during brain development. (a) Representative western blots of Apaf-1 in cortical samples at different developmental stages (E18, P7, P14, and adult). Each lane was loaded with equal amounts of total protein from the mixed samples of five individuals (n=5, unpaired t-test, *P<0.05, **P<0.01, and ***P<0.001). (b) Semiquantitative RT-PCR detection of Apaf-1 mRNA in the total RNA from cerebral cortices at different developmental stages. Quantitative RT-PCR detection of Apaf-1 mRNA (n=5, unpaired t-test, NS, not significant, **P<0.01). Error bars represent the S.E.M. (c) IHC stain of Apaf-1 in E18 and adult brain tissues. Scale bar represents 250 μm; scale bar (insert) represents 50 μm

Figure 2

The expression of miR-23-27 clusters increases during brain development. (a) Schematic of the Apaf-1 3′UTR indicating the locations of the miR-23 and miR-27 target sites that are conserved in vertebrates. The Apaf-1 3′UTR contains evolutionarily well-conserved sequences matched for the miR-23 and miR-27 families that were predicted by computer-aided algorithms. The free energies (mfes) of microRNA bindings were calculated by RNAHybrid software (BiBiServ, Bielefeld, Germany). (b) Quantitative RT-PCR detection of miR-23a, miR-23b, miR-27a, miR-27b, and miR-24 in cerebral cortex samples at different developmental stages (n=5, one-way ANOVA with Newman–Keuls multiple comparison test, *P<0.05, **P<0.01, and ***P<0.001). (c) Dot blot analyses of miR-23b and U6 snRNA in total RNA of the cerebral cortex. Each lane was loaded with equal amounts of total RNA extracted from the mixed samples of five individuals. (d) Expression of miR-23b in cortices of E18 and adult brain tissues detected by in situ hybridization. Scale bar represents 250 μm; scale bar (insert) represents 200 μm

Figure 3

The miR-23a/b and miR-27a/b suppress Apaf-1 protein expression. (a) HEK-293T cells were transfected with luciferase reporter plasmids carrying wild-type (WT) or mutant (Mut) Apaf-1 3′-UTRs. The luciferase activities were examined in the presence of pre-miR-23a/b (left) or pre-miR-27a/b (right) (n=5, unpaired t-test, NS, not significant, **P<0.01 and ***P<0.001). (b) Apaf-1 protein level in HEK-293T cells 48 h after transfection with pre-miR-23a/b or pre-miR-27a/b (n=5, unpaired t-test, *P<0.05 and **P<0.01)

Figure 4

Apaf-1 protein levels are elevated and miR-23b-27b cluster is decreased in cortices of E19.5 pups after hypoxia. (a) Coronal brain sections of E19.5 mice were stained with a cleaved caspase-3 (Cleaved-Casp3) antibody or with TUNEL staining kit. More positively stained neurons were observed in the hypoxia group (n=3) compared with the normoxia control group (n=3). Scale bar represents 100 μm. (b) Representative western blot images for Apaf-1 in the cerebral cortex under conditions of normoxia or hypoxia for 6 h followed by different recovery times (no recovery, recovery 6 h, or recovery 18 h). Relative fold changes of Apaf-1 protein level were quantified by densitometry (n=5, unpaired t-test, *P<0.05 and **P<0.01). (c) Quantitative RT-PCR detection of miR-23b and miR-27b expression in the cerebral cortices of different groups (n=5, unpaired t-test, *P<0.05, **P<0.01, and ***P<0.001). (d) Representative western blot images for Apaf-1 in primary cortical neurons under conditions of normoxia or hypoxia for 6, 12, and 24 h (n=3, unpaired t-test, **P<0.01). (e) Quantitative RT-PCR detection of miR-23b and miR-27b expression in primary cortical neurons under the conditions of normoxia or hypoxia for 6, 12, and 24 h (n=6, unpaired t-test, *P<0.05 and **P<0.01)

