Although not expressed endogenously, the presence of trogocytosed molecules on cells has the potential to significantly impact an immune response and the biology of the individual trogocytosis-positive cell. Many studies have focused on the ability of the trogocytosis-positive cells to interact with other immune cells and modulate the function of responders. Less understood and arguably equally important is the impact of these molecules on the individual trogocytosis-positive cell. Molecules that have been reported to be trogocytosed by cells include cognate ligands for receptors on the individual cell, such as activating NK cell ligands and MHC:peptide. These trogocytosed molecules have been shown to interact with receptors on the trogocytosis-positive cell and mediate intracellular signaling. In this review, we discuss the impact of this trogocytosis-mediated signaling on the biology of the individual trogocytosis-positive cell by focusing on natural killer cells and CD4+ T lymphocytes.Trogocytosis is the intercellular transfer of membrane and membrane-associated molecules. This underappreciated process has been described in a variety of biological settings including neuronal remodeling, fertilization, viral and bacterial spread, and cancer, but has been most widely studied in cells of the immune system. Trogocytosis is performed by multiple immune cell types, including basophils, macrophages, dendritic cells, neutrophils, natural killer cells, B cells, γδ T cells, and CD4 Naegleria fowleri [Trogocytosis the direct, intercellular transfer of membrane and membrane-associated molecules in a contact-dependent manner. This term was originally coined by Brown and colleagues to describe the cytopathogenicity of the parasitic amoeba  fowleri , and it  fowleri .+ [+ [Francisella tularensis [Most studies on trogocytosis have focused on interactions of immune cells. Trogocytosis has been observed in CD4+ ,9,10,11,+ [+ ,13,14,15+ [+  T cells,+ [+ ,18,19, N+ [+ ,21,22, b+ [+ , macroph+ [+ ,25, neut+ [+ ,27,28, a+ [+ ,30. Howe+ [+ . It has + [+ ,33, trog+ [+ , during + [+ ,36,37, s+ [+ , erythro+ [+ , and par+ [+ ,41,42,43larensis  and viralarensis ,46.The initial descriptions of trogocytosis date back to the early 1970s ,48,49,50+ T cells capture MHC class I molecules [For both T cells ,57,58,59olecules ,74 or irolecules . This haWith our increased understanding of trogocytosis, it has become a tool in both basic research and the clinic. The detection of trogocytosis by flow cytometry has proven to be a useful tool in the discovery of antigen and detection of antigen-specific lymphocytes. It has been used to identify antigen-specific B and T cells ,77 and v+ T cells is dependent on the affinity of the TCR for MHC:peptide, as APC loaded with lower-affinity peptide analogues leads to a significant reduction in trogocytosis . We have also reported that, while antibody blockade of CD80 had minimal impact on trogocytosis of MHC class II:peptide, blocking of MHC class II or CD3 significantly reduced trogocytosis of MHC class II:peptide [Defining the mechanism of trogocytosis has proven somewhat elusive. The efficiency of trogocytosis is impacted by a variety of factors. It is an active process, requiring both antigen recognition and cognate receptor signaling ,18,60. IThe activation state of the cell impacts trogocytosis, as activated T cells are much more efficient at performing trogocytosis than naïve T cells ,84,85,86Trogocytosis is contact-dependent and requires actin cytoskeletal rearrangement . Treatme2) membrane fragments, and blocking exosome formation in DC with the ATPase inhibitor CMA only slightly decreases the amount of trogocytosis [+ T cells [Trogocytosis is mechanistically distinct from interactions with APC-derived exosomes as it involves the transfer of large  is an RRas2- and RhoG-dependent phagocytosis event  may induce cytokine production from responders [The response initiated by trogocytosis-positive T cells presenting antigens is largely a function of the activation state of the responding T cell. Antigen presentation by trogocytosis-positive cells activates responding naïve T cells ,88,98,99sponders . TEM imasponders  and suppsponders ,102,103.+ trogocytosis-positive cells are functionally comparable to thymic-derived natural T regulatory cells (nTreg), inhibiting proliferation of responding T cells [Attenuation of the immune response via antigen presentation by trogocytosis-positive T cells has been observed using T cells from multiple myeloma patients. In vitro incubation of patient cells with myeloma cell lines led to trogocytosis of CD86 and the immunosuppressive molecule HLA-G . HLA-G+  T cells ,105. In  T cells . In anot T cells .+ T cells vs. trogocytosis-positive Treg results in differential regulation of CD4-mediated immune responses. A comparison of CD4+ T cell effector subsets is outlined in reg attenuates responses and presentation by trogocytosis-positive effector CD4+ T cells potentiates them [reg cells [reg cells themselves display high rates of Ag-specific trogocytosis [reg) and antigen-induced, peripheral induced regulatory T cells (iTreg) [reg cells use trogocytosed molecules to form connections with activated T cells, thus enhancing their suppressive activity [reg cells or TH cells displaying trogocytosed immune-suppression associated molecules, such as HLA-G, show enhanced suppressive capabilities [Presentation by trogocytosis-positive effector CD4tes them . In addieg cells ,109,110,ocytosis . CD80 an (iTreg) , and it bilities ,112,113.bilities .+ T cells capture bystander MHC class I molecules from APCs, and they can induce CD8+ T cell activation [+ T cells and subsequent presentation to CD8+ T cells may play an important role in CD4+ T cell “licensing” of CTL activity [+ T cells, dendritic cells, and CD8+T cells must interact sequentially or simultaneously to allow CD4+ licensing of CD8+ T cells. More recently, Boccasavia et al. found that trogocytosis-positive and OT-II TCR transgenic T cells can present antigen and stimulate naïve T cells in an antigen-specific manner [H17 phenotype. Interestingly, the trogocytosis-positive stimulating T cells downregulated CCR6 and upregulated Foxp3. After a 6-day coculture, the frequency of Foxp3+ cells among the trogocytosis-positive antigen-presenting T cells was almost sixfold higher than the responders, and the frequency of RORγt+ IL-17A+ cells in the responder population was sevenfold higher than in the trogocytosis-positive presenting population. This polarization of the trogocytosis-positive population to Treg cells and responders to TH17 was also observed in vivo [+CD25+ CD4 Treg cells was directly proportional to the number of antigen-presenting cells in the initial T-APC culture, while the number of TH17 cells was indirectly proportional to number of APC. The authors concluded that an abundance of antigen-bearing APC favors the differentiation of Treg, while limiting number of APC favors TH17 differentiation because that favors T–T interactions and presentation by trogocytosis-positive T cells [Antigen presentation by trogocytosis-positive T cells does not universally lead to attenuation of an immune response. Xiang and colleagues have shown that CD4tivation ,74,109. activity  and helpc manner . When th in vivo . The num T cells .+ NK cells capture the activating ligand MICA from target cells, resulting in the subsequent activation of bystander NK cells and triggering of cytolytic granule release those cells. This ultimately leads to the death of the MICA+ NK cells via fratricide [+ NK cells can trogocytose the activating ligand Rae-1δ from target cells, and the Rae-1δ+ NK cells themselves become the target of NK cells killing, both in vitro and in vivo [+ NK cells trogocytose MHC class II:peptide complexes from dendritic cells, and these trogocytosis-positive NK cells can subsequently negatively regulate CD4+ T cell activity both in vitro and in vivo [As with T cells, the presence of trogocytosed molecules on NK cells can mediate NK–NK cell interactions, leading to alterations in function and cytolytic activity of both the trogocytosis-positive NK cell and the responding cell. For example, NKG2Datricide . Similar in vivo . This ha in vivo .Thus, both trogocytosis-positive T cells and trogocytosis-positive NK cells can present antigens to bystander cells and modulate immune responses ,118,119.+ T cell surface [Trogocytosed molecules are found in a focused spot on the CD4 surface ,6,98, an surface ,120,121. surface , trogocy surface .Several lines of evidence support integration into the trogocytosis-positive cell membrane including flow cytometry and light microscopy studies, which have shown that extracellular epitopes are accessible on intact cells and intracellular epitopes are inaccessible unless the cells are detergent-permeabilized ,6,65. Si+ T cells [+ NK cells toward the CCR7 ligands CCL19 and CCL21 [+ NK cells migrated to lymph nodes at a significantly higher rate than the CCR7– NK cells [+ T cells and it efficiently binds to immune complexes, although it does not induce detectable intracellular signaling [While the ability of trogocytosis-positive T cells to present trogocytosed molecules to other T cells is well documented, much less is known about the biological consequences of trogocytosis on the individual trogocytosis-positive cell. Membrane proteins acquired from APCs are detectable for several days after removal of the APC on NK cells  and CD4+ T cells ,6,59,75. T cells ,124, as  T cells . The trond CCL21 ,124. AftNK cells . Thus, tNK cells . Hudrisiignaling .trogocytosis-mediated signaling. Trogocytosis-mediated signaling has been described in nonimmune systems. In a study using transfected CHO and HeLa cells expressing fluorescently tagged CD30 or CD30L, Nakashima et al. showed that CD30+ cells trogocytose CD30L from neighboring cells in an actin-dependent, clathrin-independent manner. This was associated with CD30 internalization, and the CD30–CD30L complexes traffic to the lysosome where they colocalize with TRAF molecules, consistent with active, trogocytosis-mediated signaling occurring within in the trogocytosis-positive cell. Ca2+ flux data helped confirm that, indeed, trogocytosis-mediated CD30 signaling is driven by trogocytosed CD30L in trogocytosis-positive cells [One underappreciated possibility is that trogocytosed molecules interact with cognate receptors on/in the trogocytosis-positive cell and mediate intracellular signaling, so-called ve cells .+ T cell surface of [+ T cells, there is specific accumulation of active signaling molecules, such as phosphorylated ZAP-70 (pZAP-70), phosphorylated Lck (pLck), and phosphorylated ERK 1/2 (pERK), at the site of trogocytosed MHC:peptide and accumulated TCR [Trogocytosed class II MHC:peptide molecules are retained in punctate areas on the CD4rface of ,6,98 forrface of ,75, and rface of ,6,59. Sirface of . Strikinated TCR ,6,59, stated TCR ,129. Eff+ NK, although perforin levels were unchanged and killing via LY49D was unaffected [+ NK cells are hyporesponsive in vitro and in vivo, and it is proposed that the trogocytosed m157 engages LY49H and sustains signaling through LY49H, thus rendering the cell hyporesponsive [Trogocytosis-mediated signaling has also been reported to occur with NK cells. On NK cells, the activating ligand MICB colocalizes with NKG2D and markedly reduces the cytolytic activity of these cells . This isaffected . Similarsponsive .