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    MicroRNA-101a-3p mimic ameliorates spinal cord ischemia/reperfusion injury
  • Figure 1|Changes of miR-101a-3p/MYCN expression and neurological function after SCII. 

    The expression of miR-101a-3p and MYCN was evaluated by real-time polymerase chain reaction and western blot at different time points after SCII. As shown in Figure 1A, miR-101a-3p expression was the lowest in the SCII 24 hours group, which was significantly reduced compared with that in the sham group (P < 0.05). Moreover, mRNA and protein expression of MYCN increased over time and peaked in the SCII 24 hours group, and the values were significantly increased compared with those in the sham group (P < 0.05; Figure 1B–D). 
    On the basis of the previous results, we selected 24 hours as the time point for the following experiments. Figure 1E shows the evaluation of hind limb function by the Tarlov scoring system. Tarlov scores were greatly reduced in the SCII group compared with those in the sham group (P < 0.05). The changes in neurological assessment and in miR-101a-3p expression occurred at the same time point after SCII, suggesting a possible relationship between motor dysfunction and miR-101a-3p. 

    Figure 2|MYCN is a target of miR-101a-3p.  

    The TargetScan 7.2 database predicted the possible binding site of miR-101a-3p to be the 3′ untranslated region (UTR) of MYCN (Figure 2A). Intrathecal injection of the synthetic miR-101a-3p mimic increased miR-101a-3p expression and reduced MYCN expression compared with those in the SCII group (P < 0.05; Figure 2B and C). These results indicated a possible relationship between miR-101a-3p and MYCN, which we investigated further via a dual-luciferase reporter assay. The luciferase activity of the WT-3′UTR reporter was significantly reduced by transfection with the miR-101a-3p mimic (P < 0.05), whereas that of the MUT-3′UTR reporter was unchanged (P > 0.05; Figure 2D and E). Additionally, there were no obvious changes through the transfection with NC (P > 0.05; Figure 2E). Therefore, we ascertained that miR-101a-3p directly targets MYCN.


    Figure 3|Immunofluorescence assay of MYCN and cell markers at 24 hours after SCII. 

    Double immunofluorescence labeling of MYCN with NeuN, Iba-1 or GFAP was performed at 24 hours after SCII. As shown in Figure 3A, MYCN fluorescent signal was mainly colocalized with the NeuN fluorescent signal but not with GFAP or Iba-1 at 24 hours after SCII, suggesting that MYCN was at least partially upregulated in neurons. As shown in Figure 3B, the ratio of MYCN/NeuN double-positive cells was upregulated in the SCII group compared with that in the sham group (P < 0.05). Furthermore, the analysis showed that SCII increased the number of MYCN-labeled neurons (P < 0.05, vs. sham; Figure 3C). 

    Figure 4|The role of synthetic miR-101a-3p on hind limb motor function and MYCN fluorescence expression in neurons. 

    As shown in Figure 4A, the Tarlov scoring system was used to assess hind limb motor function after SCII as described in our previous research (Zhang et al., 2021). Tarlov scores in the SCII group and NC group were decreased at 24 hours after SCII compared with those in the sham group (P < 0.05). Tarlov scores in the SCII and NC groups were similar (P > 0.05). Tarlov scores in the miR-101a-3p mimic group were greatly upregulated compared with those in the SCII group (P < 0.05). The data demonstrate that the miR-101a-3p mimic ameliorated hind limb motor dysfunction after SCII.
    As shown in Figure 4B, MYCN was colocalized with NeuN within the groups. The ratio and number of MYCN/NeuN double-positive cells in the miR-101a-3p mimic group were decreased at 24 hours after SCII compared with those in the SCII group (P < 0.05); whereas, there were no obvious differences between the SCII, NC and miR-101a-3p inhibitor groups (P > 0.05). Quantitative analysis in Figure 4C and D showed that SCII increased MYCN expression in neurons, which was then reduced by the miR-101a-3p mimic. These results suggested that the miR-101a-3p mimic might ameliorate SCII impairment by inhibiting MYCN expression in neurons.

    Figure 5|The effect of synthetic miR-101a-3p on cell apoptosis at 24 hours after SCII.

    TUNEL assay was used to detect apoptotic cells in the spinal cord after SCII. As shown in Figure 5A and B, apoptosis rates in the SCII, NC, and miR-101a-3p inhibitor groups were greatly upregulated compared with that in the sham group (P < 0.05), and the apoptosis rate in the miR-101a-3p mimic group was greatly decreased compared with that in the SCII group (P < 0.05). These results demonstrated that the miR-101a-3p mimic ameliorated cell apoptosis after SCII.

    Figure 6|The effect of synthetic miR-101a-3p on the MYCN-p53-caspase-9 signaling pathway and proinflammatory cytokine IL-1β at 24 hours after SCII. 

    The function of the miR-101a-3p mimic on modulating neuroapoptosis through the MYCN-p53-caspase9 signaling pathway was investigated with double immunofluorescence staining and western blot. Western blot showed that the protein expression levels of MYCN, p53 and caspase-9 were greatly increased in the SCII group compared with those in the sham group, and were greatly decreased in the miR-101a-3p mimic group compared with the SCII group (P < 0.05; Figure 6A–D). Proinflammatory cytokine IL-1β showed similar trend (Figure 6A–E). It can be concluded that the miR-101a-3p mimic protected against SCII by inhibiting proinflammatory cytokine IL-1β production.
    As shown in Figure 6F, MYCN fluorescence was colocalized with p53 fluorescence at 24 hours after SCII. The quantitative analysis in Figure 6G and H show that the number of cells double-positive for MYCN and p53 in the miR-101a-3p mimic group were decreased compared with that in the SCII group (P < 0.05), and there were no obvious differences between the SCII, NC and miR-101a-3p inhibitor groups (P > 0.05). It can be concluded that the miR-101a-3p mimic reduced the expression of caspase-9 and thus protected against SCII by inhibiting the MYCN-p53-caspase-9 signaling pathway.


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  • 发布日期: 2022-03-10  浏览: 210
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