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BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by a complex pathogenesis involving various types of cells and cytokines. Among those, the pro-inflammatory cytokine IL-23/IL-17A axis plays a crucial role in the development and rapid progression of psoriasis. Phenformin, a derivative of metformin and a member of the biguanide class of drugs, exhibits superior anti-inflammatory and anti-tumor efficacy compared to metformin. However, the potential role of phenformin in anti-psoriatic skin inflammation has not been explored. METHODS: In this study, we utilized a mouse model of psoriasis and an in vitro model using human keratinocytes to investigate whether phenformin can suppress psoriasis-like inflammatory responses. RESULTS: Our results demonstrate that the topical application of phenformin significantly inhibited acute skin inflammatory responses in the psoriasis mouse model induced by imiquimod (IMQ). Additionally, phenformin suppressed the expression of psoriasis-related cytokines IL-17, IL-23, IL-8, and S100A8/S100A9 in an in vitro psoriatic keratinocyte model induced by IMQ. Furthermore, we found that IMQ-induced psoriatic skin and IMQ-treated keratinocytes exhibited high expression of the c-Myc gene, which was downregulated by phenformin. The c-Myc inhibitor JQ1 similarly inhibited the psoriatic inflammatory response and the expression of psoriasis-related cytokines in both in vitro and in vivo models. CONCLUSION: phenformin ameliorates the psoriasis-like inflammatory response by inhibiting c-Myc expression in keratinocytes, suggesting its potential as a topical drug for the treatment of psoriasis.
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Matrix stiffness is a crucial factor in the tumor microenvironment, impacting tumor progression and development. TET2 is vital for epigenetic regulation in melanoma and is significantly reduced in advanced melanomas compared with nevi and thin melanomas. However, it is unclear how TET2 mediates the effect of matrix stiffness on melanoma cells. This study utilized A2058 cell lines and prepared different stiffness collagen hydrogels to evaluate TET2 overexpression (TET2OE) and mutant (TET2M) melanoma cells' activity, proliferation, and invasion. A2058 melanoma cells' viability and invasion decreased with increased matrix stiffness, with TET2OE cells experiencing a more significant impact than TET2M cells. Methylation analysis revealed that TET2 determines gene methylation levels, influencing cell-ECM interactions. Transcriptome analysis confirmed that TET2 promotes matrix stiffness's effect on melanoma cell fate. This research provides promising directions and opportunities for melanoma treatment.
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The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.
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Técnicas de Cultura de Células , Células Epiteliais , Glândulas Mamárias Humanas , Humanos , Células Epiteliais/citologia , Feminino , Glândulas Mamárias Humanas/citologia , Técnicas de Cultura de Células/métodos , Amidas/farmacologia , Piridinas/farmacologia , Técnicas Citológicas/métodos , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
The efficient clinical treatment of oral squamous cell carcinoma (OSCC) is still a challenge that demands the development of effective new drugs. Phenformin has been shown to produce more potent anti-tumor activities than metformin on different tumors, however, not much is known about the influence of phenformin on OSCC cells. We found that phenformin suppresses OSCC cell proliferation, and promotes OSCC cell autophagy and apoptosis to significantly inhibit OSCC cell growth both in vivo and in vitro. RNA-seq analysis revealed that autophagy pathways were the main targets of phenformin and identified two new targets DDIT4 (DNA damage inducible transcript 4) and NIBAN1 (niban apoptosis regulator 1). We found that phenformin significantly induces the expression of both DDIT4 and NIBAN1 to promote OSCC autophagy. Further, the enhanced expression of DDIT4 and NIBAN1 elicited by phenformin was not blocked by the knockdown of AMPK but was suppressed by the knockdown of transcription factor ATF4 (activation transcription factor 4), which was induced by phenformin treatment in OSCC cells. Mechanistically, these results revealed that phenformin triggers endoplasmic reticulum (ER) stress to activate PERK (protein kinase R-like ER kinase), which phosphorylates the transitional initial factor eIF2, and the increased phosphorylation of eIF2 leads to the increased translation of ATF4. In summary, we discovered that phenformin induces its new targets DDIT4 and especially NIBAN1 to promote autophagic and apoptotic cell death to suppress OSCC cell growth. Our study supports the potential clinical utility of phenformin for OSCC treatment in the future.
