MicroRNA-138-5p targets pro-apoptotic factors and favours neural cell survival

Background The central nervous system-enriched microRNA miR-138-5p becomes significantly downregulated after spinal cord injury (SCI). miR-138-5p modulates essential biological processes in the Central Nervous System (CNS). It also overcomes apoptosis by inhibiting the expression of proteins, including the effector CASP3, key in different cell death pathways. Therefore, we hypothesize that miR-138-5p downregulation following SCI underlies the overexpression of apoptotic genes and sensitizes neural cells to noxious stimuli. To confirm this hypothesis, this study aims a) to identify and validate miR-138-5p targets among the pro-apoptotic genes overexpressed following SCI; and b) to confirm that the miR-138-5p is able to modulate cell death in neural cells. Methods We employed computational tools to identify potential pro-apoptotic targets of miR-138-5p. Dysregulation of selected targets after SCI and its relationship to changes in miR-138-5p expression were analysed through qRT-PCR in a rat SCI model. Validation of the regulation of those apoptotic targets was carried out by luciferase reporter, qRT-PCR, and immunoblot assays in cultures of neural cell lines transfected with a mimetic of the microRNA. The functional effects of modifying the expression of miR-138-5p were later examined in cultures of the rat neural cell line C6 employing enzymatic assays to measure the activity of effector CASP3 and CASP7 together with MTT and flow cytometry assays to estimate cell death. Consensus among different algorithms identified 209 potential targets of miR-138-5p. A total of 176 of them become dysregulated after SCI, including proteins basic to apoptosis process such as CASP3 and CASP7, or BAK (Bcl-2 homologous antagonist/killer). Downregulation of miR-138-5p after SCI correlates with the overexpression of these three targets. Cell culture analyses confirm that miR-138-5p targets their 3’UTRs and reduces their expression after microRNA transfection. Transfection of miR-138-5p in C6 cell line results in a reduced effector caspase activity and protects cells from apoptotic stimulation. Our results demonstrate that downregulation of miR-138-5p after SCI can be deleterious to spinal neural cells. A mixture of direct effects mediated by the upregulation of apoptotic targets and indirect effects related to the upregulation of cell cycle proteins can be expected. to cell attenuate of CASP3 and CASP7 and execution of the apoptotic a


Background
Injury to the spinal cord (SCI) triggers a barrage of damaging events that spread cell death to unaffected tissue [1]. Many of these noxious stimuli activate apoptosis -a highly regulated form of programmed cell death-among neural cells of the spinal cord during the following weeks [2][3][4][5]. The different apoptotic pathways converge in the activation of effector caspases, which are responsible for cleaving structural and functional proteins and leading to cell demise [6]. The noxious events triggered by SCI also alter gene expression in neural cells, including the overexpression of pro-apoptotic mediators such as Casp3 (coding for caspase-3 or CASP3 protein), Casp7 (caspase-7 or CASP7 protein), Bak1 (Bcl-2 homologous antagonist/killer or BAK protein), Bax, or Fas [7][8][9]. Post-transcriptional regulatory mechanisms contribute to orchestrate these gene expression changes [10,11]. Among them, studies have shown that microRNA dysregulation accompanies gene expression alterations in the injured spinal cord [12][13][14]. MicroRNAs are a class of highly conserved 20-24 nucleotides long noncoding RNA molecules that function as post-transcriptional regulators of cell state in physiological and pathological conditions [15]. They are highly expressed in the mammalian Central Nervous System (CNS), including the spinal cord, and their dysregulation is associated with neurodegenerative, psychiatric, and developmental diseases [16][17][18].
MicroRNA dysregulation after SCI has been proposed to contribute to spread cell death due to their targeting on key apoptotic genes. For example, SCI-downregulated miR-29b targets the overexpressed Bad, Bim, Noxa, and Puma [19], whereas the decreased level of miR-124 agrees with the overexpression of its target calpain-1 [20], and miR-137 downregulation agrees with calpain-2 and Casp3 overexpression [21].
Microarray and bioinformatics analyses in a rat model of contusive SCI [14] allowed us to identify additional dysregulated microRNAs that may also contribute to activating the apoptotic pathways.
Among them, here we focus on miR-138-5p, a microRNA highly expressed in the CNS that becomes 5 significantly downregulated after SCI [12,14]. This microRNA controls the shape and size of dendritic spines in rat hippocampal neurons during development and thereby influences long-term memory [22].
Following injury, miR-138-5p participates in axon regeneration in peripheral nerve [23] and promotes neuroplasticity through the regulation of vimentin in the damaged spinal cord [24]. Outside the nervous system, miR-138-5p has attracted much attention in cancer research because it overcomes apoptotic cell death by simultaneous inhibition of multiple tumour suppressor pathways and proapoptotic genes, including the Casp3 [25]. Therefore, we hypothesize that, in addition to the previously described effects in the CNS, miR-138-5p downregulation after SCI underlies the overexpression of apoptotic genes and sensitizes neural cells to noxious stimuli. To confirm this hypothesis, this study aims a) to identify and validate miR-138-5p targets among the pro-apoptotic genes overexpressed following SCI; and b) to confirm that the miR-138-5p is able to modulate cell death in neural cells.

