Inhibition Of Cell Differentiation Research Articles

Electroacupuncture treatment improves postoperative ileus by inhibiting the Th1 cell-mediated inflammatory response through the vagus nerve.

Electroacupuncture (EA) has been reported to improve intestinal motility in mice with postoperative ileus (POI). Previous studies, however, have yielded heterogeneous results regarding the effect of EA on POI. Herein, a POI mouse model was constructed by intestinal manipulation. To evaluate the effect of EA treatment on colonic transit, the levels of inflammatory markers (macrophage inflammatory protein (MIP)-1α, interleukin (IL)-1β, IL-6, monocyte chemotactic protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1) were detected by enzyme-linked immunosorbent assay (ELISA); immune cell infiltration was detected by immunohistochemical staining of myeloperoxidase (MPO), ectodysplasin (ED)-1 and ED-2, and the percentage of CD4+ interferon (IFN)-γ+ Th1 cells and IFN-γ secretion levels were determined. Activated Th1 cells and pentoxifylline, a cell differentiation inhibitor, were used to assess the role of Th1 cells in EA treatment of POI. Neostigmine administration and unilateral vagotomy were performed to confirm whether the effects of EA treatment on Th1 cells were mediated by the vagus nerve (VN). The results revealed that EA treatment at ST36 improved POI, as indicated by a decreased level of inflammatory-related markers and immune cell infiltration and shortened colonic transit time. The activated Th1 cells abolished the effects of EA treatment on POI. The effects of EA treatment on POI were enhanced by stimulation of the VN along with a decreased level of Th1 cells, but these effects were abolished by vagotomy along with an increased percentage of Th1 cells; this result indicates that the VN mediates the role of Th1 cells in the effects of EA treatment of POI. Our findings showed that the effects of EA treatment of POI were mainly mediated by Th1 cells through the stimulation of the VN and inhibition of the inflammatory response.

Two‐Way Regulation of MgFe‐LDH on ESC Cell Fate via Temporal Selective Activation of Different Cell Membrane Receptors

AbstractStem cell fate regulation by biomaterials has considerable therapeutic potential; however, the mechanisms whereby biomaterials instruct stem cells for accurate fate regulation still need to be elucidated. Layered double hydroxide (LDH) nanoparticles are widely used for tissue regeneration because of their excellent physicochemical properties and biocompatibility. This study reveals that MgFe‐LDH has bidirectional regulatory effects on the differentiation of mouse embryonic stem cells into neural progenitor cells, essentially suppressing cell differentiation on the 1st day and promoting cell differentiation on the 3rd day. Single‐cell transcriptome sequencing and simulated computation of structural analysis demonstrate that MgFe‐LDH interacts with the membrane receptors leukemia inhibitory factor receptor (LIFR, 15 nm above the cell membrane) and pached1 (PTCH1, 6 nm above the cell membrane) on the 1st and 3rd day of differentiation, respectively. LIFR has more contact with MgFe‐LDH than PTCH1 and relatively weaker electrostatic interaction energies (−11010.0 and −9829.3 kJ mol−1 for PTCH1 and LIFR, respectively). These factors determine receptor binding and activation priority, followed by cell fate regulation. The results indicate that MgFe‐LDH regulate the activity and fate of stem cells dually, thus creating possibilities for personalized therapy in neurodevelopment and regenerative medicine through an in‐depth study of cell membrane receptors.

ScRNA-seq analysis discovered suppression of immunomodulatory dependent inflammatory response in PMBCs exposed to silver nanoparticles

