In bony vertebrates there are six ferlin genes encoding, in humans, dysferlin, otoferlin, myoferlin, Fer1L5 and 6 and the long noncoding RNA Fer1L4. Mutations in DYSF (dysferlin) can cause a range of muscle diseases with various clinical manifestations collectively known as dysferlinopathies, including limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. A mutation in MYOF (myoferlin) was linked to a muscular dystrophy accompanied by cardiomyopathy. Mutations in OTOF (otoferlin) can be the cause of nonsyndromic deafness DFNB9. Dysregulated expression of any human ferlin may be associated with development of cancer. This review provides a detailed description of functions of the vertebrate ferlins with a focus on muscle ferlins and discusses the mechanisms leading to disease development.Ferlins are multiple-C2-domain proteins involved in Ca Ferlins are described as calcium ions  or phosphatidylinositol 4,5-bisphosphate (PIP2), thereby mediating interaction with membranes. The proteins of this superfamily act as Ca2+-sensors and organizers of vesicular trafficking, signaling, lipid transfer and as enzymes for lipid modification. To fulfill these functions, MC2D proteins tether membranous organelles or recruit proteins to membranes. Interestingly, members of ten out of approximately twelve protein families within this superfamily function presynaptically. The scaffolding proteins Piccolo, RIM1 and RIM2 (Ras-related in brain 3 (Rab3)-interacting molecules) participate in the organization of the presynaptic active zone and recruitment of synaptic vesicles (SVs) to the membrane [2+-sensors is well described for synaptotagmins I, II and VII [2+-sensor in SV exocytosis, while both, DOC2B and Rabphilin promote priming of SVs [Drosophila, extended synaptotagmin [The superfamily of MC2D containing proteins includes members with two to seven confirmed or predicted C2 domains  Figure . A C2 domembrane ,14,15. Mmembrane ,17. The  and VII ,19,20,21g of SVs , whereastotagmin  and multtotagmin  promote totagmin ,26.Mediate tethering of the ER to the plasma membrane (PM) and lipid transfer (extended synaptotagmins) ,Recruit proteins to membranes and are involved in cell signaling (copines and RASAL1) ,29,30, aPhosphorylate inositol phospholipids, thereby influencing intracellular processes like signal transduction or clathrin-mediated endocytosis (PI3KC2s) .In addition to controlling presynaptic function in neurons and vesicle or organelle exocytosis in non-neuronal cells, MC2D proteins:Thus, MC2D proteins perform diverse functions and many of them control vesicular trafficking, in particular, neurotransmitter release, in one or the other way.Danio rerio), mice (Mus musculus) and humans (Homo sapiens)—representing important (model) organisms for the study of ferlin functions. Six ferlin genes were present in each of these organisms , FER1L2/OTOF (otoferlin), FER1L3/MYOF (myoferlin), FER1L5 (Fer1L5) and FER1L6 (Fer1L6), and the pseudogene FER1L4 encoding a long non-coding RNA [otofa and otofb, on different chromosomes [fer1l5 ortholog appears to be present [Human ferlin genes include five protein-encoding members, ding RNA ,32,33. Tomosomes , while n present   and localization of the target negatively charged lipids in the inner leaflet of PM have likely important consequences for their function. The topology of a tail-anchored protein with a large cytoplasmic domain, a single transmembrane domain at the C-terminus and a small luminal or extracellular domain, together with Ca2+-sensitivity make it likely that all ferlins directly operate in Ca2+-regulated intracellular membrane fusion and trafficking. As the functional part of the ferlins is oriented towards the cytoplasm, this may mean that cell-to-cell fusion and syncytia formation are only indirectly affected [Caiposomes ,41,42,43iposomes . In addiith PIP2 . The dysith PIP2 . C2 domaith PIP2 . DysferlPIP2 [2+ . InteresPIP2 [2+ . ProbablPIP2 [2+  and an aPIP2 [2+ . Thus, maffected . Of noteaffected . HoweverUnfortunately, 3D structures of full-length ferlins are still unknown, as it is difficult to isolate the full-length proteins. To date, structures of the C2A domains of otoferlin, dysferlin and myoferlin ,52,53, oFer1L4 long noncoding RNA was found to be selectively transcribed in human stomach tissue [Dysferlin is ubiquitously expressed in human tissues ,56. Myofh tissue , howeverh tissue ,66,67,68h tissue , pancreah tissue . Fer1L6 h tissue , but wash tissue .Although dysferlin mRNA and protein demonstrate a very broad tissue distribution, its mutations or absence cause rather specific disease phenotypes, affecting predominantly the skeletal muscle, which can be rescued by muscle-specific transgenic dysferlin expression in mice . Similarmdx mouse model of Duchenne muscular dystrophy (DMD) and in DMD patients biopsies [Four of the mammalian ferlins are expressed in muscle. Three of those, dysferlin, myoferlin and Fer1L5, are type I ferlins . Fer1L6 biopsies ,72,73. Mbiopsies . Fer1L5 biopsies . Interesbiopsies . Prominebiopsies  and its biopsies . Thus, ibiopsies .Distribution of dysferlin among the PM compartments (transverse (T)-tubules and sarcolemma) is changing during muscle maturation. In immunohistochemistry experiments, the intensity of dysferlin staining peaks on T-tubules during their development and regeneration, but redistributes predominantly to the sarcolemma in the mature muscle fibers ,77,78, wIn the following three sections , we disc2+-influx into the sarcoplasm through the lesion and depends on a set of proteins including dysferlin as one of the key players [2+-triggered interactions with negatively charged phospholipids [2+- and PS-binding proteins annexins A1, A2 and A6 [Ca2 and A6 ,86;Muscle-specific proteins mitsugumin 53 (MG53) and caveolin 3, which are important for the nucleation of the sarcolemma repair machinery and for regulating the trafficking of dysferlin to and from the PM, respectively ,88;2+ homeostasis, signaling and structure of cytoskeleton [A giant scaffolding protein AHNAK participating in the regulation of Caskeleton ,90;Myoferlin, another member of the ferlin protein family ;Affixin (β-parvin), a protein linking integrins and cytoskeleton ; and;A focal adhesion protein vinculin, cytoplasmic dynein participating in the retrograde vesicle transport along the microtubules and tubulin A ,93.The best-studied function of dysferlin is its role in repair of lesions in the surface membrane of striated muscle fibers, the sarcolemma . Muscle  players ,84. It iholipids ,45,83. D2+-sensitive membrane-binding protein important for vesicular trafficking during sarcolemma repair to cytoskeleton remodeling. It is likely that dysferlin is participating not only in exocytosis of vesicles or organelles dedicated for sarcolemma repair [These dysferlin-interacting proteins link its function as a Caa repair , but alsa repair . More tha repair .A number of cellular mechanisms have been shown to contribute to PM repair: contraction of membrane wounds, plugging (protein-based crosslinking of intracellular vesicles or membranous organelles without their fusion), patching (restoration of PM integrity by fusion of intracellular vesicles), endocytosis and externalization or membrane shedding ,95. The Relatively large (up to 4 µm) sarcolemma lesions of mature myofibers could be resealed by one of the two mechanisms called patching and plugging, while fusion of the membranous organelles within the patch or plug was not proven. Formation of a dysferlin-containing patch or plug on the sites of sarcolemma wounds in zebrafish was paralleled by an increase in a PS-sensor signal and BODIPY-cholesterol fluorescence, confirming the presence of membranous organelles in the patch or plug ,96. ThesNevertheless, the function of dysferlin in vesicular trafficking, which could underlie its role in PM repair, is supported by the observed defects in the injury triggered lysosome exocytosis across the surface of dysferlin-deficient myotubes and myoblasts ,94. Of n2+-dependent [2+-sensitive [Another mechanism of sarcolemma repair, which could depend on the function of dysferlin, is the contraction of the membrane wounds. This idea is supported by several observations: (i) dysferlin is accumulating on the rims of the lesions likely by lateral diffusion ,86,105, ensitive , (iv) dyensitive ,93,106 aensitive .Membranous repair patch or plug formation;T-tubule stabilization (see below) with T-tubule as a possible membrane reservoir;PS-sorting; recruitment of macrophages and contraction of the membrane wound, and;Lysosome exocytosis.In summary, dysferlin bears a potential to participate in sarcolemma repair by at least four mechanisms :Membran2+-metabolism of injured muscle fibers via mechanochemical stabilization of the triad junction and its Ca2+-release and thus decreasing triad and, in particular, T-tubule sensitivity to stress [Besides its function in the repair of sarcolemma of striated muscle fibers, dysferlin plays a role in T-tubule system development and in triad function upon injury. It participates in sculpturing the membranes during T-tubule biogenesis, especially in regenerating muscles and possibly also during repair of the system upon injury ,77. Moreo stress ,108,109.2+ channels (Cav1.1), also known as L-type Ca2+-channels (LTCC) or dihydropyridine receptors (DHPRs) localized to T-tubules, and the ryanodine receptors (RyRs), calcium channels mediating calcium-induced calcium release from terminal cisternae of sarcoplasmic reticulum.Anatomically, the triad is defined by three membrane compartments: one T-tubule in the center, surrounded by two terminal cisternae of the sarcoplasmic reticulum. The main function of the triad is excitation–contraction coupling of the striated muscle fibers, which is achieved by physical binding of voltage-gated Ca2+-metabolism of the injured muscle fibers inhibiting DHPRs and preventing injury-induced Ca2+-leak into the sarcoplasm through RyRs, model supported by sustained Ca2+ influxes in dysferlin-deficient muscle fibers sensitive to the DHPR inhibitor diltiazem and reduction of the extracellular Ca2+ concentration [2+ leak in injured muscle fibers in the absence of dysferlin [Dysferlin was found in a complex with DHPRs and caveolin 3, and also with RyRs ,93. Dysfntration . Howeverysferlin . Thus, dDysferlin-deficient muscles also show T-tubule system abnormalities upon regeneration . DysferlDysf [Skeletal muscles develop normally in pre-symptomatic dysferlinopathy patients and dysferlin-deficient mice. However, after reaching a certain age, several muscle groups begin to show initial signs of pathology: centrally nucleated fibers and variability in fiber size, which deteriorate with time. So, if dysferlin plays a role in muscle growth, myoblast differentiation and fusion, then it, likely, takes place after a defined developmental stage. There are contradicting reports concerning dysferlin role in skeletal muscle differentiation, grow and regeneration. Some authors present evidence for the absence of an effect of dysferlin-deficiency on myoblast differentiation in vitro ,112,113.Dysf ,115,116.Dysf ,118. AccDysf . MyoblasDysf ,118, butDysf .Impaired adult satellite cell differentiation, myoblast-to-myotube fusion and muscle growth in the absence of dysferlin can be attributed to a defect in insulin-like growth factor-1 receptor (IGF1R) trafficking, since IGFs are known to promote muscle cell differentiation ,121. FurIn summary, dysferlin could have additional functions in vesicular trafficking of growth factors receptors, secreted pro-inflammatory molecules and even fusogenic proteins that promote muscle growth and regeneration. We hypothesize that dysferlin-dependent trafficking of such signaling molecules can modulate gene expression and the function of the adult muscle stem (or satellite) cells responsible for the skeletal muscle growth and regeneration in mature individuals. Like dysferlin, myoferlin and Fer1L5 are important for skeletal muscle growth ,125,126.MYOF was reported. However, recently the first case of limb-girdle type muscular dystrophy and associated cardiomyopathy linked to MYOF mutation was described [The first function described for myoferlin was its role in skeletal muscle growth and regeneration . Interesescribed . Known Fer1L5-binding proteins are EHD1, EHD2 and GRAF1  ,129,130.Myoferlin is also involved in membrane repair in muscle fibers and during accelerated proliferation of tumor cells ,132 as wFER1L6/fer1l6 point to two functions of the gene. In humans, FER1L6 was identified as a gene linked to prostate cancer progression [The available data on gression . In zebrgression . In addigression . The absgression . It is tgression ,117,118.Mediating lysosome exocytosis in endothelial cells (dysferlin) and phagocytes, or growth factors  exocytosis in pancreas cancer cells (myoferlin) ,135,136;Recycling of endocytosed transferrin and IGF1Rs in fibroblasts (dysferlin), vascular endothelial growth factor receptor 2 (VEGFR 2) in endothelial cells and signaling of epidermal growth factor receptor (EGFR) in breast cancer cells (myoferlin) ,61,120;Clathrin- and caveolae-dependent endocytosis in endothelial cells (myoferlin) ;Membrane repair in tumor and immortalized cells (myoferlin) and, potentially, in the placenta (dysferlin), ,137,138;Regulation of activity and contact formation of such cells of the immune system, as monocytes, and of adhesion in endothelial cells (dysferlin) ,140,141;Possibly, trophoblast fusion in the placenta (dysferlin) .Dysferlin or myoferlin knock-out in mice leads to late onset muscle dystrophy or a defect in skeletal muscle growth, respectively ,124. HowProteins found to interact with these ferlins in non-muscular cells are dynamin 2 (myoferlin) , caveoliOtoferlin is important for hearing in all studied bony vertebrates, its deficiency leads to profound nonsyndromic deafness in humans, profound hearing loss in mice accompanied by a mild vestibular deficit, and a hearing defect in zebrafish coexisting with a more prominent balance and locomotion dysfunction ,26,142. pachanga (D1767G) [2+-triggered SVs exocytosis is almost completely blocked in the IHCs of otoferlin knock-out mice [pachanga mutants, indicating a defect in the reformation/maturation of SVs and thus, a role of otoferlin in this process [2+-sensitivity of C2C and C2F domains [In the otoferlin knock-out mice, the average tether length between SVs and active zone increases from 20 to 30 nm . This ob(D1767G) . The dowout mice . Followiout mice ,154,155  process ,157. The domains . In summ domains , but alsIn humans, the long non-coding RNA Fer1L4 plays a role in signaling, controlling cell proliferation, migration and apoptosis. The results of the vast majority of the studies are consistent with a function of Fer1L4 as a tumor suppressor in multiple types of cancer ,160,161.2+-triggered fusion of membranous organelles. Is the function of ferlins SNARE-dependent? Can ferlins act as Ca2+-sensitive core fusogens and fuse membranous organelles in the secretory and lysosomal pathways independent of other proteins? Or do ferlins function as Ca2+-sensors in SNARE-dependent membrane fusion, analogous to synaptotagmins? Are the C-terminal fragments comprising of two C2 domains and the transmembrane domain that result from calpain cleavage of ferlins the core functional modules? [2+-channels next to a prospective fusion pore? [One of the most intriguing questions concerning ferlins is their mechanism of action in Camodules? ,167,168.2+-triggered SNARE- and synaptotagmin-dependent membrane fusion, because in such membrane fusion reactions Ca2+-sensitivity is provided by two-C2-domain containing proteins, the synaptotagmins  a defect in sarcolemma repair; (b) changes in Ca2+-homeostasis; (c) impaired muscle growth and regeneration and (d) inflammatory processes. Below, we discussed these factors in turn. To which degree, however, these mechanisms contribute to the development of the disease is still not known. The situation becomes even more complex when considering the existence of the various clinical manifestations of dysferlinopathies.At present it is not clear how exactly loss-of-function mutations of 2+ into injured muscle fibers, (ii) leakage of the muscle fiber contents such as muscle enzymes, e.g., CK, and (iii) the probability of death of damaged myofibers [The pathomechanism could be the following: dysferlin deficiency decreases the efficiency of sarcolemma and, probably, T-tubule system repair ,83,202. yofibers ,83. The yofibers ,203. The2+ or abnormal intracellular distribution of these ions can lead to autophagic, necrotic or apoptotic cell death [2+ homeostasis and Ca2+-mediated cytotoxicity can result from (i) impaired sarcolemma and T-tubule system repair contributing to the leakage of extracellular Ca2+ into the sarcoplasm through a lesion and DHPRs as well as sarcoplasmic reticulum-stored Ca2+ through RyRs, (ii) abnormalities in the biogenesis of the T-tubule system and triads as well as a decrease in their plasticity in response to stress and (iii) enhanced X-ROS  signaling activating mechano-sensitive Ca2+ channels in the T-tubule system, coupling mechanical stress to changes in the intracellular Ca2+ concentration [Overloading of the cells with Call death . In muscntration .2+ concentrations increased in comparison to wild-type controls [Under normal physiological conditions, ROS production is linked to both signaling and metabolism (as a side product of the latter) . In the controls , implyin2+ homeostasis in dysferlinopathy can lead to myofibers death and cycles of regeneration via (i) activation of endonucleases, phospholipases and proteases like calpains leading to unwanted cleavage of cellular components; (ii) triggering multiple signaling cascades affecting gene expression or cell survival [Altered Casurvival .2+-homeostasis and inflammatory processes  or diseases resulting from dysfunction of the first auditory synapse between sensory IHCs and spiral ganglion neurons, which was evident from the analysis of DFNB9 patients and Otof knock-out or knock-in mice [Mutations in -in mice ,156,217 -in mice  for revi-in mice ,223,224.-in mice . In othe-in mice ,225, rev-in mice .OTOF mutations underlying DFNB9 are known [Currently, more than 160 pathogenic re known . Concernre known . Patientre known .2+-sensor for SV exocytosis in mature IHCs, since stimulus–secretion coupling is eliminated in otoferlin knock-out mice [http://web.jhu.edu/animalcare/procedures/mouse.html).At the moment otoferlin is considered to be the one and only Caout mice ,159,178,out mice . Of noteout mice , likely out mice  (SpeciesOncogenesis is often accompanied by enhanced mitotic signaling and a loss of factors controlling cell division, increased cell motility and invasiveness. Importantly, altered expression patterns of ferlin family members have been involved in cancer. Overexpression or downregulation of any human ferlin on the mRNA level and overexpression of myoferlin on the protein level may correlate with development of cancer (e.g., ,61,227, As a consequence, cells of several cancer types overexpress myoferlin and if this overexpression is knocked-down, decrease invasiveness or slow down proliferation ,227,234.2+ homeostasis, muscle growth and regeneration and inflammatory processes. DFNB9 phenotypes originate from defects in SV cycle/exocytosis at the first auditory synapse, while cancerogenesis can reprogram and employ normal functions of ferlins in cell proliferation and differentiation.As members of the superfamily of MC2D proteins, ferlins mediate both exo- and endocytosis of vesicles or organelles. Thereby ferlins play important roles in several aspects of human health, including locomotion and hearing. Diseases resulting from ferlin dysfunction are dysferlinopathies, mainly the muscular dystrophy LGMD2B and Miyoshi myopathy, nonsyndromic recessive deafness DFNB9 and also several types of cancer. Pathomechanistic models of dysferlinopathies include the interplay between defects in sarcolemma repair, Ca


P value: 3.55 × 10−4), MIP1b  and IL13  were significantly associated with increased risk of overall BC, as well as ER-positive BC. In addition, higher levels of MIP1b and IL13 were also significantly associated with increased risk of ER-negative BC. These findings suggest the crucial role of cytokines in BC carcinogenesis and potential of targeting specific inflammatory cytokines for BC prevention.To determine whether genetically predicted circulating levels of cytokines are associated with risk of overall breast cancer (BC), estrogen receptor (ER)-positive and ER-negative BC, we conducted two-sample MR analyses using data from the most comprehensive genome-wide association studies (GWAS) on cytokines in 8293 Finnish participants and the largest BC GWAS from the Breast Cancer Association Consortium (BCAC) with totally 122,977 BC cases and 105,974 healthy controls. We systematically screened 41 cytokines (of which 24 cytokines have available instruments) and identified that genetically predicted circulating levels (1-SD increase) of MCP1 (OR: 1.08; 95% CIs: 1.03–1.12;  Inflammatory mechanisms have been implicated in genesis, development and metastasis of BC4, and finally the prognosis and recurrence of BC7. Cytokines have indispensable functions in pathology of inflammation9 and could serve as the potential targets of anti-inflammatory intervention of BC.Breast cancer (BC) remains the main cause of cancer death and the second most common female cancer in western countries, although the death rate of BC has dropped by 40% from 1989 to 201613, etc. However, these relationships between inflammatory cytokines and BC risk identified in observational studies could be susceptible to confounding factors, limited follow-up time, small sample size and the reverse causation, which might mislead the conclusions14. For example, contrary to previous studies, plasma CRP level was not found to be significantly associated with BC risk in the Women’s Health Study with 27,919 participants15. In addition, both serum levels of IL6 and TNF-α were associated with either increased or decreased risk of BC in different samples19. Hence, the potential causality of individual cytokines in determining BC risk remains elusive.The role of cytokines in BC carcinogenesis has been widely explored by observational studies and yielded equivocal results, with suggestive associations between risk of BC and abnormal levels of cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL6), C-reactive protein (CRP), transforming growth factor β (TGF-β)21. MR uses genetic variants as instrumental variables, avoiding being misled by confounding factors, limited follow-up time, small sample size and the reverse causation of different traits20. Genome-wide association studies (GWAS), which have identified thousands of variants associated with complex traits, bring the wide usage of MR to a new climax23. Recently, a comprehensive MR analysis regarding genetically determined levels of circulating cytokines and risk of stroke gave us a good demonstration24. It was implemented leveraging the most comprehensive GWAS which evaluated 41 cytokines and growth factors in 8293 healthy subjects of Finnish ancestry25, and the largest GWAS meta-analysis on stroke and stroke subtypes to date (MEGASTROKE)26.One more robust method being widely for causal inference is Mendelian randomization (MR), which could overcome the limitations of observational research25 and the largest BC GWAS from the Breast Cancer Association Consortium (BCAC) with totally 122,977 BC cases and 105,974 healthy controls27. Totally 24 cytokines with available data, including Eotaxin, growth-regulated oncogene-a (GROa), interleukin-12, p70 (IL12p70), interleukin-13(IL13), interleukin-16(IL16), monocyte chemoattractant protein-1/C-C motif chemokine ligand 2 (MCP1/CCL2), macrophage migration inhibitory factor (MIF), macrophage inflammatory protein-1 beta/C-C motif chemokine ligand 4 (MIP1b/CCL4), and stem cell factor (SCF), beta nerve growth factor (bNGF), cutaneous T-cell attracting (CTACK), hepatocyte growth factor (HGF), interleukin-17 (IL17), interleukin-18 (IL18), interleukin-2 receptor, alpha subunit (IL2ra), interferon gamma-induced protein 10 (IP10), macrophage colony-stimulating factor (MCSF), monokine induced by interferon-gamma (MIG), platelet derived growth factor BB (PDGFbb), regulated on activation, normal T cell expressed and secreted (RANTES), stem cell growth factor beta (SCGFb), tumor necrosis factor-beta (TNFb), TNF-related apoptosis inducing ligand (TRAIL), and vascular endothelial growth factor (VEGF), were evaluated for their associations with BC risk. We predicted the circulating cytokine levels using a set of estimated SNP effects based on a GWAS to identify protein quantitative trait loci (pQTL). Estrogen receptor (ER) status is important as a BC prognostic and predictive biomarker, and could also affect the medical decision of hormonal therapy or other treatments28. Thus, we also conducted stratified analyses of ER-positive and ER-negative BC.Through checking the UKB SNP-Heritability Browser, we found the SNP-h2 for BC was 0.144. Hereby, to clarify whether genetically predicted circulating levels of cytokines are associated with risk of BC, we implemented two-sample MR analyses using data from this most comprehensive