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Treating Multiple sclerosis (MS), a well-known immune-mediated disease characterized by axonal demyelination, is challenging due to its complex causes. Naphthalenedione, present in numerous plants, is being explored as a potential medicine for MS due to its immunomodulatory properties. However, its effects on lymphocytes can vary depending on factors such as the specific compound, concentration, and experimental conditions. In this study, we aim to explore the therapeutic potential of 2-bromo-1,4-naphthalenedione (BrQ), a derivative of naphthalenedione, in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and to elucidate its underlying mechanisms. We observed that mice treated with BrQ exhibited reduced severity of EAE symptoms, including lower clinical scores, decreased leukocyte infiltration, and less extensive demyelination in central nervous system. Furthermore, it was noted that BrQ does not directly affect the remyelination process. Through cell-chat analysis based on bulk RNA-seq data, coupled with validation of flow analysis, we discovered that BrQ significantly promotes the expansion of CD8+ T cells and their interactions with other immune cells in peripheral immune system in EAE mice. Subsequent CD8+ T cell depletion experiments confirmed that BrQ alleviates EAE in a CD8+ T cell-dependent manner. Mechanistically, expanded CD8+ cells were found to selectively reduce antigen-specific CD4+ cells and subsequently inhibit Th1 and Th17 cell development in vivo, ultimately leading to relief from EAE. In summary, our findings highlight the crucial role of BrQ in modulating the pathogenesis of MS, suggesting its potential as a novel drug candidate for treating MS and other autoimmune diseases.
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Linfócitos T CD8-Positivos , Encefalomielite Autoimune Experimental , Camundongos Endogâmicos C57BL , Células Th1 , Células Th17 , Animais , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Camundongos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Feminino , Naftalenos/farmacologia , Naftalenos/uso terapêutico , Proliferação de Células/efeitos dos fármacosRESUMO
Endogenous retroviruses (ERVs) are frequently reactivated in mammalian placenta. It has been proposed that ERVs contribute to shaping the gene regulatory network of mammalian trophoblasts, dominantly acting as species- and placental-specific enhancers. However, whether and how ERVs control human trophoblast development through alternative pathways remains poorly understood. Besides the well-recognized function of human endogenous retrovirus-H (HERVH) in maintaining pluripotency of early human epiblast, here we present a unique role of HERVH on trophoblast lineage development. We found that the LTR7C/HERVH subfamily exhibits an accessible chromatin state in the human trophoblast lineage. Particularly, the LTR7C/HERVH-derived Urothelial Cancer Associated 1 (UCA1), a primate-specific long non-coding RNA (lncRNA), is transcribed in human trophoblasts and promotes the proliferation of human trophoblast stem cells (hTSCs), whereas its ectopic expression compromises human trophoblast syncytialization coinciding with increased interferon signaling pathway. Importantly, UCA1 upregulation is detectable in placental samples from early-onset preeclampsia (EO-PE) patients and the transcriptome of EO-PE placenta exhibits considerable similarities to that of the syncytiotrophoblasts differentiated from UCA1-overexpressing hTSCs, supporting up-regulated UCA1 as a potential biomarker of this disease. Altogether, our data shed light on the versatile regulatory role of HERVH in early human development and provide a unique mechanism whereby ERVs exert a function in human placentation and placental syndromes.