+ T cells preferentially survive over a 5-day culture in vitro after removal of APCs [+ cells. Over several days, the frequencies of trogocytosis-positive and trogocytosis-negative cells reverse such that trogocytosis-positive cells make up more than 82% of the viable CD4+ T cells and the trogocytosis-negative cells represent less than 18% of viable CD4+ cells on day 3. The level of trogocytosed MHC:peptide associated with the cell remains fairly constant on the trogocytosis-positive cells over 5 days, and there is minimal CFSE (5-(and 6)-carboxyfluorescein diacetate succinimidyl ester) dilution , suggesting that, under those conditions, the trogocytosis-positive cells are selectively surviving, but not dividing [After trogocytosis, trogocytosis-positive CD4 of APCs . Immedia+ T cells had elevated NF-κB (p50/p50) and AP-1 activity 24 h after APC removal [Consistent with sustained, trogocytosis-mediated signaling, Zhou et al. showed that sustained, trogocytosis-mediated signaling alters biological outcomes in trogocytosis-positive cells. In that study, trogocytosis-positive CD4 removal . These c removal , suggest+ cells increased sevenfold between 5 h and 72 h, reaching 70% of the population [H2 phenotype was not observed with anti-CD3 + anti-CD28 stimulation. Taken together, these results are in agreement with the earlier work showing that trogocytosis-mediated signaling is qualitatively different [+ cells increased, the frequency of trogocytosis-positive IFNγ-producing cells decreased from 10% at 5 h to 0.5% at 72 h after APC removal. To confirm that signaling was being driven by trogocytosed molecules and not residually from the T–APC interaction, cells were treated with the reversible Src family kinase inhibitor PP2 to halt TCR signaling. When the PP2 was subsequently washed out, signaling resumed in the trogocytosis-positive, but not trogocytosis-negative cells, consistent with signaling being induced by trogocytosed molecules, i.e., trogocytosis-mediated signaling. The frequency of trogocytosis-positive IL-4+ cells from PP2-treated cultures rebounded to levels equal to untreated cells by 24 h after PP2 removal. There was robust IL-4 production and GATA-3 expression observed in trogocytosis-positive, but not trogocytosis-negative, cells 72 h after PP2 treatment. By comparison, the expression of IFNγ did not resume in either trogocytosis-positive or trogocytosis-negative cells. Somewhat unexpectedly, in vitro polarized trogocytosis-positive TH1 cells began expressing IL-4 and GATA-3, while simultaneously decreasing expression of IFNγ and T-bet [H2 differentiation. Differentiation of trogocytosis-positive T cells to a TH2 phenotype was also observed in vivo during an immune response.When the potential impact of trogocytosis-mediated signaling on effector cytokine production was examined, it was observed that 5 h after removal of APCs, trogocytosis-positive cells expressed significantly lower levels of IFNγ and significantly higher levels of IL-4 compared to trogocytosis-negative cells in the same culture . After rpulation . Robust ifferent , particind T-bet , furtherH2 cells after APC removal partially reflects differences in trogocytosis efficiency as in vitro polarized TH2 cells are more efficient than in vitro polarized TH1 or nonpolarized cells at performing trogocytosis [H1 and TH2 cells may reflect morphological differences in their immunological synapses. At low Ag concentrations, TH1 synapses form the classical “bull’s eye” shape, while TH2 cells form multifocal synapses [H2 cells facilitate trogocytosis more efficiently than the synapses formed by TH1 cells, although this has not been formally tested.The increased frequency of Tocytosis . The difsynapses . In sepasynapses , as discH1 differentiation have been shown to require strong TCR-signaling [H1 to TH2 conversion further supports this model, as weak TCR signaling drives TH2 differentiation, even under TH1-polarizing conditions [The concept of auto-presentation is not new as, three decades ago, it was reported that T cell self-presentation of cognate peptide induces killing of the individual cell ,153. Autignaling ,165,166.nditions .H2 phenotype, continual trogocytosis-mediated signaling may aid in the generation of follicular helper T cells (TFH) or CD4+ memory cells, since sustained TCR signaling through repeated antigen encounter is required for their differentiation [H2 to a TFH phenotype takes between 5 and 7 days in vivo [FH differentiation. IL-21 appears to be polarized toward the trogocytosed molecules in a pattern similar to IL-4 . The integration of differential signaling from the immune synapse and from trogocytosed molecules may help establish asymmetric T cell division and subsequent differential T cell fate, as suggested by Reiner and colleagues [+ T cells.Interestingly, as mentioned above, trogocytosed molecules move from the T–APC interface to a region of the T cell directly opposite the immune synapse, i.e., the distal pole complex ,171, morlleagues ,173,174.+ T cell and the duration of T cell interactions with an APC. Iezzi et al. showed that full T cell activation requires up to 6 h of sustained Ag stimulation for up to 6 h [An apparent paradox exists between the duration of Ag stimulation necessary to fully activate a CD4p to 6 h . Howeverp to 6 h . In vitrp to 6 h ,156. Thep to 6 h . The Lanp to 6 h  and in vp to 6 h ; a phenop to 6 h ,179. ThuTrogocytosis-mediated signaling, by altering the activation state, survival, differentiation, and production of effector cytokines, has the potential to significantly impact the physiology and subset differentiation of the trogocytosis-positive cell and, by extension, control the immune response. This paradigm-shifting event may play a significant role in the generation and control of protective immune responses, cancer immunotherapy, and vaccine efficacy. However, we have very limited knowledge of trogocytosis-mediated signaling, and more work is necessary to improve our understanding of this underappreciated phenomenon and the role it plays in modulating the immune response.


Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) are clonal hematological diseases classified as Philadelphia chromosome-negative myeloproliferative neoplasms (MPN). MPN pathogenesis is associated with the presence of somatic driver mutations, bone marrow (BM) niche alterations, and tumor inflammatory status. The relevance of soluble mediators in the pathogenesis of MPN led us to analyze the levels of cytokines, chemokines, and growth factors related to inflammation, angiogenesis and hematopoiesis regulation in the BM niche of MPN patients.Soluble mediator levels in BM plasma samples from 17 healthy subjects, 28 ET, 19 PV, and 16 PMF patients were determined using a multiplex assay. Soluble mediator signatures were created from categorical analyses of high mediator producers. Soluble mediator connections and the correlation between plasma levels and clinic-laboratory parameters were also analyzed.status, risk stratification of disease, hemoglobin concentration, hematocrit, and red blood cell count.The soluble mediator signatures of the BM niche of PV patients revealed a highly inflammatory and pro-angiogenic milieu, with increased levels of chemokines , and growth factors . ET and PMF patients presented intermediate inflammatory and pro-angiogenic profiles. Deregulation of soluble mediators was associated with some clinic-laboratory parameters of MPN patients, including vascular events, treatment Each MPN subtype exhibits a distinct soluble mediator signature. Deregulated production of BM soluble mediators may contribute to MPN pathogenesis and BM niche modification, provides pro-tumor stimuli, and is a potential target for future therapies. Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF) are classical myeloproliferative neoplasms (MPN) also known as Philadelphia chromosome (Ph)-negative MPN. These clonal diseases are characterized by single or multilineage hyperproliferation of the bone marrow (BM) that results in spontaneous accumulation of mature myeloid cells in the BM and peripheral blood. The erythroid lineage is the mostly affected in PV patients, while the megakaryocytic lineage from ET and PMF patients exhibit hyperplasia and atypia, and BM fibrosis. PMF patients also have increased or decreased number of granulocytes, monocytosis, and erythroid dysplasia , 2.Disease pathogenesis is partially attributed to the presence of acquired driver mutations in Janus Kinase 2 (JAK2), calreticulin (CALR) or myeloproliferative leukemia virus (MPL) genes. These somatic mutations lead to abnormal activation of the JAK pathway, resulting in constitutive activation of their downstream effectors, specially STATs . JAK2V61The crosstalk between inflammation and neoplastic cells plays a crucial role in disease development and progression. The BM of MPN patients is rich in inflammatory cytokines and growth factors, which form a pro-tumorigenic microenvironment that supports neoplastic cells and favors specific clinical phenotypes –8.Cytokines and chemokines are key mediators of the immune system that regulate many complex signaling processes, and whose levels reflect the systemic and local immune activation status , 10. TheMPN are considered tumor inflammatory diseases. Over the past years, several studies have investigated how chronic inflammation contributes to MPN pathogenesis , 7, 12. Considering the relevance of cytokines, chemokines, and growth factors (hereafter referred to as soluble mediators) as mediators of inflammation, angiogenesis and hematopoiesis regulation, here we examined the soluble mediator signature in the BM niche of ET, PV, and PMF patients. We also analyzed the correlation between cytokine levels and clinic-laboratory parameters.The Ethics Committees for Human Research from the School of Pharmaceutical Sciences of Ribeirão Preto, from the University Hospital of the Ribeirão Preto Medical School , and from the Euryclides de Jesus Zerbini Transplant Hospital  approved the study protocol.The studied groups consisted of 17 healthy volunteers (CTRL group) and 63 MPN patients . The patients were recruited at the Bone Marrow Transplantation Unit of HCFMRP-USP and at the Euryclides de Jesus Zerbini Transplant Hospital. All MPN patients were diagnosed according to the 2016 World Health Organization criteria (g for 10 min at 4°C (Eppendorf 5810R centrifuge), and aliquots were stored at −80°C for further cytokine analysis.BM aspirates were collected from the left posterior iliac crest into EDTA tubes at the time of diagnosis. Plasma was separated from the BM samples by centrifugation at 400×PV patients were classified into two risk categories: high and low. High-risk patients were the ones with age >60 years and/or history of vascular complications . The patients who did not present the abovementioned two risk factors were classified as low risk .The revised IPSET-thrombosis (r-IPSET-t) risk score splits ET patients into four risk categories: very low (age <60 years and absence of JAK2 mutation), low (age <60 years and presence of JAK2 mutation), intermediate (age >60 years and absence of JAK2 mutation), and high risk (age >60 years and presence of JAK2 mutation or vascular complication) .The DIPSS-Plus scoring system was used to classify PMF patients into four categories: low, intermediate-1, intermediate-2, and high risk. The prognosis score considered the following risk factors: age, white blood cell and platelet counts, peripheral blood blast percentage, hemoglobin concentration, transfusion dependency, presence of constitutional symptoms, and unfavorable karyotype .