BRIEF RESEARCH REPORT published: 23 December 2020 doi: 10.3389/fcell.2020.584606 FAIM Is Regulated by MiR-206, MiR-1-3p and MiR-133b Elena Coccia 1,2,3 , Marc Masanas 4 , Joaquín López-Soriano 1,2,3 , Miguel F. Segura 4 , Joan X. Comella 1,2,3* and M. José Pérez-García 1,2,3* 1 Cell Signaling and Apoptosis Group, Vall d’Hebron Research Institute, Barcelona, Spain, 2 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain, 3 Institut de Neurociències, Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain, 4 Group of Translational Research in Child and Adolescent Cancer, Vall d’Hebron Research Institute (VHIR)-UAB, Barcelona, Spain Apoptosis plays an important role during development, control of tissue homeostasis and in pathological contexts. Apoptosis is executed mainly through the intrinsic pathway or the death receptor pathway, i.e., extrinsic pathway. These processes are tightly Edited by: Kai Tang, controlled by positive and negative regulators that dictate pro- or anti-apoptotic death Purdue University, United States receptor signaling. One of these regulators is the Fas Apoptotic Inhibitory Molecule Reviewed by: (FAIM). This death receptor antagonist has two main isoforms, FAIM-S (short) which Abhijit Shukla, Cornell University, United States is the ubiquitously expressed, and a longer isoform, FAIM-L (long), which is mainly Arun Upadhyay, expressed in the nervous system. Despite its role as a death receptor antagonist, FAIM Northwestern University, also participates in cell death-independent processes such as nerve growth factor- United States Hiroaki Kaku, induced neuritogenesis or synaptic transmission. Moreover, FAIM isoforms have been Western Michigan University, implicated in blocking the formation of protein aggregates under stress conditions United States or de-regulated in certain pathologies such as Alzheimer’s and Parkinson’s diseases. *Correspondence: M. José Pérez-García Despite the role of FAIM in physiological and pathological processes, little is known maria.perez@vhir.org about the molecular mechanisms involved in the regulation of its expression. Here, we Joan X. Comella seek to investigate the post-transcriptional regulation of FAIM isoforms by microRNAs joan.comella@vhir.org (miRNAs). We found that miR-206, miR-1-3p, and miR-133b are direct regulators of Specialty section: FAIM expression. These findings provide new insights into the regulation of FAIM and This article was submitted to Epigenomics and Epigenetics, may provide new opportunities for therapeutic intervention in diseases in which the a section of the journal expression of FAIM is altered. Frontiers in Cell and Developmental Biology Keywords: microRNA, neurodegenerative diseases, nervous system, death receptor, FAIM, Fas apoptotic inhibitory molecule Received: 17 July 2020 Accepted: 27 November 2020 Published: 23 December 2020 INTRODUCTION Citation: Coccia E, Masanas M, Several types of molecules are able to block apoptotic pathways, conferring cells with protection López-Soriano J, Segura MF, against threatening stimuli. The extrinsic apoptotic pathway is mediated by death receptors Comella JX and Pérez-García MJ (2020) FAIM Is Regulated by MiR-206, that integrate and transmit the extracellular apoptotic stimuli. In the last 20 years, mounting MiR-1-3p and MiR-133b. evidence has shed light on the physiological and pathological functions of these molecules and Front. Cell Dev. Biol. 8:584606. has widened the array of identified responses elicited by these receptors beyond cell death. doi: 10.3389/fcell.2020.584606 Indeed, Fas receptor and TNF receptors (TNFRs) are paradigmatic cases of receptors that can Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 December 2020 | Volume 8 | Article 584606 Coccia et al. Regulation of FAIM by miRNAs trigger apoptotic and non-apoptotic responses depending on the reported that murine Faim promoter contains three interferon cellular and molecular context (Marques-Fernandez et al., 2013). regulatory factor (IRF) binding sites, and Faim expression is The molecular response upon death receptor activation, positively regulated through IRF4 in primary B cells (Kaku and depends on the activity of proteins called death receptor Rothstein, 2009). At post-transcriptional level, FAIM can