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Retrovirus Endógenos , RNA Longo não Codificante , Animais , Humanos , Gravidez , Feminino , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Placenta/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Trofoblastos/metabolismo , Placentação , Primatas/genética , Mamíferos/genéticaRESUMO
BACKGROUND: Helicobacter pylori (H. pylori) causes chronic gastric disease. An efficient oral vaccine would be mucosa-targeted and offer defense against colonization of invasive infection in the digestive system. Proteolytic enzymes and acidic environment in the gastrointestinal tract (GT) can, however, reduce the effectiveness of oral vaccinations. For the creation of an edible vaccine, L. lactis has been proposed as a means of delivering vaccine antigens. RESULTS: We developed a plSAM (pNZ8148-SAM) that expresses a multiepitope vaccine antigen SAM-WAE containing Urease, HpaA, HSP60, and NAP extracellularly (named LL-plSAM-WAE) to increase the efficacy of oral vaccinations. We then investigated the immunogenicity of LL-plSAM-WAE in Balb/c mice. Mice that received LL-plSAM-WAE or SAM-WAE with adjuvant showed increased levels of antibodies against H. pylori, including IgG and sIgA, and resulted in significant reductions in H. pylori colonization. Furthermore, we show that SAM-WAE and LL-plSAM-WAE improved the capacity to target the vaccine to M cells. CONCLUSIONS: These findings suggest that recombinant L. lactis could be a promising oral mucosa vaccination for preventing H. pylori infection.
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Helicobacter pylori , Animais , Camundongos , Imunidade nas Mucosas , Fatores de Virulência , Vacinas Bacterianas , Urease , Vacinas Sintéticas , Camundongos Endogâmicos BALB C , Administração OralRESUMO
Tuberculosis is a worldwide contagion caused by Mycobacterium tuberculosis (MTB). MTB is characterized by intracellular parasitism and is semi-dormant inside host cells. The persistent inflammation caused by MTB can form a granuloma in lesion regions and intensify the latency of bacteria. In recent years, several studies have proven that long non-coding RNAs (lncRNAs) play critical roles in modulating autophagy. In our study, the Gene Expression Omnibus (GEO) databases were searched for lncRNAs that are associated with tuberculosis. We found that lncRNA differentiation antagonizing non-protein coding RNA (DANCR) increased in the peripheral blood samples collected from 54 pulmonary tuberculosis patients compared to 23 healthy donors. By constructing DANCR overexpression cells, we analyzed the possible cellular function of DANCR. After analyzing our experiments, it was found that the data revealed that upregulation of DANCR facilitated the expression of signal transducer and activator of transcription 3, autophagy-related 4D cysteine peptides, autophagy-related 5, Ras homolog enriched in the brain, and microtubule-associated protein 1A/1B light chain 3 (STAT3, ATG4D, ATG5, RHEB, and LC3, respectively) by sponging miR-1301-3p and miR-5194. Immunofluorescence analysis indicated that DANCR played a positive role in both autophagosome formation and fusion of autolysosomes in macrophages. The colony-forming unit (CFU) assay data also showed that the cells overexpressing DANCR were more efficient in eliminating the intracellular H37Ra strain. Consequently, these data suggest that DANCR restrained intracellular survival of M. tuberculosis by promoting autophagy via miR-1301-3p and miR-5194.
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Purpose: Due to the natural advantages of spermidine in immunity, we investigated the effects of spermidine pretreatment on nanobody-based CAR-T cells (Nb CAR-T) mediated cytotoxicity and potential mechanism. Patients and Methods: The optimal concentration of spermidine was determined by detecting its impact on viability and proliferation of T cells. The phenotypic characteristic of CAR-T cells, which were treated with spermidine for 4 days, was examined by flow cytometry. The expansion ability of CAR-T cells was monitored in being cocultured with tumor cells. Additionally, CAR-T cells were stimulated by lymphoma cells to test its cytotoxicity in vitro, and the supernatant in co-culture models were collected to test the cytokine production. Furthermore, xenograft models were constructed to detect the anti-tumor activity of CAR-T cells in vivo. Results: The optimal concentration of spermidine acting on T cells was 5µM. The antigen-dependent proliferation of spermidine pretreatment CD19 CAR-T cells or Nb CAR-T cells was increased compared to control. Central memory T cells(TCM) dominated the CAR-T cell population in the presence of spermidine. When spermidine pretreatment CAR-T cells were stimulated with Daudi cells, the secretion of IL-2 and IFN-γ has been significantly enhanced. The ability of CAR-T cells to lysis Daudi cells was enhanced with the help of spermidine, even at higher tumor loads. Pre-treated Nb CAR-T cells with spermidine were able to control tumor cells in vivo, and therefore prolong mice survival. Conclusion: Our results revealed that spermidine could promote Nb CAR-T mediated cytotoxicity to lymphomas cells through enhancing memory and proliferation, and provided a meaningful approach to strengthen the anti-tumor effect of CAR-T cells.