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Autofagia , Carcinoma de Células Escamosas , Proliferação de Células , Estresse do Retículo Endoplasmático , Neoplasias Bucais , Fenformin , Fatores de Transcrição , Fenformin/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Neoplasias Bucais/tratamento farmacológico , Autofagia/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Fatores de Transcrição/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Camundongos , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Western BlottingRESUMO
The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.
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Azoospermia , Retroelementos , Masculino , Animais , Humanos , Retroelementos/genética , RNA , Azoospermia/genética , Diferenciação Celular/genética , Metiltransferases/genética , Metiltransferases/metabolismo , RNA Mensageiro/genética , Mamíferos/metabolismoRESUMO
Wound research has typically been performed without regard for where the wounds are located on the body, despite well-known heterogeneities in physical and biological properties between different skin areas. The skin covering the palms and soles is highly specialized, and plantar ulcers are one of the most challenging and costly wound types to manage. Using primarily the porcine model, we show that plantar skin is molecularly and functionally more distinct from nonplantar skin than previously recognized, with unique gene and protein expression profiles, broad alterations in cellular functions, constitutive activation of many wound-associated phenotypes, and inherently delayed healing. This unusual physiology is likely to play a significant but underappreciated role in the pathogenesis of plantar ulcers as well as the last 25+ years of futility in therapy development efforts. By revealing this critical yet unrecognized pitfall, we hope to contribute to the development of more effective therapies for these devastating nonhealing wounds.
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Fenótipo , Pele , Cicatrização , Animais , Cicatrização/fisiologia , Suínos , Pele/patologia , Pele/lesões , Pele/metabolismo , Modelos Animais de Doenças , Úlcera do Pé/fisiopatologia , Úlcera do Pé/patologia , Humanos , Feminino , Fenômenos Fisiológicos da Pele , PéRESUMO
The management of skin wound healing is still a challenge. MicroRNA-21 (miR-21) has been reported to play important roles in wound repair; however, the underlying mechanism needs to be further clarified. The present study aimed to study the direct role of miR-21 in skin wound healing in miR-21 KO mice and to investigate the role of miR-21 in controlling the migration and proliferation of primary human skin cells and its underlying mechanism(s). miR-21 KO and wild-type (WT) mice were used for in vivo wound healing assays, while mouse and human primary skin cells were used for in vitro assays. miR-21 inhibitors or mimics or negative control small RNAs were transfected to either inhibit or enhance miR-21 expression in the human primary dermal fibroblasts or epidermal cells. RNA sequencing analysis was performed to identify the potential molecular pathways involved. We found that the loss of miR-21 resulted in slower wound healing in miR-21 KO mouse skin and especially delayed the healing of dermal tissue. In vitro assays demonstrated that the reduced expression of miR-21 caused by its inhibitor inhibited the migration of human primary dermal fibroblasts, which could be enhanced by increased miR-21 expression caused by miR-21 mimics. RNA-sequence analysis revealed that the inhibition of miR-21 expression downregulated the inflammatory response pathways associated with the decreased expression of inflammatory cytokines, and the addition of IL-1ß into the culture medium enhanced the migration and proliferation of dermal fibroblasts in vitro. In conclusion, miR-21 in dermal fibroblasts can promote the migration and growth of epidermal and dermal cells to enhance skin wound healing through controlling the expression of inflammatory cytokines.
