miR-29a-3p and TGF-β Axis in Fanconi Anemia: Mechanisms Driving Metabolic Dysfunction and Genome Stability

miR-29a-3p and TGF-β Axis in Fanconi Anemia: Mechanisms Driving Metabolic Dysfunction and Genome Stability | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results miR-29a-3p and TGF-β Axis in Fanconi Anemia: Mechanisms Driving Metabolic Dysfunction and Genome Stability Nadia Bertola , Stefano Regis , Vanessa Cossu , Matilde Balbi , Martina Serra , Fabio Corsolini , Cristina Bottino , Paolo Degan , Carlo Dufour , Filomena Pierri , Enrico Cappelli , Silvia Ravera doi: https://doi.org/10.1101/2025.01.14.632746 Nadia Bertola 1 IRCCS Ospedale Policlinico San Martino , Largo Rosanna Benzi, 10, 16132 Genova, Italy ; Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stefano Regis 2 Laboratory of Clinical and Experimental Immunology, IRCCS Istituto Giannina Gaslini , Via Gerolamo Gaslini 5, 16147 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Vanessa Cossu 1 IRCCS Ospedale Policlinico San Martino , Largo Rosanna Benzi, 10, 16132 Genova, Italy ; 3 Experimental Medicine Department, University of Genova , Via De Toni 14, 16132 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Matilde Balbi 3 Experimental Medicine Department, University of Genova , Via De Toni 14, 16132 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Martina Serra 3 Experimental Medicine Department, University of Genova , Via De Toni 14, 16132 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Fabio Corsolini 4 Haematology Unit, IRCCS Istituto Giannina Gaslini , Via Gerolamo Gaslini 5, 16147 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Cristina Bottino 2 Laboratory of Clinical and Experimental Immunology, IRCCS Istituto Giannina Gaslini , Via Gerolamo Gaslini 5, 16147 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Paolo Degan 2 Laboratory of Clinical and Experimental Immunology, IRCCS Istituto Giannina Gaslini , Via Gerolamo Gaslini 5, 16147 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carlo Dufour 4 Haematology Unit, IRCCS Istituto Giannina Gaslini , Via Gerolamo Gaslini 5, 16147 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Filomena Pierri 4 Haematology Unit, IRCCS Istituto Giannina Gaslini , Via Gerolamo Gaslini 5, 16147 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Enrico Cappelli 4 Haematology Unit, IRCCS Istituto Giannina Gaslini , Via Gerolamo Gaslini 5, 16147 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: EnricoCappelli{at}gaslini.org Silvia Ravera 1 IRCCS Ospedale Policlinico San Martino , Largo Rosanna Benzi, 10, 16132 Genova, Italy ; 3 Experimental Medicine Department, University of Genova , Via De Toni 14, 16132 Genoa, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and cancer susceptibility due to defective DNA double-strand break repair. However, FA cells are also characterized by mitochondrial dysfunction and redox imbalance. To identify a common factor among these alterations, we focused on miR-29a-3p, a microRNA involved in hematopoiesis. Our data show that miR-29a-3p is downregulated in lymphoblasts and fibroblasts mutated for the FANC-A gene, causing the overexpression of its target genes, FOXO3, SGK1, and IGF1, which results in PI3K/AKT pathway hyperactivation, altered mitochondrial metabolism and insufficient antioxidant response. Furthermore, miR-29a-3p downregulation appears associated with hyperactivation of the TGF-β signal. However, restoring miR-29a-3p expression improves mitochondrial metabolism, oxidative stress response, and DNA damage repair by inhibiting the PI3K/AKT pathway and modulating TGF-β signaling by a feedback mechanism. Based on these findings, miR-29a-3p appears as a promising molecular target to address several mechanisms based on FA pathogenesis. INTRODUCTION Fanconi anemia (FA) is a genetic disease, typically with pediatric onset, characterized by aplastic anemia and predisposition to several types of cancer, such as acute myeloid leukemia, gynecological carcinoma, and head and neck squamous cell carcinoma (HNSCC) 1 , 2 . DNA repair has long been considered the primary defect in FA cells 3 , 4 . However, in the last 20 years, research has shown that FA proteins are involved in several other cellular processes, including defective energy metabolism 5 , 6 , impaired antioxidant response 7 – 9 , and the overproduction of pro-inflammatory and cytotoxic cytokines 10 – 13 . In detail, FA cells are characterized by altered electron transport between respiratory complexes I and III 14 , 15 , leading to the uncoupling of oxidative phosphorylation (OxPhos) 16 – 18 . As a result, FA cells exhibit increased reactive oxygen species (ROS) production 19 , 20 , not counteracted by endogenous antioxidant defensesbecause FA cells are not able to trigger an adaptative response to the oxidative stress increment 21 – 23 . This imbalance in redox status promotes DNA damage accumulation 24 and the development of a pro-inflammatory environment 7 , triggering a vicious circle. Additionally, unrepaired DNA damage is reported to contribute to the pro-inflammatory conditions observed in FA patients 25 , 26 , increasing the expression of cytotoxic cytokines. This heightened expression is associated with increased cellular sensitivity and leads to the death of hematopoietic progenitors 13 , 27 .In this context, several pro-inflammatory cytokines have already been associated with defective hematopoiesis in patients with FA 28 . For example, bone marrow mononuclear cells (BM-MNC) isolated from FA patients have shown hypersensitivity to TNF-α, which inhibits erythropoiesis in vitro 12 . Additionally, hyperactivity of the TGF-β/SMAD3 pathway inhibits DNA repair via homologous recombination (HR), activating non-homologous end joining (NHEJ), which is toxic to FA HSCs 29 . Although DNA damage accumulation, impaired energy metabolism and antioxidant defenses, and increased production of proinflammatory cytokines in FA have been extensively described in the literature, no mechanism has yet been proposed yet to explain their interconnection and mutual influence. To address this gap, we focused our attention on the microRNA (miRNA) profile in FA cells, because, to date, few studies have explored the miRNA profile in FA cells 30 , 31 . In this study, we investigate the role of miR-29a-3p, a member of the miR-29 family, which modulates numerous biochemical and physiological pathways, including the maturation, differentiation, and survival of hematopoietic stem and progenitor cells (HSPCs) 32 . Notably, miR-29a-3p has been shown to regulate DNA methyltransferase 3 (DNMT3), supporting self-renewal in HSPCs and guiding lineage commitment and differentiation 32 , 33 . Our findings reveal that FA lymphoblasts and primary fibroblasts exhibit significantly reduced expression of miR-29a-3p compared to healthy controls and isogenic corrected cells. This reduction is mediated by negative feedback involving TGF-β pathway activity. Furthermore, transfection of FA cells with miR-29a-3p restores mitochondrial function, enhances the oxidative stress response and reduces DNA damage by modulating the TGF-β pathway through decreasingSMAD3phosphorylation. RESULTS miR-29a-3p expression is downregulated in Fanc-A cells Although several miRNAs may potentially be involved in the pathogenesis of FA 30 , 31 ,we focused our attention on miR-29a-3p, due to its role in modulating mitochondrial activity and redox balance 32 , two features altered in FA cells 17 , 21 . Our data show a significant reduction in miR-29a-3p expression in Fanc-A lymphoblasts( Figure 1A ) and fibroblasts ( Figure S1 ) compared to the corresponding isogenic corrected cells, suggesting a possible role of miR-29a-3p in mitochondrial dysfunction and associated oxidative stress production in FA cells. Download figure Open in new tab Figure 1. miR-29a-3p expression in Fanc-A cells. The graph