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In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An in vitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development.
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Regulação da Expressão Gênica no Desenvolvimento , Zigoto , Blastocisto , Desenvolvimento Embrionário/genética , Genoma , HumanosRESUMO
Background: Circular RNAs (circRNAs) have been discovered in colorectal cancer (CRC), but there are few reports on the expression distribution and functional mining analysis of circRNAs. Methods: Differentially expressed circRNAs in CRC tissues and adjacent normal tissues were screened and identified by microarray and qRT-PCR. ROC curves of the six circRNAs were analyzed. A series of bioinformatics analyses on differentially expressed circRNAs were performed. Results: A total of 207 up-regulated and 357 down-regulated circRNAs in CRC were screened, and three top up-regulated and down-regulated circRNAs were chosen to be verified in 33 pairs of CRCs by qRT-PCR. 6 circRNAs showed high diagnostic values (AUC = 0.6860, AUC = 0.8127, AUC = 0.7502, AUC = 0.9945, AUC = 0.9642, AUC = 0.9486 for hsa_circRNA_100833, hsa_circRNA_103828, hsa_circRNA_103831 and hsa_circRNA_103752, hsa_circRNA_071106, hsa_circRNA_102293). A circRNA-miRNA-mRNA regulatory network (cirReNET) including six candidate circRNAs, 19 miRNAs and 210 mRNA was constructed, and the functions of the cirReNET were predicted and displayed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on these mRNAs and protein-protein interaction (PPI) network of the hub genes acquired by string and CytoHubba. Conclusion: A cirReNET containing potential diagnostic and predictive indicators of CRCs and several critical circRNA-miRNA-mRNA regulatory axes (cirReAXEs) in CRC were mined, and may provide a novel route to study the mechanism and clinical targets of CRC.
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Biomarcadores Tumorais , Neoplasias Colorretais , MicroRNAs , RNA Circular , Humanos , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , MicroRNAs/análise , MicroRNAs/genética , Mapas de Interação de Proteínas , RNA Circular/análise , RNA Circular/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genéticaRESUMO
BACKGROUND: Mycobacterium tuberculosis (M. tuberculosis), which parasitizes host macrophages and lead to cellular immunologic responses, such as autophagy and apoptosis. Several studies had indicated that autophagy played important roles in alleviating intracellular survival of M. tuberculosis by accelerating the maturation of phagosome. Previously, we found miR-142-3p was significantly down-regulated in the macrophages after infection with M. tuberculosis. However, the role of miR-142-3p in the regulation of autophagy and M. tuberculosis survival is elusive. METHODS: Bioinformatics analysis was used to obtain target genes of miR-142-3p; the binding sites of ATG16L1 and ATG4c were further confirmed with dual luciferase reporter assay; RAW264.7 cells were infected with H37Ra and the expression of miR-142-3p was measured by qRT-PCR; the autophagic marker protein was detected by western blot as well as immunofluorescence microscopy and transmission electron microscopes analysis. RESULTS: Overexpression of miR-142-3p significantly inhibited H37Ra-induced activation of autophagy, blocked the maturation of phagosome in macrophages and promoted M. tuberculosis survival in macrophages. Furthermore, miR-142-3p negatively-regulated expression of ATG16L1 and ATG4c by directly targeting its 3'-UTR, and meaningfully abated the level of autophagy. CONCLUSION: These findings suggested that miR-142-3p inhibited M. tuberculosis-induced activation of autophagy and promoted H37Ra survival in RAW264.7 cells by targeting ATG16L1 and ATG4c.