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The undulating microstructure rete ridge (RR) located at the junction between the dermis and epidermis plays a crucial role in improving skin mechanical properties and maintaining skin homeostasis. However, the investigation of RR microstructures is usually neglected in current tissue engineering for skin regeneration. Here, to create an epidermal model with RR microstructures, keratinocytes were cultured on a patterned GelMA-PEGDA hydrogel constructed using molding technology. Furthermore, grafting acryloylated Arg-Gly-Asp (RGD) peptides on the hydrogel surface significantly improved cell adhesion, fusion, and development. RT-PCR, Western blot, and immunofluorescence staining confirmed that cells on RR microstructures exhibited higher gene and protein expression associated with epidermal stem cells. RNA sequencing analysis of cells on RR microstructure showed higher gene expression profiles related to stem cell maintenance, basement membrane formation, and epidermal development. Furthermore, RT-PCR analysis of epidermal models of various dimensions demonstrated that smaller microstructures were more conducive to epidermal stem cell marker gene expression, which is analogous to human skin. Overall, we have successfully developed a method for integrating RR microstructures into an epidermal model that mimics natural skin to maintain epidermal stem cell niche, providing a valuable reference for researching skin regeneration within the fields of tissue engineering and regenerative medicine. STATEMENT OF SIGNIFICANCE: This study presents a method for precisely fabricating microstructures of skin rete ridges using composite hydrogels, thereby creating a skin model that mimics natural human skin. The findings reveal that this microstructure provides a stem cell niche that regulates the pathways and promotes the expression of proteins related to epidermal stem cells. This work advances the functional properties of tissue engineered skin and holds promise for improving the therapeutic efficacy of artificial skin grafts for the skin wounds.
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Hidrogéis , Nicho de Células-Tronco , Humanos , Hidrogéis/farmacologia , Células Cultivadas , Epiderme , Engenharia Tecidual/métodos , Transdução de SinaisRESUMO
Spatiotemporal regulation of the cellular transcriptome is crucial for proper protein expression and cellular function. However, the intricate subcellular dynamics of RNA remain obscured due to the limitations of existing transcriptomics methods. Here, we report TEMPOmap-a method that uncovers subcellular RNA profiles across time and space at the single-cell level. TEMPOmap integrates pulse-chase metabolic labeling with highly multiplexed three-dimensional in situ sequencing to simultaneously profile the age and location of individual RNA molecules. Using TEMPOmap, we constructed the subcellular RNA kinetic landscape in various human cells from transcription and translocation to degradation. Clustering analysis of RNA kinetic parameters across single cells revealed 'kinetic gene clusters' whose expression patterns were shaped by multistep kinetic sculpting. Importantly, these kinetic gene clusters are functionally segregated, suggesting that subcellular RNA kinetics are differentially regulated in a cell-state- and cell-type-dependent manner. Spatiotemporally resolved transcriptomics provides a gateway to uncovering new spatiotemporal gene regulation principles.
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RNA , Transcriptoma , Humanos , RNA/genética , Cinética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Análise de Célula Única/métodosRESUMO
Rete ridges (RRs) are distinct undulating microstructures at the junction of the dermis and epidermis in the skin of humans and certain animals. This structure is essential for enhancing the mechanical characteristics of skin and preserving homeostasis. With the development of tissue engineering and regenerative medicine, artificial skin grafts have made great progress in the field of skin healing. However, the restoration of RRs has been often disregarded or absent in artificial skin grafts, which potentially compromise the efficacy of tissue repair and regeneration. Therefore, this review collates recent research advances in understanding the structural features, function, morphogenesis, influencing factors, and reconstruction strategies pertaining to RRs. In addition, the preparation methods and limitations of tissue-engineered skin with RRs are discussed. STATEMENT OF SIGNIFICANCE: The technology for the development of tissue-engineered skin (TES) is widely studied and reported; however, the preparation of TES containing rete ridges (RRs) is often ignored, with no literature reviews on the structural reconstruction of RRs. This review focuses on the progress pertaining to RRs and focuses on the reconstruction methods for RRs. In addition, it discusses the limitations of existing reconstruction methods. Therefore, this review could be a valuable reference for transferring TES with RR structure from the laboratory to clinical applications in skin repair.