shows the comparison of miR-29a-3p expression in isogenic Fanc-A corrected lymphoblasts (Fanc-A corr, used as a control) and Fanc-A lymphoblasts (Fanc-A). RNU44 was used as a reference control. Data are expressed as mean ± SD and are representative of three independent experiments. ** indicates a significant difference for p < 0.01 between Fanc-A corr and Fanc-A. Fanc-A cells display an alteration of several putative miR-29a-3p-regulated genes involved in DNA repair, mitochondrial function, redox balance, and apoptosis Based on the results of miR-29a-3p expression, we interrogated the miRPathDB v2.0 database to identify putative associations between miRNAs, target genes, and cellular pathways. Specifically, we selected genes potentially regulated by miR-29a-3p and involved in DNA damage response, oxidative stress, mitochondrial metabolism, lipid metabolism, and apoptosis, all of which are pathways potentially altered in FA cells. Furthermore, TargetScan was used to define the gene list based on the strength of predicted miRNA-target gene interactions. The list was then refined by analyzing the role and subcellular location of each gene using NCBI Gene and GeneCards. The final list is reported in Table 1 . View this table: View inline View popup Download powerpoint Table 1. Putative target genes of miR-29a-3p in DNA damage response processes, mitochondrial function, oxidative stress, lipid metabolism, and apoptosis miR-29a-3p transfection reduced the oxidative damage and improved the oxidative phosphorylation in Fanc-A cells Since low expression of miR-29a-3p in FA cells suggests possible altered expression of genes involved in oxidative stress response ( Table 1 ), catalase (CAT) activity, and intracellular malondialdehyde (MDA) levels have been evaluated as markers of antioxidant defenses and oxidative damage, in FA cells transfected with miR-29a-3p. Additionally, 8-hydroxy-2’-deoxyguanosine (8-OHdG) content and histone H2AX phosphorylation have been assayed as DNA damage markers. Fanc-A cells displayed reduced CAT activity ( Figure 2A and Figure S2A ) and increased concentrations of MDA ( Figure 2B and Figure S2B ) and 8-OHdG ( Figure 2C and Figure S2C ) compared to the corrected cells, indicating increased oxidative damage to lipids and DNA due to defective antioxidant defenses. In addition, histone H2AX was hyper-phosphorylated in Fanc-A lymphoblasts ( Figure 2D ), confirming DNA damage accumulation. However, these detrimental effects appeared partially recovered after miR-29a-3p transfection, suggesting a role of this miRNA in the unbalanced oxidative stress of FA cells. Download figure Open in new tab Figure 2. Antioxidant defenses, oxidative stress, and energy metabolism were modulated by miR-29a-3p expression in Fanc-A lymphoblasts. All analyses were conducted on Fanc-A lymphoblasts corrected with the WT Fanc-A gene (Fanc-A corr), Fanc-A lymphoblasts (Fanc-A), Fanc-A lymphoblasts transfected with amiRNA mimic negative control for 48h (Fanc-A scr), and Fanc-A lymphoblasts transfected with miR-29a-3p for 48h (Fanc-A + miR-29a-3p). (A) Catalase activity as an antioxidant defense marker. (B) Intracellular concentration of malondialdehyde (MDA) as a lipid peroxidation marker. (C) 8-hydroxy-2’-deoxyguanosine (8-OHdG) content as a DNA oxidation marker. (D) WB signal and relative densitometric analysis of p-H2AX. The densitometric analysis was normalized to the actin signal and used as a housekeeping protein. (E) ATP synthesis through F o F 1 -ATP synthase. (F) Oxygen consumption rate (OCR). (G) P/O value, an OxPhos efficiency marker. For Panels E, F, and G, the analyses were conducted in the presence of pyruvate plus malate (P/M) or succinate (Succ) to induce the OxPhos pathways led by Complex I or Complex II, respectively. (H) Electron transfer between Complexes I and III. (I) Intracellular ATP content. (J) Intracellular AMP content. (K) Cellular energy status is obtained by calculating the ATP/AMP ratio. Data are expressed as mean ± SD and are representative of three independent experiments for Panels E-G and six independent experiments for Panels A-D and H-K. **, ***, and **** indicate a significant difference for p < 0.01, 0.001, or 0.0001, respectively, between Fanc-A corr and Fanc-A or Fanc-A scr. ##, ###, and #### indicate a significant difference for p < 0.01, 0.001, or 0.0001, respectively, between Fanc-A + miR-29a-3p and Fanc-A or Fanc-A scr. Considering that miR-29a-3p could also regulate genes involved in energy metabolism and that FA cells display an altered OxPhos associated with increased oxidative stress production 14 , 17 , 34 , the ATP synthesis through F o F 1 -ATP synthase, the oxygen consumption rate (OCR), and the OxPhos efficiency were investigated before and after miR-29a-3p transfection in Fanc-A lymphoblasts and fibroblasts. As expected, data show that Fanc-A cells were characterized by defective ATP production ( Figure 2E and Figure S2D ) and OCR ( Figure 2F and Figure S2E ) when OxPhos was induced by pyruvate/malate addition but not with succinate. This impairment resulted in a decrease of OxPhos efficiency via complexes I-III-IV, as indicated by the reduction of theP/O value ( Figure 2G and Figure S2F ). All these metabolic parameters showed a significant improvement after miR-29a-3p transfection. This recovery depended on the restoration of electron transport between respiratory complexes I and III ( Figure 2H and Figure S2G ), leading to an increase in intracellular ATP content ( Figure 2I and Figure S2H ) and a reduction in AMP concentration ( Figure 2J and Figure S2I ), ultimately improving the energy status ( Figure 2K and Figure S2J ). miR-29a-3p transfection restores FOXO3, SGK1, and IGF1 genes expression in Fanc-A cells The data presented in Table 1 highlight that FOXO3, SGK1, and IGF1 are implicated in multiple pathways, including DNA damage response, mitochondrial activity regulation, oxidative stress management, and apoptosis, all hallmark features of FA pathology. Consequently, the expression of these genes was assessed in Fanc-A lymphoblasts ( Figure 3 ) and Fanc-A fibroblasts ( Figure S3 ). The results revealed significantly elevated expression levels for all three genes compared to their corrected counterparts. By contrast, in Fanc-A cells transfected with miR-29a-3p, the expression of the FOXO3, SGK, and IGF1 genes decreased by about 40%, 34%, and 30%, respectively, compared to the FA cells, reaching a comparable level compared to control cells. Download figure Open in new tab Figure 3. FOXO3, SGK1, and IGF1 expression in Fanc-A lymphoblasts. Graphs show the comparison of FOXO3 (A), SGK1 (B), and IGF1 (C) expression in (i) Fanc-A cells corrected with the WT Fanc-A gene (Fanc-A corr), (ii) Fanc-A cells (Fanc-A), (iii) Fanc-A cells transfected with a miRNA mimic negative control for 48h (Fanc-A scr), and (iv) Fanc-A cells transfected with miR-29a-3p for 48h (Fanc-A + miR-29a-3p). GAPDH was used as the reference control. Data are expressed as mean ± SD and are representative of three independent experiments. * or **** indicate a significant difference for p < 0.05 or 0.0001, respectively, between Fanc-A corr and the other samples. ## and ### indicate a significant difference for p < 0.01 or 0.001, respectively, between Fanc-A + miR-29a-3p and Fanc-A or Fanc-A scr. Treatment with miR-29a-3p restores the nuclear translocation of FOXO3a in Fanc-A lymphoblasts Since FOXO3a function is closely linked to its translocation from the cytoplasm to the nucleus 35 , FOXO3a localization has been evaluated by WB analysis, observing thatFanc-A lymphoblasts predominantly expressed FOXO3a in the cytoplasmic fraction while Fanc-A corr cellsexhibited higher protein expression in the nucleus. Conversely, miR-29a-3p transfection in Fanc-A cells significantly promoted FOXO3a translocation to the nucleus, enhancing its transcriptional activity ( Figure 4 ). Download figure Open in new tab Figure 4. miR-29a-3p changed FOXO3a intracellular localization in Fanc-A lymphoblasts. All analyses were conducted on cell homogenate (H), nuclear fraction (N), and cytoplasmic fraction (C) derived from Fanc-A lymphoblasts corrected with the WT Fanc-A gene (Fanc-A corr), Fanc-A lymphoblasts transfected with a miRNA mimic negative control for 48h (Fanc-A scr), and Fanc-A lymphoblasts transfected with miR-29a-3p for 48h (Fanc-A + miR-29a-3p). (A)Representative WB signals of FOXO3a, GAPDH (used as a cytoplasmic marker), and Histone H3 (used as a nuclear marker). The virtual absence of the GAPDH signal in the nuclear fraction (N) and the Histone H3 signal in the cytoplasmic fraction (C) demonstrates the correct separation of the two cellular fractions. (B) Densitometric analysis of the FOXO3a signal. Data in panel B are expressed as mean ± SD and are representative of three independent experiments. **** indicates a significant difference of p < 0.0001 between the same cellular fractions in Fanc-A corr and Fanc-A scr. ### indicates a significant difference of p < 0.0001 between the same cellular fractions in Fanc-A + miR-29a-3p and Fanc-A scr. The miR-29a-3p-induced nuclear translocation of FOXO3a depends on the modulation of its hyperphosphorylation through AKT and SGK1 pathways FOXO3a migration from cytoplasm to nucleus depends on different post-transcriptional modifications 36 – 38 , including the phosphorylation on Ser253 by AKT phosphorylated on Ser473, which is the principal post-translational modification that prevents FOXO3a entry into the nucleus 39 , 40 . Therefore, the phosphorylation levels of these two proteins were evaluated by WB analysis. Our results show that Fanc-A lymphoblasts displayed higher phosphorylation of Ser253 p-FOXO3a ( Figure 5A and 5B ) and Ser473 p-AKT ( Figure 5A and 5C ) compared to Fanc-A corrected cells, which was reverted by miR-29a-3p treatment. Furthermore, since SGK1 shares the same phosphorylation site on FOXO3a as AKT 41 , 42 when phosphorylated on Ser422, the expression of phosphorylated and total forms of SGK1 in Fanc-A cells has been evaluated, observing a hyperphosphorylation in Fanc-A cells compared to Fanc-A corr, which was reduced after miR-29a-3p transfection( Figure 5A and 5D ). Download figure Open in new tab Figure 5. miR-29a-3p transfection modulates the FOXO3a,AKT, and SGK1 phosphorylation in Fanc-A lymphoblasts. All analyses were conducted on Fanc-A lymphoblasts corrected with the WT Fanc-A gene (Fanc-A corr), Fanc-A lymphoblasts (Fanc-A), Fanc-A lymphoblasts transfected with a miRNA mimic negative control for 48h (Fanc-A scr), and Fanc-A lymphoblasts transfected with miR-29a-3p for 48h (Fanc-A + miR-29a-3p). (A)Representative WB signals of:phospho-FOXO3a (Ser253); total FOXO3a; phospho-AKT (Ser473); total AKT; phospho-SGK1 (Ser422); total SGK1. Actin signal was used as housekeeping. (B) The ratio ofphosphorylated and total forms of FOXO3a signals. (C) The ratio ofphosphorylated and total forms of AKT signals. (D) The ratio of phosphorylated and total forms of SGK1 signals. Data in panels B, C, and D are expressed as mean ± SD and are representative of three independent experiments. * and**** indicate a significant difference for p <0.05 or 0.0001 between Fanc-A corr and other samples. ### and #### indicate a significant difference for p <0.001 or 0.0001, respectively, between Fanc-A + miR-29a-3p and Fanc-A scr. miR-29a-3p and TGF-β pathway modulate each other Since the miR-29a-3p expression is negatively regulated by the TGF-β pathway hyperactivation 43 , which is a hallmark of FA cells 44 , its expression has been evaluated in the presence of Luspatercept, a TGF-β pathway inhibitor acting on SMAD2/3 signaling 45 . Data show that FA lymphoblast treated with Luspatercept increased the miR-29a-3p expression, reaching the level of control cells ( Figure 6 ). Download figure Open in new tab Figure 6. miR-29a-3p expression in Fanc-A lymphoblasts treated with Luspatercept The graph shows the miR-29a-3p expression in Fanc-A lymphoblasts corrected with the WT Fanc-A gene (Fanc-A corr), Fanc-A lymphoblasts (Fanc-A), Fanc-A lymphoblasts treated with Luspatercept (TGF-beta pathway inhibitor) for 48h (Fanc-A + Luspatercept). Data are expressed as mean ± SD and are representative of three independent experiments **indicates a significant difference for p <0.01 between Fanc-A corr and Fanc-A. ## indicates a significant difference for p <0.01between Fanc-A and Fanc-A + Luspatercept. On the other hand, miR-29a-3p also affects TGF-β signaling, as miR-29a-3p transfection in Fanc-A lymphoblasts reduced SMAD3 hyperphosphorylation to levels comparable to those observed in corrected cells ( Figure 7 ). Download figure Open in new tab Figure 7. SMAD3 phosphorylation in Fanc-A lymphoblasts transfected with miR-29a-3p All analyses were conducted on Fanc-A lymphoblasts corrected with the WT Fanc-A gene (Fanc-A corr), Fanc-A lymphoblasts (Fanc-A), Fanc-A lymphoblasts transfected with a miRNA mimic negative control for 48h (Fanc-A scr), and Fanc-A lymphoblasts transfected with miR-29a-3p for 48h (Fanc-A + miR-29a-3p). (A)Representative WB signals of phospho-SMAD3 (Ser423/425) and total SMAD3; Actin signal was used as housekeeping. (B) The ratio ofphosphorylated and total forms of SMAD3 signals. Data in panel B are expressed as mean ± SD and are representative of three independent experiments. **** indicates a significant difference for p<0.0001 between Fanc-A corr and other samples. ## indicates a significant difference for p < 0.01 between Fanc-A + miR-29a-3p and Fanc-A scr. Inhibition of TGF-βor IGF1 signaling reduces the oxidative damage and improves the oxidative phosphorylation in Fanc-A lymphoblasts As shown in Figure 6 , the TGF-βsignal reduction leads to a recoveryof miR-29a-3p content in FA lymphoblasts, suggesting the evaluation of possible effects of Luspatercept on antioxidant defenses, DNA damage, and energy metabolism. Moreover, since FA cells displayed elevated IGF1 expression compared to the control, which, however, decreased after transfection with miR-29a-3p ( Figure 3 ), the same investigation was considered appropriate for Klotho, an IGF1 signaling inhibitor 46 . The data show that both the inhibition of the TGF-β pathway and IGF1 signaling exhibit the same trend observed after transfecting FA cells with miR-29a-3p. In particular,Luspatercept and Klothotreatmentsincreased AO response ( Figure 8A ), an evident reduction in oxidative stress accumulation( Figure 8B-C ),a recovery of mitochondrial function( Figure 8D-G ), and of cellular energy status ( Figure 8H-L ). Download figure Open in new tab Figure 8. Antioxidant defenses, oxidative stress, and energy metabolism were modulated by Luspaterceptor Klothotreatment in Fanc-A lymphoblasts. All analyses were conducted on Fanc-A lymphoblasts corrected with the WT Fanc-A gene (Fanc-A corr), Fanc-A lymphoblasts (Fanc-A), Fanc-A lymphoblasts treated with Luspatercept (TGF-β pathway inhibitor) for 48h(Fanc-A + Luspatercept), and Fanc-A lymphoblasts treated with Klotho (IGF1 signaling inhibitor) for 48h (Fanc-A + Klotho). (A)Catalase activity as an antioxidant defense marker. (B) Malondialdehyde (MDA) intracellular concentration, as a lipid peroxidation marker. (C) 8-hydroxy-2’-deoxyguanosine (8-OHdG) content as a DNA oxidation marker. (D) ATP synthesis through F o F 1 -ATP synthase. (E) Oxygen consumption rate (OCR). (F) P/O value, an OxPhos efficiency marker. For Panels D, E, and F, the analyses were conducted in the presence of pyruvate plus malate (P/M) or succinate (Succ) to induce the OxPhos pathways led by Complex I or Complex II, respectively. (G) Electron transfer between Complexes I and III. (H) Intracellular ATP content. (I) Intracellular AMP content. (J) Cellular energy status is obtained by calculating the ATP/AMP ratio. Data are expressed