Immunoblot assay
Levels of CASP3 and CASP7, and BAK were analysed using standard immunoblot procedures. Briefly, cell lysates were incubated with radioimmunoprecipitation assay lysis buffer (RIPA) containing a complete EDTA-free protease inhibitor cocktail (Roche) and centrifugated (10000xg for 15 min at 4°C).

Measurement of CASP3 and CASP7 activity
Briefly, C6 cells were seeded at a density of 20,000 cells per well in white 96-well plates and 24 hours after transfected with either miR-138-5p mimic or cel-miR-67 negative control microRNA employing Dharmafect 1.0 (Dhamarcon). The next day cell death was induced by treating the cultures with 0.3 μM STS or 127.5 μM ETO overnight. Effector caspases activity was assessed 24 hours after, employing the luminescent Caspase-Glo® 3/7 assay kit (Promega), according to the manufacturer's instructions.
Luminescence was measured in a Infinite M200 plate reader.

Quantification of cell death using Flow Cytometry
For cell death analysis, C6 cells were plated in duplicate for each transfection condition (cel-miR-67 negative control or miR-138-5p microRNAs) in p24 plates and cultured until reaching 50% confluence before transfection. Then, miRNA mimics were administered for transfection using Dharmafect 1.0 and, 24 hours later, cells were exposed to 127. Committee (ref# 63/2010). Animals were divided into two groups: one group without surgery prior to extraction (control) and one injured group (contusion). Animals were sacrificed at 3 and 7 days postinjury (dpi). 5 animals were randomly allocated in each time or control group using the sequence generator utility of the online random number generator random.org (https://www.random.org). SCI surgery followed the methodology described in Yunta and col. [14]. 15 s at 95°C plus 1 min at 60°C using the 2 -ΔΔCt method [36]. Briefly, the difference (ΔCt) between the cycle threshold of the target mRNA or miRNA and their respective endogenous loading controls -U6 small nuclear RNA for miRNAs and 18S ribosomal RNA for mRNA-was estimated together with its associated variance following the standard propagation of error method from Headrick and col. [37].
Then, we compared the ΔCt value from different times post-injury with the ΔCt from non-injured animals (0 dpi) to calculate the ΔΔCt and the correspondent fold increase (2 -ΔΔCt ), indicating also the 95% confidence interval.

Data analysis
Statistical significance of the treatment effects was tested using paired or non-paired Student's t-test depending on the characteristics of the data. Normality and homocedasticity of the data were assessed Statistical analyses and graphic design were conducted in Prism Software 5 (GraphPad Software Inc.) and R version 3.4.3 (https://www.R-project.org/) [39]. Differences were considered statistically significant when p-value was below 0.05.

Results
Predicting novel targets of miR-138-5p among the genes dysregulated after SCI To explore the contribution of miR-138-5p dysregulation on the gene expression changes observed after SCI, we initially carried out an in silico analysis to predict potential targets of miR-138-5p. Since the various available programs can yield rather different predictions, we combined TargetScan 7.1, miRWalk 2.0, miRmap and miRanda-programs to search for rat miR-138-5p gene targets ( Figure 1A).

Casp7, and Bak1
Previous studies based on murine SCI models [12,14] showed that miR-138-5p becomes downregulated 3 and 7 days after SCI. Present qPCR analysis of rat spinal cord samples confirmed the downregulation of miR-138-5p at 3 dpi and 7 dpi (Figure 2A). Additional qPCR analyses of the predicted miR-138-5p targets confirmed the previously described [7] overexpression of Casp3, Casp7, and Bak1 following SCI ( Figure 2B). Comparison between miRNA and mRNA expression data suggests that the increase in target expression negatively correlates with the downregulation of miR-138-5p. Correlation was significant for Casp3 (Spearman correlation rs: -0.8, p<0.01, Figure 2C) and Casp7 (rs: -0.85, p<0.01, figure 2C) employing available paired data for miRNA and mRNA expression. Although a similar trend was observed when employing the mean daily values of miR-138-5p and Bak1 (see Figure 2C), lack of paired data and the number of available values (corresponding to 0, 3 and 7 dpi) precluded their analyses.