The assessment of AgNPs toxicity in vitro and in vivo models are frequently conflicting and inaccurate. Nevertheless, single cell immunological responses in a heterogenous environment have received little attention. Therefore, in this study, we have performed in-depth analysis which clearly revealed cellular-metal ion association as well as specific immunological response. Our study didn’t show significant population differences in PMBC between control and AgNPs group implying no toxicological response. To confirm it further, deep profiling identified differences in subsets and differentially expressed genes (DEGs) of monocytes, B cells and T cells. Notably, monocyte subsets showed significant upregulation of metallothionein (MT) gene expression such as MT1G, MT1X, MT1E, MT1A, and MT1F. On the other hand, downregulation of pro-inflammatory genes such as IL1β and CCL3 in both CD16 + and CD16- monocyte subsets were observed. This result indicated that AgNPs association with monocyte subsets de-promoted inflammatory responsive genes suggesting no significant toxicity observed in AgNPs treated group. Other cell types such as B cells and T cells also showed negligible differences in their subsets suggesting no toxicity response. Further, AgNPs treated group showed upregulation of cell proliferation, ribosomal synthesis, downregulation of cytokine release, and T cell differentiation inhibition. Overall, our results conclude that treatment of AgNPs to PMBC cells didn’t display immunological related cytotoxicity response and thus motivate researchers to use them actively for biomedical applications.

PHF6 loss reduces leukemia stem cell activity in an acute myeloid leukemia mouse model

Acute myeloid leukemia (AML) is a malignant hematologic disease caused by gene mutations and genomic rearrangements in hematologic progenitors. The PHF6 (PHD finger protein 6) gene is highly conserved and located on the X chromosome in humans and mice. We found that PHF6 was highly expressed in AML cells with MLL rearrangement and was related to the shortened survival time of AML patients. In our study, we knocked out the Phf6 gene at different disease stages in the AML mice model. Moreover, we knocked down PHF6 by shRNA in two AML cell lines and examined the cell growth, apoptosis, and cell cycle. We found that PHF6 deletion significantly inhibited the proliferation of leukemic cells and prolonged the survival time of AML mice. Interestingly, the deletion of PHF6 at a later stage of the disease displayed a better anti-leukemia effect. The expressions of genes related to cell differentiation were increased, while genes that inhibit cell differentiation were decreased with PHF6 knockout. It is very important to analyze the maintenance role of PHF6 in AML, which is different from its tumor-suppressing function in T-cell acute lymphoblastic leukemia (T-ALL). Our study showed that inhibiting PHF6 expression may be a potential therapeutic strategy targeting AML patients.

LILRB4 Checkpoint for Immunotherapy: Structure, Mechanism and Disease Targets.

LILRB4, a myeloid inhibitory receptor belonging to the family of leukocyte immunoglobulin-like receptors (LILRs/LIRs), plays a pivotal role in the regulation of immune tolerance. LILRB4 primarily mediates suppressive immune responses by transmitting inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs (ITIMs). This immune checkpoint molecule has gained considerable attention due to its potent regulatory functions. Its ability to induce effector T cell dysfunction and promote T suppressor cell differentiation has been demonstrated, indicating the therapeutic potential of LILRB4 for modulating excessive immune responses, particularly in autoimmune diseases or the induction of transplant tolerance. Additionally, through intervening with LILRB4 molecules, immune system responsiveness can be adjusted, representing significant value in areas such as cancer treatment. Thus, LILRB4 has emerged as a key player in addressing autoimmune diseases, transplant tolerance induction, and other medical issues. In this review, we provide a comprehensive overview of LILRB4, encompassing its structure, expression, and ligand molecules as well as its role as a tolerance receptor. By exploring the involvement of LILRB4 in various diseases, its significance in disease progression is emphasized. Furthermore, we propose that the manipulation of LILRB4 represents a promising immunotherapeutic strategy and highlight its potential in disease prevention, treatment and diagnosis.

MiR-129 Regulates Yak Intramuscular Preadipocyte Proliferation and Differentiation through the PI3K/AKT Pathway.

miR-129 plays a crucial role in regulating various cellular processes, including adipogenesis; however, its downstream molecular mechanisms remain unclear. In this study, we demonstrated that miR-129 promotes yak adipogenesis in vitro via the PI3K/AKT pathway. Overexpression and interference of miR-129 in yak intramuscular preadipocytes (YIMAs) enhanced and inhibited cell differentiation, respectively, with corresponding changes in cell proliferation. Further investigation revealed that miR-129 enhances AKT and p-AKT activity in the AKT pathway without affecting cell apoptosis, and a specific inhibitor (LY294002) was used to confirm that miR-129 regulates YIMAs proliferation and differentiation through the PI3K/AKT pathway. Our findings suggest that miR-129 promotes yak adipogenesis by enhancing PI3K/AKT pathway activity. This study provides the foundation to precisely elucidate the molecular mechanism of miR-129 in YIMAs adipogenesis and develop advanced miRNA-based strategies to improve meat nutrition and obesity-related ailments in beef production.