® Assay, R&D Systems) performed on the Luminex1 MAGPIX1 System (Luminex Corporation). The cytokines and chemokines measured were interleukins (IL) IL-6, IL-1β, IL-12p70, IL-10, IL-17a, and IL-18; interferon gamma (IFN-γ); tumor necrosis factor alpha (TNF-α); IL-6 receptor subunit alpha (IL-6Ra); C–X–C motif ligands (CXC) CXCL8 , CXCL12 , CXCL10 ; and C–C motif chemokine ligands (CCL) CCL2  and CCL5 (known as RANTES). The growth factors granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and VEGF receptor 2 (VEGF-R2) were also quantified. Data were analyzed using the Milliplex Analyst software v3.5 .Soluble mediator levels were determined using a customized microbeads multiplex assay  and patients’ JAK2V617F status. Spearman test was used for the correlation analysis of hematological parameters. GraphPad Prism 6.0 software (GraphPad Software) was used for statistical analysis. Significance was set at 0.05.r-values (correlation coefficient) from the Spearman test that had p-value <0.05.Cytoscape 3.7.2 software  was used to construct the soluble mediator signatures , using tOverall soluble mediator profile was obtained by characterizing the general cytokine pattern of each group , 19. Eacstatus, risk stratification, fibrosis rate, transfusion dependency, treatment status, hematological parameters, and reticulin rate are summarized in The median age of MPN patients was 65.5 years (20–85), distributed as 63 (31–78), 62 (20–85), and 68.5 (54–80) years for ET, PV, and PMF patients, respectively. The median age of CTRL subjects was 49 (19–83) years. Male–female proportions in the studied CTRL, ET, PV, and PMF groups were 6–11, 5–23, 12–7, and 12–4, respectively. Demographic and clinic-laboratory characteristics of the MPN cohort, including age, gender, mutation Compared with CTRL, the BM niche of PV patients presented increased levels of inflammatory cytokines and angiogenesis- and hematopoiesis-related factors, including CCL2, CCL5, CXCL8, CXCL10, CXCL12, GM-CSF, HGF, IFN-γ, IL-1β, IL-6Ra, IL-12p70, IL-17a, IL-18, M-CSF, TNF-α, VEGF, and VEGF-R2 Table S5ET patients exhibited augmented levels of CCL2, CCL5, CXCL8, CXCL10, GM-CSF, IFN-γ, IL-1β, IL-17a, IL-18, TNF-α, and VEGF, when compared with CTRL. MF patients displayed higher levels of only CXCL8, CXCL10, IL-6Ra, and IL-18, as compared with CTRL Table S5All MPN categories presented high production of the chemokines IL-18, CXCL10 and CXCL8. Compared with PV patients, ET patients had lower levels of G-CSF, HGF, IFN-γ, IL-10, and IL-17a in the BM niche, while PMF patients presented lower levels of CCL2, CCL5, CXCL12, G-CSF, GM-CSF, HGF, IFN-γ, IL-1β, IL-10, IL-17a, IL-12p70, M-CSF, TNF-α, and VEGF. The BM niche of ET patients exhibited higher levels of CCL5, IL-6 and VEGF than PMF patients.The present results indicated that the BM levels of IL-17, IFN-γ, G-CSF, and HGF in PV patients were higher than those detected in PMF and ET patients. Hence, PV patients seem those with more unique soluble mediators profile as compared to the other MPN.Categorical analyses were performed to better comprehend the soluble mediator production patterns and the differences among MPN subtypes. Patients were stratified into high and low producers of soluble mediators using the overall median as cut-off point. High producer was the individual whose soluble mediator production value was higher than the overall median, while low producer was the individual whose soluble mediator production level was equal to or lower than the overall median. The frequency of high producers of each mediator was calculated for all disease and CTRL subsets. The production of each mediator was considered relevant when the frequency of high producers exceeded 50% , 19, 20.The CTRL group did not produce any of the mediators in relevant amounts, since less than 50% of the individuals were high producers , weak (r ≤0.35), moderate (r ≥0.36 and r ≤0.67) or strong (r ≥0.68). The CTRL group exhibited the highest number of strong correlations, and similar findings were obtained in ET patients . PMF patients also showed many strong interactions , while PV patients were highly different from the CTRL group and showed the lowest number of strong correlations . These results highlighted the deregulation of soluble mediator network in PV patients versus other MPN categories and CTRL group.After identifying the soluble mediator signature for each disease  and CTRL group, we analyzed the existence of correlation between the mediator levels Figure 2We analyzed the potential correlation between soluble mediator levels and the major clinic-laboratory parameters  higher CXCL8 levels than those detected in low risk ET patients (n = 4)  had lower G-CSF levels than untreated PV patients  exhibited lower levels of IL-17, CCL5, GM-CSF, and VEGF than ET patients with no vascular event (n = 17)  for JAK2V617F and CALR. ET CALR mutated patients displayed elevated M-CSF levels than DN; and JAK2V617F- patients showed lower levels of M-CSF and CXCL12 than DN patients  of soluble mediators according to the presence of driver mutations was also analyzed in ET  complex lead to subsequent activation of JAK/STAT, MAPK and PIK pathways, and it can be activated in all types of cells, including cells not responsive to IL-6 alone . An obse+ MPN patients had higher M-CSF levels than JAK2V617F+ patients. It is well-known that CALR+ patients have better prognosis and higher overall survival than JAK2V617F+ patients . Most of the soluble mediators interaction found in our study presented biological relevance, and resides in their synergistic interactions, which could be observed between: 1) IL-1β and IL-12 inducing IFN-γ secretion ; 2) IL-1status, occurrence of vascular events and oncoinflammation.PV patients display very strong positive correlations only between a few cytokine and chemokine molecules . This data suggests that the immune imbalance in BM microenvironment is more prominent in PV than in ET and PMF patients. Literature reported that these molecules are associated with a pro-inflammatory status in MPN patients’ serum , a cytoredutor drug indicated for MPN treatment, is capable of lowering serum inflammatory markers such as TNF-α, IL-6, CXCL8, and IL-1β in sickle cell disease patients , 57. ThiThe frequency of vascular events was associated with different setups of soluble mediators among MPN subtypes. PV patients exhibited increased TNF-α, CXCL12, and VEGFR2 levels; ET patients displayed increased IL-17, CCL5, GM-CSF, and VEGF levels; and PMF patients had increased IL-6Ra levels. Elevated GM-CSF and IL-12 serum levels are associated with the lack of vascular complications in ET and PV patients; these cytokines may also help to select the treatment regimen . In addiActivated platelets are the mainly secretors of CXCL12. Upregulated CXCL2 expression and secretion may favor the development of cardiovascular diseases, while the fast increase of CXCL12 in peripheral blood platelets can be used as biomarker for cardiac injury . PatientCXCL10 and CCL2 gene expression and increased fibrosis in BM niche , by the São Paulo Research Foundation , and by the National Council for Scientific and Technological Development . MC and MB-C are recipients from FAPESP scholarships .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.


The lncRNA BACE1-AS was identified as a plasma molecular marker in the early diagnosis of Alzheimer's disease, but its role in tumors remains poorly defined.  The expression patterns, genomic mutation, and prognostic significance of BACE1-AS in pan-cancers were compared by analyzing 32 types of tumors from The Cancer Genome Atlas and cBioPortal databases. The relationships between BACE1-AS expression levels and the degree of immune cell infiltration, immune components, and immune-related genes were explored. The possible molecular mechanisms of BACE1-AS in tumors were explored using gene set enrichment analysis (GSEA). Finally, the role of BACE1-AS in hepatocellular carcinoma was confirmed via quantitative real-time polymerase chain reaction (qRT-PCR).  BACE1-AS expression levels were significantly upregulated in LIHC, GBM, KIRC, CHOL, STAD, KICH, COAD, and PRAD. Higher expression levels of BACE1-AS were associated with worse overall survival in patients with HNSC and LIHC, while the opposite was found in PCPG and THCA. The overall mutation rate of BACE1-AS in pan-cancer was only approximately 0.9%, and it occurred mainly in uveal melanoma and uterine carcinoma. Generally, BACE1-AS expression was negatively correlated with the immune microenvironment. BACE1-AS expression was mainly related to naïve B cells, activated memory CD4 T cells, monocytes, M1 macrophages, M2 macrophages, and resting mast cells. The potential mechanisms of BACE1-AS in tumors were mainly via regulating the activities of B cell-mediated immunity, immune response regulating cell surface receptor signaling, RNA binding in posttranscriptional gene silencing, B cell receptor signaling pathways, and immune receptor activity. Finally, the qRT-PCR results confirmed that the expression levels of BACE1-AS in hepatocellular carcinoma cell lines were upregulated.  Overall, our results suggest that BACE1-AS is associated with the expression, prognosis, and rate of immune cell infiltration of most tumors. Thus, BACE1-AS may be a potential target for immunotherapies aimed at improving cancer patient outcomes. Malignant tumors are a major global public health problem that seriously threatens human health, and their overall incidence is rising worldwide . In manyThe theory that the genesis of a tumor is primarily determined by its genetic material is based on the tumor-centric view. Traditional methods such as surgical resection, radiotherapy, and chemotherapy are all derived from this theory; these methods all inevitably have the same destructive effect on normal tissues . Recentlβ-secretase 1 (BACE1) and can pair with BACE1 mRNA to change its spatial structure and increase its stability to promote protein translation in a positive feed-forward fashion [β-secretase family and is thought to play an important role in Alzheimer's disease via the processing of amyloid precursor protein (APP) in neurotoxic Aβ peptides. Recently, BACE1 has been reported to be abnormally expressed in a variety of tumors and is associated with poor prognosis in colon cancer, non-small-cell lung cancer, invasive ductal carcinoma of the breast, glioblastoma, and gastric cancer [Long noncoding RNAs (lncRNAs), a group of RNAs with a length of more than 200 nucleotides, do not contain protein-coding transcripts due to the lack of an open reading frame . However fashion , 13. In c cancer –18.Whether BACE1-AS has a similar effect in malignant tumors is unknown. Therefore, we conducted an in-depth analysis of 32 human tumors from The Cancer Genome Atlas (TCGA) database to further explore the role of BACE1-AS in tumors and its relationship with tumor immunity.https://portal.gdc.cancer.gov/), an open database covering 32 human cancer types, containing more than 30,000 tumor samples and information on the expression of more than 20,000 genes [http://www.cbioportal.org).The expression matrix of BACE1-AS for 32 human tumors was obtained from TCGA database , disease-specific survival (DSS), progression-free interval (PFI), and disease-free interval (DFI) were analyzed to evaluate prognostic value. The log-rank test was used to evaluate the statistical significance between them, and http://xena.ucsc.edu/), and tumor mutation load, the number of mutations per megabase, was calculated. The correlation between the expression of BACE1-AS and tumor mutation burden (TMB) or microsatellite instability (MSI) was calculated by the Spearman test. The results were represented by radar map drawn by the R “fmsb” package.Mutation data for 32 cancer types were downloaded by UCSC XENA (The tumor microenvironment (TME), a special environment surrounding the presence of tumor cells, is composed of tumor stroma, adjacent cells, blood vessels, peripheral immune cells, and immune molecules . The chaThe relative proportion of infiltrating immune cells in a pan-cancer patient was calculated by the CIBERSORT algorithm. The correlation between BACE1-AS expression and immune cells was analyzed by the Spearman correlation test. The R “ggpubr” package was used to analyze the correlation between 32 kinds of human tumors and 22 kinds of immune cells. The correlation between the expression of BACE1-AS and immune-related genes was performed by the R “reshape2” and the “RColorBrewer” packages.org.Hs.eg.db,” and “clusterProfiler” packages based