miR-138-5p directly targets Casp3, Casp7, and Bak1 3'UTRs
To experimentally validate the apoptotic targets of miR-138-5p predicted in silico, we carry out reporter assays as well as gene and protein expression analyses. In order to choose appropriate cell lines for these assays, we compared the endogenous levels of miR-138-5p, and CASP3, CASP7, and BAK proteins in different cell lines (Human: SH-SY5Y and HEK293T; Rat: PC12 and C6). We selected the human HEK293T and rat C6 cell lines for the following experiments due to their high CASP3, CASP7, and BAK protein expression level and their low to moderate endogenous expression of miR-138-5p (Supplementary Material 5). To evaluate whether miR-138-5p presents binding sites in Casp3, Casp7, and Bak1 3'UTRs, we prepared luciferase reporter constructs containing the rat wild-type 3'UTR of the three genes. We employed the 3'UTR of Fadd (apoptotic adaptor protein) with no predicted or validated miR-138-5p binding sites to control for unspecificity of reporter constructs. We cotransfected these constructs with miR-138-5p mimic or cel-miR-67 negative control (negative control) into rat C6 or human HEK293T cells to perform a dual-luciferase reporter assay. Analysis of the normalized activity values (Firefly/Renilla) revealed no effect of miR-138-5p on the luciferase activity of the control construct (pmirGLO without 3'UTR subcloned) which yielded values similar to cultures treated with the negative control miRNA ( Figure 3A), thus confirming that miR-138-5p did not alter luciferase activity. Conversely, co-transfection of the negative control miRNA with any of the 3'UTRs luciferase constructs in C6 cells significantly downregulated the luciferase activity ( Figure 3A), suggesting that the expression of the three genes is subjected to an endogenous regulation. A similar reduction of luciferase activity was observed in human HEK293T cell cultures, except when cotransfecting Casp3 3'UTR construct ( Figure 3B). Despite this endogenous downregulation of the three targets, transfection with the miR-138-5p mimic was able to further reduce the luciferase activity (compared to the negative control microRNA) in C6 cells when co-transfected with the 3'UTRs of Casp3 As for C6 cells, the expression of Fadd construct remained unaffected by the transfection of the miR-138-5p mimetic (-1.58±27.6 n=3, t(2)=1.28, p=0.86) ( Figure 3B). Altogether, the results from these reporter assays confirm that miR-138-5p regulates the 3'UTRs of Casp3, Casp7, and Bak1. These results also reveal that the endogenous and miR-138-5p regulation of the 3'UTRs of these proteins depends on cell type, being highly active in C6 neural cells compared to HEK293T kidney cells.

miR-138-5p attenuates caspase-dependent apoptosis
Since CASP3, CASP7, and BAK actively participate in apoptosis, we investigated whether miR-138-5p regulation of their expression also affects their activity in this cell death process. We focused these analyses on the effector CASP3 and CASP7, leaving BAK unanalyzed because its apoptotic activity ultimately relies on (and is reflected by) the cleavage and activation of both effector proteases [40]. For these functional studies, we first extended the immunoblot analysis of the pro-CASP3 and pro-CASP7 to examine the effects of transfecting miR-138-5p mimic in C6 cells under apoptotic conditions. It is well established that apoptotic stimulation leads to the proteolytic cleavage of the effector caspases and, concomitant, to the reduction in the levels of their proforms. Accordingly, stimulation of control C6 cells (i.e. transfected with negative control miRNA) with 350 µM ETO reduced the level of pro-pro-CASP3 and pro-CASP7 relative to unstimulated cells (pro-CASP3 reduction=46.59%±4.9, n=5; pro-CASP7 reduction=75.4%±13.8, n=4; see Figure 5A). ETO stimulation also reduced the levels of both procaspases when C6 cells were previously transfected with miR-138-5p mimics (pro-CASP3 reduction=18.02%±7.8, n=5; pro-CASP7 reduction=16.75%±1.8, n=4; Figure 5A). However, the reduction was significantly smaller than that measured in C6 cells transfected with the negative control miRNA (pro-CASP3: t(4)=3.577, p=0.023; pro-CASP7: t(3)=3.960, one-tailed p=0.029), indicating that miR-138-5p modulation reduces caspase cleavage in C6 cells under apoptotic stimulation which ultimately should result in diminished activity of these proteases. To verify this hypothesis, we quantified the enzymatic activity of effector CASP3 and CASP7 in C6 cell cultures transfected with either miR-138-5p mimic or the negative control miRNA and exposed to apoptotic stimulation. As shown in Figure 5B, the enzymatic assay revealed that basal activity of the effector CASP3 and CASP7 was slightly but significantly lower when C6 cell cultures were transfected with the miR-138-5p mimic than when transfected with the negative control miRNA (ratio miR-138-5p mimic/negative control