as mean ± SD and are representative of three independent experiments for Panels A and E-G and six independent experiments for Panels B-D and H-K. *, **, ***, and **** indicate a significant difference for p <0.05, 0.01, 0.001, and 0.0001, respectively, between Fanc-A corr and Fanc-A. ##, ###, and ### indicate a significant difference for p < 0.01, 0.001, and 0.0001, respectively, between Fanc-A and Fanc-A + Luspatercept. Luspatercept and Klotho treatments reduce the hyperphosphorylation of FOXO3a, SGK1, and AKT Since Luspatercept and Klotho treatments modulate miR-29a-3p expression and downstream function, respectively, their effects on the hyperphosphorylation of FOXO3a, AKT, and IGF1 have been investigated. Data reported in Figure 9 show that bothtreatments can reduce the phosphorylation level of miR-29a-3p targets, restoring levels similar to the control. Download figure Open in new tab Figure 9. Luspatercept and Klothotreatmentsmodulate the FOXO3a, AKT, and SGK1 phosphorylation in Fanc-A lymphoblasts. All analyses were conducted on Fanc-A lymphoblasts corrected with the WT Fanc-A gene (Fanc-A corr), Fanc-A lymphoblasts (Fanc-A), Fanc-A lymphoblasts treated with Luspatercept (TGF-beta pathway inhibitor) for 48h(Fanc-A + Luspatercept), and Fanc-A lymphoblasts treated with Klotho (IGF1 signaling inhibitor) for 48h (Fanc-A + Klotho). (A) Representative WB signals of: phospho-FOXO3a (Ser253); total FOXO3a; phosphor-AKT (Ser473); total AKT; phosphor-SGK1 (Ser422); total SGK1. Actin signal was used as housekeeping. (B) The ratio of phosphorylated and total forms of FOXO3a signals. (C) The ratio of phosphorylated and total forms of AKT signals. (D) The ratio of phosphorylated and total forms of SGK1 signals. Data in panels B, C, and D are expressed as mean ± SD and are representative of three independent experiments. **** indicate a significant difference for p <0.0001 between Fanc-A corr and other samples. ### and #### indicate a significant difference for p <0.001 or 0.0001, respectively, between Fanc-A + miR-29a-3p and Fanc-A scr. DISCUSSION The data presented herein provide novel insights into the role of the miR-29a-3p and TGFβ axis in FA pathogenesis, particularly concerning mitochondrial dysfunction, redox balance, and DNA damage accumulation. These findings suggest that, although the defect in DNA damage repair is the principal cause of FA, additional molecular mechanisms contribute to maintaining an altered cellular state, perpetuating a vicious cycle that exacerbates DNA damage over time. Previous studies have highlighted the dysregulation of several miRNAs in FA cells 30 , 31 . However, to the best of our knowledge, this is the first study to demonstrate that the downregulation of miR-29a-3p impacts energetic and redox imbalances typical of FA cells. In detail, we report that the miR-29a-3p transfection improved OxPhos in terms of function and coupling and promoted the antioxidant enzyme expression and activity in FA lymphoblasts and fibroblasts. This enhancement of redox balance prevented membrane lipoperoxidation, as evidenced by reduced MDA levels, and mitigated DNA damage, as indicated by decreased 8-OHdG content and reduced H2AX phosphorylation. On the other hand, miR-29a-3p has emerged as a critical regulator, modulating the expression of genes involved in mitochondrial activity 47 , redox balance 32 , DNA damage 32 , and cell proliferation 48 . To understand the mechanism underlying the influence of miR-29a-3p on the energy metabolism and redox balance of FA cells, we focused our attention on FOXO3a, a miR-29a-3p-targeted transcription factor playing a pivotal role in the mitochondrial metabolism modulation 47 . On the other hand, miR-29a-3p appears essential for the self-renewal of hematopoietic stem cells just by controlling mitochondrial function 49 . Our data show that FA cells displayed a threefold higher expression of the FOXO3 gene compared to control cells. This elevated expression in FA cells could explain the metabolic alterations characterizing FA cells as FOXO3a activation causes a reduction in mitochondrial DNA copy number, mitochondrial protein expression, respiratory complexes, and mitochondrial respiratory activity 50 . This hypothesis is confirmed by the recovery of OxPhos activity and efficiency as well as the cellular energy status improvement observed after the reduction of FOXO3 gene expression following the transfection with miR-29a-3p both in FA lymphoblasts and fibroblasts. In addition, behind the FOXO3 gene expression, it is necessary to consider that FOXO3a protein functiondepends on its cellular localization, which is regulated by various post-translational modifications; for example, FOXO3a nuclear translocation is promoted by the macrophage stimulating 1 (MST1)-induced phosphorylation while it is inhibited by thephosphorylation on Thr32 and Ser253 by Ser473-phosphorylated AKT 41 . Since AKT hyperphosphorylation is a hallmark of FA cells 51 , it is quite surprising to observe that, in FA lymphoblasts, FOXO3a appears hyperphosphorylated at Ser253 and accumulates in the cytoplasmic fraction. Interestingly, both FOXO3a and AKT hyperphosphorylation were reduced in cells transfected with miR-29a-3p, leading to an increase in FOXO3a levels in the nuclear fraction, suggesting that the AKT pathway is also regulated by miR-29a-3p 52 . Indeed, a recent study by Pang et al. proposes that the FOXO3a localization in nuclei depends on the mono-ubiquitination of FANCD2 and is independent of AKT phosphorylation. However, this discrepancy suggests that the regulation of FOXO3a nuclear localization is highly complex and involves additional post-translational modifications 53 . Since FOXO3a also promotes ROS detoxification by increasingcellular antioxidant defense 54 and genome stability 35 , it is plausible to speculate that the miR-29a-3p-induced FOXO3 gene and protein modulation also leadsto the increase in catalase activity and the decrease of the DNA damage accumulation observed in transfected FA lymphoblasts and fibroblasts. The SGK1, another miR-29a-3p target gene, also modulates FOXO3a phosphorylation and the consequent cellular localization. In detail, SGK1 phosphorylated on Ser422 leads to the FOXO3a negative modulation 41 . Regarding this, our data show that FA lymphoblasts are characterized by elevated SGK1 gene expression and hyperphosphorylation of the SGK1 protein, which return to levels similar to those of controls following transfection with miR-29a-3p. Therefore, these data suggest that miR-29a-3p transfection exerts a dual effect on FOXO3a: it acts as a direct modulator of its gene expression while also influencing its cellular localization and function through the regulation of AKT and SGK1, another target gene of miR-29a-3p. To investigate the mechanism underlying the downregulation of miR-29a-3p in FA cells, we focused on the TGF-β signaling, as this pathway is hyperactivated in FA cells 55 . In addition, the miR-29a-3p expression is modulated by TGF-β through the signal transducer SMAD3 56 . Indeed, our data show that inhibition of TGF-β signaling following treatment with Luspatercept leads to an increased expression of miR-29a-3p in FA lymphoblasts, which, in turn, exerts positive effects on mitochondrial metabolism, cellular energy status, and the activation of antioxidant defenses. The same results were also observed in the presence of Klotho, an inhibitor of IGF1 signaling, which is an effector of the TGF-β pathway 57 . IGF1 expression is elevated in FA cells and is regulated by miR-29a-3p. This finding is particularly interesting given that FA patients are characterized by borderline hyperglycemia 58 , which could lead to increasing in IGF1 signaling. The inhibition of the TGF-β signal throughLuspatercept or Klotho also causes a reduction of DNA damage accumulation, as demonstrated by the decrement in 8-OHdG content and