on “c2.cp.kegg.v7.1.symbols.gmt gene set” and “c5.all.v7.1.symbols.gmt”. In order to obtain the normalized enrichment fraction, nominal (NOM) P < 0.05 and false discovery rate (FDR) < 0.05 were considered to be significantly enriched.The potential mechanism of BACE1-AS in pan-cancer was mainly revealed by gene set enrichment analysis (GSEA) including Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms. The GSEA was performed by R “ggplot2,” “enrichplot,” “The prognostic value of BACE1-AS combined with coexpression immune-related checkpoint genes for hepatocellular carcinoma was analyzed, and a prognostic model was established by Cox regression analysis. Furthermore, the expression level of BACE1-AS in HCC was detected by real-time quantitative reverse transcription PCR (qRT-PCR) at cell level. The sensitivity analysis of the common compounds was performed by R “pRRophetic” package.P value < 0.05 was considered statistically significant.OS, DFI, DSS, and PFI were analyzed through R packages (R version 4.1.0) “survival” and “survminer” by the Kaplan-Meier method. The correlations between BACE1-AS expression level and TMB, MSI, stromal, tumor-infiltrating immune cells, and immune marker sets were calculated by the Spearman test. The correlation between BACE1-AS expression and clinical characteristics by the sensitivity analysis of the common compounds was analyzed by the Wilcoxon test. All statistics were achieved by SPSS statistical software (version 25.0.0). The pan-cancer data of 32 primary tumors are described in The correlations between BACE1-AS expression levels and the prognosis of different cancers are depicted in P < 0.05). BACE1-AS expression in male patients (P < 0.05). In the other types of tumors, BACE1-AS expression was not significantly different between the genders. BACE1-AS expression levels were correlated with tumor stage in BRCA, KIRP, CHOL, COAD, and LIHC , stromal scores , and estimate scores .The correlations between BACE1-AS expression and stromal scores and estimate score are shown in Supplementary Figures R = −0.45, P < 0.05), while monocytes and macrophages M2 have the most significant positive correlation . The correlation analysis between 22 immune cell types and pan-cancer as based on the TIMER database is illustrated in Figures Immune checkpoints are one of the most promising targets for cancer therapy. They protect the immune system by preventing T cell overactivation from causing damage to the body. The upregulation of these immune marker sets can induce immune escape to inhibit the antitumor response of the immune system. Thus, we analyzed the relationship between 46 immune checkpoint genes and pan-cancer.TNFRSF25, PDCD1LG2, CD276, CD80, HAVCR2, LAIR1, CD160, TNFRSF14, ADORA2A, and CD48 were significantly associated with BACE1-AS expression in more than half of the tumor types. Notably, KIR3DL1 was the gene negatively correlated with BACE1-AS expression only in PRAD . ACC was significantly correlated with TNFRSF25  and TNFRSF14  only. Finally, DLBC was significantly correlated with CD276  and BTNL2 .We found that THCA, TGCT, OV, GBM, and KIRC were the top five tumors significantly correlated with most immune marker genes . BACE1-AWe applied Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on different BACE1-AS expression levels in pan-cancer. We found that KEGG function between the high- and low-expression BACE1-AS groups was significantly different in 15 types of tumors and was significantly different in GO enrichment across all tumor types.BACE1-AS expression mainly affected pathways including complement and coagulation cascades, RRAR signaling pathway, intestinal immune network for immunoglobulin A (IgA) production, cancer pathways, autophagy regulation, cytokine-cytokine receptor interaction, and antigen processing and presentation, which are mainly related to tumorigenesis and immune activity . The resP < 0.05).The role of BACE1-AS in tumorigenesis and its relationship with immunity in pan-cancer cannot be ignored by the above analysis. However, experimental evidence is lacking. We further validated the role of BACE1-AS in HCC at cellular levels using quantitative real-time polymerase chain reaction (qRT-PCR). CD276, TNFRSF14, ADORA2A, TNFSF15, BTNL2, TNFRSF4, PDCD1, TNFSF4, NRP1, TNFRSF8, TNFRSF25, LGALS9, HHLA2, TNFSF14, TNFSF9, TNFRSF18, VTCN1, CD70, and CTLA4) were identified significantly associated with BACE1-AS expression .19 immune checkpoint genes . However, the expression levels of BACE1-AS in UVM were not significantly correlated with these immune cells. Neutrophils were significantly negatively correlated with BACE1-AS expression in the most common tumor types. Notably, infiltrating neutrophils may promote tumor-associated inflammation or, through the expression of antitumor and cytotoxic mediators, inhibit tumor growth [γ signaling pathway. Based on their phenotypes and functions, macrophages can be divided into classically activated macrophages (M1 type) and alternatively activated macrophages (M2 type), also known as tumor-associated macrophages (TAMS). TAMs can prevent T cells from attacking tumor cells, secrete growth factors to nourish tumor cells, promote the formation of blood vessels in tumor tissues, and further promote the metastasis and proliferation of tumor cells [Immune infiltrating cells in tumor tissues can change the metabolism and function of tumor cells and promote tumor immunosuppression and immune escape . We analr growth . This chr growth . We alsoor cells , 32. We κB and MARK signaling pathways [Immune checkpoint molecules, such as CTLA4, NRP1, TNFSF15, CD44, and BTLA, are present in the TME or on the surface of tumor cells, and they negatively regulate T cell activation. Under normal conditions, these molecules mainly maintain T cell homeostasis, but during tumor evolution, these molecules can help tumor cells escape the immune response , 34. CTLpathways –39. ThusWe found that BACE1-AS expression affects pathways related to tumorigenesis and immune activity. These pathways include the complement and coagulation cascades, RRAR signaling pathway, intestinal immune network for IgA production, cancer pathways, autophagy regulation, cytokine-cytokine receptor interaction, and antigen processing and presentation. These results account for the association between BACE1-AS expression and tumor immunity from the perspective of internal mechanisms, but further experimental investigation is needed for verification.Overall, our results suggest that BACE1-AS is abnormally expressed in most tumor types, and its expression level is correlated with clinical features, prognosis, TMB, and MSI. Moreover, BACE1-AS expression may affect the TME, the content of infiltrating immune cells, the immune molecular checkpoints, and the immune activity-related pathways. Altogether, our data indicate that BACE1-AS can be a potential immunotherapy target in improving cancer patient outcomes.


Scutellariae baicalensis Georgi. with various beneficial effects, including anti-inflammatory and neuroprotective effects. A previous study showed that OA promotes retinal ganglion cell (RGC) survival after optic nerve (ON) crush injury. The purpose of this research was to further explore the potential actions of OA in ischemic injury in an experimental anterior ischemic optic neuropathy (rAION) rat model induced by photothrombosis. Our results show that OA efficiently attenuated ischemic injury in rats by reducing optic disc edema, the apoptotic death of retinal ganglion cells, and the infiltration of inflammatory cells. Moreover, OA significantly ameliorated the pathologic changes of demyelination, modulated microglial polarization, and preserved visual function after rAION induction. OA activated nuclear factor E2 related factor (Nrf2) signaling and its downstream antioxidant enzymes NAD(P)H:quinone oxidoreductase (NQO-1) and heme oxygenase 1 (HO-1) in the retina. We demonstrated that OA activates Nrf2 signaling, protecting retinal ganglion cells from ischemic injury, in the rAION model and could potentially be used as a therapeutic approach in ischemic optic neuropathy.Nonarteritic anterior ischemic optic neuropathy (NAION) is the most common cause of acute vision loss in older people, and there is no effective therapy. The effect of the systemic or local application of steroids for NAION patients remains controversial. Oroxylin A (OA)  is a bioactive flavonoid extracted from  Nonarteritic anterior ischemic optic neuropathy (NAION) is one of the most common acute optic neuropathies in the elderly population , and it Scutellariae baicalensis Georgi., exerts anti-apoptotic and anti-inflammatory effects in an optic nerve crush model [Neuroinflammation and microglial activation have been considered the main pathogenic mechanisms in the NAION model. Previous studies demonstrated that the invasion of extrinsic macrophages into the optic nerve (ON) triggered the release of proinflammatory cytokines or chemokines and increased intrinsic microglial activation, leading to RGC dysfunction and death in a rodent model of NAION ,19,20. Osh model . OA has sh model ,23, amelsh model ,25, decrsh model , inhibitsh model , and impsh model ,29,30,31Four- to six-week-old male Wistar rats (weighing 100–125 g), obtained from BioLASCO.Co. Taiwan, were used in this study. All the animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Tzu Chi University (No. 107008). The rats were divided into three groups: sham, AION induction treated with PBS, or AION treated with the subcutaneous injection of OA . Every effort was made to minimize distress and suffering in the animals.We performed all the animal experiments under general anesthesia, accomplished by an intramuscular injection of a mixture of ketamine and xylazine  . The aniAfter general anesthesia, we performed AION induction via photodynamic thrombosis with an injection of 2.5 mM rose bengal   in pH 7.4 phosphate buffered saline (PBS) ,32. Immen = 6 per group).The FVEPs were measured at 28 days after rAION induction in 18 experimental rats using a visual electrodiagnostic system  as previously described ,33. We m2; 225 by 170 mm2) in the central and mid-peripheral regions of each retina (n = 6 rats for each group) were counted.The retrograde labeling procedures were described in detail in our previous reports . BrieflyThe frozen sections of ONs and retina were rinsed with PBS and then blocked with 5% normal goat serum containing 1% bovine serum albumin (BSA) for 30 min. The ON sections were labeled with anti-ED1 , anti-2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) , and anti-Ym1  primary antibodies, and the retinal sections were labeled with anti-ionized calcium binding adaptor molecule 1 (Iba1)  and anti-interleukin 6 (IL-6)  primary antibodies. Those sections were then incubated with corresponding Alexa Fluor conjugated secondary antibodies. Photographs were taken using a Zeiss LSM 900 confocal system . At least six images per eye by 20× magnification were taken for quantification the ED-1 or Ym1 staining positive cell in the optic nerve and for quantification of Iba1 and IL-6 staining in the retina. To quantify the intensity of CNPase staining in optic nerve, we opened CNPase and DAPI channel in ImageJ, the intensity of CNPase and DAPI in optic nerve were measured and the CNPase generated per DAPI-positive cells were calculated.n = 6 rats for each group).Apoptotic cells in the ganglion cell layer (GCL) of the retina were detected by a TUNEL assay according to the manufacturer’s protocol  as described previously ,5,6,7. SThe optic disc width and retinal nerve fiber layer (RNFL) thickness were evaluated using a Phoenix Micron IV retinal microscope with an image-guided OCT system as previously described ,32. We s® Imaging System . The signal intensity was measured and analyzed using ImageJ 1.8.0_172.The experimental procedures for immunoblotting were described in detail for previous studies ,34. Briep-values less than 0.05 were considered to indicate statistical significance.All the data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using the Mann–Whitney U test and the Kruskal–Wallis test for comparisons between groups via GraphPad Prism 7 . 