Assessment of myeloid-derived suppressor cell differentiation ex vivo.

Myeloid-derived suppressor cells (MDSCs) are major promoters of progression and metastasis in cancer. MDSCs inhibit the anti-tumor immune response through multiple mechanisms. The main MDSC functions in cancer are related to the inactivation of T cells and the establishment of an immunosuppressive tumor microenvironment (TME) through the production of pro-inflammatory cytokines, among other mechanisms. MDSCs are phenotypically similar to conventional myeloid cells, so their identification is challenging. Moreover, they infiltrate the tumors in limited numbers, and their purification from within the tumors is technically difficult and makes their study a challenge. Therefore, several ex vivo differentiation methods have been established. Our differentiation method leads to MDSCs that closely model tumor-infiltrating counterparts. In this protocol, MDSCs are differentiated from bone marrow precursors by incubation in differentiation medium produced by murine tumor cell lines engineered to constitutively express granulocyte-monocyte colony stimulating factor (GM-CSF). These ex vivo-generated MDSC subsets show high fidelity compared to their natural tumor-infiltrated counterparts. Moreover, the high yields of purification from these ex vivo differentiated MDSC enable their use for validation of new treatments in high-throughput assays. In this chapter we describe the engineering of a stable cell line overexpressing GM-CSF, followed by production and collection of conditioned media supporting MDSC differentiation. Finally, we detail the isolation procedure of bone marrow cells and the specific MDSC differentiation protocol.

Antimicrobial and Cell-Friendly Properties of Cobalt and Nickel-Doped Tricalcium Phosphate Ceramics.

β-Tricalcium phosphate (β-TCP) is widely used as bone implant material. It has been observed that doping the β-TCP structure with certain cations can help in combating bacteria and pathogenic microorganisms. Previous literature investigations have focused on tricalcium phosphate structures with silver, copper, zinc, and iron cations. However, there are limited studies available on the biological properties of β-TCP containing nickel and cobalt ions. In this work, Ca10.5-xNix(PO4)7 and Ca10.5-xCox(PO4)7 solid solutions with the β-Ca3(PO4)2 structure were synthesized by a high-temperature solid-state reaction. Structural studies revealed the β-TCP structure becomes saturated at 9.5 mol/% for Co2+ or Ni2+ ions. Beyond this saturation point, Ni2+ and Co2+ ions form impurity phases after complete occupying of the octahedral M5 site. The incorporation of these ions into the β-TCP crystal structure delays the phase transition to the α-TCP phase and stabilizes the structure as the temperature increases. Biocompatibility tests conducted on adipose tissue-derived mesenchymal stem cells (aMSC) using the (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) assay showed that all prepared samples did not exhibit cytotoxic effects. Furthermore, there was no inhibition of cell differentiation into the osteogenic lineage. Antibacterial properties were studied on the C. albicans fungus and on E. coli, E. faecalis, S. aureus, and P. aeruginosa bacteria strains. The Ni- and Co-doped β-TCP series exhibited varying degrees of bacterial growth inhibition depending on the doping ion concentration and the specific bacteria strain or fungus. The combination of antibacterial activity and cell-friendly properties makes these phosphates promising candidates for anti-infection bone substitute materials.

Genomic characterization of a unique Panton-Valentine leucocidin-positive community-associated methicillin-resistant Staphylococcus aureus lineage increasingly impacting on Australian indigenous communities.