Figure 5

Overexpression of miR-23b or miR-27b represses Apaf-1 protein levels in primary cortical neurons and attenuates neuronal apoptosis caused by hypoxia. (a) Western blot analysis of Apaf-1 in primary cortical neurons 48 h after transfection with Apaf-1 siRNA. Relative amounts of Apaf-1 protein were quantified by densitometry (n=3, unpaired t-test, **P<0.01). (b) Immunofluorescence staining of Cleaved-Casp3 or TUNEL staining. Neurons were stained with Cleaved-Casp3 antibody or with TUNEL stain kit after hypoxia treatment (24 h) at DIV5. Statistical analysis of the percentage of Cleaved-Casp3-positive neurons or TUNEL-positive neurons to DAPI-stained cells (cell numbers=400–500, unpaired t-test, ***P<0.001). (c) Western blot analysis of Apaf-1 in primary cortical neurons 48 h after transfection with pre-miR-23b or pre-miR-27b. Relative amounts of Apaf-1 protein were quantified by densitometry (n=3, unpaired t-test, **P<0.01). (d) Immunofluorescence staining of Cleaved-Casp3 or TUNEL staining (cell numbers=600–700, unpaired t-test, **P<0.01). Scale bar represents 100 μm

Figure 6

Overexpression of Apaf-1 re-activates the sensitivity of neurons overexpressing miR-23b/27b to apoptosis induced by hypoxia. (a) Western blot analysis of Apaf-1 in primary cortical neurons 48 h after co-transfection with plasmids (pCMV6 or pCMV6-Apaf-1) and miRNAs (pre-miR-23b or pre-miR-27b). Relative amounts of Apaf-1 protein were quantified by densitometry (n=3, unpaired t-test, ***P<0.001). (b) Immunofluorescence staining of Cleaved-Casp3 or TUNEL staining. Cultured cortical neurons co-transfected with plasmids (pCMV6 or pCMV6-Apaf-1) and miRNAs (pre-miR-23b or pre-miR-27b) were stained with Cleaved-Casp3 antibody or with TUNEL staining kit after hypoxia treatment (24 h) at DIV5. (c) Statistical analysis of the percentage of Cleaved-Casp3-positive neurons or TUNEL-positive neurons to DAPI-stained cells (cell numbers=700–800, unpaired t-test, **P<0.01 and ***P<0.001). Scale bar represents 100 μm

Figure 7

The miR-23b-27b transgenic mice show more resistance to hypoxia-induced apoptosis. (a) A cartoon of generation of the miR-23b-27b cluster transgenic mice. The Tubb3 gene promoter was employed because of its high expression capability and specificity in neurons in the early embryonic stage. Quantitative RT-PCR detection of miR-23b and miR-27b in primary cortical neurons 24 h after transfection with the constructed plasmid. (b) Quantitative RT-PCR detection of miR-23b-27b (top) and western blot analysis of Apaf-1 protein levels (bottom) in cultured primary cortical neurons from wild-type (WT) mice and transgenic (TG) mice (n=3, unpaired t-test, *P<0.05 and **P<0.01). (c) Cultured cortical neurons isolated from E15.5 pups were stained with Cleaved-Casp3 antibody after hypoxia treatment (24 h) at DIV5 and counterstained with DAPI. Statistical analysis of the percentage of Cleaved-Casp3-positive neurons to DAPI-stained cells (cell numbers=400–500, unpaired t-test, ***P<0.001). Scale bar represents 100 μm. (d) Cultured cortical neurons were stained with TUNEL staining kit after hypoxia treatment. Statistical analysis of the percentage of TUNEL-positive neurons to DAPI-stained cells (cell numbers=400–500, unpaired t-test, **P<0.01). (e) Quantitative RT-PCR detection of miR-23b-27b (top) and western blot analysis of Apaf-1 protein levels (bottom) in the cerebral cortices from E19.5 mice (TG mice n=4 and WT mice n=6, unpaired t-test, *P<0.05 and **P<0.01). (f) Coronal sections of WT and transgenic neocortex at E19.5 were stained with Cleaved-Casp3 antibody after 6 h of hypoxia followed by a 6-h recovery or with TUNEL staining kit after a 18-h recovery. Scale bar represents 100 μm