GWAS on cytokinesp < 5 × 10−8), false discovery rate (FDR < 5%), F-statistics (>10), and linkage disequilibrium , 229 independent SNPs were finally selected as the proxy of circulating levels of 24 cytokines. Figure R2, F-statistics for the instruments, and the results of MR analyses of circulating levels of 24 cytokine with risk of BC and subgroups. F-statistics for their respective genetic instruments ranged from 29 to 636, suggesting that our analyses were unlikely to suffer from weak instrument bias. Among them, nine cytokines, including Eotaxin, GROa, IL12p70, IL13, IL16, MCP1, MIF, MIP1b, and SCF, showed significant associations with risk of BC in either total participants or subgroups , three cytokines, including MCP1, MIP1b and IL13, still showed statistically significant associations with risk of total BC or subtype BC.In the current study, we evaluated whether genetically predicted circulating levels of cytokines are associated with risk of overall BC, ER-positive and ER-negative BC. Through filtering the threshold of significance p < 5 × 0−8, falsP value: 3.55 × 10−4), MIP1b  and IL13  were associated with increased BC risk. For ER-positive BC cases, genetically raised MCP1 , MIP1b  and IL13  were consistently associated with increased BC risk. For ER-negative BC cases, genetically raised MIP1b  and IL13  were consistently associated with increased BC risk. In alternative analyses . However, significant heterogeneity was detected for MIP1b . We also ran the MR analysis including the pleiotropic SNPs in the sensitivity analyses, and found results were not changed for IL13 and MIP1b. Genetically raised MCP1  was still associated with increased BC risk, basing on 14 instrumental SNPs. During the bidirectional MR, results showed no evidence of BC causally influencing the levels of MCP1 , MIP1b  and IL13 .The results of primary analyses for genetically predicted circulating levels (1-SD increase) of MCP1, MIP1b, and IL13 and risk of BC were presented in Fig. P value: 0.200, 0.112, and 0.224, respectively). Second, MR-PRESSO analyses were conducted and revealed no potential outliers for MCP1 and IL13 . One outlier variant (rs7621046) was detected for MIP1b. Following exclusion of SNP rs7621046 in the restrictive MR analysis, genetically raised MIP1b was still associated with higher risk of any BC , while Cochran Q test for the heterogeneity didn’t reject the null hypothesis . Further, comprehensive lookup of the MR-Base, PhenoScanner and the GWAS catalog didn’t reveal significant associations of the selected variants in current study with other traditional BC risk factors, including height, obesity, age of menarche and menopause.To remove the potential influence of the possible pleiotropy effect, a series of measures have been adopted. At the stage of instrument construction, SNPs those were associated with levels of more than one cytokine were removed. At the stage of data analysis, first there was no indication for directional pleiotropy effects as assessed by the MR-Egger intercept for MCP1, MIP1b, and IL13  of MCP1, MIP1b, and IL13 were all significantly associated with increased risk of overall BC and ER-positive BC. In addition, higher levels of MIP1b and IL13 were also significantly associated with increased risk of ER-negative BC. No obvious pleiotropy effect was detected. Besides, we also found suggestive evidence (which didn’t reaching the Bonferroni-corrected threshold) for associations between Eotaxin, GROa, IL12p70, IL16, MIF, SCF, and BC risk. To our knowledge, this study should be the largest and most comprehensive MR assessment of associations between genetically inflammatory cytokines and BC risk to date.P value: 3.55 × 10−4), and ER-positive BC cases . Our systematic retrieval only identified limited observational studies reporting association between MCP1 level and BC risk. Some conclusions were null, possibly because our study had increased statistical power and previous reports might be false negative33. Some studies reported positive correlation between MCP1 level and BC risk, however, the conclusions were still underpowered or possibly biased due to the inherent defect of traditional observational studies39. Among the potentially modifiable cytokine factors, MIP1b which has the most instrumental SNPs, contributes to higher BC risk , especially in ER-negative BC cases . MIP1b plays a central role in the recruitment of regulatory T cells to B cells and antigen-presenting cells, and failure to do this would cause autoimmune activation40. IL13 is another cytokine associated with higher BC risk, especially in ER-negative BC cases . Autocrine IL13 plays an important role in the pathophysiology of BC, through inhibiting estrogen-induced proliferation and favoring acquisition of breast cancer cell differentiation markers41. Cytokines are vital to many biological processes, and its serum concentration is tightly regulated. We additionally found suggestive evidence for significant associations between Eotaxin, GROa, IL12p70, IL16, MIF, SCF, and BC risk. Among them, Eotaxin, GROa, IL12p70, IL16, and SCF took part in the regulation of inflammatory pathways in BC carcinogenesis, and contributed to higher BC risk45. Interestingly, genetically raised level of MIF was associated with decreased risk of BC , especially in ER-negative BC cases . This was contradictory to the results of observational studies, which showed higher MIF level in BC patients47. Possible reason might be the dual role of MIF in human BC carcinogenesis48. Further research is required to decipher the biological pathways underpinning these associations.Genetically raised MCP1 was associated with increased overall BC risk , GROa (76.0%), IL12p70 (71.2%%), IL16 (84.2%), MIF (91.6%) has also reached more than 70.0%. The interpretation and generalizability of the study findings are also affected by several limitations. First should be the limited cytokine instruments. To pursue the homogeneity of research cytokine variables, the instrument selection was based on a single GWAS. Although we implemented the most comprehensive and largest GWAS on cytokines, several cytokines being implicated in BC risk previously, such as CRP, TGF-β, were still missed in current analysis. Second, we didn’t obtain reliable genetic instruments for 17 cytokines as no SNPs meeting the strict filtering procedures. Third, we assumed the sex difference of cytokine was small in the current study, as no sex specific results were reported. Strictly speaking, the MR analyses in the study were testing the associations between female cytokines and female BCs. Fourth, we didn’t validate our findings in an independent GWAS, as there were no GWAS with sample sizes that match the BACA study . Fifth, collider bias remains an issue in MR analysis. Sixth, we still can’t remove the possible pleiotropy effect  that might be concealed by small sample size or the small number of SNP instruments, although we have used a series of methods, including MR-Egger, MR-PRESSO analyses, and checking whether the SNPs used as instruments have any known pleiotropic effects in curated genotype to phenotype databases. However, its impact is likely to be less than other biases, and it could be substantial only when the effects of the risk factor and confounders on selection are particularly largeConclusively, using a two-sample MR approach, we find evidence that higher genetically predicted circulating level of MCP1, MIP1b, and IL13 are associated with increased risk of overall BC and ER-positive BC. These findings suggest the crucial role of cytokines in BC carcinogenesis and potential of targeting specific inflammatory cytokines for BC prevention. Further research is necessary to assess the viability of these cytokine biomarkers as drug targets for BC prevention and treatment. Nonetheless, these findings have potential implications for changing the inflammation status of the general women to reduce the burden of BC and improve female health.25. For BC, we obtained the data from the largest BC consortia—BCAC, which provided summary association statistics for overall , ER-positive  and ER-negative BC 27. The consortia results included the OncoArray  and iCOGS datasets , and the combined results from eleven additional GWAS . To minimize the ancestral mismatch, current analyses are restricted to women of European ancestry only. All studies included rigorous quality control, imputation to the 1000 Genomes Project panel. As all analyses were based on summary statistics and not individual-level data, no ethical approval was required.For the genetic instruments of the cytokines, the summary statistics were taken from the most comprehensive and largest GWAS for cytokines, which genotyped up to 8293 Finnish participants from three independent population cohorts: the Cardiovascular Risk in Young Finns Study, FINRISK1997, and FINRISK200225. For each cytokine, the single nucleotide polymorphisms (SNPs) were filtered according to the following procedures: (1) a genome-wide threshold of significance (p < 5 × 10-8) and a significance threshold of a FDR < 5% were adopted. This caused totally 7262 SNPs were selected. (2) To avoid the pleiotropic effect, 809 SNPs those were associated with levels of more than one cytokine were removed, leaving 6,453 SNPs for further step. (3) then, 5983 SNPs were identified to be available in the BCAC dataset. (4) We computed F-statistics to quantify the strength of the selected instruments, and all were well above the threshold of F statistics > 10 typically recommended for MR analyses. (5) Linkage disequilibrium , which retaining SNPs with the lowest p-value as independent instrument, were exerted to remove the superposition effect of correlated SNPs using LDlink50. Finally, we identified 229 SNPs not in LD and to be significantly associated with circulating cytokine levels. These instruments related to the circulating levels of 24 cytokines, whereas for 17 cytokines no SNPs meeting the above conditions  difference of the risk factor, as all effect sizes were in SD-scaled units in Ahola-Olli’s report25. Causal estimates are thus presented per genetically predicted SD, and a log-linear association with odds of BC is implicit across the range of intermediate risk exposure. In current study, inverse-variance weighted (IVW) method was used as the primary method for the MR analyses to test the potential causal associations between circulating levels of selected cytokines and BC risk. In the absence of directional pleiotropy, it provides robust causal estimates. Further, penalized IVW, robust IVW, penalized robust IVW, MR-Egger, simple median, weighted median, and penalized weighted median method were used for alternative analyses. Among these methods, MR-Egger allows free estimation of the intercept, and a statistically significant intercept term implies the presence of unbalanced pleiotropy and causal estimates in MR Egger are less precise than those in IVW. Meanwhile, we also conducted bidirectional MR to test the potential causal effect of BC 53. All statistical analyses were conducted using the R (version 3.6.3) or SAS 9.4 statistical software. All P values are two-tailed, and P < 0.05 was considered significant.In current study, Further information on research design is available in the SUPPLEMENTAL MATERIALsReporting Summary Checklist FLAT