H2AX phosphorylation. On the other hand, in FA cells, the hyperactivation of TGF-β signaling promotes DNA repair via non-homologous end joining (NHEJ), an error-prone repair pathway that contributes to toxicity in FA hematopoietic stem cells 55 . Conversely, inhibition of the TGF-β pathway through silencing of the SMAD3 gene in FA mice and human cells modulates the expression of DNA repair genes in favor of homologous recombination (HR) over NHEJ, resulting in increased growth of hematopoietic progenitors and rescue of bone marrow failure 29 . In addition, TGF-β signaling inhibition plays a pivotal role in modulating the PI3K/AKT pathway 59 , 60 also influencing FOXO3a, as demonstrated by the decreased phosphorylation of AKT, SGK1, and FOXO3a following treatment with Luspatercept and Klotho. Interestingly, miR-29a-3p is also a modulator of the TGF-β pathway, as its transfection into FA cells reduces the phosphorylation of SMAD3, a key effector of the TGF-β pathway. In other words, the data suggest that miR-29a-3p and TGF-β are connected through a negative feedback loop that mutually regulates their expression. Therefore, if increased miR-29a-3p expression can restore mitochondrial functionality and redox balance while reducing DNA damage accumulation in FA cells through the modulation of AKT, SGK1, and FOXO3a, it is plausible to hypothesize that thismodulation, in turn, reduces the release of pro-inflammatory cytokines, leading to a consequent decrease of the TGF-β signaling, further promoting miR-29a-3p expression. CONCLUSIONS The data presented in this study demonstrate the central role of miR-29a-3p and the TGF-β pathway in the pathogenesis of FA, as the overexpression of the former and the reduction of the latter promote the recovery of metabolic functionality, the restoration of redox balance, and the reduction of DNA damage accumulation. Furthermore, the literature reports that miR-29a-3p plays a pivotal role in hematopoiesis, supporting self-renewal, lineage commitment, and HSC differentiation 49 . In addition, altered miR-29a-3p expression is associated with the development of Head and Neck Squamous Cell Carcinoma 61 , a type of cancer highly prevalent in individuals with FA 62 . These findings also suggest that the cellular dysfunctions characteristic of FA, which lead to DNA damage accumulation, depend on the activation of multiple, partially redundant signaling pathways. Consequently, any potential therapeutic approach should aim to modulate these pathways at multiple levels to ensure effectiveness. MATERIALS AND METHODS Cell Culture and treatments Fanc-A lymphoblast cell lines and FANC-A primary fibroblast cell lines (Fibro FA) that carried out different mutations of the FANC-A gene were obtained from the ‘‘Cell Line and DNA Biobank from Patients affected by Genetic Diseases’’ (G. Gaslini Institute) - Telethon Genetic Biobank Network (Project No. GTB07001). As controls, isogenic FA-corr cell lines generated by the same FANC-A lymphoblast and fibroblast cell lines corrected with S11FAIN retrovirus (Lympho FA-corr and Fibro FA-corr) were employed 34 . The study was conducted following the Declaration of Helsinki and approved by the regional ethics committee, protocol JS002, register number 037-21/01/2019. All the subjects or their legal guardians gave written informed consent to the investigation. For lymphoblast cell lines, RPMI-1640 medium (GIBCO, Billing, MT, USA) containing 10% fetal bovine serum (FBS, Euroclone, Milano, Italy), 100 U/mL penicillin, and 100 μg/mL streptomycin (Euroclone, Milano, Italy) was used, and the cells were grown at 37 °C with a 5% CO 2 34 . Primary fibroblasts were cultured as a monolayer in DMEM high glucose with glutamax® (GIBCO, Billing, MT, USA), containing 10% FBS (Euroclone, Milano, Italy), 100 U/mL penicillin, and 100 μg/mL streptomycin (Euroclone, Milano, Italy) at 37 °C with a 5% CO 2 34 . FA lymphoblast and fibroblast cells transfection with miR-29a-3p FA lymphoblast cells were transfected with miR-29a-3p mimic (ThermoFisher Scientific, Waltham, MA, USA), using the lipofectamineRNAiMAXTransfectionReagent(Invitrogen, Waltham, MA, USA) according to the manufacturer’s instruction. In detail, for subsequent RNA extraction, 500.000 cells grown in 6-well plates were transfected with 25 pmol of miR-29a-3p mimic or negative control mimic using 7.5μl lipofectamine. After 48h, cells were harvested, washed with PBS, lysed, and RNA was extracted.For the subsequent biochemical analysis, 7.5×10 6 cells grown in 75 cm 2 flasks were transfected with 187.5 pmol of miR-29a-3p mimic or negative control mimic using 56.25 μl lipofectamine. Cells were processed 48h post-transfection. FA fibroblast cells were transfected with miR-29a-3p mimic (Thermo Fisher Scientific, Waltham, MA, USA) using the same transfectionreagent(Invitrogen, Waltham, MA, USA). In detail, for the subsequent RNA extraction, 75.000 cells grown in 6-well plates were transfected with 25 pmol of miR-29a-3p mimic or negative control mimic using 7.5 μl lipofectamine. After 48h, cells were harvested, washed with PBS, lysed, and RNA was extracted.For subsequent biochemical analysis, 500.000 cells grown in 175 cm 2 flasks were transfected with 437.5 pmol of miR-29a-3p mimic or negative control mimic using 131,25 μllipofectamine. Cells were processed 48h post-transfection. FA lymphoblast cell treatments To inhibit the TGF-β pathway, lymphoblasts were treated with Luspatercept, an inhibitor acting on the SMAD2/3 signaling 45 . In detail, 500.000 cells were plated in a 2 ml culture medium in the presence of 10 μg/ml Luspatercept for 48h. To inhibit IGF1 signaling, 500.000 lymphoblasts were treated with 400 pM Klotho 46 for 48h. In silico selection of pathway-related miR-29a-3p target genes To identify putative miR-29a-3p target genes potentially involved in Fanconi cell metabolism impairment, we utilized the miRPathDB v2.0 database ( https://mpd.bioinf.uni-sb.de ). A curated list of miR-29a-3p-regulated genes associated with DNA damage response, oxidative stress, mitochondrial metabolism, lipid metabolism, and apoptosis was generated by evaluating the strength of predicted miRNA-target gene interactions using TargetScan ( https://mpd.bioinf.uni-sb.de ). Further refinement was conducted by assessing the role and the subcellular localization of each gene with the help of the NCBI Gene database ( https://www.ncbi.nlm.nih.gov/gene ) and GeneCards ( https://www.genecards.org ). The final list is presented in Table 1 . RNA isolation to evaluate the expression of miR-29a-3p and FOXO3, SGK1, and IGF1 genes RNA, including the small RNA fraction, was extracted using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The expression of miR-29a-3p was assessed using a specific TaqMan MicroRNA Assay (Applied Biosystems, Waltham, MA, USA). Briefly, 10 ng of RNA was reverse-transcribed using the TaqMan MicroRNA Reverse Transcription Kit and the specific RT primer. Real-time PCR was performed in triplicate with specific primers. miRNA expression levels were normalized to RNU44 expression. To evaluate the expression of the FOXO3, SGK1, and IGF1 genes, 100 ng of RNA were reverse transcribed using the SuperScript VILO IV cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA). The resulting cDNA was used for real-time PCR with primers provided in the specific TaqMan Gene Expression Assays (Applied Biosystems, Waltham, MA, USA). Gene expression levels were normalized to GAPDH expression. All experiments were performed in triplicate. Catalase activity evaluation Catalase (CAT) activity was assayed as a marker of cellular antioxidant defenses. For each spectrophotometric assay, 50 μg of total proteins were used, following the H 2 O 2 decomposition at 240 nm. The assay mix contained 50 mM phosphate buffer (pH 7.0) and 5 mM H 2 O 2 (Sigma-Aldrich, St. Louis, MO, USA) 21 . Oxidative stress markers evaluation