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Epiderme , Pele , Animais , Humanos , Cicatrização , Células Epidérmicas , Engenharia Tecidual/métodos , MorfogêneseRESUMO
BACKGROUND: Sperm, during epididymal transit, acquires microRNAs(miRNAs), which are crucial for embryonic development. However, whether sperm miRNAs influenced by an obstructive epididymal environment affect embryonic development remains unknown. METHOD: The sham operation and vasectomy were performed in C57BL/6 J mice to create the control group (CON) and the obstructive epididymal environment group(OEE) group, respectively. The morphology of the testis and epididymis was observed using hematoxylin and eosin staining (HE staining) to establish the OEE mice model. The sperm quality test, intracytoplasmic sperm injection (ICSI), and epididymosomes fusion were employed to observe the effect of the obstructive epididymal environment on sperm and resultant embryonic development. The alteration of the sperm small RNA (sRNA) profile was analyzed by sRNA sequencing. RT-qPCR and DNA methylation were applied to observe the effect of obstructive epididymis on the expression of sperm miRNAs. The miRNAs microinjection was used to explore the impacts of sperm miRNAs on embryonic development. RESULTS: We confirmed postoperative 8-week mice as the OEE mice model by examining the morphology of the testis and epididymis. In the OEE group, we observed that sperm quality degraded and the development potential of embryos was reduced, which can be saved by the normal epididymal environment. The sperm sRNA sequencing revealed that the expression of the developmental miR-17-92 cluster and the Sfmbt2 miRNA cluster was downregulated in the OEE group. The expression of these two miRNA clusters in epididymis was also downregulated and regulated by DNA methylation. However, the downregulation of either the miR-17-92 cluster or the Sfmbt2 miRNA cluster in normal zygotes did not impair embryonic development. CONCLUSION: The obstructive epididymal environment influences sperm quality and resultant embryonic development, as well as the abundance of the developmental miR-17-92 cluster and the Sfmbt2 miRNA cluster in sperm, but these miRNA clusters are not the cause of abnormal embryonic development. It implies that epididymis is important in early embryonic development and may play a potential role in sperm epigenome.
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Epididimo , MicroRNAs , Masculino , Feminino , Gravidez , Camundongos , Animais , MicroRNAs/genética , Camundongos Endogâmicos C57BL , Sêmen , Espermatozoides , Desenvolvimento Embrionário/genética , Modelos Animais de Doenças , Proteínas RepressorasRESUMO
Human dermal fibroblasts (HDFs) have the potential to differentiate into endothelial cells (VECs). In our previous research, we reported that a hypochlorous acid (HOCl) probe CPP efficiently induced the differentiation of HDFs into VECs, however, the mechanism of differentiation was not clear. As an HOCI probe, CPP binds HOCI to modulate its effects. In this study, through Western blotting, qPCR, and PHD2 enzyme activity assay, we found that CPP inhibited the enzyme activity of prolyl-4-hydroxylase 2 (PHD2), thereby stabilizing HIF-1α. To further clarify the mechanism by which CPP inhibits PHD2 enzyme activity, we constructed plasmids, and found that CPP inhibited PHD2 activity to increase the HIF-1α level through the modulation of PHD2 at Cys302 by HOCl in HDFs. Furthermore, RNA-seq experiments showed that CPP could induce the expression of HEY1, which is not only a target gene regulated by HIF1α, but also a key transcription factor for VECs. We used siRNA transfection and in vivo experiments to confirm that CPP could induce HDFs to differentiate into VECs by HEY1. In summary, we identified a new inhibitor of PHD2, demonstrated the new role of HOCl in cell differentiation, and elucidated the mechanism by which HOCl probe CPP induced the differentiation of HDFs into VECs.
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Células Endoteliais , Prolina Dioxigenases do Fator Induzível por Hipóxia , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Ácido Hipocloroso/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolil Hidroxilases/metabolismo , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
BACKGROUND: Human dermal fibroblasts (HDFs) have the potential to differentiate into vascular endothelial cells (VECs), but their differentiation rate is low and the mechanism involved is not clear. The small molecule pathway controls the phenotype of fibroblasts by activating cellular signaling pathways, which is a more convenient method in the differentiation strategy of HDFs into VECs. METHODS: In this study, HDFs were treated with the different doses of CPP ((E)-4-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl) piperazin-1-yl) styryl)-1-methylpyridin-1-ium iodide), and the mRNA and protein levels of HDFs were detected by qPCR, Western blot, flow cytometry and immunofluorescent staining. The matrigel assays, acetylated-LDL uptake and angiogenesis assays of chick embryo chorioallantoic membrane (CAM) and hindlimb ischemia model of nude mice were performed to evaluate the functions of VECs derived from HDFs. RESULTS: Here, we report that the small chemical molecule, CPP, can effectively induce HDFs to differentiate into VECs. First, we observed the morphological changes of HDFS treated with CPP. Flow cytometry, Western blot and qRT-PCR analyses showed that CPP effectively decreased the level of the HDFs-marker Vimentin and increased levels of the VEC-markers CD31, CD133, TEK, ERG, vWF, KDR and CDH5. Detection of the percentage of CD31-positive cells by immunofluorescent staining confirmed that CPP can effectively induce HDFs to differentiate into VECs. The results of Matrigel assays, DiI-ac-LDL uptake, angiogenesis assays on CAM and hindlimb ischemia model of nude mice showed that CPP-induced HDFs have the functions of VECs in vitro and in vivo. Western blot and qRT-PCR analysis showed that CPP induces HDFs to differentiate into VECs by promoting the expression of pro-angiogenic factors (VEGF, FGF-2 and PDGF-BB). CONCLUSIONS: Our data suggest that the small chemical molecule CPP efficiently induces the differentiation of HDFs into VECs. Simultaneously, this new inducer provides a potential to develop new approaches to restore vascular function for the treatment of ischemic vascular diseases.