Discussion
We designed the present study to search for evidence of the participation of miR-138-5p downregulation in the cell death processes that characterize SCI pathophysiology. MicroRNAs are known to regulate the identity, state, and fate of neural cells [15]. Not surprisingly, microRNA dysregulation accompanies multiple neurological pathologies [41], including SCI [42]. In search for microRNAs that could contribute to cell death progression during SCI pathophysiology, we identified the CNS-specific microRNA miR138-5p [43] as a potential candidate due to its dysregulation after SCI [12,14] and its targeting on the SCI-upregulated pro-apoptotic protein CASP3 [25]. miR-138-5p modulates essential biological processes in the CNS, including oligodendrocyte differentiation and myelin maintenance, or controlling dendritic spines morphogenesis of hippocampal neurons [22,44].
Before analyzing the effects of miR-138-5p dysregulation, we employed qRT-PCR to confirm the previously observed downregulation of this miRNA in a rat model of moderate contusive SCI. In agreement with the results from previous microarray analyses [12,14], our qRT-PCR data indicate that miR-138-5p reduces its expression in the spinal cord during the first week after injury. The decrease in miR-138-5p abundance may result from transcriptional changes in the neurons and oligodendrocytes that mainly express this miRNA [44], but also from the loss of these cells during the secondary injury.
According to Bicker and colleagues [45], miR-138-5p expression in neurons is highly responsive to pathologies and activity changes, suggesting that this miRNA and its targets may be involved in cellular processes that protect neurons from disturbance.
Once we confirmed the downregulation of miR-138-5p in our animal model of SCI, we explored how this dysregulation affects target expression in the damaged spinal cord, focusing on proteins involved in cell death. In a previous study [14], we already described that injury to the spinal cord leads to a significant enrichment of gene expression changes among miR-138-5p targets, which supposes clear evidence of the downregulation of this miRNA and indicates some of its potential effects in the damaged spinal cord. The in silico analyses carried out here extended the identified miR-138-5p targets to include additional cell death proteins among those upregulated after SCI according to available transcriptomic data [9,32]. Specifically, our in silico data identified the pro-apoptotic proteins CASP3, CASP7, and BAK as potential or validated miR-138-5p targets upregulated after SCI (see references above and [7,[46][47][48]). Reporter and immunoblot assays confirmed miR-138-5p post-transcriptional regulation of CASP3 (previously shown by [25]), and established for the first time its regulation of CASP7 and BAK. Expression analyses revealed that the overexpression of the three targets in the injured spinal cord follows the downregulation of miR-138-5p, providing additional evidence of its regulation of the three pro-apoptotic targets. Previous studies have identified other miRNAs targeting them, such as miR-106b and miR-337-3p on CASP7 [49,50], miR-17-5p, miR-132-3p and miR-212-3p on Casp3 [50], and miR-125b on BAK [51]. Interestingly, except for miR-106b and miR-337-3p, all these miRNAs appear dysregulated during the first week after injury according to our prior microarray analyses [14] or similar studies [12,52] and, therefore, may cooperate to regulate the apoptotic pathway in the damaged spinal cord.
Apoptosis is a major mechanism of neural cell dismissal during the secondary damage of SCI [2]. This cell death process has been described to be induced by either downregulation of miRNAs aiming at pro-apoptotic genes, such as miR29b and the BH3 genes or upregulation of miRNAs that target antiapoptotic genes, such as miR200c and FAP1 [53]. mir-138-5p's target BAK is a member of the BCL2 20 protein family that contributes to the activation of apoptosis through the permeabilization of the mitochondrial outer membrane to release apoptogenic factors, including cytochrome c [40]. The other two miR-138-5p targets, CASP3 and CASP7, are cysteine-aspartic acid proteases which, upon activation, execute the apoptotic program and play a central role in the extension of apoptosis after SCI [54,55]. It is our hypothesis that targeting on these targets will allow miR-138-5p to regulate cell death. In agreement, the results from our analyses demonstrate that the changes in miR-138-5p expression attenuate the enzymatic activity of CASP3 and CASP7 and conditions the execution of the apoptotic program. All these results suggest that miR-138-5p can be considered a neuroprotectant whose downregulation contributes to the secondary death of neural cells following SCI.
Even though the deleterious effects of miR-138-5p downregulation in the damaged CNS are explored here for the first time, the cytoprotective properties of this miRNA are well documented in astrocytes injured under ischemic conditions [56], glioblastoma [57], glioma stem cells [25], as well as in neural stem cells [58], pulmonary artery smooth muscle cells [59] and cardiomyocytes under hypoxia [60,61], and in microglial cells exposed to oxidative levels of H2O2 [62]. According to these studies, cytoprotection by miR-138-5p relies on its targeting and inhibition of the cell death-related genes Casp3, BLCAP and MXD1 [25], LCN2 [56,63], BIM [57], Mst-1 [64], and Mlk3 [61,62], or even in the inhibition of the JNK and p38MAPK pathways [58]. Some of these targets -including Casp3, Mst-1, BIM, and MLK3 (but also HIF-1α [65,66])-contribute to the cell death processes of secondary injury [67,68], suggesting that miR-138-5p can be considered a cytoprotectant whose downregulation contributes to extend neural cell death after SCI, as described by Tang and colleagues [63] for cerebral ischemia/reperfusion injury. In apparent opposition with this possibility, many cancer researchers consider miR-138-5p a tumour suppressor that becomes downregulated in different cancer processes, such as leukemia [69], lung cancer [70] and hepatocellular carcinoma [71]. Even within the nervous system, the ectopic expression of miR-138-5p in glioma multiforme cells seems to be tumour suppressive [72]. The antitumoral effects attributed to miR-138-5p mainly rely on its targeting on proliferative and cell cycle 21 proteins such as EZH2, E2F2, E2F3, CDK6, CCND3, or ABL1 [69,71,72] rather than on its direct induction of cell death. Interestingly, the antiproliferative and cell cycle arresting activity of miR-138-5p may be protective for neurons and oligodendrocytes, for which reentry in the cell cycle is linked to apoptosis after DNA damage in Alzheimer and other neurodegenerative diseases (see for review [73]). In fact, available evidence indicates that EZH2, E2F2, E2F3, and CCND3 become upregulated in the damaged spinal cord [74][75][76] where they can induce death among postmitotic neural cells during the secondary damage that follows SCI [9,74,77].