The thiobarbituric acid reactive substances (TBARS) assay was employed to quantify malondialdehyde (MDA), an indicator of lipid peroxidation. The TBARS reagent was prepared using 0.25 M HCl, 0.25 mM trichloroacetic acid, and 26 mM thiobarbituric acid (all sourced from Merck, Darmstadt, Germany). A total of 50 µg of protein, dissolved in 300 µl of Milli-Q water, was mixed with 600 µl of the TBARS solution. The reaction mixture was incubated at 95 °C for 1 hour and the absorbance was measured spectrophotometrically at 532 nm. Standard solutions of MDA, with concentrations ranging from 1 to 20 µM, were used to generate a calibration curve 21 . To measure 8-hydroxy-2-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage, an ELISA kit (#ab201734, Abcam, Cambridge, UK) was utilized according to the manufacturer’s instructions. Evaluation of aerobic metabolism function and efficiency The OxPhos function was assessed by measuring the oxygen consumption rate (OCR) and F o F 1 -ATP synthase activity. OCR was determined using an amperometric electrode (Unisense Microrespiration, Aarhus, Denmark) in a closed chamber. For each test, 10 5 cells were permeabilized for 1 minute with 0.03 mg/ml digitonin and then used in the assay. To activate the respiratory pathway led by Complex I or Complex II, 10 mM pyruvate with 5 mM malate (Merck, Darmstadt, Germany) or 20 mM succinate Merck, Darmstadt, Germany) were added, respectively 21 . F o F 1 -ATP synthase activity was measured in 10 5 cells suspended in PBS plus 0.6 mM ouabain Merck, Darmstadt, Germany) and 0.25 mM di(adenosine)-5-penta-phosphate (an adenylate kinase inhibitor, Merck, Darmstadt, Germany). After 10 minutes of incubation, 10 mM pyruvate with 5 mM malate (Merck, Darmstadt, Germany) or 20 mM succinate (Merck, Darmstadt, Germany) were added to stimulate pathways mediated by Complex I or II, respectively. ATP production was quantified using a luminometer (GloMax® 20/20 Luminometer, Promega Italia, Milan, Italy),employing the luciferin/luciferase chemiluminescent method (ATP bioluminescence assay kit CLS II, #11699695001 Roche, Basel, Switzerland). Measurements were taken at 30-second intervals over 2minutes 21 . The OxPhos efficiency was determined by calculating the P/O ratio, which represents the amount of ATP synthesized aerobically per oxygen molecule consumed. Mitochondria with optimal efficiency exhibit P/O ratios around 2.5 for pyruvate and malate or 1.5 for succinate. Ratios below these thresholds suggest incomplete oxygen utilization for ATP production, potentially reflecting increased ROS generation 21 . Assessment of ATP and AMP intracellular concentration and the consequent cellular energy status ATP and AMP concentrations were assayed in 50 μg of total protein. ATP content spectrophotometric analysis was performed following the NADPreduction at 340 nm. The assay solution contained 100 mMTris-HCl (pH 8.0; Merck, Darmstadt, Germany), 0.2 mM NADP(Merck, Darmstadt, Germany), 5 mM MgCl 2 (Merck, Darmstadt, Germany), 50 mM glucose (Merck, Darmstadt, Germany), and 3 μg of pure hexokinase and glucose-6-phosphate dehydrogenase (Merck, Darmstadt, Germany) 20 . AMP was measured spectrophotometrically following the NADH oxidation at 340 nm. The reaction medium was composed of 100 mMTris-HCl (pH 8.0; Merck, Darmstadt, Germany), 5 mM MgCl 2 (Merck, Darmstadt, Germany), 0.2 mM ATP (Merck, Darmstadt, Germany), 10 mMphosphoenolpyruvate (Merck, Darmstadt, Germany), 0.15 mM NADH (Merck, Darmstadt, Germany), 10 IU adenylate kinase, 25 IU pyruvate kinase, and 15 IU lactate dehydrogenase (Merck, Darmstadt, Germany) 20 . The cellular energy status was calculated as the ratio between intracellular concentration of ATP and AMP (ATP/AMP ratio) 20 . Evaluation of electron transfer between Complex I and Complex III To evaluate the electron transfer between Complex I and Complex III, a spectrophotometric assay was performed following the reduction of oxidized cytochrome c (cyt c ) at 550 nm in the presence of NADH. The assay medium contained 50 mMTris-HCl (pH 7.4; Merck, Darmstadt, Germany), 5 mMKCl (Merck, Darmstadt, Germany), 2 mM MgCl 2 (Merck, Darmstadt, Germany), 0.5 M NaCl (Merck, Darmstadt, Germany), 0.03% oxidized cyt c (Merck, Darmstadt, Germany), and 0.6 mM NADH (Merck, Darmstadt, Germany) 20 . Cellular fraction separation To obtain nuclei-enriched fractions (N), cells were homogenated in a 0.25 M sucrose solution. This homogenate (H) was centrifuged at 800 g for 10 min. The supernatant was collected and used as a cytoplasmic fraction (C), and the pellet was resuspended in 0.25 M sucrose solution and centrifuged again at 800 g for 10 min to obtain N. Western Blot Analysis Denaturing electrophoresis (SDS-PAGE) was performed on 30 μg of proteins employing a 4–20% gradient gel (BioRad, Hercules, CA, USA). The following primary antibodies were used: phospho-H2AX (#05-636, Merck, Darmstadt, Germany). phospho-FOXO3a (#SAB5701786 Merck, Darmstadt, Germany), FOXO3a (#2497S, Cell Signaling Technology, Beverly, MA, USA), GAPDH (#2118, Cell Signaling Technology, Beverly, MA, USA), H3 (#4499, Cell Signaling Technology, Beverly, MA, USA), phospho-AKT (#4060S, Cell Signaling Technology, Beverly, MA, USA), AKT (#4691S, Cell Signaling Technology, Beverly, MA, USA), phospho-SGK1 (#5599, Cell Signaling Technology, Beverly, MA, USA), SGK1 (#711183, Thermo Fisher Scientific, Waltham, MA, USA), phospho-SMAD3 (#9520S, Cell Signaling Technology, Beverly, MA, USA), SMAD3 (#9523S, Cell Signaling Technology, Beverly, MA, USA), and β-Actin (#MA1-140, ThermoFisher Scientific, Waltham. MA, USA). All primary antibodies were diluted following the manufacturer’s instructions in PBS plus 0.15% Tween 20 (PBSt; Roche, Basel, Switzerland). Specific secondary antibodies were employed (Merck, Darmstadt, Germany, all diluted 1:10,000 in PBSt). Bands were detected in the presence of an enhanced chemiluminescence substrate (ECL, BioRad, Hercules, CA, USA), bya chemiluminescence system (Alliance 6.7 WL 20M, UVITEC, Cambridge, UK). Band intensity was evaluated by UV1D software (UVITEC, Cambridge, UK). All bands of interest were normalized versus the actin signal detected on the same membrane. Statistical Analysis Data were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test by Prism 9 Software (GraphPad Software Inc., Boston, MA, USA). Data are expressed as mean ± SD and are representative of at least three independent experiments. An error with a probability of p<0.05 was considered significant. FUNDING This research was funded by Associazione Italiana Ricerca sull’anemia di Fanconi ODV— AIRFA (Grant number: #AIRFA2019 to Si.R.). AUTHOR CONTRIBUTIONS Conceptualization, S.R., E.C., P.D., and Si.R.; investigation, N.B., S.R., V.C., M.B., M.S:, F.C., E.C., and Si.R.; resources, C.D., E.C., and Si.R.; data curation, S.R., E.C., Si.R.; writing, N.B., S.R., V.C., P.D., E.C., Si.R; supervision, C.B., F.P., C.D. All authors have read and agreed to the published version of the manuscript. CONFLICTS OF INTEREST The authors declare no conflict of interest. DATA AVAILABILITY All data supporting the conclusions of this study can be found in the Article and Supplementary Material. SUPPLEMENTARY MATERIAL Download figure Open in new tab Figure S1. miR-29a-3p expression in Fanc-A fibroblasts. The graph shows the comparison of miR-29a-3p expression between Fanc-A fibroblasts corrected with the WT Fanc-A gene (Fanc-A corr) and Fanc-A fibroblasts (Fanc-A). RNU44 was used as a reference control.Data are expressed as mean ± SD and are representative of three independent experiments. *indicates a significant difference for p < 0.05 between Fanc-A corr and Fanc-A. Download figure Open in new tab Figure S2. Antioxidant defenses, oxidative stress, and energy metabolism were modulated by miR-29a-3p expression in Fanc-A fibroblasts. All analyses were conducted on Fanc-A fibroblasts corrected with