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Células Endoteliais , Fator A de Crescimento do Endotélio Vascular , Animais , Becaplermina/metabolismo , Células Cultivadas , Embrião de Galinha , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Humanos , Iodetos/metabolismo , Isquemia/terapia , Camundongos , Camundongos Nus , Neovascularização Patológica/metabolismo , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vimentina/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Exosomes secreted by cancer cells are important components in the tumor microenvironment, enabling cancer cells to communicate with each other and with noncancerous cells to play important roles in tumor progression and metastasis. Phenformin, a biguanide antidiabetic drug, has been reported to have a strong antitumor function in multiple types of cancer cells, however little research has been reported about whether phenformin can regulate the secretion of exosomes by cancer cells to regulate the tumor microenvironment and contribute to its antitumor function. Here we found that exosomes (Phen-Exo) derived from phenformin-treated oral squamous cell carcinoma (OSCC) cells significantly suppress the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. The inhibition of angiogenesis by Phen-Exo was verified in vivo by matrigel plug angiogenesis assays and by chick chorioallantoic membrane assays. Mechanistically, we discovered that the expression of microRNA-1246 (miR-1246) and microRNA-205 (miR-205) was significantly increased in exosomes secreted by OSCC cells treated with phenformin, while high expression levels of miR-1246 or miR-205 in vascular endothelial cells inhibited their angiogenic effects and decreased expression of the angiogenic factor VEGFA. In conclusion, these results reveal that phenformin can inhibit angiogenesis by regulating the levels of miR-1246 and miR-205 in exosomes secreted by OSCC cells, suggesting that phenformin has the potential to alter the tumor microenvironment to antagonize the growth of OSCCs, which provides a theoretical basis for developing new strategies to treat OSCCs in the future.
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The treatment of many skin inflammation diseases, such as psoriasis and atopic dermatitis, is still a challenge and inflammation plays important roles in multiple stages of skin tumor development, including initiation, promotion and metastasis. Phenformin, a biguanide drug, has been shown to play a more efficient anti-tumor function than another well-known biguanide drug, metformin, which has been reported to control the expression of pro-inflammatory cytokines; however, little is known about the effects of phenformin on skin inflammation. This study used a mouse acute inflammation model, ex vivo skin organ cultures and in vitro human primary keratinocyte cultures to demonstrate that phenformin can suppress acute skin inflammatory responses induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in vivo and significantly suppresses the pro-inflammatory cytokines IL-1ß, IL-6 and IL-8 in human primary keratinocytes in vitro. The suppression of pro-inflammatory cytokine expression by phenformin was not directly through regulation of the MAPK or NF-κB pathways, but by controlling the expression of c-Myc in human keratinocytes. We demonstrated that the overexpression of c-Myc can induce pro-inflammatory cytokine expression and counteract the suppressive effect of phenformin on cytokine expression in keratinocytes. In contrast, the down-regulation of c-Myc produces effects similar to phenformin, both in cytokine expression by keratinocytes in vitro and in skin inflammation in vivo. Finally, we showed that phenformin, as an AMPK activator, down-regulates the expression of c-Myc through regulation of the AMPK/mTOR pathways. In summary, phenformin inhibits the expression of pro-inflammatory cytokines in keratinocytes through the down-regulation of c-Myc expression to play an anti-inflammation function in the skin.