Serum and glucocorticoid-induced protein kinase 1 (SGK1) is a member of the “AGC” subfamily of protein kinases, which shares structural and functional similarities with the AKT family of kinases and displays serine/threonine kinase activity. Aberrant expression of SGK1 has profound cellular consequences and is closely correlated with human cancer. SGK1 is considered a canonical factor affecting the expression and signal transduction of multiple genes involved in the genesis and development of many human cancers. Abnormal expression of SGK1 has been found in tissue and may hopefully become a useful indicator of cancer progression. In addition, SGK1 acts as a prognostic factor for cancer patient survival. This review systematically summarizes and discusses the role of SGK1 as a diagnostic and prognostic biomarker of diverse cancer types; focuses on its essential roles and functions in tumorigenesis, cancer cell proliferation, apoptosis, invasion, metastasis, autophagy, metabolism, and therapy resistance and in the tumor microenvironment; and finally summarizes the current understanding of the regulatory mechanisms of SGK1 at the molecular level. Taken together, this evidence highlights the crucial role of SGK1 in tumorigenesis and cancer progression, revealing why it has emerged as a potential target for cancer therapy. Serum and glucocorticoid-induced protein kinase 1 (SGK1) was initially cloned as an immediate early gene transcriptionally activated by serum and glucocorticoids in rat mammary tumor cells . In humaSGK1 belongs to the “AGC” subfamily of protein kinases, containing 60 members, including PKA, PKG, and PKC, which all share a conserved catalytic kinase domain . And SGKIn recent years, as one of the laboratories studying the role and function of SGK1 in human cancer, we identified SGK1 as a crucial key molecule involved in prostate cancer (PCa) progression through regulation of cell apoptosis, cell cycle, invasion/migration, and autophagy , 20. TheA growing number of investigations suggest that SGK1 is a potential predictor of tumor type, tumor grade and lymph node metastasis in multiple human cancer types . MoreoveThe expression pattern of SGK1 has been extensively investigated and compared in nontumor and tumor human tissues Table 1.Decreased SGK1 expression was also observed in adrenocortical carcinoma , which iAs stated above, aberrant expression of SGK1 is closely related to the clinical characteristics of human cancer. Moreover, SGK1 expression was suggested to indicate a later clinical stage, as well as the extent of metastasis. For instance, a high copy number of the SGK1 gene was positively correlated with short-interval recurrence and metastasis in high-grade Müllerian adenosarcoma . SGK1 mRSGK1 not only has great potential in indicating the clinical features of human cancer but also plays an important role in predicting the progression and prognosis of cancer patients. Abbruzzese et al.  showed tTherapy resistance is a major risk factor for poor prognosis in cancer patients who undergo chemo- and radio-therapy. In prostate cancer, despite new treatments for castration-resistant prostate cancer (CRPC), the prognosis of patients with CRPC remains bleak due to acquired resistance to androgen receptor (AR)-directed therapy. Isikbay et al.  found thTumor development is a multistep process that includes sustained proliferation signaling, evasion of growth suppressors, cell death resistance, enabling of replicative immortality, angiogenesis induction, invasion and metastasis activation, energy metabolism reprogramming, evasion of immune destruction, and creation of a “tumor microenvironment” . A growiin vivo , while SGK1 shRNA and inhibitors showed inverse effects -deficient mice, an effect at least partially due to enhanced beta-catenin protein abundance . In vitr effects . Similar effects . In glio in vivo , 64. Fur in vivo . SGK1 in in vivo . Recentl in vivo . Convers in vivo .via downregulation of p53 expression by enhancing its mono- and polyubiquitylation in RKO colorectal cancer cells  . Bone metastasis .via vinculin dephosphorylation  and MMP-9 levels, which were induced at least partially by repression of epithelial-to-mesenchymal transition (EMT) through downregulation of Snail -LC3 and pFOXO3a(S253)-p27 interactions -positive HeyA8 and SKOV3 cells, dexamethasone (100 nM) treatment upregulated the pro-survival genes SGK1, and MKP1/DUSP1 and inhibited carboplatin/gemcitabine-induced cell death  . SGK1 alin vivo toxicity test  also play critical roles in differentiation of T helper 17 cells/regulatory T cells (TH17/Treg)  . In addi17/Treg) . In grow17/Treg) , which a17/Treg) . Recentl17/Treg)  showed tvia SGK1 activation. Fagerli et al.  in kidney cancer . IL-2 isi et al.  determini et al. . On the via both transcriptional factors and epigenetic factor-induced mechanisms in cancer cells , which may act as a ceRNA on level . A similon level . Moreoveon level .via targeting its 3’-UTR, thus affecting the occurrence and progress of cancer.In human gastric cancer, serum Lnc-SGK1 expression in T cells in combination with H. pylori (Hp) infection and/or a high-salt diet (HSD) was associated with poor prognosis of GC patients . FurtherIn light of the emerging evidence highlighting multiple roles for SGK1 in mediating tumorigenesis and progression, several specific and selective inhibitors of SGK1 have been developed, including GSK650394 , EMD638650) values of 62 nM based on an in vitro scintillation proximity assay (SPA). GSK650394 functionally inhibited SGK1 in an LNCaP growth assay, with an IC50 value of approximately 1 μM (which is similar to the previously measured IC50 in other cell-based assays) , Janus kinase isoforms , insulin-like growth factor1 receptor (IGF1R), Rho-associated protein kinase (ROCK), dual-specificity tyrosine phosphorylation-regulated kinase (DYRK1A) and PDK1, and less than 10-fold more selective for SGK1 than for Aurora and c-Jun N-terminal kinase  . More re assays) , squamou assays) , 144, lu assays) , CRC 7250 values assays) . In addi assays) , 20. Howl kinase , 146.In vitro testing identified EMD638683 as an SGK1 inhibitor with an IC50 of 3 μM in human cervical carcinoma HeLa cells. Originally, MD638683 served as a template for drugs counteracting hypertension in individuals with type II diabetes and metabolic syndrome in vivo   . Subsequ µmol/L) , 67. Spe µmol/L) , or syne in vivo . Remarka in vivo , 71, 14750 values were determined, and only compound QGY-5-114-A showed a significantly lower IC50 value of 122.9 μM, indicating better inhibitory potency than the inhibitor GSK650394. Furthermore, QGY-5-114-A could dramatically restrain colonic tumor cell proliferation via activating cell apoptosis and inducing cell cycle arrest at G0/G1 phase, and significantly impede colonic tumor cell migration in vitro .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metopograpsus frontalis Miers, 1880 . The circular genome is 15,587 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, as well as a control region. Both atp8/atp6 and nad4L/nad4 share 7 nucleotides in their adjacent overlapping region, which is identical to those observed in other Grapsidae crabs. The genome composition and gene order follow a classic crab-type arrangement regulation. The phylogenetic analysis suggested that Grapsidae crabs formed a solid monophyletic group. The newly described mitochondrial genome may provide genetic marker for studies on phylogeny of the grapsid crabs.In this study, we sequenced and analyzed the whole mitochondrial genome of  Metopograpsus frontalis lives in mangroves and intertidal areas of eastern Indian and western Pacific Oceans, from Singapore to southern China , Guangxi province, China, and they were deposited at the Marine Biology Institute, Shantou University, Shantou, China. Total genomic DNA was isolated from the muscle tissue. Long and conventional PCRs were employed to obtain the whole mitochondrial genome sequence. The mitogenome was assembled, annotated and finally submitted into GenBank database under the accession number of MH028874.Specimens of M. Frontalis is 15,587 bp in length, containing 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 1 putative control region. Of the 37 genes, twenty-three are encoded by the plus strand and the remainders by the minus strand. Both atp8/atp6 and nad4L/nad4 share 7 nucleotides in their adjacent overlapping region, which is identical to those observed in other Grapsidae crabs, such as Pachygrapsus crassipes , ATT (atp6 and nad6), GTG (nad2 and nad4) and ATA (nad3) are used as starting codons; TAA , TA (cox3) and T (cytb and cox1-2) serve as terminal codons. The mitogenome shows a classic crab-type gene arrangement regulation  from 12 crab species , using maximum likelihood (ML) method. Harpiosquilla harpax was used as an outgroup for tree rooting (A phylogenetic tree was constructed based on 12 concatenated protein-coding genes (except  rooting . It was


Background: Bronchoalveolar lavage (BAL) is a specific type of air-way fluid. It is a commonly used clinical specimen for the diagnosis of benign diseases and cancers of the lung. Although previous studies have identified several disease-associated proteins in the BAL, the potential utility of BAL in lung cancer is still not well-studied. Based upon the fact that the majority of secreted proteins are glycoproteins, we have profiled N-glycoproteins in BAL collected from lung cancers, and investigated the expression of glycoproteins such as the matrix N-glycoprotein, periostin, in lung cancers.Methods: BAL specimens (n = 16) were collected from lung cancer patients, and analyzed using mass spectrometry-based quantitative N-glycoproteomic technique. Additional BAL specimens (n = 39) were independently collected to further evaluate the expression of periostin by using an enzyme-linked immunosorbent assay (ELISA).Results: A total of 462 glycoproteins were identified in BAL samples using N-glycoproteomic technique, including 290 in lung adenocarcinoma , 376 in squamous cell carcinoma , 309 in small cell lung carcinoma , and 316 in benign lung disease (n = 3). The expressions of several glycoproteins were elevated, including 8 in ADC, 12 in SQCC, and 17 in SCLC, compared to benign BALs. The expression of periostin was detected in all subtypes of lung cancers. To further investigate the expression of periostin, an ELISA assay was performed using additional independently collected BALs (n = 39) The normalized levels of periostin in benign disease, ADC, SQCC, and SCLC were 255 ± 104 (mean ± SE) and 4,002 ± 2,181, 3,496 ± 1,765, and 1,772 ± 1,119 ng/mg of total BAL proteins.Conclusion: Our findings demonstrate that proteomic analysis of BAL can be used for the study of cancer-associated extracellular proteins in air-way fluid from lung cancer patients. Lung cancer is a heterogeneous group of tumors, including non-small cell carcinoma (NSCLC) and small cell carcinoma (SCLC). In the NSCLC, squamous cell carcinoma (SQCC) and adenocarcinoma (ADC) are the two dominant subtypes , 2. The Currently, we commonly use the combination of imaging and histological assessment of the lesion for the diagnosis of lung cancer. Several types of specimens can be used for histomorphological evaluation, such as bronchial brushing and bronchoalveolar lavage (BAL) samples, and transbronchial fine-needle aspiration (EBUS-TBNA) biopsy with or without ultrasound guidance specimens –13. In aN-glycoprotein (SPEG) and liquid chromatography-tandem mass spectrometry (LC-MS/MS)  contains proteins released from lung parenchyma that may be useful for the detection and monitory of certain benign lung diseases, as well as lung cancer progression –16, 18. biomarkers have been characterized in cancers  network .In this study, we investigated the profile of n = 5), SQCC (n = 4), SCLC (n = 4), and benign (n = 3) were used for the proteomic analysis. For a validation study, additional BAL specimens were collected for the ELISA assay, including six cases of benign lung diseases, 18 cases of primary lung ADCs, 6 cases of primary lung SCLCs, and 9 cases of primary lung SQCCs. To remove the cellular debris, all BAL specimens were centrifuged and were then stored at −80°C freezer before the analysis.The discarded BAL specimens were collected from the cytologic laboratory after the evaluation of lung cells. Briefly, 5–10 cc normal saline was applied to rinse the lung during the bronchoscopic procedure, the bronchoscopy was used to collect the fluids into a sterilized test tube and sent to the cytological laboratory. After carefully examined the pulmonary cells, the BAL fluids of ADC (This study was approved by the Institution Review Board (IRB) of Johns Hopkins Medical Center.All the BAL specimens were sonicated on an ice bath for 5 min and then centrifuged at 12,600 rpm for 10 min. One-hundred microliters of trifluoroethanol (TFE) was added into each specimen and incubated for 2 h. The protein concentration of the BAL samples was calculated using the BCA protein assay kit . Then the proteins were reduced by Tris (2-carboxyethyl) phosphine (TCEP)  at room temperature for 1 h. BAL specimens were incubated with the sequencing grade trypsin  at 37°C overnight. The digested peptides were used for further analysis.N-linked glycopeptides  in 100 mM NH4HCO3 at 37°C overnight. After the C18 cartridge, the purified glycopeptides were dried by Speed-Vac and resuspended with 0.4% acetic acid for further use of LC-MS/MS analysis.As previously described, the SPEG method was used to capture peptides . C18 colm/z at a resolution of 70,000 followed by data-dependent higher-energy collisional dissociation (HCD) MS/MS, with a 2 m/z isolated mass window.Peptides were analyzed by LC-MS/MS on an Orbitrap Velos  and a nanoLC system . One microgram of peptides was subjected to a self-packed C18 column with a 120 min gradient. Mobile phase flow rate was set as 300 nL/min with 0.2% FA (A) and 0.2% FA, 95% ACN (B), respectively. The de-glycosylated peptides were separated with gradient