In 2010 a single isolate of a trimethoprim-resistant multilocus sequence type 5, Panton-Valentine leucocidin-positive, community-associated methicillin-resistant Staphylococcus aureus (PVL-positive ST5 CA-MRSA), colloquially named WA121, was identified in northern Western Australia (WA). WA121 now accounts for ~14 % of all WA MRSA infections. To gain an understanding of the genetic composition and phylogenomic structure of WA121 isolates we sequenced the genomes of 155 WA121 isolates collected 2010-2021 and present a detailed genomic description. WA121 was revealed to be a single clonally expanding lineage clearly distinct from sequenced ST5 strains reported outside Australia. WA121 strains were typified by the presence of the distinct PVL phage φSa2wa-st5, the recently described methicillin resistance element SCCmecIVo carrying the trimethoprim resistance (dfrG) transposon Tn4791, the novel β-lactamase transposon Tn7702 and the epidermal cell differentiation inhibitor (EDIN-A) plasmid p2010-15611-2. We present evidence that SCCmecIVo together with Tn4791 has horizontally transferred to Staphylococcus argenteus and evidence of intragenomic movement of both Tn4791 and Tn7702. We experimentally demonstrate that p2010-15611-2 is capable of horizontal transfer by conjugative mobilization from one of several WA121 isolates also harbouring a pWBG749-like conjugative plasmid. In summary, WA121 is a distinct and clonally expanding Australian PVL-positive CA-MRSA lineage that is increasingly responsible for infections in indigenous communities in northern and western Australia. WA121 harbours a unique complement of mobile genetic elements and is capable of transferring antimicrobial resistance and virulence determinants to other staphylococci.

IgA nephropathy.

IgA nephropathy (IgAN), the most prevalent primary glomerulonephritis worldwide, carries a considerable lifetime risk of kidney failure. Clinical manifestations of IgAN vary from asymptomatic with microscopic or intermittent macroscopic haematuria and stable kidney function to rapidly progressive glomerulonephritis. IgAN has been proposed to develop through a 'four-hit' process, commencing with overproduction and increased systemic presence of poorly O-glycosylated galactose-deficient IgA1 (Gd-IgA1), followed by recognition of Gd-IgA1 by antiglycan autoantibodies, aggregation of Gd-IgA1 and formation of polymeric IgA1 immune complexes and, lastly, deposition of these immune complexes in the glomerular mesangium, leading to kidney inflammation and scarring. IgAN can only be diagnosed by kidney biopsy. Extensive, optimized supportive care is the mainstay of therapy for patients with IgAN. For those at high risk of disease progression, the 2021 KDIGO Clinical Practice Guideline suggests considering a 6-month course of systemic corticosteroid therapy; however, the efficacy of systemic steroid treatment is under debate and serious adverse effects are common. Advances in understanding the pathophysiology of IgAN have led to clinical trials of novel targeted therapies with acceptable safety profiles, including SGLT2 inhibitors, endothelin receptor blockers, targeted-release budesonide, B cell proliferation and differentiation inhibitors, as well as blockade of complement components.

In vitro naive CD4+T cell differentiation upon treatment with miR-29b-loaded exosomes from mesenchymal stem cells.

Gene regulation by microRNA (miRNA) is central in T lymphocytes differentiation processes. Here, we investigate miRNA-29b (miR-29b) roles in the reprogramming of T cell differentiation, which can be a promising therapeutic avenue for various types of inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Adipose Mesenchymal Stem Cell-derived exosomes (AMSC-Exo) enriched with miR-29b were delivered into naive CD4+ T (nCD4+) cells. The expression level of important transcription factors including RAR-related orphan receptor gamma (RORγt), GATA3 binding protein (GATA3), T-box transcription factor 21, and Forkhead box P3 was determined by quantitative Real-Time PCR. Moreover, flow cytometry and Enzyme-linked Immunosorbent Assay were respectively used to measure the frequency of T regulatory cells and the levels of cytokines production (Interleukin 17, Interleukin 4, Interferon-gamma, and transforming growth factor beta. This study indicates that the transfection of miR-29b mimics into T lymphocytes through AMSC-Exo can alter the CD4+ T cells' differentiation into other types of T cells. In conclusion, AMSC-Exo-based delivery of miR-29b can be considered as a new fascinating avenue for T cell differentiation inhibition and the future treatment of several inflammatory disorders.