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Citocinas , Dermatite Atópica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Citocinas/metabolismo , Dermatite Atópica/metabolismo , Humanos , Inflamação/metabolismo , Queratinócitos/metabolismo , Camundongos , Fenformin/farmacologia , Fenformin/uso terapêuticoRESUMO
Mesenchymal stem cells (MSCs) within the periodontal ligament (PDL), termed periodontal ligament stem cells (PDLSCs), have a self-renewing capability and a multidirectional differentiation potential. The molecular mechanisms that regulate multidirectional differentiation, such as the osteogenic differentiation of PDLSCs, remain to be elucidated. Cullin 4B (CUL4B), which assembles the CUL4B-RING ubiquitin ligase (CRL4B) complex, is involved in regulating a variety of developmental and physiological processes including the skeletal development and stemness of cancer stem cells. However, nothing is known about the possible role of CUL4B in the osteogenic differentiation of PDLSCs. Here, we found that knockdown of CUL4B decreased the proliferation, migration, stemness and osteogenic differentiation ability of PDLSCs. Mechanistically, we demonstrate that CUL4B cooperates with the PRC2 complex to repress the expression of miR-320c and miR-372/373-3p, which results in the upregulation of RUNX2, a master transcription factor (TF) that regulates osteogenic differentiation. In brief, the present study reveals the role of CUL4B as a new regulator of osteogenic differentiation in PDLSCs.
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Physiological root resorption of deciduous teeth is a normal phenomenon. How the angiogenesis process is regulated to provide adequate levels of oxygen and nutrients in hypoxic conditions when the dental pulp tissue is reduced at the stage of root resorption is not fully understood. In this study, we designed hypoxic preconditioning (2%) to mimic the physiological conditions. We isolated exosomes from hypoxic-preconditioned SHED (Hypo-exos) cells and from normally cultured SHED cells (Norm-exos). We found that treatment with Hypo-exos significantly enhanced the growth, migration and tube formation of endothelial cells in vitro compared with Norm-exos. We also performed matrigel plug assays in vivo and higher expression of VEGF and higher number of lumenal structures that stained positive for CD31 were found in the Hypo-exos treated group. To understand the potential molecular mechanism responsible for the positive effects of Hypo-exos, we performed exosomal miRNA sequencing and validated that Hypo-exos transferred both let-7f-5p and miR-210-3p to promote the tube formation of endothelial cells. Further study revealed that those two miRNAs regulate angiogenesis via the let-7f-5p/AGO1/VEGF and/or miR-210-3p/ephrinA3 signal pathways. Finally, we found that the increased release of exosomes regulated by hypoxia treatment may be related to Rab27a. Taking these data together, the present study demonstrates that exosomes derived from hypoxic-preconditioned SHED cells promote angiogenesis by transferring let-7f-5p and miR-210-3p, which suggests that they can potentially be developed as a novel therapeutic approach for pro-angiogenic therapy in tissue regeneration engineering.
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HYPOTHESIS: The drug release efficiency of microneedle is usually slower than that of oral delivery or hypodermic injection, which severely restricts its widespread use. Herein, a Fe3O4-loaded photothermal microneedle (Fe3O4@MN) patch is developed for controlled drug delivery. Under near infrared (NIR) irradiation, the drug loaded on Fe3O4@MN can be quickly released, achieving an enhanced drug release efficiency. EXPERIMENTS: The mechanical property and characterization of Fe3O4@MN were systematically investigated, and the photothermal performance of Fe3O4@MN was also conducted. Moreover, the model-drug-releasing tests and doxycycline hydrochloride releasing tests were carried out to evaluate the drug release performance of Fe3O4@MN under NIR irradiation. FINDINGS: Fe3O4@MN has enough mechanical strength to pierce into skins, and the temperature of Fe3O4@MN patch could rapidly increase by 40 â in 1 min under NIR irradiation. In vitro experiment, the release rate of model drug in Fe3O4@MN reached â¼ 80 % in 20 min and the doxycycline hydrochloride release rate of Fe3O4@MN reached â¼ 70 % after 20 min of NIR irradiation, indicating the potential application of the synthesized microneedle patch for transdermal drug delivery. Further penetration test showed that the penetration depth of model drugs carried by Fe3O4@MN patch on the porcine skin under NIR irradiation was 150 - 200 µm longer than that of the patch without Fe3O4 nanoparticles.