2, respectively  retrograde labeling of RGCs was performed to determine the neuroprotective effect of OA on the RGCs after infarction. The mean RGC counts in the central retina in the sham, PBS-treated, and OA-treated groups were 2056 ± 361, 538 ± 144, and 1315 ± 490 cells/mmectively A–C,G. Thectively D–F,H. ThTo determine the visual function, the P1–N2 amplitudes in the flash visual evoked potentials (FVEPs) were recorded 28 days after the ischemic injury in each group. The average P1–N2 amplitudes in the sham, PBS-treated, and OA-treated groups were 44.34 ± 8.05, 16.3 ± 6.32, and 42.56 ± 10.91 μV, respectively . SignifiThe optic nerve width (ONW) profiles of the sham, PBS-treated, and OA-treated groups were recorded on Days 1, 3, 7, 14, and 28 . At the In situ TUNEL assay on retinal cross sections was performed to evaluate whether OA can protect RGCs from apoptosis . The numBlood-borne macrophages  infiltrated the ON tissue and induced tissue inflammation after rAION induction. Immunostaining for ED1 in ON tissue was performed to evaluate whether OA treatment will attenuate the extrinsic macrophage infiltration in ONs 28 days after rAION A,D. The The upregulation of ionized calcium binding adapter molecule 1 (Iba1), a microglial marker, indicates microglial activation during neuroinflammation ,36. The 2′,3′-Cyclic nucleotide 3′-phosphodiesterase (CNPase) is a myelination-associated enzyme and is highly expressed in the myelin-producing oligodendrocyte cells and Schwann cells . Some stTo elucidate the molecular mechanisms involved in the neuroprotective effect of OA on RGCs, the expression of genes regulating cytoprotective responses to inflammation and oxidative stress was determined by immunoblotting analysis. We analyzed the expression of Nrf2, NQO-1, and HO-1 in the retinas after rAION. rAION induction suppressed Nrf2′s expression and that of its downstream regulated genes, NQO-1 and HO-1. OA significantly enhanced the protein expression of Nrf2, NQO-1, and HO-1 in the retinas 28 days after rAION .Microglia and macrophages are polarized to M1 or M2 phenotypes and mediate corresponding signaling pathways in response to different pathophysiological states ,46,47. FOur morphologic data from this study clearly show that OA treatment enhanced RGC survival and significantly reduced the recruitment of inflammatory cells into the optic nerve after infarction. Furthermore, OA not only prevented RGC apoptosis but also preserved RGC function as demonstrated by FVEP, confirming its beneficial effect on the ischemic injury of the optic nerve. In addition, we observed that OA treatment ameliorated neuroinflammation and selectively polarized microglia toward an anti-inflammatory M2 status, resulting in a neuroprotective effect.Post-ischemic inflammatory responses were identified in the rAION model, including the release of proinflammatory cytokines, extrinsic macrophage infiltration, intrinsic microglial activation, and the breakdown of the blood–optic nerve barrier ,18,20,48Axon demyelination and/or focal damage to the ON has been identified in clinical NAION patients and NAION animal models ,54,55. RMicroglia/macrophage-mediated neuroinflammation has been considered to be an important contributing factor in the pathogenesis of ischemic optic neuropathy ,19,20,48Oxidative stress resulting from mitochondrial dysfunction, the overproduction of ROS, and an impaired antioxidant system contributes to the pathogenesis of many ocular diseases ,65,66. TBase on the evidence from this study and our previous study , the datIn summary, this study provides evidence that OA promotes retinal ganglion cell survival and preserves visual function by preventing the apoptosis of RGCs, maintaining myelin sheath integrity, reducing the levels of proinflammatory cytokines, and modulating microglial polarization. These neuroprotective effects were achieved by the activation of Nrf2 signaling in the retina . Our dat


Fibrosis, a major cause of morbidity and mortality, is a histopathological manifestation of many chronic inflammatory diseases affecting different systems of the human body. Two types of transforming growth factor beta (TGF-β) signaling pathways regulate fibrosis: the canonical TGF-β signaling pathway, represented by SMAD-2 and SMAD-3, and the noncanonical pathway, which functions without SMAD-2/3 participation and currently includes TGF-β/mitogen-activated protein kinases, TGF-β/SMAD-1/5, TGF-β/phosphatidylinositol-3-kinase/Akt, TGF-β/Janus kinase/signal transducer and activator of transcription protein-3, and TGF-β/rho-associated coiled-coil containing kinase signaling pathways. MicroRNA (miRNA), a type of non-coding single-stranded small RNA, comprises approximately 22 nucleotides encoded by endogenous genes, which can regulate physiological and pathological processes in fibrotic diseases, particularly affecting organs such as the liver, the kidney, the lungs, and the heart. The aim of this review is to introduce the characteristics of the canonical and non-canonical TGF-β signaling pathways and to classify miRNAs with regulatory effects on these two pathways based on the influenced organ. Further, we aim to summarize the limitations of the current research of the mechanisms of fibrosis, provide insights into possible future research directions, and propose therapeutic options for fibrosis. Fibrosis is a histopathological manifestation of many chronic inflammatory diseases . Some fiTransforming growth factor beta (TGF-β), a key molecule in the development of fibrosis, can regulate a series of biological processes, including the cell growth cycle, cell differentiation, immune regulation, and extracellular matrix (ECM) deposition . Two typMicroRNA (miRNA), a type of non-coding single-stranded small RNA, comprises approximately 22 nucleotides encoded by endogenous genes, which can regulate physiological and pathological processes in fibrotic diseases . Its cenIn the canonical TGF-β signaling pathway, active TGF-β is first isolated from the latent TGF-β complex in the extracellular space, which maintains TGF-β in the inactive form . The inaDuring fibrotic progression, complex regulatory networks composed of multiple TGF-β-related signaling pathways work together . TherefoUnlike the canonical ALK5-SMAD-2/3 pathway, the TGF-β type I receptor ALK1 forms a complex with ALK5 under stimulation by TGF-β, leading to activation of SMAD-1/5 and suppression of ALK5-SMAD-2/3 signaling . Moreovevia activations of MKK4 and MKK3/6, respectively. Finally, the activated JNK and p38 continuously activate the downstream transcription factors c-jun and ATF-2, which then regulate SMAD activity through phosphorylation  signaling cascade, which comprises the core units of phosphatidylinositide 3-kinases and their downstream mediators AKT and mTOR, mediate cell proliferation, survival, and metabolism and control cellular differentiation . TGF-β rROCK is the eventful cellular regulator of rho GTPases that organizes the actin cytoskeleton and facilitates myofibroblast differentiation and ECM production in fibroblasts of diverse tissues . RhoA a. The rhoJAK, which is a receptor-associated tyrosine kinase, acts as regulators of cytokine and growth factor signaling. JAK2 is a regulator of the profibrotic results of TGF-β in many fibrotic diseases . STAT3, in vivo and in vitro through the regulation of the TGF-β-SMAD-2/3 pathway. Overexpression of miR-29 can downregulate Adam12 and Adam19 expressions, partially mitigating fibrosis  and Runt-related protein 2 (RUNX2)  . As for  (RUNX2) . Thus, d (RUNX2) . In the  (RUNX2) .In cardiac fibrosis, miRNAs related to the regulation of the canonical TGF-β/Smad2/3 signaling pathway should first be miR-150-5p . In a stIn the non-canonical TGF-β/MAPKs signaling pathway, a study on miR-433 characterized its crucial role in regulating cardiac fibrosis . In thatHypertrophic scar (HS) is a pathological scar resulting from abnormal wound healing . A studyBladder outlet obstruction (BOO) is commonly encountered in the field of urology, often accompanying fibrosis of the bladder structure . Wang etOral submucous fibrosis (OSMF) is a chronic, progressive, pre-malignant condition mainly related to the consumption of betel nuts . In recevia canonical or non-canonical TGF-β signaling, including miR-21, miR-101, miR-29, miR-200, miR-942, and miR-193. Among them, miR-21 has been widely confirmed to promote the occurrence and development of fibrosis of organs, such as the kidney (Previous studies have suggested that the TGF-β signaling pathway, which plays a major regulatory role in fibrotic diseases, is widely regulated by miRNAs. Some of these miRNAs can affect various fibrotic diseases e kidney , liver (e kidney , lung (We kidney  and heare kidney , by targe kidney , hepatice kidney , or pulme kidney  and blade kidney , by diree kidney  and HS (e kidney  by targee kidney  by regule kidney  and lunge kidney  by direce kidney  but playe kidney . In addie kidney  and hepae kidney  by regulFibrosis offers the advantage of promoting tissue repair and healing. However, excessive fibrosis can cause tissue and organ dysfunctions . During Fibrosis is often the final histopathological change in the development of chronic inflammatory diseases and can occur in almost all tissues and organs throughout the body. According to the current research results of fibrotic diseases of various organs, the developmental mechanisms of tissue and organ fibrosis mainly involve the canonical TGF-β/SMAD2/3 signaling pathway and the non-canonical TGF-β pathway, including the TGF-β/ALK1/Smad1/5 pathway, the TGF-β/MAPK pathway, the TGF-β/PI3K-Akt pathway, the TGF-β/JAK2/STAT3 pathway, and the TGF-β/ROCK pathway. MiRNA, which is involved in various physiological and pathological processes, with important roles in the canonical or noncanonical TGF-β signaling pathways, has received widespread attention in recent years. The regulating function of miRNAs in fibrosis is mainly to promote or ameliorate fibrosis by inhibiting the expression of effect molecules in the fibrosis signal pathway at the mRNA level. However, current research on the regulation of miRNA in fibrotic diseases mainly focuses on fibrosis of organs such as the liver, the kidneys, the lungs, and the heart, whereas the fibrosis-related research of other tissues and organs is limited and not in-depth. In addition, for the current mechanism research, the specific targets of many miRNAs and the upstream and downstream regulatory relationships are not comprehensive or sufficiently thorough.In the future, many questions concerning the mechanisms of the pathogenesis of fibrotic diseases should be solved. For example, in addition to the few aforementioned organs, fibrosis of other tissues and organs has unique features and developmental progression, carrying research value. Therefore, more in-depth research should be performed. There may be more than five non-canonical TGF-β signaling pathways that affect the development of fibrosis. The related miRNAs are not limited to pathways, and long-term continuous exploration is required. The pathogenesis of fibrotic diseases is complex. Therefore, a single target therapy cannot have a complete effect, and combination treatment with multiple targets and signaling pathway may be more reasonable. Hence, the next step should be to explore the mechanisms of fibrotic diseases from different tissue and organs, investigating more signaling pathways related to fibrosis and more functional miRNAs in these diseases and trying combination therapeutic methods with multiple targets and multiple pathways.