elution of phase B from 5%, and ramped to 30% in 86 min. LC-MS/MS data acquisition used a data-dependent analysis (DDA) method of the top 10 most abundant ions. The MS spectra were collected from 350 to 1,800 m/z width of 10 ppm, frame retention time width for 2.5 min, the intensity threshold of 5 ×105. After the SIEVE framing, the results from Proteome Discoverer were filtered with 1% false-discovery rate (FDR) and quantified using the unique peptide for a protein. In SIEVE analysis, the frames for formal glycopeptides  were targeted for quantification. Data were normalized by total ion current (TIC).Proteome Discoverer software  was applied for protein annotation. The dataset was searched against a human protein database . The MS and MS/MS tolerances were set as 20 ppm and 0.06 Da, respectively. For de-glycosylated peptides identification, the cysteine residues were modified by carbamidomethyl group  as a stable modification, and methionine oxidation  and asparagine deamidation  were set as dynamic modifications. A maximum of 2 miss cleavages was allowed. Then we used SIEVE software  for the label-free, semi-quantitative analysis of glycopeptides and glycoproteins. The chromatograms of raw files were aligned after importing into the SIEVE. Frame parameters were set as follows: mass range from 350 to 1,800, retention time from 0 to 120 min, a maximum frame of 100,000, frame In the assay, 100 μl of BAL specimens were incubated with periostin capture antibody in a 96-well-microtiter plate overnight at 4°C, and then incubated with HRP-conjugated mouse anti-human periostin antibody at 4°C for 30 min. Tetramethylbenzidine was added and interacted at room temperature for 20 min. Then using an absorbance reader  to acquire the absorbance readings at 450 nm. Recombinant human periostin (concentration range: 0–250 ng/mL) was used to generate a standard curve from a four-parameter logistic curve fit. All samples were analyzed duplicated and calculated the average of the two values. The total protein concentration of all BAL samples was determined using the BCA protein assay. The levels of periostin were expressed as ng/mg total proteins in BAL specimens.2 and Fisher's exact tests were used to calculate the P-value. The relationship between periostin and pathological characteristics of NSCLS patients was using Spearman rank correlation analysis. All tests were determined using bilateral 95% confidence intervals (CI). If the alpha value was <0.05, it was considered statistically significant (P <0.05).Characteristics of clinicopathological information were evaluated using the SPSS statistical software  The χn = 5), SQCC (n = 4), SCLC (n = 4), and benign lung diseases (n = 3). All cancer cases were primary lung cancers. For 16 proteomic BAL samples, the median age of patients was 63 years, ranging from 23 to 88 years; and the female and male ratio was: 1:1. In 13 cancer cases, 1 case was pT1, 3 cases were pT2, 5 cases were pT3, and 4 cases were pT4. The smoking status revealed that six patients were non-smokers, and 10 were former or current smokers . The differentially expressed glycoproteins included periostin (POSTN), vascular cell adhesion protein 1 (VCAM1), TNF Receptor Superfamily Member 19 (TNFRSF19), and mucin-13 (MUC13). Overall data are summarized in In total, 462 glycoproteins from 1,081 glycopeptides were identified in BAL samples, whereas, there were 290, 376, 309, and 316 glycoproteins identified in ADCs, SQCCs, SCLCs, and benign samples, respectively. Compared to the benign samples, there were 8, 12, and 17 glycoproteins differentially expressed in ADCs, SQCCs, and SCLCs, respectively  and 4,002 ± 2,181, 3,496 ± 1,765, and 1,772 ± 1,119 ng/mg of total BAL proteins . The perN-linked glycosylation, is commonly identified on a large number of cellular proteins, including cell surface proteins, transmembrane proteins, and extracellular proteins secreted in body fluids  is a 90-kDa glycoprotein, and frequently expressed in endothelial cells, which is participated in the regulation of cellular adhesion and inflammation-related activities , 37. TheN-glycoprotein, that was first identified in osteoblast-like cells. It is present in many healthy tissues and involved in bone formation, cardiac development, wound healing, and other cellular functions  can be found at: MassIVE database, This study was reviewed by the Institutional Review Board of the Johns Hopkins Medical Institution. Ethical approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent was not required to participate in this study in accordance with the national legislation and the institutional requirements.YZ, WY, HZ, and QL: conceptualization. YZ, MA, EG, and QL: project administration and methodology. YZ, WY, MA, and NH: data curation and software. YZ, WY, and HZ: formal analysis. MA and NH: validation. YZ and NH: writing—original draft. EG, DC, HZ, and QL: writing—review and editing. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lipid peroxides (LOOHs) abound in processed food and have been implicated in the pathology of diverse diseases including gut, cardiovascular, and cancer diseases. Recently, RNA Sequencing (RNA-seq) has been widely used to profile gene expression. To characterize gene expression and pathway dysregulation upon exposure to peroxidized linoleic acid, we incubated intestinal epithelial cells (Caco-2) with 100 µM of 13-hydroperoxyoctadecadienoic acid (13-HPODE) or linoleic acid (LA) for 24 h. Total RNA was extracted for library preparation and Illumina HiSeq sequencing. We identified 3094 differentially expressed genes (DEGs) in 13-HPODE-treated cells and 2862 DEGs in LA-treated cells relative to untreated cells. We show that 13-HPODE enhanced lipid metabolic pathways, including steroid hormone biosynthesis, PPAR signaling, and bile secretion, which alter lipid uptake and transport. 13-HPODE and LA treatments promoted detoxification mechanisms including cytochrome-P450. Conversely, both treatments suppressed oxidative phosphorylation. We also show that both treatments may promote absorptive cell differentiation and reduce proliferation by suppressing pathways involved in the cell cycle, DNA synthesis/repair and ribosomes, and enhancing focal adhesion. A qRT-PCR analysis of representative DEGs validated the RNA-seq analysis. This study provides insights into mechanisms by which 13-HPODE alters cellular processes and its possible involvement in mitochondrial dysfunction-related disorders and proposes potential therapeutic strategies to treat LOOH-related pathologies. Dietary lipids, including vegetable oils, contain different quantities of the most common dietary polyunsaturated fatty acid (PUFA), linoleic acid (LA), in the form of triglycerides, which are hydrolyzed by bile and lipases. This process releases large amounts of free fatty acids (FFAs), often in millimolar quantities, that are absorbed by intestinal cells . DependiLOOHs have been strongly linked to disorders of the digestive system, including inflammatory bowel disease (IBD) and malignancy . StudiesThe Caco-2 cell line is a human intestinal epithelial cell line that is derived from human colorectal adenocarcinoma . Under cA recently published study demonstrated similar results between Caco-2 cells treated with 13-HPODE and mice fed with 13-HPODE , which mCaco-2 cells were purchased from American Type Culture Collection (ATCC) . Cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 15% fetal bovine serum , 2 mM L-glutamine , and 1% penicillin-streptomycin . After attaining confluence, cells were cultured in the same medium supplemented with 7.5% FBS and the same concentration of other constituents. Confluent cells were trypsinized using 0.25% Trypsin-EDTA solution . Caco-2 cells were seeded in 6-well plates and experiments were carried out on fully differentiated cells .Stock solution of linoleic acid (LA)  was prepared in ethanol, and LA (200 μM) in phosphate-buffered saline  was prepared for LA treatment of cells. 13-hydroperoxyoctadecadienoic acid (13-HPODE) was freshly prepared in PBS as previously described ,14,30. BAs previously described , Dif Cacg for 15 min. The upper aqueous phase, containing RNA, was transferred into fresh tubes. Isopropyl alcohol was added to the samples and centrifuged for 10 min at 12,000× g to precipitate RNA from the aqueous phase. Total RNA was washed with ethanol at 7500× g for 5 min and air-dried for 2–3 min. RNA was resuspended in RNase-free water, then sample concentration, purity, and quality were determined using a NanoDrop spectrophotometer , which showed absorbance ratios of 1.8–2 at 260 nm and 280 nm. Any co-extracted DNA was removed from RNA samples using the TURBO DNA-free kit , following the manufacturer’s instructions.Cells were lysed directly in the 6-well culture plates using TRIzol reagent . Cell lysate was transferred to tubes and chloroform was added. The samples were vortexed and incubated at room temperature for 3 min then centrifuged at 12,400× We isolated mRNA from total RNA samples using the NEBNext Poly(A) mRNA Magnetic Isolation Module  with an approximate input of 500 ng of total RNA per sample. RNA-seq libraries were prepared from mRNA samples using the NEBNext Ultra II Directional RNA Library Prep kit for Illumina  according to the manufacturer’s instructions, including NEBNext Sample Purification Bead cleanups to remove unincorporated primers and adapters. Samples were fragmented for 15 min at 94 °C to achieve a target fragment size of 200 base pairs (bp). Indexed adaptors for Illumina sequencing  were ligated to libraries through 8 cycles of PCR. Library quality was assessed using High Sensitivity D1000 reagents  on a TapeStation 2200 instrument . Library concentrations were determined using the NEBNext Library Quant Kit for Illumina  following the manufacturer’s instructions. Three dilutions of each library were prepared  and plated in triplicate on a 96-well qPCR plate along with manufacturer-supplied standards (20 µL reactions). Concentration data were used to ensure equimolar pooling across libraries for multiplexing. The final library pool was checked for quality using the High Sensitivity D1000 ScreenTape assay, which showed good quality with a maximum peak size of 337 bp , and senp < 0.05) between different groups [The following pipeline was used to analyze the paired-end sequencing reads. We used FastQC (version v0.11.7) to check the quality of reads, which were all of good quality scores > 30, and the presence of adapters or overrepresented sequences . Trimmomt groups . Gene syp < 0.05) (from DESeq2) in the treated cell group to identify enriched GO terms including biological processes, molecular functions, and cellular components. Pathway and gene set enrichment analyses (GSEA) were conducted by running the gage function on the list of DEGs and their log2 fold change scores. This generated a list of dysregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, their p values, and their direction (downregulated or upregulated).Differentially expressed genes were evaluated further using gene ontology and enrichment analyses using the enrichGO function of the clusterProfiler (version 3.18.0) R package , and GenTM Universal SYBR® Green Supermix , 2.5 μM of primer (forward and reverse each), and 0.1 ng of cDNA was used in a 10 μL PCR reaction. The protocol was set as 95 °C for 30 s , and 40 cycles of 95 °C for 15 s (denaturation) and 60 °C for 30 s . GAPDH was used as a housekeeping gene. Relative mRNA expression levels were determined using the t-test was used to determine the statistical differences between the treated and untreated groups and the data were presented as means ± SD. Primer sequences are provided in RNA-seq results were technically confirmed via qRT-PCR using the same RNA samples used for the RNA-seq study. Fourteen representative DEGs were chosen to perform qRT-PCR for 13-HPODE-treated cells and eleven DEGs for LA-treated cells. SsoAdvancedWe incubated Dif Caco-2 cells with 100 µM of 13-HPODE or LA for 24 h. We chose these concentrations as the proximal intestine is exposed to millimolar concentrations of FA and our preliminary results showed little or no cytotoxicity . The conp < 0.05) between 13-HPODE-treated cells and untreated cells  synthesis, which might indicate increased GSH contents. Reduced glutathione peroxidase activity has been linked with colon cancer [HMOX1, CAT, UGT2B4, and TXNRD1. Several solute carrier (SLC) transporters were upregulated, such as SLC26A3, a chloride transporter; SLC38A4, an amino acid transporter; and SLC5A3, a myo-inositol transporter, indicating changes in substrate transport across the cell membrane. In addition, SLC transporters have been implicated in various diseases, including IBD and metabolic diseases [COL7A1, which forms fibrils between epithelial cell basement membrane and extracellular matrix, and PDZK1, which regulates epithelial cell surface proteins and is involved in cholesterol metabolism. Among the downregulated genes were ODC1 and POLD2, which are important for polyamine synthesis and DNA replication, respectively. GPCPD1, which is involved in glycerophospholipid biosynthesis, and PTGES2, which is involved in prostaglandin E synthesis, were also downregulated. 