Effect of pregnancy-specific β1-glycoprotein on myeloid-derived suppressor cell differentiation

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population that primarily suppress T lymphocytes in healthy pregnancies and pathologies. MDSCs are one of the key regulators of immune responses. Finding ways to control them is important for the treatment of cancer, autoimmune diseases, miscarriage, and post-transplant complications. The mechanisms of immune suppression by MDSC are: expression of CD73, ADAM17, PD -L1, production of Arg 1, iNOS, IDO, IL -10 and TGF-b1.Pregnancy-specific b1-glycoprotein (PSG) has modulatory effects on dendritic cells and macrophages that mediate the shift of T cell phenotypes toward Th2 and Treg. We have previously shown that native PSG suppresses Th17 differentiation and cytokine production, stimulates the production of IDO by monocytes and the differentiation of Tregs.Considering the immunomodulatory properties of PSG and the key role of MDSCs in pathologies, the aim of our work was to investigate the effect of native and recombinant PSG on the differentiation of MDSCs in vitro.MDSCs were differentiated from CD11b+ peripheral blood cells. Cells were cultured for 7 days and received stepwise GM-CSF, IL-1b, and LPS. Native (n) (1; 10 and 100 mg/mL) and recombinant (r) (1 and 10 mg/mL) PSG were introduced into the cultures three days before the end of incubation. Flow cytometry was used to determine the percentage of MDSC among the cells in culture and the percentage of M-, PMN-, and e-MDSC among the total number of MDSCs.It was found that rPSG (1 mg/mL) increased the percentage of MDSCs in culture. Both nPSG (1 and 10 mg/mL) and rPSG (10 mg/mL) increased the proportion of M-MDSC, whereas rPSG (10 mg/mL) decreased the number of PMN-MDSC.Thus, the cytokine background in CD11b+ cell cultures favored the differentiation of predominantly M-MDSC, similar to the tumor microenvironment, whereas native and recombinant PSG enhanced this effect. Thus, nPSG and rPSG are able to modulate the differentiation of MDSCs by increasing their number, mainly due to the monocytic subpopulation. This fact opens perspectives for new research on targeted manipulation of MDSCs.

Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.

Sucrose Signaling Contributes to the Maintenance of Vascular Cambium by Inhibiting Cell Differentiation.

Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named 'Vascular Cell Induction Culture System Using Arabidopsis Leaves' (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation, among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation in cambial cells. Physiological and genetic analyses suggested that sucrose might function through the BRI1-EMS-SUPPRESSOR1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

1203 Increased horizontal cell division and suppressed cell differentiation of pigmented basal keratinocytes in senile lentigo

1203 Increased horizontal cell division and suppressed cell differentiation of pigmented basal keratinocytes in senile lentigo

Abstract 4424: Immunoregulatory role of club cell secretory proteins in non-small cell lung cancer

Abstract Introduction. Immune checkpoint inhibitors (ICI) particularly those targeting the PD-1/PD-L1 axis are approved for NSCLC patients. However, most patients experience little clinical benefit due to immunosuppressive barriers in the tumor microenvironment. Our goal to identify immunomodulatory agents that may overcome these barriers, led to the finding that low dose stereotactic body radiation therapy (SBRT) in combination with ICI resulted in marked tumor regression and improved survival by generating durable anti-tumor immunity (Ban et al., Nature Cancer, 2021). Unexpectedly, we uncovered that the immune-modulating functions of SBRT was acutely dependent on the secretory function of lung resident Scgb1a1+ club cells that protect the bronchioles from xenobiotic agents. Blocking club cell secretome in vivo blunted the efficacy of the combination treatment and altered myeloid cell phenotypes. These findings led to the hypothesis that radiation-induced club cell factors may synergize with ICI to attenuate immunosuppressive barriers by reducing myeloid suppressive cells. Methods and Data. Expression of 8 highest expressed club cells secretory factors (CC10, SP-A, SP-B, SP-D, Hp, Secretoglobin 3A1, 3A 2, and 1C 1) in combination with PD-1 blockade elicited significant tumor control and improved survival of NSCLC mice. Mechanistically, the 8 club cell factors inhibit myeloid suppressor cell phenotypes as determined by reduced pSTAT3, pERK and concomitant inhibition of immunosuppressive mediators Arg1, iNOS, etc. Further screening of individual club cell factors showed that CC10 alone effectively blunted myeloid suppression activity as determined by flow cytometry for Arg1 and iNOS. Hence, CC10 can functionally alter the immunosuppressive nature of myeloid suppressor cells. The ability of CC10 in improving the efficacy of ICI in impairing tumor growth and improving survival is currently being examined. Conclusions. Myeloid suppressor cells inhibit immune cells like T cells or NK cells leading to poor prognosis in cancer treatment. Increase in their numbers has been linked to tumor aggressiveness and low efficacy of immunotherapy. Hence, there is an unmet clinical need to specifically target these cells. Agents targeting myeloid suppressor cell accumulation and differentiation (e.g., ATRA, Vitamin D, Sunitinib, Gemcitabine, Bevacizumab, Tadalafil), are being tested in human clinical trials. However, these agents lack specificity and selectivity for myeloid suppressor cells in vivo. Therefore, our work provides the rationale for the development of CC10 as selective inhibitor of myeloid suppressor cells and has the potential for achieving a robust and durable anti-tumor immunity in NSCLC. Citation Format: Aakanksha Rajiv Kapoor, Vivek Mittal. Immunoregulatory role of club cell secretory proteins in non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4424.