the WT Fanc-A gene (Fanc-A corr), Fanc-A fibroblasts (Fanc-A), Fanc-A fibroblasts transfected with empty vector for 48h (Fanc-A scr), and Fanc-A fibroblasts transfected with miR-29a-3p for 48h (Fanc-A + miR-29a-3p). (A) Catalase activity, as an antioxidant defenses marker. (B) Malondialdehyde (MDA) intracellular concentration, as a lipid peroxidation marker. (C) 8-hydroxy-2’-deoxyguanosine (8-OHdG) content, as a DNA oxidation marker. (D) ATP synthesis through F o F1-ATP synthase. (E) Oxygen consumption rate (OCR). (F) P/O value, an OxPhos efficiency marker. For Panels D, E, and F, the analyses were conducted in the presence of pyruvate plus malate (P/M) or succinate (Succ) to induce the OxPhos pathways led by Complex I or Complex II, respectively. (G) Electron transfer between Complexes I and III. (H) Intracellular ATP content. (I) Intracellular AMP content. (J) Cellular energy status is obtained by calculating the ATP/AMP ratio. Data are expressed as mean ± SD and are representative of three independent experiments for Panels D-F and six independent experiments for Panels A-C and G-J. **, ***, and **** indicate a significant difference for p < 0.01, 0.001, or 0.0001, respectively, between Fanc-A corr and Fanc-A or Fanc-A scr. ##, ###, and ### indicate a significant difference for p < 0.01, 0.001, or 0.0001, respectively, between Fanc-A + miR-29a-3p and Fanc-A or Fanc-A scr. Download figure Open in new tab Figure S3. FOXO3, SGK1, and IGF1 expression in Fanc-A fibroblasts. Graphs show the comparison of FOXO3 (A), SGK1 (B), and IGF1 (C) expression in (i) Fanc-A cells corrected with the wt Fanc-A gene (Fanc-A corr), (ii) Fanc-A cells (Fanc-A), (iii) Fanc-A cells transfected with a miRNA mimic negative control for 48h (Fanc-A scr), and (iv) Fanc-A cells transfected with miR-29a-3p for 48h (Fanc-A + miR-29a-3p). GAPDH was used as the reference control. Data are expressed as mean ± SD and are representative of three independent experiments. *, ** or **** indicate a significant difference for p < 0.05, 0.01, or 0.0001, respectively, between Fanc-A corr and the other samples. # and ### indicates a significant difference for p < 0.05 or 0.001, respectively, between Fanc-A + miR-29a-3p and Fanc-A or Fanc-A scr. ACKNOWLEDGEMENTS We want to acknowledge ERG SpA, Cambiaso and Risso, Rimorchiatori Riu-niti, and Saar Depositi Oleari Portuali for supporting the activity of the Clinical and Experimental Hematology Unit of the G. Gaslini Institute. REFERENCES 1. ↵ de Winter , J. P. & Joenje , H. The genetic and molecular basis of Fanconi anemia . Mutat Res 668 , 11 – 19 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ Svahn , J. & Dufour , C . Fanconi anemia - learning from children . Pediatr Rep 3 Suppl 2 , e8 – e8 ( 2011 ). OpenUrl PubMed 3. ↵ Grompe , M. & D’Andrea , A . Fanconi anemia and DNA repair . Hum Mol Genet 10 , 2253 – 2259 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 4. ↵ Yang , Y.-G. et al. The Fanconi anemia group A protein modulates homologous repair of DNA double-strand breaks in mammalian cells . Carcinogenesis 26 , 1731 – 1740 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 5. ↵ Ravera , S. , Dufour , C. , Degan , P. & Cappelli , E . Fanconi anemia: From DNA repair to metabolism . European Journal of Human Genetics 26 , ( 2018 ). 6. ↵ Cappelli , E. et al. Mitochondrial respiratory complex I defects in Fanconi anemia . Trends Mol Med 19 , ( 2013 ). 7. ↵ Du , W. , Adam , Z. , Rani , R. , Zhang , X. & Pang , Q . Oxidative stress in Fanconi anemia hematopoiesis and disease progression . Antioxid Redox Signal 10 , 1909 – 1921 ( 2008 ). OpenUrl CrossRef PubMed 8. Li , J. et al. Fanconi Anemia Links Reactive Oxygen Species to Insulin Resistance and Obesity . Antioxid Redox Signal 17 , 1083 – 1098 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 9. ↵ Zhang , X. , Sejas , D. P. , Qiu , Y. , Williams , D. A. & Pang , Q . Inflammatory ROS promote and cooperate with the Fanconi anemia mutation for hematopoietic senescence . J Cell Sci 120 , 1572 – 1583 ( 2007 ). OpenUrl Abstract / FREE Full Text 10. ↵ Vanderwerf , S. M. et al. TLR8-dependent TNF-α overexpression in Fanconi anemia group C cells . Blood 114 , 5290 – 5298 ( 2009 ). OpenUrl Abstract / FREE Full Text 11. Svahn , J. et al. P38 mitogen-activated protein kinase inhibition enhances invitro erythropoiesis of Fanconi anemia, complementation group A-deficient bonemarrow cells . Exp Hematol 43 , ( 2015 ). 12. ↵ Dufour , C. et al. TNF-alpha and IFN-gamma are overexpressed in the bone marrow of Fanconi anemia patients and TNF-alpha suppresses erythropoiesis in vitro . Blood 102 , 2053 – 2059 ( 2003 ). OpenUrl Abstract / FREE Full Text 13. ↵ Korthof , E. T. et al. Immunological profile of Fanconi anemia: a multicentric retrospective analysis of 61 patients . Am J Hematol 88 , 472 – 476 ( 2013 ). OpenUrl CrossRef PubMed 14. ↵ Ravera , S. et al. Mitochondrial respiratory chain Complex i defects in Fanconi anemia complementation group A . Biochimie 95 , ( 2013 ). 15. ↵ Bottega , R. et al. Hypomorphic FANCA mutations correlate with mild mitochondrial and clinical phenotype in Fanconi anemia . Haematologica 103 , ( 2018 ). 16. ↵ Cappelli , E. et al. Defects in mitochondrial energetic function compels Fanconi Anaemia cells to glycolytic metabolism . BiochimBiophys Acta Mol Basis Dis 1863 , ( 2017 ). 17. ↵ Cappelli , E. et al. The passage from bone marrow niche to bloodstream triggers the metabolic impairment in Fanconi Anemia mononuclear cells . Redox Biol 36 , ( 2020 ). 18. ↵ Lyakhovich , A . Damaged mitochondria and overproduction of ROS in Fanconi anemia cells . Rare Diseases 1 , e24048 – e24048 ( 2013 ). OpenUrl CrossRef 19. ↵ Pagano , G. et al. Oxidative stress as a multiple effector in Fanconi anaemia clinical phenotype . Eur J Haematol 75 , 93 – 100 ( 2005 ). OpenUrl CrossRef PubMed 20. ↵ Cappelli , E. et al. A Multidrug Approach to Modulate the Mitochondrial Metabolism Impairment and Relative Oxidative Stress in Fanconi Anemia Complementation Group A . Metabolites 12 , 6 ( 2021 ). OpenUrl CrossRef PubMed 21. ↵ Bertola , N. et al. Effects of Deacetylase Inhibition on the Activation of the Antioxidant Response and Aerobic Metabolism in Cellular Models of Fanconi Anemia . Antioxidants (Basel) 12 , ( 2023 ). 22. Pagano , G. & Youssoufian , H. Fanconi anaemia proteins: major roles in cell protection against oxidative damage . Bioessays 25 , 589 – 595 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 23. ↵ Kumari , U. , Ya Jun , W. , Huat Bay , B. & Lyakhovich , A. Evidence of mitochondrial dysfunction and impaired ROS detoxifying machinery in Fanconi anemia cells . Oncogene 33 , 165 – 172 ( 2014 ). OpenUrl CrossRef PubMed 24. ↵ Degan , P. et al. In vivo accumulation of 8-hydroxy-2’-deoxyguanosine in DNA correlates with release of reactive oxygen species in Fanconi’s anaemia families . Carcinogenesis 16 , 735 – 741 ( 1995 ). OpenUrl CrossRef PubMed Web of Science 25. ↵ Brosh , R. M. , Bellani , M. , Liu , Y. & Seidman , M. M . Fanconi Anemia: a DNA Repair Disorder Characterized by Accelerated Decline of the Hematopoietic Stem Cell Compartment and Other Features of Aging . Ageing Res Rev 33 , 67 ( 2017 ). OpenUrl CrossRef PubMed 26. ↵ Helbling-Leclerc , A. , Garcin , C. & Rosselli , F . Beyond DNA repair and chromosome instability—Fanconi anaemia as a cellular senescence-associated syndrome . Cell Death Differ 28 , 1159 ( 2021 ). OpenUrl CrossRef PubMed 27. ↵ Landelouci , K. , Sinha , S. & Pépin , G . Type-I Interferon Signaling in Fanconi Anemia . Front Cell Infect Microbiol 12 , 820273 ( 2022 ). OpenUrl CrossRef PubMed 28. ↵ Wang , J. , Erlacher , M. & Fernandez-Orth , J . The role of inflammation in hematopoiesis and bone marrow failure: What can we learn from mouse models? Front Immunol 13 , ( 2022 ). 