also antagonists. Among these, FAIM (Fas apoptosis inhibitory be regulated by MicroRNAs (miRNAs) (Patron et al., 2012; molecule) was first identified as a negative regulator of Fas Santosa et al., 2015). MiRNAs are short non-coding RNA of signaling (Schneider et al., 1999). It was later found to play 18–25 base pairs in length that are involved in the regulation of multifaceted roles in other physiological processes such as the gene expression at the post-transcriptional level. Mature miRNAs protective or deleterious effects of TNFα in neurodegenerative repress gene expression through binding to the 30 UTR of the disorders (Carriba et al., 2015), regulating axon-selective mRNA with the miRNA seed region, a 6–8 bases located at pruning, hippocampal long-term depression (LTD) (Martinez- the 50 end of the mature miRNA and perfectly complementary Marmol et al., 2016) and opposition to stress-induced to the target mRNA sequence (Mullany et al., 2016), thereby accumulation of protein aggregates (Kaku and Rothstein, 2020). inhibiting mRNA translation or inducing mRNA degradation Two main FAIM isoforms generated by alternative splicing (Alvarez-Garcia and Miska, 2005; Shingara et al., 2005). Thus far, have been found at the protein level. While the shorter isoform, the evidence of FAIM being regulated by miRNA was reported named FAIM-S, is ubiquitously expressed, FAIM-L is expressed by Patron and colleagues who showed that miR-133b directly exclusively in neurons and testes (Zhong et al., 2001; Segura et al., impairs the expression of FAIM, thereby enhancing Fas-induced 2007). In the nervous system, FAIM-S participates in neurite cell death in HeLa and PC3 cells (Patron et al., 2012). outgrowth by activating Ras-ERK and NF-κB pathways. On the Owing to the pathological consequences that FAIM de- other hand, FAIM-L has been shown to modulate death receptor- regulation may have for certain human diseases like are those induced apoptosis and caspase activation by binding to the involved in neurodegeneration, we sought to screen for other receptor (Segura et al., 2007), as well as through interaction with miRNA that could bind to the FAIM 30 UTR and modulate its X-linked inhibitor of apoptosis (XIAP) (Moubarak et al., 2013). expression. Our study identified miR-206, miR-1-3p and miR- Alterations in the expression of FAIM may be relevant in 133b as direct regulators of FAIM, thereby providing a deeper several types of human diseases. For example, in multiple knowledge on the FAIM regulation mechanisms and opening up myeloma (MM) patients, FAIM expression is increased in B new opportunities for therapeutic intervention. lymphocyte cells compared with normal individuals and its expression is higher in symptomatic MM patients compared with asymptomatic and premalignant individuals (Huo et al., 2013). MATERIALS AND METHODS FAIM expression is also elevated in CD34 hematopoietic stem cells and leukocytes. This deregulation is associated with chronic FAIM 30 UTR Analysis myeloproliferative pathogenesis (Tognon et al., 2011). The miRWalk 2.0 database using five miRNA-target Other results show FAIM as an important molecule in prediction algorithms (miRDB (RRID:SCR_010848), miRWalk metabolic processes. When both isoforms of FAIM are knocked (Vlachos et al., 2015), miRanda (Betel et al., 2010), miRMap out, mice spontaneously develop non-hyperphagic obesity, as (RRID:SCR_016508) and TargetScan; version 6.2 (Agarwal well as also manifest hepatosteatosis, adipocyte hypertrophy, et al., 2015) were used for the computational miRNA target dyslipidemia, hyperglycemia, and hyperinsulinemia. In obese prediction analysis. The miRNA target search was restricted to patients, FAIM expression is lower in blood cells and is inversely the 30 UTR of FAIM and with a minimum complementarity of correlated with insulin resistance biomarkers (Huo et al., 2016). 