The term RASopathy was originally created to describe a phenotypically similar group of medical genetic syndromes caused by germline pathogenic variants in components of the RAS/mitogen-activated protein kinase (RAS/MAPK) pathway. In defining a RASopathy syndrome, one needs to consider the complex nature of the RAS/MAPK pathway, the numerous genes and regulatory components involved, its crosstalk with other signaling pathways and the phenotypic spectrum among these syndromes. Three main guiding principles to the definition should be considered. First, a RASopathy is a clinical syndrome with overlapping phenotypic features caused by germline pathogenic variants associated with the RAS/MAPK pathway. Second, a RASopathy is caused by multiple pathogenetic mechanisms, all of which lead to a similar outcome of RAS/MAPK pathway activation/dysregulation. Finally, because a RASopathy has dysfunctional germline RAS/MAPK pathway activation/dysregulation, it may, therefore, be amenable to treatment with pathway modulators. Summary: RASopathy is a term used to unify a phenotypically similar group of medical genetic syndromes, yet its definition is challenging and should consider the perspectives of clinicians, scientists and the patients. PTPN11, SOS1, RAF1/CRAF, KRAS, NRAS, SHOC2, CBL, RRAS, RIT1, RASA2, SOS2, MAP3K8, SPRY1, MYST4/KAT6B, LZTR1, A2ML1, PPP1CB, MRAS, RALA, RRAS2 and ERK2/MAPK1. Cardio-facio-cutaneous syndrome (CFC) is caused by activating mutations in multiple genes, including BRAF, MAP2K1/MEK1, MAP2K2/MEK2, KRAS and possibly YWHAZ. Other RASopathies, including neurofibromatosis type 1 (NF1), Noonan syndrome with multiple lentigines (NSML), Costello syndrome (CS), Legius syndrome, SYNGAP1 and central conducting lymphatic anomalies (CCLA), are caused by alterations in one or two genes in the RAS pathway  pathway . Collect pathway  , RAS guanine nucleotide exchange factors (RAS-GEFs), kinases, scaffolding/adaptor proteins, ubiquitin ligases, phosphatases and pathway inhibitors – with all identified mutations culminating in the activation of ERK1/2 . AlthougFrom an oncologist's perspective, the RASopathies are cancer syndromes due to the presence of potentially oncogenic mutations in the germline . RAS/MAPThe RASopathies provide a unique perspective in the study of cancer genes because individuals harbor a single germline mutation, unlike a somatic cancer, which typically acquires many oncogenic mutations. Understanding the function of RASopathy-associated genes in the RAS/MAPK pathway, and their role in development, provides clues as to its function in cancer and vice versa. NF1 is the best-studied cancer syndrome caused by germline haploinsufficiency of the tumor suppressor gene neurofibromin 1. NF1 individuals have more than twice the risk of cancer of the general population . MalignaHras mouse model  within a gene(s) that encodes a component or regulator of the RAS/MAPK pathway, which causes the activation of ERK1 and/or ERK2. The pathogenic variant may be


Skeletal muscle atrophy in an inevitable occurrence with advancing age, and a consequence of disease including cancer. Muscle atrophy in the elderly is managed by a regimen of resistance exercise and increased protein intake. Understanding the signaling that regulates muscle mass may identify potential therapeutic targets for the prevention and reversal of muscle atrophy in metabolic and neuromuscular diseases. This review covers the major anabolic and catabolic pathways that regulate skeletal muscle mass, with a focus on recent progress and potential new players. Biological aging is broadly defined as the time-dependent loss of functionality and robustness, and is associated with increased rates of cancer ,2, cardiThe cellular and molecular mechanisms linking biological aging with the loss of muscle mass, strength, and functionality are complex. In its simplest form, the maintenance of muscle mass, and strength, is due to the balance of anabolic and catabolic processes. Stem cell exhaustion, cellular senescence, altered intercellular communication, losses of proteostasis and genome stability, telomere attrition, mitochondrial dysfunction, epigenetic alterations, and dysregulation of gene expression and alternative splicing are hallmarks of, and causal for, the age-associated loss of cellular and biological robustness ,21,22,23GHRH (lit/lit) dwarf mice have reduced growth hormone (GH) signaling and reduced serum IGF-1 levels, but have up to a 70% extension of their lifespans [Igf1r) have a modest ~6–8% reduction in bodyweight by 7 weeks of age, but live up to 25% longer [Insulin, and insulin-like growth factor 1 (IGF-1) signaling (IIS) is ubiquitous to multicellular animals, connecting nutrient availability to development, longevity, and growth in response to hypothalamic-pituitary growth signals . Activatifespans ,36,37,38% longer ,40. Howe% longer ,41,42. C% longer . In cont% longer ,45,46,47PI3K/Akt/mTOR signaling is a key regulator of glycolytic muscle homeostasis, hypertrophic growth, and metabolism through its activation of mTOR-dependent anabolic processes while simultaneously inhibiting both autophagy and Foxo1/3 catabolic processes ,49. SkelTsc2 null MEFs expressing a phospho-deficient form of TSC2 failed to induce mTORC1 signaling despite the phosphorylation of PRAS40 at T246 [Activated Akt acts directly on multiple targets to modulate cellular growth and metabolic processes (reviewed in ). Akt ph at T246 , suggest at T246 ,85. FoxO at T246 , in part at T246 . FoxO pr at T246 . Akt pho at T246 ,90 and F at T246 ,92 to inMTHDF2 by eIF2α-independent activation of ATF4 [FGF21, SQSTM1, ATG3, ATG12, and Sestrin2 (Sesn2) via ATF4 [The mTOR-containing complex 1 (mTORC1) is a major effector of activated Akt, stimulating the synthesis of proteins, lipids, and nucleotides, while inhibiting protein degradation pathways . Recruit of ATF4 . In respvia ATF4 ,108,109.via ATF4 . Under gvia ATF4  and Atg1via ATF4 ,113.Nutrient and energy availability is a critical regulator of mTORC1. Increases and decreases in amino acids leucine and arginine positively and negatively modulate mTORC1 kinase activity via CASTOR1, Sestrin2, and SLC38A9 ,117,118,Multiple human studies have indicated significantly reduced rates of mixed, myofibrillar and/or mitochondrial protein synthesis in the muscles of elderly subjects ,128,129.Resistance training increases muscle protein synthesis in both young and elderly men and women ,139,140.In vivo mechanistic studies in animal models add further support to this notion. Reductions in Akt phosphorylation at S473 and S6K1 phosphorylation at T389 are observed in the gastrocnemius of old mice as compared to the young controls . This stTsc1 knockout) in mice induces progressive myofiber atrophy, phenocopying many of the histological features of aged muscles including increases in both myofiber vacuolation and the number of caspase-3 positive myofibers [Raptor knockout) in mice induced a leftward shift in the myofiber size distribution curve and histological features including centralized nuclei and vacuoles, and myofiber degeneration [Ccl2, Ccl3, Il10) and metabolic stress indicators  in the hypertrophic muscles of Akt transgenic mice [The percentages of histologically abnormal myofibers, and myofibers staining positive for both phosphorylated rpS6 and active caspase-3 are increased in the muscles of both old mice and humans . Counteryofibers ,154. Simyofibers . In contyofibers . Consistneration . These aneration ,52 in paneration . Importaneration . These onic mice , and in nic mice , furtherTsc1 mutant mice [Treatment with rapamycin both extends the lifespan of mice and fruit flies ,162,163 ant mice ,171 and ant mice , in partAging and aging-related diseases are linked to impaired proteome integrity or proteostasis ,173. AcuCells have evolved multiple protein degradation processes to maintain proteome integrity or proteostasis in response to endogenous and exogenous stresses that negatively impact the protein functionality due to damage or misfolding. Approximately 60–80% of protein degradation in growing mammalian cells occurs through 26S proteasome catalysis ,183,184.Eif4ebp1), transcription factors Atf4, Foxo1, and Foxo3 [Trim63) and Atrogin-1/MAFbx (Fbxo32) [Map1lc3a) and Gabarap [In humans and animal models, these proteolytic processes become dysregulated with pathologies associated with muscle atrophy including cancer cachexia, chronic renal failure, diabetes, denervation, and age ,188,189.nd Foxo3 ,195, the(Fbxo32) ,197, and Gabarap ,198,199. Gabarap ,202,203  Gabarap  proteolyATF4, DDIT3 (CHOP), and PPP1R15A (GADD34) [ER) [Autophagy and ATF4 are both positively regulated in response to cellular stress  and majo(GADD34) . The Eif34) [ER) ,207, dsR34) [ER) ,209, and34) [ER) ,211, res34) [ER) , and a c34) [ER) , CASTOR134) [ER)  and SLC334) [ER) ,215,216.Foxo1 specifically in skeletal muscle results in mice with decreases in muscle mass, functionality, and sarcomeric protein expression [Foxo1 knockout mice were largely protected from nephrectomy and dexamethasone-induced muscle atrophy [Foxo1, Foxo3, and Foxo4) abolished both fasting [Foxo3 and overexpression of a dominant negative form of FoxO3 blocked fasting-induced autophagy [FoxO transcription factors are positive regulators of Atrogin-1, MuRF1, LC3, GABARAPs, ULK2, ATG12, and Beclin ,217,218,pression , while m atrophy . Muscle- fasting  and insu fasting  induced  fasting . Similarutophagy .Nlrp3 blunted both the age-associated changes in muscle mass and caspase-1 activity, respectively [c levels or cytosolic caspase-3 activity from 6-month old controls [c stimulation of muscle homogenates from 6- and 24-month old rats was unable to increase caspase-3 enzymatic activity, suggesting that additional factors may be necessary for caspase-3 activation in muscle. Acute insulinopenia stimulates non-lysosomal, ATP-, and proteasome-dependent proteolysis in rats [Proteins may also be cleaved by members of the caspase family of proteases which are known to play key roles in regulating apoptosis, necrosis, and inflammation ,224. Perectively , thus decontrols , suggest in rats . Subsequ in rats . Subsequ in rats  and foll in rats . Importa in rats . Collect+ T cells, in the endomysium [Sporadic inclusion body myopathy (sIBM) is the most common degenerative muscle disease in patients aged 50 years or more, and is often referred to as sporadic inclusion-body myositis due to high levels of immune cells, including cytotoxic CD8domysium . sIBM padomysium  suggestidomysium ,236,237.domysium . Consistdomysium . Treatmedomysium , indicatRNA-binding proteins (RBPs) play essential roles in nearly every aspect of cell biology through their direct influence on post-transcriptional gene expression ,242,243,ADARB1 and ADARB2 are associated with longevity in both humans and Caenorhabditis elegans [Adar induced lethality around ED11.5 due to severe defects in hematopoiesis and disintegration of the liver [Adarb2 is genetically deleted develop normally, but die between P0 and P20, becoming progressively seizure prone by P12 [Adar) and ADAR2 T553 (Adarb2) as being direct targets of Akt kinase activity in cultured human cell lines [NEIL1, CCNI, AZIN1, and CYFIP2. Interestingly, millions of RNA editing sites have been identified in human cells [Alu elements largely found in noncoding regions of the genome [Altered post-transcriptional gene expression regulation, in part through changes in miRNA expression, play causal roles in the cellular and tissue morphological changes in the cardiovascular systems