13-HPODE treatment also caused the reduction of NOX1, a NADPH oxidase that generates ROS, as well as DKK1, which is an inhibitor of Wnt signaling [Using DESeq2, we identified 3094 DEGs  involved in translation, ribosome biogenesis, RNA processing, response to hypoxia and oxidative stress, mitochondrial translation, and gene expression. Purine nucleoside monophosphate, alcohol, and amino acid metabolic processes  in Caco-2 cells treated with 13-HPODE were ATPase, oxidoreductase, antioxidant, electron transfer, RNA polymerase I, and helicase activities. Additionally, coenzyme, carboxylic acid, heat shock protein, and cadherin and chaperone binding functions were enriched (Among the enriched molecular functions (adjusted enriched .p < 0.05) included the mitochondrial inner membrane, matrix, ribosome, and protein complex, as well as ribosomal subunits, the spliceosomal complex, focal adhesion, and the cell-substrate adherens junction (The enriched cellular components (adjusted junction . The brup < 0.05) in 13-HPODE-treated cells, relative to untreated cells, were steroid hormone biosynthesis, bile secretion, and protein digestion and absorption. We also observed upregulation of importantly relevant pathways such as metabolism of xenobiotics by cytochrome P450, focal adhesion, and PPAR signaling, but with slightly higher p values.We used the gage R-package to perform pathway enrichment analysis . Among tp < 0.05) observed for 13-HPODE-treated cells, relative to untreated cells, were spliceosome, RNA transport, ribosome biogenesis, and RNA polymerase pathways, as well as purine and pyrimidine metabolism. Oxidative stress can cause base modification and DNA damage [Among the downregulated pathways (A damage . Pathwayp < 0.05) in LA-treated cells compared to untreated cells ; SLC2A1, a glucose transporter; and SLC20A1, a phosphate transporter [We identified 2862 DEGs  of metabolic processes including retinol, xenobiotic, and drug metabolism by cytochrome P450, relative to untreated cells. The protein digestion and absorption pathway was also upregulated.Linoleic acid treatment of Dif Caco-2 caused upregulation (p < 0.05) in LA-treated cells, relative to untreated cells, were ribosome biogenesis, spliceosome, RNA transport, and RNA polymerase pathways. As in 13-HPODE-treated cells, purine and pyrimidine metabolism, oxidative phosphorylation, proteasome, cell cycle, and excision repair pathways were also suppressed in LA-treated cells. Toll-like receptor signaling, which activates innate immunity, was downregulated (Among the pathways downregulated (egulated .p < 0.05) between 13-HPODE-treated and LA-treated cells , the reduced form of 13-HPODE . LOOHs hAKR1C2, that are essential for steroid hormone synthesis. Studies have reported the ability of the intestine to synthesize steroid hormones [BAAT gene, which is responsible for bile acid conjugation that enhances fat solubility, was upregulated in 13-HPODE-treated cells. This could propose another mechanism by which 13-HPODE increases lipid solubilization. A previous study demonstrated that bile secretion in response to a high-fat diet could injure the intestinal epithelium and that secondary bile acids produced by intestinal flora may induce tumor formation [BAAT gene was not differentially expressed in LA-treated cells relative to untreated cells. It is also worth mentioning that both 13-HPODE and LA treatments enhanced the expression of APOB, apolipoprotein B, which is a major component of chylomicron and LDL, along with A1CF, a complementation factor of ApoB mRNA editing enzyme complex essential for the generation of ApoB48, which promotes dietary fat absorption [In the current study, we have demonstrated that 13-HPODE increased the expression of aldo/keto reductases, such as hormones ,56. Sterhormones ; on the hormones , but mayhormones , which mhormones ,60. Uprehormones . In addihormones . Oxidizehormones . Althougormation ; this misorption .p-value of 0.1, several downstream genes involved in fatty acid and lipid homeostasis were among the top differentially upregulated genes (adjusted p < 0.05) in 13-HPODE-treated cells relative to untreated cells. Among those were PLIN2, which forms the lipid droplet coat, and FABP1, which may promote cholesterol uptake and protect against oxidative stress caused by LOOH [HMGCS2, which is involved in ketogenesis and has been found to promote cellular differentiation of Caco-2 cells [CPT1A and PCK1 were also among the differentially upregulated genes of PPAR signaling which may indicate promoted fatty acid oxidation, as well as gluconeogenesis. As oxidized LA is considered a ligand that activates PPAR signaling, which plays multiple roles ranging between metabolism, anti-inflammatory, and anti- and pro-carcinogenic effects, PPAR signaling should be considered a powerful form of crosstalk between 13-HPODE and peroxidized lipid-related diseases and should be studied further. Moreover, CREB3L3, a transcription factor that works with PPARPLIN2, PCK1, and CPT1A in LA-treated cells, the expression of these genes appeared to be higher in 13-HPODE-treated cells compared to LA-treated cells. This suggests that 13-HPODE may have a more powerful effect on PPAR signaling and FA oxidation than non-peroxidized LA. We also observed higher expression of G6PD, required for FA and cholesterol biosynthesis, and a lower expression of atheroprotective APOH in 13-HPODE-treated cells compared to LA-treated cells, which could propose a mechanism by which LOOHs contribute to the development of cardiovascular disease.Although upregulation of the PPAR signaling pathway upon treating cells with 13-HPODE had a slightly higher  by LOOH . HMGCS2,-2 cells , was alsp manner  and is iCYP1B1/2C9/2B6 may be a protective response against 13-HPODE treatment as it is considered a harmful metabolite of oxidized LA. in addition to aldo/keto reductases’ function in steroid hormone synthesis, they are also involved in xenobiotic metabolism by cytochrome P450, as well as bile acid synthesis and transport [CYP2C9. Moreover, LA treatment resulted in higher expression of alcohol and aldehyde dehydrogenases ADH6 and ALDH6A1 in LA-treated cells compared to 13-HPODE-treated cells, which could indicate a protective effect of LA against lipid peroxidation products, including aldehydes. This may also suggest that LA could undergo oxidation in the intestinal cells to which the cells respond by enhancing the detoxification mechanism.Our results indicate that 13-HPODE may enhance detoxification by cytochrome P450 enzymes, which play a role in the metabolism of ingested drugs and toxins, and in the synthesis of steroid hormones, bile acids, and some other fats ,69. Upreransport ,71. In tGPX1 and GPX7 and upregulation of the catalytic subunit of glutamate-cysteine ligase, GCLC, essential for glutathione (GSH) synthesis, the cells could accumulate GSH. The latter has previously shown to convert the tocopheroxyl radical back to TP to maintain its scavenging effect on reactive oxygen species (ROS) via oxidation of vitamin E, thus preventing further lipid oxidation and inhibiting cell proliferation [CXCL1 [CXCL1, CXCL8 (IL-8), and CCL20 chemokines in 13-HPODE-treated cells. It has been suggested that tocopheryl hydroquinone (TQH) is a more powerful antioxidant than TQ [POR gene) [CASP3 and a number of Bcl family members that have apoptotic activity, such as BMF, BCL2L11, and BNIP5 (this gene has not been studied yet), and downregulation of the anti-apoptotic BAG1 gene in 13-HPODE cells. This is consistent with a recently published study [As 13-HPODE comes in contact with the cell membrane, we could expect oxidation of vitamin E  and the formation of tocopheryl quinone (TQ), which is anti-androgenic  and has feration . TP regeferation , which wferation , and hasn [CXCL1 . In our  than TQ ,79. In oOR gene) , which iOR gene) . In thised study  which deNFE2L2 (Nrf2), we did observe upregulation of some target genes that play a role in the antioxidant defense systems, including HMOX1, CAT, UGT2B4, and TXNRD1, as well as downregulation of SOD1 in 13-HPODE-treated cells. This indicates that a degree of the antioxidant protective response was enhanced by 13-HPODE. Moreover, we observed increased expression of FOXO3 and FOXO4 transcription factors, by which 13-HPODE could modulate insulin signaling and the antioxidant response and could trigger apoptosis [CAT, another FOXO-regulated gene, CDKN1A (p21), which inhibits cell proliferation in response to DNA damage, was also upregulated in 13-HPODE-treated cells.Although we did not observe expression changes in members of activator protein-1 (AP-1) or poptosis . In addiPOLD2, MCM7, and PCNA, which are involved in DNA replication; CDC25A, which is important for cell cycle progression; and RPP40 and EIF5A, which are involved in RNA processing and translation. On the other hand, tumor suppressor genes including APC, KLF4, and FOXO4 were upregulated in 13-HPODE-treated cells. In LA-treated cells, we observed downregulation of the proto-oncogene, JUN, and upregulation of the tumor suppressor genes KLF7 and FOXO4. Interestingly, LA treatment caused a reduction in the expression of cell adhesion molecules CEACAM1/M5/M6, which are considered biomarkers of tumor progression [13-HPODE treatment caused the downregulation of events that have been reported to be reduced during Caco-2 cell differentiation . Among tgression ; this magression  due to tgression . Despitep-value 0.06) in 13-HPODE-treated cells relative to untreated cells. Among the related induced genes was COL7A1. In the intestine, this pathway plays a role in epithelial barrier homeostasis and repair and tight junction organization [VIL1 gene in 13-HPODE-treated cells. In addition to the upregulation of VIL1, the sucrase-isomaltase gene, SI, which is another marker of intestinal differentiation [UGT2B4, a detoxifying enzyme that plays a role in the retinol metabolic pathway, was detected and upregulated in LA-treated cells.In support of shifting towards differentiation, the focal adhesion pathway, which is involved in cell motility, differentiation, and other processes, was enhanced  signaling was downregulated in LA-treated Caco-2 cells relative to untreated cells, which supports the previously reported inhibitory effect of PUFAs on TLR activation and inflammatory gene expression . SpecifiThe results of our transcriptomic profiling of Caco-2 cells carried out under standard in vitro conditions shed light on the response of intestinal epithelial cells to 13-HPODE or LA in terms of gene expression and pathway enrichment. The results presented in this study suggest that the most common dietary peroxidized lipid, 13-HPODE, may, on the one hand, enhance bile acid conjugation, which alters lipid uptake and might contribute to intestinal injury. On the other hand, 13-HPODE may affect multiple pathways of lipid metabolism including steroid hormone synthesis, which might affect intestinal barrier and immune function; and PPAR signaling, which alters fatty acid and glucose metabolism, energy production, and mitochondrial function leading to alteration of gut physiology. In addition, 13-HPODE as well as LA may have the ability to promote the antioxidant response and detoxification by cytochrome P450 in intestinal epithelial cells. Moreover, both treatments could reduce proliferative potential and could play a role in the absorptive cell differentiation and survival fates as they might suppress pathways involved in the cell cycle, DNA synthesis/repair, and enhance focal adhesion pathways. While similar effects could be seen in liver cells, it is conceivable that dietary peroxidized LA could have an impairment effect on energy production and lipid storage. In conclusion, this in vitro study using Caco-2 cells provides insights into the physiological changes that might occur in the intestinal epithelium upon exposure to 13-HPODE and the possible mechanisms by which it contributes to disease. Future directions of this research include studying the response of Caco-2 cells to LOOH using an experimental setting that simulates in vivo intestinal environment, such as the INFOGEST method .