7 nucleotides in the seeding region. Probability distribution of Moreover, FAIM-L levels have been found to be relevant in random matches was set at 0.05 (Poisson p-value). MiRNAs neurodegenerative diseases. FAIM-L was found to be reduced with p ≤ 0.05 predicted by all five algorithms were selected for in the hippocampus of Alzheimer’s disease patients (Carriba further analysis. et al., 2015) and in the entorhinal and hippocampal cortex of Alzheimer’s disease mouse models (APP-PS1) (Carriba et al., Cell Culture and Transfection 2015). In Parkinson’s disease, the expression of FAIM-L was SH-SY5Y (Cat# CRL-2266, RRID:CVCL_0019), SK-N-AS (Cat# found to be reduced in midbrain dopaminergic neurons after CRL-2137, RRID:CVCL_1700), HEK293T (Cat# CRL-3216, trophic factor deprivation, as well as to sensitize them to Fas- RRID:CVCL_0063) and HeLa (CLS Cat# 300194/p772_HeLa, induced cell death (Yu et al., 2008). Recent findings also show RRID:CVCL_0030) cell lines were purchased from American that FAIM could play a role in Amyotrophic Lateral Sclerosis Type Culture Collection (ATCC, Rockville, MD, United States). inhibiting the aggregation of mutant SOD1, suggesting that FAIM SH-SY5Y, SK-N-AS and HEK293T were grown in Dulbecco’s participates in maintaining cell homeostasis (Kaku and Rothstein, modified Eagle’s medium (DMEM, Thermo Fisher Scientific, 2020). Kaku et al. (2020) also described that FAIM is recruited Waltham, MA, United States) containing 10% fetal bovine to cellular stress-induced ubiquitinated proteins, and the levels serum (HEK293T, SK-N-AS) or 15% fetal bovine serum (SH- of stress-induced protein aggregates are much greater in FAIM- SY5Y) (Thermo Fisher Scientific, Waltham, MA, United States). deficient cell lines. HeLa cells were cultured in Roswell Park Memorial Institute Despite the pathological consequences of FAIM de-regulation, (RPMI) 1640 (Thermo Fisher Scientific) supplemented with little is known about how its expression is modulated. Kaku et al. 10% fetal bovine serum, sodium pyruvate 1 mM (Thermo Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 December 2020 | Volume 8 | Article 584606 Coccia et al. Regulation of FAIM by miRNAs Fisher Scientific) and 1% of non-essential amino acids (Thermo sample and incubated in optical 384-well reaction plates. FAIM Fisher Scientific). All media were supplemented with 100 U/mL mRNA expression level was calculated as (Rao et al., 2013). penicillin, 100 µg/mL streptomycin (Thermo Fisher Scientific) and 5 µg/mL PlasmocinTM (InvivoGen). Culture conditions were Western Blot maintained at 37◦ C in a humidified atmosphere containing 5% Proteins were extracted using SET lysis buffer [10 mM Tris– CO2 . For miRNA transfection, SH-SY5Y, SK-N-AS and HeLa HCl pH7.4, 150 mM NaCl, 1 mM EDTA and 1% sodium were seeded at 6 × 105 , 4.5 × 105 , and 4 × 105 cells in dodecyl sulfate (SDS)] and then quantified using a modified 60 mm dishes, respectively, and transfected 24 h later with Lowry assay (DC protein assay, Bio-Rad). Equal amounts of the indicated miRIDIAN microRNA mimic oligonucleotides protein (30 µg per lane) were separated by 10% sodium (25 nM, Dharmacon), GE Healthcare using Lipofectamine 2000 dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis, transfection reagent (Thermo Fisher Scientific, Waltham, MA, and then transferred onto a polyvinylidene fluoride membrane United States), following the manufacturer’s instructions. Mimic (PVDF, Merck Millipore, MA, United States). Membranes were Transfection Control with Dy547 was used as a negative control. blocked with 5% non-fat milk at room temperature for 1 h and then incubated with the primary antibodies against FAIM Luciferase Reporter Assay (1:1000) (Segura et al., 2007) and α-tubulin (1:10000, Cat# Wild type and mutated 30 UTR sequences of FAIM were T9026, RRID:AB_477593;Sigma-Aldrich) overnight at 4◦ C. The synthetized using the GeneArt Gene synthesis platform (Thermo membranes were then incubated with horseradish peroxidase- Fisher Scientific, Waltham, MA, United States) and cloned into conjugated goat anti-rabbit IgG secondary antibody (1:10000, the psiCheckTM -2 dual luciferase reporter vector (Promega, Cat# AP132, RRID:AB_11214051;Sigma-Aldrich) and anti- C8021). For luciferase assays, HEK293T (Cat# CRL-3216, mouse (1:20000, Cat# AP124, RRID:AB_92455;Sigma-Aldrich) RRID:CVCL_0063) cells were co-transfected with 50 ng of for 1 h at room temperature. An enhanced chemiluminescence psiCheckTM -2 vectors containing wild type or mutated FAIM detection System, EZ-ECL detection kit (Biological Industries) 30 UTR and 25 nM of the indicated miRNAs, using Lipofectamine was used to develop signals, using α-tubulin as a loading control. 