Riboflavin mediates m6A modification targeted by miR408, promoting early somatic embryogenesis in longan.

Plant somatic embryogenesis (SE) is an in vitro biological process wherein bipolar structures are induced to form somatic cells and regenerate into whole plants. MicroRNA (miRNA) is an essential player in plant SE. However, the mechanism of microRNA408 (miR408) in SE remains elusive. Here, we used stable transgenic technology in longan (Dimocarpus longan) embryogenic calli to verify the mechanism by which miR408 promotes cell division and differentiation of longan early SE. dlo-miR408-3p regulated riboflavin biosynthesis by targeting nudix hydrolase 23 (DlNUDT23), a previously unidentified gene mediating N6-methyladenosine (m6A) modification and influencing RNA homeostasis and cell cycle gene expression during longan early SE. We showed that DlMIR408 overexpression (DlMIR408-OE) promoted 21-nt miRNA biosynthesis. In DlMIR408-OE cell lines, dlo-miR408-3p targeted and downregulated DlNUDT23, promoted riboflavin biosynthesis, decreased flavin mononucleotide (FMN) accumulation, promoted m6A level, and influenced miRNA homeostasis. DNA replication, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, the pentose phosphate pathway, and taurine and hypotaurine metabolism were also closely associated with riboflavin metabolism. In a riboflavin feeding assay, dlo-miR408-3p and pre-miR408 were upregulated and DlNUDT23 was downregulated, increasing the m6A level and cell division and differentiation in longan globular embryos. When riboflavin biosynthesis was inhibited, dlo-miR408-3p was downregulated and DlNUDT23 was upregulated, which decreased m6A modification and inhibited cell division but did not inhibit cell differentiation. FMN artificial demethylated m6A modification affected the homeostasis of precursor miRNA and miRNA. Our results revealed a mechanism underlying dlo-miR408-3p-activated riboflavin biosynthesis in which DlNUDT23 is targeted, m6A modification is dynamically mediated, and cell division is affected, promoting early SE in plants.

Quercetin Reprograms Immunometabolism of Macrophages via the SIRT1/PGC-1α Signaling Pathway to Ameliorate Lipopolysaccharide-Induced Oxidative Damage.

The redox system is closely related to changes in cellular metabolism. Regulating immune cell metabolism and preventing abnormal activation by adding antioxidants may become an effective treatment for oxidative stress and inflammation-related diseases. Quercetin is a naturally sourced flavonoid with anti-inflammatory and antioxidant activities. However, whether quercetin can inhibit LPS-induced oxidative stress in inflammatory macrophages by affecting immunometabolism has been rarely reported. Therefore, the present study combined cell biology and molecular biology methods to investigate the antioxidant effect and mechanism of quercetin in LPS-induced inflammatory macrophages at the RNA and protein levels. Firstly, quercetin was found to attenuate the effect of LPS on macrophage proliferation and reduce LPS-induced cell proliferation and pseudopodia formation by inhibiting cell differentiation, as measured by cell activity and proliferation. Subsequently, through the detection of intracellular reactive oxygen species (ROS) levels, mRNA expression of pro-inflammatory factors and antioxidant enzyme activity, it was found that quercetin can improve the antioxidant enzyme activity of inflammatory macrophages and inhibit their ROS production and overexpression of inflammatory factors. In addition, the results of mitochondrial morphology and mitochondrial function assays showed that quercetin could upregulate the mitochondrial membrane potential, ATP production and ATP synthase content decrease induced by LPS, and reverse the mitochondrial morphology damage to a certain extent. Finally, Western blotting analysis demonstrated that quercetin significantly upregulated the protein expressions of SIRT1 and PGC-1α, that were inhibited by LPS. And the inhibitory effects of quercetin on LPS-induced ROS production in macrophages and the protective effects on mitochondrial morphology and membrane potential were significantly decreased by the addition of SIRT1 inhibitors. These results suggested that quercetin reprograms the mitochondria metabolism of macrophages through the SIRT1/PGC-1α signaling pathway, thereby exerting its effect of alleviating LPS-induced oxidative stress damage.