of the elderly ,249,250. elegans , and alt elegans ,253. In he liver . Similare by P12 . In addie by P12 , suggeste by P12 . A recenll lines . Akt inhan cells ,260. Howe genome . AlthougAlterations in RNA processing steps including splicing, poly-adenylation, and 5′ capping) are associated with both physiological aging and age-associated disease ,264,265.RBFOX1 have been observed in patients with neurological disorders [Rbfox1 has been specifically deleted in skeletal muscle have decreases in muscle mass and maximal force generation [Rbfox1 and Rbfox2 are specifically deleted in adult mice, experienced rapid losses of muscle mass, myofiber cross sectional area, and strength, due in part, to reductions in autophagy and the aberrant expression of calpain-3 [Rbfox1 and/or Rbfox2 significantly impaired myogenesis in the C2C12 model system through its direct role in promoting the muscle-specific splicing of Mef2d [Genetic mutations in the splicing factor gene isorders  and in aisorders . Mice inneration , thus esalpain-3 . Furtherof Mef2d . CollectSFPQ introns, are observed in motor neurons that were differentiated from ALS patient-derived iPSCs [Sfpq in mice led to impaired transcriptional elongation, an effect most pronounced on long genes (>100 kb), and gross developmental abnormalities in the brain [Amyotrophic lateral sclerosis (ALS) is a progressive age-related neurodegenerative disease in which motor neurons are selectively degenerated. Both reductions in nuclear/cytoplasmic ratio of the splicing factor SFPQ, and the aberrant retention of ed iPSCs , thus suhe brain . Skeletahe brain . Betweenhe brain ,289,290,HNRNPU are associated with intellectual disability and muscle weakness [Hnrnpu is deleted in striated muscles (Ckm-Cre) die around P14 due to a dilated cardiomyopathy (DCM) phenotype, whereas mice in which Hnrnpu is deleted specifically within cardiomyocytes (Myh6-Cre) die around P10 due to a similar DCM phenotype [Hnrnpu is deleted specifically in skeletal muscle (ACTA1-Cre) are born phenotypically normal, but by 3-months of age develop a myopathy-like phenotype characterized by selective muscle atrophy of glycolytic muscles and histopathological observations of central nuclei, myofiber degeneration, intracellular inclusions, and extensive fibrosis [Ckm-Cre) and skeletal muscles (ACTA1-Cre). Interestingly, the atrophied skeletal muscles (ACTA1-Cre) exhibit signs of metabolic stress. Akt is observed to be constitutive active with its phosphorylation at S473 being desensitized to IGF-1 stimulation, SQSTM1 and Ulk1 phosphorylation at S757 are significantly increased, and multiple genes involved in the transport/import and biosynthesis of amino acids, as well as the repression of oxidative metabolism are significantly reduced in expression [A rare cohort of patients have been identified in which heterozygous mutations in weakness ,293,294.weakness ,296,297,weakness , X-chromweakness , and is weakness . In miceweakness  and skelweakness , suggesthenotype . Mice infibrosis . Changespression . Though pression  and S6K1pression  phosphorINSR is regulated in a cell-type specific context that is dependent upon the development stage or disease condition. Alternative splicing of exon 11 gives rise to protein isoforms INSR-A (exclusion of exon 11) and INSR-B (inclusion of exon 11). INRS-A, which is prevalent during fetal development and in the nervous system, preferentially binds IGF-2 over IGF-1 and insulin, while INRS-B, which is prevalent in adulthood and highly expressed in adipose tissue, liver and skeletal muscle, is more sensitive to insulin [INSR is regulated by multiple RBPs including CELF1, SRSF3, MBNL1, and hnRNPs A1, F, and H [INSR splicing occurs in the insulin target tissues of patients with insulin resistance [INSR splicing in the pathogenesis of type 2 diabetes is unclear [Splicing of the insulin receptor gene  insulin ,303,304.F, and H ,307,308.F, and H  and ZNF9F, and H , respectF, and H . MoleculF, and H ,313,314.F, and H , and dyssistance ,313, the unclear  or uncha unclear ,318 are  unclear . To the FUS, HNRNPM, SFPQ, and SRSF2 were significantly increased, while coimmunoprecipitation and cosedimentation of hnRNP-M with core spliceosomal proteins were significantly enhanced following treatment. Importantly, siRNA knockdown of HNRNPM blocked a subset of Dactolisib induced changes in cassette exon splicing for exons containing proximal hnRNP-M consensus motifs [Rictor [Rictor or HNRNPM repressed insulin-stimulated Akt S473 and SGK1 S422 phosphorylation to a similar degree, the molecular mechanism underlying these observations is unknown. hnRNP-E2 (PCBP2) was identified in a two-hybrid screen of a human bone marrow cDNA library with SIN1 (MAPKAP1), while siRNAs targeting either SIN1 (MAPKAP1) or hnRNP-E2 (PCBP2) similarly potentiate an increase in the percentage of apoptotic cells treated with either H2O2 or TNFα [2O2 and TNFα induced apoptosis, suggesting their participation in a common signaling pathway, the molecular mechanism, presumably activating mTORC2, is unknown. SR protein SF2/ASF (SFRS1) positively modulates protein translation through an Akt-independent mechanism [Treatment of Ewing sarcoma cells with the ATP-competitive PI3K and mTOR inhibitor Dactolisib induced significant reductions in both cell growth and proliferation . Genes fs motifs . Interes [Rictor . Though  or TNFα . Though echanism . MEF andechanism  suggestiWorldwide, the number of individuals aged 65 years or older is expected to more than double by the year 2050 . In the


DNAJB1, HSPA1B and HSPB1 genes were commonly upregulated across all brain regions and cell types except for medium spiny neurons (MSNs) at symptomatic disease stage, and HSPH1 and SAT1 genes were altered in expression in all symptomatic brain datasets, indicating early and sustained changes in the expression of genes related to heat shock response as well as response to misfolded proteins. Specifically in indirect pathway medium spiny neurons (iMSNs), mitochondria related genes were among the top uniquely dysregulated genes. Interestingly, blood from HD patients showed commonly differentially expressed genes with a number of brain regions and cells, with the highest number of overlapping genes with MSNs and BA9 region at symptomatic stage. We also found the differential expression and predicted altered activity of a set of transcription factors and epigenetic regulators, including BCL6, EGR1, FOSL2 and CREBBP, HDAC1, KDM4C, respectively, which may underlie the observed transcriptional changes in HD. Altogether, our work provides a complete overview of the transcriptional studies in HD, and by data synthesis, reveals a number of common and unique gene expression and regulatory changes across different cell and tissue types in HD. These changes could elucidate new insights into molecular mechanisms of differential vulnerability in HD.Huntington’s disease (HD) is a chronic neurodegenerative disorder caused by an expansion of polyglutamine repeats in exon 1 of the Huntingtin gene. Transcriptional dysregulation accompanied by epigenetic alterations is an early and central disease mechanism in HD yet, the exact mechanisms and regulators, and their associated gene expression programs remain incompletely understood. This systematic review investigates genome-wide transcriptional studies that were conducted using RNA sequencing (RNA-seq) technology in HD patients and models. The review protocol was registered at the Open Science Framework (OSF). The biomedical literature and gene expression databases, PubMed and NCBI BioProject, Array Express, European Nucleotide Archive (ENA), European Genome-Phenome Archive (EGA), respectively, were searched using the defined terms specified in the protocol following the PRISMA guidelines. We conducted a complete literature and database search to retrieve all RNA-seq-based gene expression studies in HD published until August 2020, retrieving 288 articles and 237 datasets from PubMed and the databases, respectively. A total of 27 studies meeting the eligibility criteria were included in this review. Collectively, comparative analysis of the datasets revealed frequent genes that are consistently dysregulated in HD. In postmortem brains from HD patients, Systematic Review Registration:https://osf.io/pm3wq Huntingtin gene (HTT)  is a dominantly inherited neurodegenerative disorder caused by an expansion in CAG repeats in the exon I of the ne (HTT) . The mutogresses . The disogresses . Althougogresses , a diseaBdnf), dopamine receptor 2 (Drd2), dopamine receptor 1a (Drd1a), preproenkephalin (Penk1), adenosine A2a Receptor (Adora2a) and protein phosphatase 1 regulatory subunit 1B (Ppp1r1b).While the genetic cause of HD has been known for almost 3 decades, the molecular mechanisms underlying the disease pathogenesis are complex, involving disruption of multiple cellular processes, among which transcriptional dysregulation is one of the earliest and central pathogenic mechanisms. Transcriptional profiling studies demonstrated progressive changes in gene expression in HD human brain and in experimental disease models . TranscrHTT has been proposed to cause transcriptional dysregulation include sequestration as well as soluble interactions with regulators of transcription leading to perturbation of their activities  between the presymptomatic and symptomatic brain. We observed that the heat shock response genes were significantly upregulated in presymptomatic brain. In particular, three common genes among all the postmortem brain regions, except for symptomatic MSNs, DNAJB1, HSPA1B and HSPB1 were upregulated. However, this upregulation was less prominent in the symptomatic brain regions, and was not observed in the striatum in mouse studies. Within the striatum, MSNs exhibit dysregulation of mitochondrial function-related genes in addition to SAT1 gene that is common with all the late postmortem brain categories. Additionally, blood shared more DEGs with symptomatic postmortem brain than with presymptomatic. Furthermore, non-human datasets also shared some key DEGs like, Adora2a, Adcy5, CamkV, Penk that are associated with HD.Here, we reviewed 27 independent studies on cellular models in human, human brain and blood as well as in non-human models. Within the human models, we observed large differences in gene expression, enrichment of gene sets and the related processes and pathways. While cellular models showed enrichment of cellular and developmental processes, postmortem brain models showed enrichment of genes involved in heat shock response, response to protein unfolding, synaptic transmission among others. One of the human studies that compared presymptomatic HTT knockout/-down study, 8) Treatment study, 9) Microarray, 10) Non-RNA-seq study, 11) Non-peer reviewed and 12) DEG not available. The screening process is presented in a PRISMA flow diagram  AND  NOT (review [pt])) AND ((“2013/01/01” [Date - Publication]: “2020/08/15” [Date-Publication])). For data repositories, search terms were adapted to each database: 1) huntingt*[title] AND “transcriptome gene expression” [Filter] for BioProject (SRA and GEO); 2) huntingt* AND exptype:“RNA-seq of coding RNA” for ArrayExpress; 3) huntingt* AND RNA-seq, with filter “study” for European Nucleotide Archive; 4) huntingt* for European Genome-Phenome Archive. In total, 288 articles were identified through PubMed and 237 datasets were identified through data repositories. Then, the articles and datasets were screened with the following exclusion criteria in order of priority: 1) Non-HD