Conclusions
Data from present and previous studies agree that downregulation of miR-138-5p after SCI can be deleterious to postmitotic neurons and oligodendrocytes through a mixture of direct effects mediated by the upregulation of apoptotic targets and indirect effects related to the upregulation of cell cycle proteins. Targeting of miR-138-5p on Sirt1, a regulator of autophagy initiation [78], may also contribute to the deleterious blockage of this process in neurons and oligodendrocytes following SCI (see [79] and references therein). In addition, miR-138-5p downregulation may also limit cytoprotective treatments due to its targeting on MDR1, a channel that confers drug resistance in cancer [80], which becomes upregulated in the damaged spinal cord to limit the effects of Riluzole [81]. Further studies will be neccessary to determine the precise contribution of miR-138-5p dysregulation on the activation of these cell death pathways after CNS injury. However, up to date, the available evidence suggests that miR-138-5p may be a valid neuroprotective target for SCI. Establishing the therapeutical value of miR-138-5p modulation for SCI will require additional information. On the one hand, it will be neccessary to carry out histological studies to identify which cells are expressing miR-138-5p in physiological conditions and which cells become dysregulated after injury both in animal models and particularly in humans. On the other hand, in vivo studies will be neccessary to evaluate the confirm of the therapeutical modulation of miR-138-5p in the damaged CNS. LGA: L-Glutamic Acid.

Disclosure Statement
The authors have no conflicts of interest to declare

Funding Sources
Research was funded by grants from the Fundación Tatiana   All authors read and approved the final manuscript.

Availability of data and materials
All data generated during this study are included in the published article and its supplementary information files.    with miR-138-5p or the negative control mimics and analyzed using immunoblot and densitometry.

Figure Legends
Comparison between densitometry values was carried out through a paired t-test (n=4 experiments for CASP7; n=6 for CASP3 and BAK). The graph underneath shows the densitometry data of each apoptotic protein following transfection with miR-138-5p relative to their respective values after transfection with the negative control. * and ** indicate p<0.05 and p<0.01 relative to its corresponding negative control.