29. ↵ Zhang , H. et al. TGF-β Inhibition Rescues Hematopoietic Stem Cell Defects and Bone Marrow Failure in Fanconi Anemia . Cell Stem Cell 18 , 668 ( 2016 ). OpenUrl CrossRef PubMed 30. ↵ Degan , P. et al. A Global MicroRNA Profile in Fanconi Anemia: A Pilot Study . MetabSyndrRelatDisord 17 , ( 2019 ). 31. ↵ Cappelli , E. et al. Advanced Analysis and Validation of a microRNA Signature for Fanconi Anemia . Genes 2024, Vol. 15, Page 820 15 , 820 ( 2024 ). OpenUrl CrossRef 32. ↵ Jung , Y. deunet al . Epigenetic regulation of miR-29a/miR-30c/DNMT3A axis controls SOD2 and mitochondrial oxidative stress in human mesenchymal stem cells . Redox Biol 37 , 101716 ( 2020 ). OpenUrl CrossRef PubMed 33. ↵ Yang , Y. L. , Kuo , H. C. , Wang , F. S. & Huang , Y. H . MicroRNA-29a Disrupts DNMT3b to Ameliorate Diet-Induced Non-Alcoholic Steatohepatitis in Mice . International Journal of Molecular Sciences 2019, Vol. 20, Page 1499 20 , 1499 ( 2019 ). OpenUrl CrossRef PubMed 34. ↵ Bertola , N. et al. Altered Mitochondrial Dynamic in Lymphoblasts and Fibroblasts Mutated for FANCA-A Gene: The Central Role of DRP1 . International Journal of Molecular Sciences 2023, Vol. 24, Page 6557 24 , 6557 ( 2023 ). OpenUrl CrossRef PubMed 35. ↵ Fasano , C. , Disciglio , V. , Bertora , S. , Signorile , M. L. & Simone , C . FOXO3a from the Nucleus to the Mitochondria: A Round Trip in Cellular Stress Response . Cells 2019, Vol. 8, Page 1110 8 , 1110 ( 2019 ). OpenUrl CrossRef 36. ↵ Skurk , C. et al. The Akt-regulated Forkhead Transcription Factor FOXO3a Controls Endothelial Cell Viability through Modulation of the Caspase-8 Inhibitor FLIP . Journal of Biological Chemistry 279 , 1513 – 1525 ( 2004 ). OpenUrl Abstract / FREE Full Text 37. Boccitto , M. & G. Kalb , R. Regulation of Foxo-dependent transcription by post-translational modifications . Curr Drug Targets 12 , 1303 – 1310 ( 2011 ). OpenUrl CrossRef PubMed 38. ↵ Liu , Y. et al. Critical role of FOXO3a in carcinogenesis . Molecular Cancer 2018 17:1 17 , 1 – 12 ( 2018 ). OpenUrl PubMed 39. ↵ Feehan , R. P. & Shantz , L. M . Negative regulation of the FOXO3a transcription factor by mTORC2 induces a pro-survival response following exposure to ultraviolet-B irradiation . Cell Signal 28 , 798 ( 2016 ). OpenUrl CrossRef PubMed 40. ↵ Brunet , A. et al. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor . Cell 96 , 857 – 868 ( 1999 ). OpenUrl CrossRef PubMed Web of Science 41. ↵ Wang , X. , Hu , S. & Liu , L . Phosphorylation and acetylation modifications of FOXO3a: Independently or synergistically? Oncol Lett 13 , 2867 – 2872 ( 2017 ). OpenUrl CrossRef PubMed 42. ↵ Brunet , A. et al. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a) . Mol Cell Biol 21 , 952 – 965 ( 2001 ). OpenUrl Abstract / FREE Full Text 43. ↵ Xu , X. , Hong , P. , Wang , Z. , Tang , Z. & Li , K . MicroRNAs in Transforming Growth Factor-Beta Signaling Pathway Associated With Fibrosis Involving Different Systems of the Human Body . Front Mol Biosci 8 , 707461 ( 2021 ). OpenUrl CrossRef PubMed 44. ↵ Zhang , H. et al. Bone Marrow Failure in Fanconi Anemia from Hyperactive TGF-β Signaling . Blood 124 , 356 ( 2014 ). OpenUrl CrossRef 45. ↵ Hatzimichael , E. , Timotheatou , D. , Koumpis , E. , Benetatos , L. & Makis , A . Luspatercept: A New Tool for the Treatment of Anemia Related to β-Thalassemia, Myelodysplastic Syndromes and Primary Myelofibrosis . Diseases 2022, Vol. 10, Page 85 10 , 85 ( 2022 ). OpenUrl CrossRef PubMed 46. ↵ Olejnik , A. , Radajewska , A. , Krzywonos-Zawadzka , A. & Bil-Lula , I . Klotho inhibits IGF1R/PI3K/AKT signalling pathway and protects the heart from oxidative stress during ischemia/reperfusion injury . Scientific Reports 2023 13:1 13 , 1 – 15 ( 2023 ). OpenUrl CrossRef PubMed 47. ↵ Dalgaard , L. T. , Sørensen , A. E. , Hardikar , A. A. & Joglekar , M. V . The microRNA-29 family: role in metabolism and metabolic disease . Am J Physiol Cell Physiol 323 , C367 – C377 ( 2022 ). OpenUrl CrossRef PubMed 48. ↵ Xiao , Z. , Wang , Y. & Ding , H . XPD suppresses cell proliferation and migration via miR-29a-3p-Mdm2/PDGF-B axis in HCC . Cell Biosci 9 , 1 – 12 ( 2019 ). OpenUrl CrossRef PubMed 49. ↵ Hu , W. et al. miR-29a maintains mouse hematopoietic stem cell self-renewal by regulating Dnmt3a . Blood 125 , 2206 ( 2015 ). OpenUrl Abstract / FREE Full Text 50. ↵ Ferber , E. C. et al. FOXO3a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression . Cell Death & Differentiation 2012 19:6 19 , 968 – 979 ( 2011 ). OpenUrl PubMed 51. ↵ Ibáñez , A. et al. Elevated levels of IL-1beta in Fanconi anaemia group A patients due to a constitutively active phosphoinositide 3-kinase-Akt pathway are capable of promoting tumour cell proliferation . Biochem J 422 , 161 – 170 ( 2009 ). OpenUrl Abstract / FREE Full Text 52. ↵ Li , X. et al. Fancd2 is required for nuclear retention of Foxo3a in hematopoietic stem cell maintenance . J Biol Chem 290 , 2715 – 2727 ( 2015 ). OpenUrl Abstract / FREE Full Text 53. ↵ Wang , Z. , Yu , T. & Huang , P . Post-translational modifications of FOXO family proteins (Review) . Mol Med Rep 14 , 4931 – 4941 ( 2016 ). OpenUrl CrossRef PubMed 54. Martins , R. , Lithgow , G. J. & Link , W . Long live FOXO: unraveling the role of FOXO proteins in aging and longevity . Aging Cell 15 , 196 – 207 ( 2016 ). OpenUrl CrossRef PubMed 55. ↵ Rodríguez , A. et al. TGFβ pathway is required for viable gestation of Fanconi anemia embryos . PLoS Genet 18 , e1010459 ( 2022 ). OpenUrl CrossRef PubMed 56. ↵ Blahna , M. T. & Hata , A . Smad-mediated regulation of microRNA biosynthesis . FEBS Lett 586 , 1906 – 1912 ( 2012 ). OpenUrl CrossRef PubMed 57. ↵ Danielpour , D. & Song , K. Cross-talk between IGF-I and TGF-beta signaling pathways . Cytokine Growth Factor Rev 17 , 59 – 74 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 58. ↵ Petryk , A. et al. Endocrine Disorders in Fanconi Anemia: Recommendations for Screening and Treatment . J Clin Endocrinol Metab 100 , 803 ( 2015 ). OpenUrl CrossRef PubMed 59. ↵ Suwanabol , P. A. et al. TGF-β and Smad3 modulate PI3K/Akt signaling pathway in vascular smooth muscle cells . Am J Physiol Heart Circ Physiol 302 , H2211 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 60. ↵ Zhang , L. , Zhou , F. & ten Dijke , P . Signaling interplay between transforming growth factor-β receptor and PI3K/AKT pathways in cancer . Trends Biochem Sci 38 , 612 – 620 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 61. ↵ Liu , Y. Bin , Wang , Y. , Zhang , M. De , Yue , W. & Sun , C. N. MicroRNA-29a functions as a tumor suppressor through targeting STAT3 in laryngeal squamous cell carcinoma . Exp Mol Pathol 116 , 104521 ( 2020 ). OpenUrl CrossRef PubMed 62. ↵ Kutler , D. I. et al. High incidence of head and neck squamous cell carcinoma in patients with Fanconi anemia . Arch Otolaryngol Head Neck Surg 129 , 106 – 112 ( 2003 ). OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted January 15, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following miR-29a-3p and TGF-β Axis in Fanconi Anemia: Mechanisms Driving Metabolic Dysfunction and Genome Stability Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share miR-29a-3p and TGF-β Axis in Fanconi Anemia: Mechanisms Driving Metabolic Dysfunction and Genome Stability Nadia Bertola , Stefano Regis , Vanessa Cossu , Matilde Balbi , Martina Serra , Fabio Corsolini , Cristina Bottino , Paolo Degan , Carlo Dufour , Filomena Pierri , Enrico Cappelli , Silvia Ravera bioRxiv 2025.01.14.632746; doi: https://doi.org/10.1101/2025.01.14.632746 Share This Article: Copy Citation Tools miR-29a-3p and TGF-β Axis in Fanconi Anemia: Mechanisms Driving Metabolic Dysfunction and Genome Stability Nadia Bertola , Stefano Regis , Vanessa Cossu , Matilde Balbi , Martina Serra , Fabio Corsolini , Cristina Bottino , Paolo Degan , Carlo Dufour , Filomena Pierri , Enrico Cappelli , Silvia Ravera bioRxiv 2025.01.14.632746; doi: https://doi.org/10.1101/2025.01.14.632746 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)