Incretins are gut hormones that potentiate glucose-stimulated insulin secretion (GSIS) after meals. Glucagon-like peptide-1 (GLP-1) is the most investigated incretin hormone, synthesized mainly by L cells in the lower gut tract. GLP-1 promotes β-cell function and survival and exerts beneficial effects in different organs and tissues. Irisin, a myokine released in response to a high-fat diet and exercise, enhances GSIS. Similar to GLP-1, irisin augments insulin biosynthesis and promotes accrual of β-cell functional mass. In addition, irisin and GLP-1 share comparable pleiotropic effects and activate similar intracellular pathways. The insulinotropic and extra-pancreatic effects of GLP-1 are reduced in type 2 diabetes (T2D) patients but preserved at pharmacological doses. GLP-1 receptor agonists (GLP-1RAs) are therefore among the most widely used antidiabetes drugs, also considered for their cardiovascular benefits and ability to promote weight loss. Irisin levels are lower in T2D patients, and in diabetic and/or obese animal models irisin administration improves glycemic control and promotes weight loss. Interestingly, recent evidence suggests that both GLP-1 and irisin are also synthesized within the pancreatic islets, in α- and β-cells, respectively. This review aims to describe the similarities between GLP-1 and irisin and to propose a new potential axis–involving the gut, muscle, and endocrine pancreas that controls energy homeostasis. Incretins are gut hormones that potentiate insulin secretion after meal ingestion in a glucose-dependent manner. In particular, the “incretin effect” refers to the ability of oral glucose to elicit a greater insulin secretory response than does intravenous glucose, despite inducing similar levels of glycaemia . The twoOn the other hand, irisin is a myokine firstly described in 2012 by Boström et al.  as a perInterestingly, irisin and GLP-1 show comparable pancreatic and pleiotropic effects and activate similar intracellular pathways.In light of this evidence, the current review aims to describe the similarities between GLP-1 and irisin and to propose a new potential axis—involving the gut, muscle, and endocrine pancreas—relevant for the control of energy homeostasis.GLP-1 is synthesized and secreted from enteroendocrine L cells scattered throughout the small and large intestine, following the post-translational processing of proglucagon by prohormone convertase 1/3 (PC1/3) a.2, that is biologically equipotent to GLP-1(7–37) and represents the major form in humans . AlIn addition to physical activity, the nutritional composition of the diet could also impact irisin secretion. We have previously demonstrated that a high-fat diet  causes a rapid and persistent increase of blood irisin concentrations, in healthy, wild-type mice . In partIt has been amply demonstrated that the “incretin effect” is typically reduced or even absent in people with impaired glucose tolerance or diabetes, and this contributes to defective insulin secretion and hyperglycaemia in patients with type 2 diabetes (T2D) . As no cConversely, in a state of obesity, GIP levels are elevated during both fasting and after an oral glucose challenge, whereas GLP-1 levels are unchanged during fasting and reduced in response to an oral glucose challenge . OverallLikewise, despite the existence of conflicting results, most clinical studies, including the meta-analyses thereof, agree that circulating irisin levels are lower in patients with T2D ,39,40,41Numerous interventional studies in animal models of diabetes and/or obesity have shown that the exogenous administration of recombinant irisin can restore glucose and lipid homeostasis, thus exerting antidiabetic and antiobesity effects ,55,56,57Interestingly, metformin, the current first-line antidiabetic drug, is able to promote both the secretion of GLP-1, likely through indirect mechanisms  and the A deficit of β-cell functional mass is recognized as a necessary and early condition for the development of T2D. Thus, β-cell regeneration or the preservation of functional pancreatic islet integrity should be highly considered for T2D treatment and, possibly, cure .In pancreatic β-cells, in addition to their known ability to induce glucose-stimulated insulin secretion (GSIS), GLP-1R activation results in increased glucose sensitivity, insulin biosynthesis, and β-cellular neogenesis and proliferation, while decreasing apoptosis  Figure a. IndeedATP], increase in intracellular Ca2+ levels) [ATP, and the cAMP-Epac2 pathway endorses the synthesis of ATP, while both signaling pathways promote the increase of intracellular Ca2+ levels [GLP-1 stimulates insulin secretion by intervening in nearly all steps involved in the exocytosis of insulin secretory granules  . These e levels)  , insulin receptor substrate-2 (IRS-2), mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinases (ERK), leading to the upregulation of transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1) and CREB, and of the cell cycle regulator cyclin D1, all involved in the proliferation process b. These In pancreatic islets, irisin exerts effects that are very similar to those of GLP-1 a. We havIn addition, irisin promoted β-cell proliferation through the activation of the ERK1/2 pathway . AdditioRecently, it has been suggested that β-cells themselves could produce irisin, which could enhance β-cell survival and function by acting in autocrine-manner .The advent of GLP-1RAs represented a revolution in the treatment of T2D, because in addition to ensuring good glycemic control with a low risk of hypoglycemia and weight loss, they showed cardiovascular protective effects. These effects are in part dependent on the reduction of hyperglycemia but also involve direct effects of GLP-1 on several organs and tissues, such as the heart and vessels, CNS, and, to a lesser extent, kidney, liver, skeletal muscle, and adipose tissue. Likewise, irisin has shown similar pleiotropic effects .2O2-induced oxidative stress in human cardiac progenitor cells [Although best known for their insulinotropic and weight-lowering actions, GLP-1 and GLP-1RAs also confer a series of beneficial effects on the cardiovascular system. These include the inhibition of cardiomyocyte apoptosis induced by different harmful stimuli ,72,73,74or cells , which adb/db mice characterized by depression in cardiac function [2O2 [Similarly, several recent reports have shown that irisin protects the heart against ischemia/reperfusion injury, improves cardiac function, and reduces infarct size, both in diabetic and nondiabetic rodents ,103,104.function . Interesfunction . Importafunction . Accordifunction , H2O2 [1ion [2O2 , hypoxiaion [2O2 ,110, andion [2O2 , as wellion [2O2 ,112,113 ion [2O2 . The indion [2O2 ,115, as ion [2O2 ,111,116 ion [2O2 ,114 coulion [2O2 . Additioion [2O2 ,120,121.ion [2O2 ,123 throion [2O2 ,124, action [2O2 , and augFurthermore, in vitro studies conducted on human umbilical vein endothelial cells (HUVECs) or human microvascular endothelial cells (HMEC-1) have shown the irisin’s ability to exert proangiogenic effects through activation of the ERK-regulated proliferation signaling pathway ,127,128,Both central and peripheral administration of GLP-1 and its analogs are able to lower body weight via centrally regulated inhibition of food intake in both healthy individuals and patients with T2D ,137,138.On the other hand, the ability of irisin to modulate food intake in diabetic or obese mice/rats remains little investigated and the few existing results are conflicting ,54. We hIn addition to affecting feeding behavior, central GLP-1R agonism has been demonstrated to have neuroprotective effects ,156 and Similarly, several studies have demonstrated that irisin plays a developmental role in regulating the process of neuronal differentiation and maturation , inducesThe GLP-1R is expressed in the kidney and intravenous infusion of GLP-1 in animal models produces natriuretic and diuretic responses that are associated with increased glomerular filtration rate and inhibition of sodium reabsorption in the proximal tubule, thus exerting an antihypertensive effect . The fewob/ob mice [It is not definitively clear if the effects of GLP-1 on liver, skeletal muscle, and adipose tissue depend on a direct effect or are the result of the overall amelioration in whole-body insulin sensitivity. Indeed, the presence of GLP-1R in these tissues is equivocal ,3,12, al/ob mice ,174. Oth/ob mice ,176, andConversely, numerous studies have confirmed the ability of irisin to directly act on liver, skeletal muscle, and adipose tissue. Indeed, irisin has been demonstrated to inhibit lipogenesis, cholesterol synthesis, and gluconeogenesis ,182,183,It has been reported that GLP-1 can promote bone formation and inhibit bone resorption in animal models, thus enhancing bone mineral density and quality  Figure . These eIn this review we have focused on the similarities between irisin and GLP-1 because of the therapeutic potential of GLP-1, rather than GIP, in T2D. Nevertheless, there are similarities and differences also between irisin and GIP that should not be underestimated.GIP is released by intestinal K cells and, similar to GLP-1, is secreted in response to nutrient ingestion and activates GIP-R in pancreatic β-cells, enhancing meal-stimulated insulin secretion in a glucose-dependent manner and stimulating β-cell survival and, especially in rodents, proliferation . In addiMoreover, GIP also targets adipose tissue, stimulating adipogenesis, lipogenesis, and lipid storage in fat cells ,217. It By definition, incretins are hormones with low basal plasma concentrations that are released after the ingestion of physiological nutrients to reach concentrations that augment the insulin secretory responses at a permissive degree of hyperglycaemia, although they are ineffective at low glucose concentrations . AccordiAdditionally, like GIP, irisin promotes bone formation while, unlike GIP, it reduces lipid accumulation in adipose tissue . Irisin In conclusion, due to its modalities of secretion and its pancreatic and extra-pancreatic effects, irisin could be considered an incretin-like hormone with an action similar to that of GLP-1, with the addition of an anabolic GIP-like effect at the bone level and a marked antilipogenic action. This evidence highlights the promising antidiabetic and antiobesity potential of irisin, possibly in combination therapeutic regimens with GLP-1RAs.