study, 2) Erratum/Superseries, 3) Review, 4) Different analysis of same data, 5) Non-RNA study, 6) RNA isoform/noncoding RNA study, 7)  diagram .n = 12), lowly expressed genes were removed from the expression matrix based on the selection criteria defined in each paper. For each subcategory, background gene lists were created by taking the union of all expressed genes in the datasets within the corresponding category.For human HD datasets for which all filtered expressed genes were not provided  for all mouse and human categories were defined based on how often a gene was observed as significantly changed across the datasets in each category. Genes that are differentially expressed in at least two datasets were included in the category-representative DEGs. For subcategories with single dataset, all the genes were considered. As an exception, for the human blood category, union of DEGs from the two datasets within the blood category were considered as blood category-representative DEGs as the number of overlapping genes between the two blood datasets is 1.Gene overlap analysis between pairwise comparisons were performed by using one-sided Fisher’s exact test. To test gene overlap among more than two categories, we used the SuperExactTest  package n = 44) with the GSEA method in the WebGestaltR package v.0.4.2  , for dif v.0.4.2  for the Common or unique gene sets were analyzed by online Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GOrilla) . For paiTo identify potential regulators for DEGs in each HD human dataset, we used hTFtarget database in which human TFs were curated from Chip-Seq experiments in different cell lines, tissues and cells . For eacThe overlap analysis between the TFs obtained from H3K27ac motif data from the HD iPSC n = 2,765) and epigenetic modifiers (n = 167) were curated from The Human Transcription Factors website  datasets from 14 human HD studies  Table 1Table 1. We classified 44 human DEG datasets into five categories based on sample source and disease stage . The fivIGDCC3) and XK Related 4 (XKR4) were common in all four subcategories (p = 2.3E-4)  . Overall 2.3E-4) .p = 5.7E-28) overlapped, with the most significant enrichment of GO terms related to low-density lipoprotein transport. We observed an overlap of 156 genes (p = 1.7E-49) between pluripotent and differentiated_neuronal while there were 285 common genes (p = 5.3E-48) between pluripotent and differentiated_non-neuronal. The overlapping genes between pluripotent and progenitor  showed enrichment of GO terms such as regulation of cell differentiation, regulation of developmental process, regulation of nervous system development, and regulation of cellular component organization. There were 115 overlapping genes (p = 5.8E-15) between differentiated_neuronal and differentiated_non-neuronal while, we obtained 551 unique genes in differentiated_neuronal subcategory , late_ striatal_interneuron and late_ striatal_non-neuronal brain tissues, there were 226, 2,806, 70, 3414, 1988 and 1914 frequently differentially expressed genes, respectively .p = 1.2E-35), such as HSPA6, DNAJB1, HSPA1B, HSPA1A, HSPB1, JUN  between early and late cortical tissues , showing enrichment of various GO terms such as regulation of neuron projection development, regulation of neurogenesis, regulation of neuron differentiation, regulation of nervous system development, regulation of synapse organization, biological adhesion, axon guidance, vesicle localization and cyclic nucleotide catabolic process (p = 0) between MSNs and interneurons in striatum showed enrichment of GO terms such as modulation of chemical synaptic transmission, regulation of neuron projection development, regulation of synaptic plasticity, regulation of neurotransmitter levels, glutamate receptor signaling pathway, axon guidance, regulation of synaptic vesicle exocytosis, regulation of NMDA receptor activity, regulation of cation channel activity, cytosolic calcium ion transport, lysosomal transport, glutamate secretion, receptor localization to synapse, metal ion homeostasis, and regulation of GTPase activity. On the other hand, 2,127 unique genes in MSNs compared to interneurons show enrichment of cytoskeleton organization, cytoskeleton-dependent intracellular transport, cell projection organization, movement of cell or subcellular component, microtubule-based process as well as transport, plasma membrane bounded cell projection organization, positive regulation of GTPase activity, macromolecule modification and nervous system process to name some (p = 1.4E-152), interneurons and non-neuron cells within striatum showed an overlap of 694 genes (p = 2.2E-143).Within the three subcategories of the late striatal group from  process . Meanwhiame some . While tMT-CO2, MT-ND4L, MT-ND4, MT-ATP6, MT-CO3, SLC26A3, INO80D, and MT-ND1, were related to mitochondrial complex I, cytochrome c oxidase and ATP synthase, while the top downregulated genes were OTOF, TAC1, PILRB, JPH4, DDX24, MPHOSPH8 and KNOP1  are the most vulnerable cell types to degeneration in HD patients , we examnd KNOP1  that arend KNOP1  with othnd KNOP1 . Among 3onocytes ; Table 3DNAJB1), Heat Shock Protein Family A (Hsp70) Member 1B (HSPA1B) and Heat Shock Protein Family B  Member 1 (HSPB1) were common. These genes encode for HSP40, HSP70 and HSPB1 proteins, respectively, showing an enrichment of GO terms like chaperone-mediated protein folding, response to unfolded protein and negative regulation of inclusion body assembly. The expression of DNAJB1, HSPA1B and HSPB1 was upregulated in early BA9 and caudate nucleus. In late brain subcategories related datasets, the expression was less upregulated in comparison to the presymptomatic datasets (HSPH1) and Spermidine/Spermine N1-Acetyltransferase 1 (SAT1) were common for all late-brain categories  Member B1  and Prostaglandin-Endoperoxide Synthase 2 (PTGS2) (p = 0.07). Moreover, we observed significant overlap of 27 frequently dysregulated genes (p = 0.004) with late cortical subcategory   on 44 huGSEA analysis showed strong negative enrichment of genes associated with p53 signaling pathway and cancer in pluripotent cells in Switonska_hiPSC_PSC_109Q . ContrasIn brain, presymptomatic striatum and cortex showed positive regulation of gene sets associated with intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress, immune response, transcriptional misregulation in cancer, regulation of protein maturation, heat shock response. In symptomatic brain subcategory, the late cortex was positively enriched for various gene sets related to metal ion homeostasis, protein folding, p53 signaling, cancer and positive regulation of peptidyl-tyrosine phosphorylation. Late cortex and late_striatal_neuronal_MSNs showed common positive enrichment of glutathione metabolism, RNA polymerase, biosynthesis of unsaturated fatty acids, alanine, aspartate and glutamate metabolism, protein localization to chromosome/centromeric region, DNA replication, acidic amino acid transport, postsynapse assembly, purine metabolism, and synaptic vesicle cycle, while sharing negative enrichment in gene sets related to amyloid precursor protein metabolic process, cAMP signaling pathway, nerve development, alcoholism, Huntington disease, synaptic transmission, morphine addiction, long-term depression, Alzheimer disease and circadian entrainment . In striIL-17 signaling pathway, JAK-STAT signaling pathway and pathways in cancer  database . An acti, SP4, NRF1, BRD4, RYBP, ZFP64, HDAC1, FOXO1, CREB1, NFKB1, BRD2, SP1, CTCF and JUN some of which were previously implicated in HD pathology  from the available H3K27ac motif data from a previous publication . For this, three studies were excluded as they were cell culture models and could not be confidently assigned to a disease stage. Then, we categorized the mouse datasets as early, intermediate and late stages , in whicand Wnt4 . In early others . The topy others .Serpina1 in the cortex . Our systematic review protocol has been registered at the Open Science Framework (OSF) . We retrIGDCC3 and XKR4 genes were commonly dysregulated in all studies. IGDCC3 takes part in nervous system development  gene, which has been reported to have positive influence on recognition and memory in a rodent model of HD , Parkinson’s Disease (PD) and amyotrophic lateral sclerosis (ALS). Ruffinni et al. (2020) have identified an overlap of 139 genes among HD, AD, PD and ALS, which were also involved in the response to heat and hypoxia . In thisnditions . Labbadinditions . Moreoveel of HD . Vivo etel of HD . Additioel of HD . In MSNs in MSNs .HTT toxicity, we observed that dMSNs and Foxp2/Olfm3-expressing neurons (FON) share most genes with iMSNs. In contrast, only a few genes were in common with blood monocytes, platelets as well as with presymptomatic BA9 and caudate nucleus . Among these, binding of HSF1 to their loci was observed to be depleted in in vitro HD studies. In vivo, both the expression of heat shock genes as well as HSF1 binding were reported to be modulated in HD (TCF4 is involved in regulation of synaptic plasticity, DNA methylation and memory function (SON is known to participate in transcription, pre-messenger RNA splicing and cell cycle regulation (INO80 complex an ATP-dependent nucleosome remodeling complex conserved from yeast to human (PRMT2 an arginine methyltransferase and a coactivator of the androgen- and estrogen-mediated transactivation (KDM4B, SMARCD1 and KMT2C were downregulated and two of the top downregulated epigenetic modifiers, PRMT8 and SETD5 were upregulated in MSNs (TCF4, FOSL2, BCL6, RXRA, CUX1, CREBBP, E2F3, BCL11A, EGR1, FOXG1, TP53, JMJD1C and STAT4, which were both changed in expression and were predicted to be the regulators of DEGs in HD (Furthermore, to search for key regulators underlying the transcriptional dysregulation in HD, we examined both the regulators predicted by DEGs and regulators that were differentially expressed themselves in HD. In the first analysis, we found potential regulators like in blood . The sec factors , FOS rel factors , CBP (Giors (CBP , EGR1 (C (, EGR1  and ELK1R1 (ELK1 . Additioed in HD . Among ted in HD , TCF4 isfunction , SON is gulation , INO80 cto human  and PRMTtivation . Interes in MSNs . MoreoveGs in HD .Adora2a, Adcy5, CamkV and Penk (. In particular, the mouse data showed relatively fewer dysregulated transcripts in the early stages, which expanded tremendously during the progression of the disease at later stages (In non-human models, we observed some of the well reported DEGs in HD such as, and Penk . In partr stages . We founr stages . This isDNAJB1, HSPA1B and HSPB1, that were upregulated in both early and late stages of HD (SAT1 gene may be related to early neurodegeneration of MSNs, which might also be the consequence of unique dysregulation of mitochondrial function related genes. We also found differential expression and predicted activity of a number of transcription factors such as BCL11A, BCL6, EGR1 and FOSL2, and epigenetic regulators like CREBBP, HDAC1, KAT2B, KDM4C and KDM5B in human HD. This review hence summarizes and confirms reports from previous studies as well as presents new observations by systematically reviewing all the literature on transcriptional analysis using RNA sequencing in HD.Nevertheless, in this study, we were able to show that MSNs exhibit unique dysregulation of genes in comparison to the other brain regions. Except for MSNs, all cortex and striatum categories shared three heat shock response and unfolded protein response genes, es of HD . In iMSNes of HD . Additio