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Doxiciclina , Óxidos , Animais , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Preparações Farmacêuticas , SuínosRESUMO
BACKGROUND: Anti-angiogenic therapy has been shown to be a promising strategy for anti-tumor treatment. Increasing evidence indicates that tumor angiogenesis is affected by exosomes that are secreted by mesenchymal stem cells (MSCs), but whether exosomes derived from MSCs suppress or promote angiogenesis remain paradoxical. The purpose of this study focused on understanding the potential role of exosomes derived from stem cells of human deciduous exfoliated teeth (SHED-Exos) in regulating angiogenesis and the underlying molecular mechanism. METHODS: Exosomes were isolated from supernatants of SHED cells using an exosome purification kit and were characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. Cell Counting Kit-8, flow cytometric assays, western blots, wound healing and transwell migration assays were performed to characterize the roles of SHED-Exos on cell proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs). The anti-angiogenic activity of SHED-Exos was assessed via a tube formation assay of endothelial cells and angiogenesis-related factors were analyzed by western blotting. In vivo, we used the chick chorioallantoic membrane (CAM) assay and an oral squamous cell carcinoma (OSCC) xenograft transplantation model with nude mice that received multi-point injections at three-day intervals to evaluate the effects on angiogenesis. Furthermore, the sequencing of microRNAs (miRNAs) in SHED-Exos was performed to investigate the underlying anti-angiogenic mechanism. RESULTS: The results showed that SHED-Exos inhibit cell proliferation and migration and induce apoptosis in HUVECs. SHED-Exos suppress the tube-like structure formation of HUVECs in vitro. SHED-Exos downregulate several angiogenesis-related factors, including VEGFA, MMP-9 and ANGPT1. In vivo, the chick CAM assay verified that treatment with SHED-Exos inhibits micro-vascular formation, and importantly, significantly reduces the micro-vascular formation of tumors generated from xenografted OSCC cells, which was associated with the inhibition of tumor growth in vivo. Mechanistically, our data suggested that SHED-Exos are enriched with miR-100-5p and miR-1246 and are transferred to endothelial cells, which results in decreased tube formation via the down-regulation of VEGFA expression. CONCLUSIONS: These results demonstrate that SHED-Exos inhibit angiogenesis in vitro and in vivo, which suggests that SHED-Exos could potentially serve as a novel and effective therapeutic approach for anti-angiogenic treatment.
Assuntos
Carcinoma de Células Escamosas , Exossomos , MicroRNAs , Neoplasias Bucais , Animais , Carcinoma de Células Escamosas/metabolismo , Proliferação de Células , Exossomos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Bucais/metabolismo , Células-Tronco/metabolismoRESUMO
The 3D bioprinting technologies have attracted increasing attention due to their flexibility in producing architecturally relevant tissue constructs. Here, a vertical embedded extrusion bioprinting strategy using uniaxial or coaxial nozzles is presented, which allows formation of vertical structures of homogeneous or heterogeneous properties. By adjusting the bioprinting parameters, the characteristics of the bioprinted vertical patterns can be precisely controlled. Using this strategy, two proof-of-concept applications in tissue biofabrication are demonstrated. Specifically, intestinal villi and hair follicles, two liner-shaped tissues in the human body, are successfully generated with the vertical embedded bioprinting method, reconstructing some of their key structures as well as restoring partial functions in vitro. Caco-2 cells in the bioprinted intestinal villus constructs proliferated and aggregated properly, also showing functional biomarker expressions such as ZO-1 and villin. Moreover, preliminary hair follicle structures featuring keratinized human keratinocytes and spheroid-shaped human dermal papilla cells are formed after vertical bioprinting and culturing. In summary, this vertical embedded extrusion bioprinting technique harnessing a uniaxial or coaxial format will likely bring further improvements in the reconstruction of certain human tissues and organs, especially those with a linear structure, potentially leading to wide utilities in tissue engineering, tissue model engineering, and drug discovery.