Targeting HDAC3 to overcome the resistance to ATRA or arsenic in acute promyelocytic leukemia through ubiquitination and degradation of PML-RARα.

Acute promyelocytic leukemia (APL) is driven by the oncoprotein PML-RARα, which recruits corepressor complexes, including histone deacetylases (HDACs), to suppress cell differentiation and promote APL initiation. All-trans retinoic acid (ATRA) combined with arsenic trioxide (ATO) or chemotherapy highly improves the prognosis of APL patients. However, refractoriness to ATRA and ATO may occur, which leads to relapsed disease in a group of patients. Here, we report that HDAC3 was highly expressed in the APL subtype of AML, and the protein level of HDAC3 was positively associated with PML-RARα. Mechanistically, we found that HDAC3 deacetylated PML-RARα at lysine 394, which reduced PIAS1-mediated PML-RARα SUMOylation and subsequent RNF4-induced ubiquitylation. HDAC3 inhibition promoted PML-RARα ubiquitylation and degradation and reduced the expression of PML-RARα in both wild-type and ATRA- or ATO-resistant APL cells. Furthermore, genetic or pharmacological inhibition of HDAC3 induced differentiation, apoptosis, and decreased cellular self-renewal of APL cells, including primary leukemia cells from patients with resistant APL. Using both cell line- and patient-derived xenograft models, we demonstrated that treatment with an HDAC3 inhibitor or combination of ATRA/ATO reduced APL progression. In conclusion, our study identifies the role of HDAC3 as a positive regulator of the PML-RARα oncoprotein by deacetylating PML-RARα and suggests that targeting HDAC3 could be a promising strategy to treat relapsed/refractory APL.

Metformin regulates myoblast differentiation through an AMPK-dependent mechanism.

This study aims to investigate how metformin (Met) affects muscle tissue by evaluating the drug effects on proliferating, differentiating, and differentiated C2C12 cells. Moreover, we also investigated the role of 5'-adenosine monophosphate-activated protein kinase (AMPK) in the mechanism of action of Met. C2C12 myoblasts were cultured in growth medium with or without Met (250μM, 1mM and 10mM) for different times. Cell proliferation was evaluated by MTT assay, while cell toxicity was assessed by Trypan Blue exclusion test and Lactate Dehydrogenase release. Fluorescence Activated Cell Sorting analysis was performed to study cell cycle. Differentiating myoblasts were incubated in differentiation medium (DM) with or without 10mM Met. For experiments on myotubes, C2C12 were induced to differentiate in DM, and then treated with Met at scalar concentrations and for different times. Western blotting was performed to evaluate the expression of proteins involved in myoblast differentiation, muscle function and metabolism. In differentiating C2C12, Met inhibited cell differentiation, arrested cell cycle progression in G2/M phase and reduced the expression of cyclin-dependent kinase inhibitor 1. These effects were accompanied by activation of AMPK and modulation of the myogenic regulatory factors. Comparable results were obtained in myotubes. The use of Compound C, a specific inhibitor of AMPK, counteracted the above-mentioned Met effects. We reported that Met inhibits C2C12 differentiation probably by blocking cell-cycle progression and preventing cells permanent exit from cell-cycle. Moreover, our study provides solid evidence that most of the effects of Met on myoblasts and myotubes are mediated by AMPK.