COL1A2 is the most overexpressed gene, which may be due to circHIPK3/miR-29b-c/COL1A2 axis dysregulation. We identified 27 GC-related pathways that may be affected mainly by circPVT1, circHIPK3 and circNF1. Our results indicate that circHIPK3/miR-107/BDNF/LIN28 axis may mediate chemoresistance in GC, and that circPVT1, circHIPK3, circNF1, ciRS-7 and circ_0000096 appear to be involved in gastrointestinal cancer development. Lastly, circHIPK3, circNRIP1 and circSMARCA5 were identified in different ethnic populations and may be ubiquitous modulators of gastric carcinogenesis. Overall, the studied sponge circRNAs are part of a complex RBP-circRNA-miRNA-mRNA interaction network, and are involved in the establishment, chemoresistance and progression of GC. Circular RNAs (circRNAs) are a new class of long noncoding RNAs able to perform multiple functions, including sponging microRNAs (miRNAs) and RNA-Binding Proteins (RBPs). They play an important role in gastric carcinogenesis, but its involvement during gastric cancer (GC) development and progression are not well understood. We gathered miRNA and/or RBPs sponge circRNAs present in GC, and accessed their biological roles through functional enrichment of their target genes or ligand RBPs. We identified 54 sponge circRNAs in GC that are able to sponge 51 miRNAs and 103 RBPs. Then, we evaluated their host gene expression using The Cancer Genome Atlas (TCGA) database and observed that  Gastric cancer (GC) is the fifth most frequently diagnosed cancer and the third leading cancer-related deaths worldwide . It is aGenomic studies have uncovered key changes that result in the development of GC and, the most studied and characterized epigenetic alteration is the tumor-associated DNA methylation profile ,4. HowevCircRNAs are a new class of long noncoding RNAs that have their 5′ and 3′ ends covalently joined ,8. CurreA large number of studies have demonstrated the role of circRNAs as miRNA-sponges in GC ,67,68,69More recently, it has been found that circHIPK3/miR-107/BDNF imbalance leads to proliferation and migration of GC cells, and circHIPK3 has been suggested as a potential prognostic biomarker in GC . AdditioSOX4 oncogene and promote gastric tumorigenesis [circRNAs can also sponge RBPs in GC and can negatively affect the activity of the anchored protein or favor the expression and/or activity of others. For instance, circ-DONSON has been shown to interact directly with proteins from the NURF chromatin remodeling complex to regulate the transcription of igenesis . On the igenesis . AlthougIn this study, we gathered experimentally validated sponge circRNAs in GC, in order to understand and to have a broader view of their significance during the development of this type of tumor. Hence, we evaluated the biological roles of these circRNAs, considering their associated RBPs and/or the target genes of their sponged miRNAs, and thus, identified their involvement in GC-related pathways. Our results indicate that the sponge circRNAs in GC are part of a complex RBP-circRNA-miRNA-mRNA interaction network, and may be involved in the establishment, chemoresistance and progression of the tumor. Our search identified 56 papers reporting 54 dysregulated sponge circRNAs in GC, of which 38 were upregulated and 16 were downregulated . We explCOL1A2, FAT1, NF1, FN1, NOTCH1, OSBPL10, PDSS1, PTK2, RANGAP1 and SKA3) presented |fold change|>2, being COL1A2, PVT1, RBMS3 and SERPINB5 the ones that showed expression levels higher than three-fold in GC (We used GC samples from The Cancer Genome Atlas (TCGA) to access the expression of the sponge circRNAs’ host genes. We found 38 differentially expressed host genes in GC (37 upregulated and one downregulated) when compared to the adjacent tissue . Ten genld in GC . In geneThe 54 circRNAs are able to sponge 51 different miRNAs . These sIn general, we observed 46 circRNAs sponging a single microRNA and eight circRNAs regulating at least two microRNAs simultaneously . On the BCL2, CDK6, SP1, IGF1R, VEGFA, CCND2, HMGA2, EGFR, CDKN1A, MCL1 and PTEN genes can be regulated by more than five miRNAs each  pathways . Of thesp ≤ 0.05 and |fold change| ≥1.5, By performing differential expression analysis of the 966 miRNAs’ target genes using the TCGA database, we found 45 DE genes  and, consequently, the gene associated with the highest number of sponge circRNAs  is composed of 147 main genes, of which 53 can be regulated by 24 miRNAs evaluated in this study . ThroughCDKN1A, EGFR, SMAD4 and TP53 genes can be regulated by four to five miRNAs each, and by six to eight sponge circRNAs each one , circAKT3, ciRS-7, circ_SPECC1, circSMARCA5, hsa_circ_0000467, hsa_circ_0000267, hsa_circ_0000673, hsa_circ_0001368, circ_0000096 and hsa_circ_0000993), and that together, they are able to bind to 103 different proteins . circFATAlthough genomic big data have found key genetic modifications that are capable of leading to GC onset ,4, studiIn this study, we gathered circRNAs that can sponge miRNAs and RBPs, and observed that many of them may break homeostasis and disturb biological pathways in GC. An overview of our analyses and results is presented in COL1A2 was the circRNAs’ host gene with the highest fold change in GC , AKT2 (circHIPK3/miR-29b-3p) and AKT3 (cirNF1/miR-16-5p) may also be under the regulatory influence of these axes  that are directly involved in PI3K/Akt pathway , we noticed that it can be more affected by circNF1, circHIPK3, circPVT1 and circ_0000096, since they can regulate many target genes . These ccircRNAs can act as an anchoring point for RBPs and this interaction can block these binding protein’s activity . We founAlthough LIN28 can inhibit the maturation of miR-107, this miRNA can also negatively regulate LIN28 . LIN28 iLastly, of the 54 circRNAs identified in this study, all were tested in the Asian/Chinese population. However, we observed that circHIPK3, circNRIP1 and circSMARCA5 were also identified in the Brazilian population . The BraThe sponge function of the circRNAs has been debated since most of them harbor only a few miRNA-binding sites and effective target repression may often require many binding sites . HoweverIn general, comprehensive bioinformatics analysis of regulatory networks has pointed out potential interactions and new biological roles between small and long noncoding RNAs associated with complex diseases, such as gastric and colorectal cancers ,98,99. WSponge circRNAs were extracted from previous studies selected from PubMed and/or Google Scholar repository databases. Only original research articles published until March 2020 were selected. Keywords that relate and/or associate sponge circRNA, miRNA and gastric cancer were used in the search. In both repositories, we used the following entries: “circRNA sponge and Gastric Cancer”, “circRNA sponge in Gastric Cancer” and “circRNA-miRNA in Gastric Cancer”. Only studies that conducted experimental validation  of the analyzed sponge circRNA were included in the analyzes. Studies based only on in silico predictions of circRNA-miRNA interaction were excluded. Restrictions by year of publication or language were not applied.http://ualcan.path.uab.edu/index.html). This tool is connected to The Cancer Genome Atlas (TCGA) database and provides OMICS expression data of 415 gastric cancer and 34 controls (adjacent to gastric cancer) samples in transcripts per million (TPM). To analyze the differential expression of sponge circRNAs’ host genes, we used the “UALCAN: Analyze, Integrate, Discovery” online tool  (http://https://ccb-web.cs.uni-saarland.de/mirtargetlink/). This tool is connected to the miRTarBase database (release 7.0) [http://miRTarBase.cuhk.edu.cn/), which provides information on experimentally validated interaction between the miRNA and its target gene. We only considered target genes that were validated with “strong evidence” .Target genes of the sponged miRNAs were analyzed using miRTargetLink Human online tool  (https:/ase 7.0)  (http://https://www.r-project.org/) [Functional enrichment analysis of the sponged miRNAs’ target genes was performed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and/or Reactome pathways in STRING online tool (v.11.0). In this analysis, we considered the total number of target genes of each miRNA. Graphs were made in R statistical package  ,104. http://starbase.sysu.edu.cn/starbase2/index.php), which provides large-scale CLIP-Seq data of circRNA-RBP interaction. Its functional analysis was performed using Reactome pathways in STRING online tool (v.11.0). Graphs were made in R statistical package  [Target RBPs of the sponge circRNAs were accessed using the starBase (v. 2.0) online tool (ct.org/) ,104.COL1A2 was the host gene with the highest fold change and circHIPK3/miR-29b-c/COL1A2 axis may be the one that maintains high levels of this gene in GC, favoring the severe clinicopathological characteristics. Thirteen circRNAs may interact with 103 RBPs that are mostly associated with RNA metabolism. Among them, Lin28 stands out for being previously associated with GC. In this context, circHIPK3 may control the balance between LIN28/miR-107 to favor the activity of Lin28 in a negative feedback loop, promoting chemoresistance of GC cells. circHIPK3, circNRIP1 and circSMARCA5 are involved with GC in different ethnic populations and may be ubiquitous regulators of this type of tumor. Finally, circHIPK3, circPVT1, ciR-7, circNF1 and circ_0000096 are potentially promoting gastrointestinal tumors. Therefore, the sponge circRNAs are favoring gastric carcinogenesis by affecting complex mRNA-miRNA-circRNA-RBP interaction networks, and future studies are required to validate their potential as therapeutic targets for this type of tumor. In this context, the identified sponge circRNAs appear to play a central role in the background of gastric tumors and may be considered major players in tumorigenesis.The functional roles of the sponge circRNAs in the pathophysiology of GC remains poorly understood. However, in this study, we pointed to new directions for understanding this context. Overall, the 54 sponge circRNAs studied are capable of interfering in 27 GC-related biological pathways by modulating the interaction of 51 different miRNAs and their target genes, with a more pronounced effect on PI3K/Akt signaling pathway.