2000 (Invitrogen, Carlsberg, CA, United States), following the manufacturer’s protocol. Luciferase activity was measured 24 h Statistical Analysis post-transfection using the Dual Luciferase Reporter Assay Statistical analysis was performed using GraphPad Prism 7.0. System (Promega Corporation, Madison, WI, United States). All data in this study were shown as the mean of three Luminescence was measured in an Appliskan (Thermo Fisher independent experiments ± SEM. Statistical differences in Scientific) microplate reader. Renilla luciferase activity was multiple groups were examined by one-way ANOVA followed by normalized to corresponding firefly luciferase activity and plotted Dunnett’s multiple range test. P value < 0.05 was considered as as a percentage of the control. statistically significant. Quantitative Real-Time PCR RESULTS Total RNA, including small RNA, was isolated from human cell lines using the miRNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. Equal amounts of RNA (1 µg) were Five MiRNAs Are Predicted to Target converted to cDNA using the High Capacity RNA-to-cDNA Kit FAIM by Five Different MiRNA-Binding (Applied Biosystems), following the manufacturer’s instructions. Algorithms The quantitative real-time PCR (RT-qPCR) was performed In order to screen for potential miRNAs able to modulate the using TaqMan Universal PCR Master Mix Kit (Thermo Fisher expression of FAIM, we compared the prediction of putative Scientific). Samples were subjected to a PCR amplification miRNA-binding sites in the 30 UTR of FAIM from five different protocol using an AB7900HT Real Time PCR System (Thermo prediction algorithms, i.e., TargetScan, miRanda, miRWalk, Fisher Scientific, Waltham, MA, United States) using the miRMap, and miRDB (see Supplementary Table 1). When following primers for FAIM-L (Hs00992098_m1;Thermo predictions from the different algorithms were overlapped, five Fisher Scientific, Waltham, MA, United States) and for FAIM miRNAs were commonly found, namely miR-140-3p, miR-206, (Hs00216756_m1; Thermo Fisher Scientific, Waltham, MA, miR-1-3p, miR-133a-3p, and miR-133b (Figure 1A). The two United States). The PCR conditions were: 94◦ C for 3 min, main isoforms codified by FAIM gene, FAIM-S and FAIM-L, 40 cycles of 45 s at 94◦ C, followed by 30 s at 55◦ C, 72◦ C for differ in their 50 UTR composition, and in the inclusion of the 1 min and 72◦ C for 10 min. The data were analyzed using the exon 2b in the neuronal isoform FAIM-L. On the other hand, SDS 2.3 software (Thermo Fisher Scientific, Waltham, MA, the 30 UTR which includes the predicted target sites of these 5 United States) and normalized using GAPDH as a housekeeping miRNAs, is common to both isoforms (Figure 1B). Of note, while gene. TaqMan MicroRNA Assay (Applied Biosystems) was used the protein sequence is highly conserved during the evolution, to convert miRNA to cDNA for the analysis of mature miRNAs. the 30 UTR, and more precisely, the identified miRNA-binding cDNA was quantified by Taqman Universal Master Mix (Applied sites are conserved only among vertebrates, thereby suggesting Biosystems). MiRNA expression was normalized against RNU-44 that this mechanism of regulation was incorporated lately in the small RNA. The reactions were performed in triplicate for each evolution (Supplementary Figure 1). Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 December 2020 | Volume 8 | Article 584606 Coccia et al. Regulation of FAIM by miRNAs FIGURE 1 | In silico screening for potential FAIM targeting miRNAs. (A) Venn diagram showing the overlap of potential FAIM 30 UTR binding miRNAs using five target prediction tools. (B) Schematic representation of the two main FAIM isoforms. Labels shows the miR-140-3p (blue), miR-206/miR-1-3p (pink) and miR-133a-3p/miR133b (green) binding sites in the 30 UTR of FAIM. UTR: untranslated region; CDS: coding sequence. Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 December 2020 | Volume 8 | Article 584606 Coccia et al. Regulation of FAIM by miRNAs MiR-206, MiR-1-3p and MiR-133b Can FAIM acts as a death receptor antagonist by binding directly to Bind Directly to the 30 UTR of FAIM the death receptor (Segura et al., 2007) or by interacting with downstream effectors such as X-linked inhibitor of apoptosis To confirm whether the identified miRNAs are truly direct protein (XIAP) (Moubarak et al., 2013). De-regulation of regulators of FAIM expression, a luciferase-reporter vector FAIM is associated with the pathophysiology of cancer and containing the wild type 30 UTR was cloned and co-transfected neurodegenerative diseases among others. In Alzheimer’s with control mimic oligonucleotides or the indicated miRNA disease (AD), the levels of FAIM-L were shown to be decreased mimics. Since miR-133a-3p and miR-133b are almost identical according to Braak stages in AD patients (Braakman et al., 1991; (20/21 nucleotides) and share the exact same seed region, we Carriba et al., 2015). At molecular level, FAIM-L levels reduction proceeded with our analyses only with miR-133b. A remarkable abolished TNFα protection against amyloid-β neurotoxicity reduction in luciferase activity was observed upon transfection of (Carriba et al., 2015). Thus, a better understanding on how miR-206, miR-1-3p, and miR-133b but not with the transfection FAIM levels are modulated can be paramount for better of miR-140-3p (Figure 2A). The 30 UTR region of FAIM contains characterization of human disease and for the design of new one binding site common to miR-206 and miR-1-3p, and one therapeutic approaches. binding site for miR-133b. In order to confirm the interactions MiRNAs have important roles in regulating diverse biological were sequence specific we engineered specific mutations in the processes, such as cell proliferation, immunity, development, 30 UTR, giving rise to correspondingly 30 UTR mut206/1-3p and differentiation, metabolism and cell death, and generally they 30 UTR mut 133b, respectively (Figure 2B). Luciferase activity act as a negative feedback factor in cell signaling (Ha, 2011). reduction found in the wild type- 30 UTR was almost completely Furthermore, miRNA deregulation is a frequent event in human restored to control levels when miRNAs were co-transfected with disease, and they can be used as therapeutic tools to treat the respective 30 UTR mutated forms (Figure 2C). Of note, Clip- pathologies with unbalanced cell death and survival pathways seq data mining also showed binding of Ago2 in the 30 UTR of (Paul et al., 2018). FAIM for miR-133a-3p, miR-133b, miR-206, and miR-1-3p but We found that miR-206, miR-1-3p, and miR-133b directly not for miR-140-3p (Supplementary Table 2). Overall, we were regulate FAIM by binding to 30 UTR, decreasing the mRNA able to show that miR-206, miR-1-3p, and miR-133 have the and protein levels. MiR-133b has already been described to capacity to directly bind FAIM 30 UTR. target FAIM in PC3 and HeLa cells (Patron et al., 2012). The authors showed that FAIM silencing or miR-133b overexpression MiR-206, MiR-1-3p, and MiR-133b exacerbated death receptor-induced cell death. Our results Modulate FAIM Expression confirmed that miR-133b is a direct regulator of FAIM in To elucidate whether the direct binding of miRNAs to the a broader spectrum of cell types including the neuronal 30 UTR causes a downregulation of FAIM expression, we decided lineage. As regards the potential connection of miR-133b-FAIM to transfect miRNA mimics oligonucleotides into human cells in neurodegenerative diseases, Jimenez-Jimenez et al. (2014) lines that could represent different tissues where one or both reported that variations in miR-133b could contribute to the risk FAIM isoforms are expressed (Figure 3A). On the one hand, we of developing Parkinson’s disease. In this regard, the expression selected the neuroblastoma cell line SH-SY5Y that express both of FAIM-L was also described to be reduced in dopaminergic FAIM-L and FAIM-S, and SK-N-AS that only express FAIM-S. neurons in Parkinson’s disease (Yu et al., 2008), thus making Furthermore, we added HeLa cells, since is one of the few models this type of neurons more vulnerable to Fas-induced death. where the functionality of FAIM in human models has been tested Thus, high levels of miR-133b could contribute to lowering the (Patron et al., 2012). The expression at mRNA and protein levels expression of FAIM in these neurons. However, in Alzheimer’s was measured in the indicated cell lines after transfection of miR- disease, miR-133b was found to be significantly downregulated 140-3p, miR-206, miR1-3p, and miR-133b (Figures 3B,C). While after Aβ25-35 treatment (Yang et al., 2019). In a different study, miR-140-3p did not modulate the levels of FAIM in any of the cell FAIM levels also appear to be reduced in hippocampal samples lines tested, transfection of miR-206, miR-1-3p, and miR-133b from AD patients (Carriba et al., 2015), thus suggesting that caused a ∼2-fold reduction in FAIM mRNA levels (Figure 3B). the miR-133b-FAIM axis would not be relevant in this disease Similarly, FAIM protein levels decreased in presence of miR- and opens up the question of whether other miRNAs could be 206, miR1-3p, and miR-133b overexpression in the three cell responsible for FAIM downregulation. lines tested (Figure 3C). Overall, we were able to confirm that Here, we report, for the first time, that miR-206 and miR- among the predicted miRNAs targeting FAIM 30 UTR, miR-206, 1-3p can also be direct modulators of FAIM. Interestingly, miR-1-3p, and miR-133b regulate FAIM isoforms levels, while miR-206 and miR-1-3p belong to the same miRNA family, miR-140 does not. which means that, they share the same seed region, thereby suggesting a major overlap in their targets. Furthermore, miR- 206, is clustered with miR-133b in the short arm of chromosome DISCUSSION 6, indicating that these miRNAs can be co-regulated and provide a strong mechanism in the regulation of FAIM. MiR- Death receptor-induced cell death is essential during 206 was found to be significantly upregulated in blood samples development due to its role regulating tissue homeostasis from Alzheimer’s disease patients compared with age-matched and differentiation. In the adult, death receptor signaling can be normal controls. Furthermore, upregulation of miR-206 has important under physiological or pathological circumstances. been detected in serum from patients with mild cognitive Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 December 2020 | Volume 8 | Article 584606 Coccia et al. Regulation of FAIM by miRNAs FIGURE 2 | miR-206, miR-1-3p, and miR-133b bind directly to FAIM 30 UTR. (A) Dual luciferase activity assay in HEK293T cells after the transfection of 25 nM of the indicated miRNAs using psiCheckTM -2 vectors encoding the wild type 30 UTR of FAIM. (B) Schematic representation of the indicated miRNA binding sites in the wild type (wt) and mutated (mut) 30 UTR of FAIM. The mutated nucleotides are indicated in red. (C) Dual luciferase activity assay in HEK293T cells after the transfection of 25 nM of the indicated miRNAs using psiCheckTM -2 vectors encoding the wild type and mutated forms of FAIM 30 UTR. Graph represents the values of luciferase activity and are the mean of three independent experiments ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, compared with control vector. ##P < 0.05 compared with the wild type 30 UTR of FAIM. Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 December 2020 | Volume 8 | Article 584606 Coccia et al. Regulation of FAIM by miRNAs FIGURE 3 | FAIM expression is modulated by miR-206, miR-1-3p, and miR-133b. (A) MiRNAs expression levels were assessed by RT-qPCR. (B) SK-N-AS, SH-SY5Y and HeLa cells were transfected with 25 nM of miR-140-3p, miR-206, miR-1-3p, miR-133b or a control miRNA (miR-Scr) and FAIM mRNA expression levels were assessed by RT-PCR. (C) Representative western blots of FAIM in neuroblastoma and HeLa cells transfected with the indicated miRNAs. Lower panels show the quantification of the band intensity of the western blots normalized to the control miRNA (miR-Scr) condition. *Unspecific band. Graphs represent the mean of four independent experiments ± SEM. Tubulin was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.005. Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 December 2020 | Volume 8 | Article 584606 Coccia et al. Regulation of FAIM by miRNAs impairment (Xie et al., 2015), and in the temporal cortex of manuscript. EC, MM, MS, and JL-S analyzed data. All human AD brains (Lee et al., 2012). Previous studies using authors contributed to the article and approved the microglial BV-2 cells and miR-206 mimics demonstrated that submitted version. a pro-inflammatory stimulus (LPS treatment), increased miR- 206 expression and enhanced the release of pro-inflammatory cytokines, including IL-1β and TNFα. Thus, in a scenario with FUNDING high levels of TNFα and low levels of FAIM as reported in some neurodegenerative diseases, TNFα signaling can be switched This work was funded by grants awarded by the from a pro-survival to a pro-apoptotic response. Previous results Spanish “Ministerio de Economía y Competitividad” from our lab showing that Aβ treatment decreased the levels (SAF2016-80236-R and PID2019-107286RB-I00), CIBERNED of FAIM-L and blocked TNFα protection against Aβ toxicity (CB06/05/1104) and Generalitat de Catalunya (2017SGR996) (Carriba et al., 2015) would support this hypothesis. to JC. EC was supported by a pre-doctoral fellowship To date, there are no effective therapies for these diseases from the Vall d’Hebron Research Institute (VHIR) and new strategies are needed. Given the encouraging results and MM was awarded by the FPU-Ph.D. fellowship of profiling studies and preclinical testing, miRNAs are now (Grant No. FPU16/01099) from the Spanish Ministry being integrated into human clinical trials. For example, of Education. miR-122 has successfully reached clinical trials as a targeted therapy for hepatitis C (Lanford et al., 2010). Disrupting the miRNA-mediated reduction of anti-apoptotic proteins such as FAIM, could represent a new neuroprotective strategy against SUPPLEMENTARY MATERIAL neurodegenerative diseases such as Alzheimer’s or Parkinson’s. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2020. DATA AVAILABILITY STATEMENT 584606/full#supplementary-material Supplementary Figure 1 | Multiple sequence alignment of 30 UTR FAIM from The raw data supporting the conclusions of this article will be different species. Sequences included in the alignment are those of Homo made available by the authors, without undue reservation. sapiens, Mus musculus, Rattus norvegicus, and Sus scrofa. The sequences of miRNA are boxed in blue (miR-140-3p), pink (miR-206;miR-1-3p), and green (miR-133b;miR-133a-3p). AUTHOR CONTRIBUTIONS Supplementary Table 1 | MiRNA-binding sites in the 30 UTR of FAIM. EC and MM performed the experiments. MS, JC, Supplementary Table 2 | AGO2-ClipSeq studies (Li et al., 2014 and Zhou, and MP-G designed the experiments and wrote the ENCORI: the encyclopedia of RNA interactomes). REFERENCES IRF4 expression in multiple myeloma. Leukemia 27, 1165–1171. doi: 10.1038/ leu.2012.326 Agarwal, V., Bell, G. W., Nam, J. W., and Bartel, D. P. (2015). Predicting effective Jimenez-Jimenez, F. J., Alonso-Navarro, H., Garcia-Martin, E., and Agundez, J. A. microRNA target sites in mammalian mRNAs. eLife 4:e05005. (2014). 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Hyperosmotic stress activates the expression of members of the miR- 15/107 family and induces downregulation of anti-apoptotic genes in rat liver. Conflict of Interest: The authors declare that the research was conducted in the Sci. Rep. 5:12292. absence of any commercial or financial relationships that could be construed as a Schneider, T. J., Fischer, G. M., Donohoe, T. J., Colarusso, T. P., and Rothstein, T. L. potential conflict of interest. (1999). A novel gene coding for a Fas apoptosis inhibitory molecule (FAIM) isolated from inducibly Fas-resistant B lymphocytes. J. Exp. Med. 189, 949–956. Copyright © 2020 Coccia, Masanas, López-Soriano, Segura, Comella and Pérez- doi: 10.1084/jem.189.6.949 García. This is an open-access article distributed under the terms of the Creative Segura, M. F., Sole, C., Pascual, M., Moubarak, R. S., Perez-Garcia, M. J., Gozzelino, Commons Attribution License (CC BY). The use, distribution or reproduction in R., et al. (2007). The long form of Fas apoptotic inhibitory molecule is expressed other forums is permitted, provided the original author(s) and the copyright owner(s) specifically in neurons and protects them against death receptor-triggered are credited and that the original publication in this journal is cited, in accordance apoptosis. J. Neurosci. 27, 11228–11241. doi: 10.1523/jneurosci.3462-07. with accepted academic practice. No use, distribution or reproduction is permitted 2007 which does not comply with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 December 2020 | Volume 8 | Article 584606