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Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/metabolismo , MicroRNAs/metabolismo , Mycobacterium tuberculosis , Tuberculose/imunologia , Animais , Autofagia , Western Blotting , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência , Células RAW 264.7 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tuberculose/metabolismoRESUMO
Colorectal cancer (CRC) is one of the most malignant cancers with high morbidity and mortality. MicroRNAs (miRNAs) are small non-coding RNAs that affect biological processes by binding to mRNAs and regulating their expression, and epigenetic alterations including miRNA dysregulation are significantly involved in CRC development. Determining the effect of the miRNA-mRNA network on CRC could be helpful for developing novel therapeutic targets and prognostic biomarkers, and even improving survival. In this study, microarray assays were used to screen differentially expressed miRNAs (DE miRNAs) and mRNAs (DE mRNAs) in CRC and the adjacent normal tissues. Among the detected genes, 42 miRNAs and 142 mRNAs were significantly upregulated in CRC, while 23 miRNAs and 279 mRNAs were significantly downregulated. Through overlapping of predicted targets of DE miRNAs and anti-expressed DE mRNAs, networks of DE miRNAs and DE mRNAs in CRC were established. Additionally, the formation of a protein-protein interaction network of DE mRNAs possibly targeted by DE miRNAs, functional annotation and pathway analysis, stable subnetwork mining, and determination of hub genes provided the probable mechanism used by DE miRNAs and DE mRNAs to regulate CRC growth. Finally, validation of expression and prognostic potential of hub genes provided further support for the results above and indicated that CCL-28, GPR15, PNOC, NUSAP1, and their interacted miRNAs may be a potential signature for prognosis of CRC patients. In sum, we successfully established miRNA-mRNA regulatory networks based on microarray results targeting CRC, and these findings may elucidate the mechanisms used for CRC growth and identify miRNA-related signatures for prognosis and treatment of CRC.
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Objective To optimize the culture system of chimeric antigen receptor T (CAR-T) cells in vitro and lentivirus infection conditions. Methods Peripheral blood mononuclear cells (PBMCs) of healthy people and umbilical cord blood mononuclear cells (UCBMCs) of healthy pregnant women were isolated and purified by CD3 magnetic beads, and then they were cultured in different cell culture systems. There were eight cell culture systems containg different combinations of the following components: recombinant human interleukin 2 (rhIL-2), rhIL-12, rhIL-18, rhIL-7, rhIL-21, TWS119. Cell proliferation was detected by counting the cells at 0, 3, 5, 7, 10, 18 days after the cells were seeded into cell plates. Flow cytometry was used to detect the expression of programmed death 1 (PD-1), and ELISA was used to detect the expression of interferon-γ (IFN-γ). Cell culturing plates were coated with serial concentrations of recombinant human fibronectin fragment (RetroNectinr) (0, 20, 50 µg/mL), and antibodies against human CD3/CD28 (250, 500, 1 000 ng/mL). Then T cells cultured in the above plates were infected with negative control lentivirus at different multiplicity of infection (MOI=3, 5); 72 hours later, expression of green fluorescent protein (GFP) was observed under a fluorescence microscope to preliminarily determine virus infection efficiency. Flow cytometry was used to detect CD3/GFP positive rate to obtain lentivirus infection conditions. CD19 CAR lentivirus was packaged. Real-time quantitative PCR and Western blotting were performed to detect whether the CD19 CAR vector was successfully constructed. Finally, T cells were cultured in 1 µg/mL anti-human CD3/CD28 and 20 µg/mL RetroNectinr-coated culture plates, and rhIL-2, rhIL-12, rhIL-18 were added in the culture medium, then the cells were infected with CD19 CAR lentivirus at the optimized virus infection conditions. Results The cell culture system with the best proliferation ability was rhIL-2 combining with rhIL-18; the cell culture system with the strongest release of IFN-γ was rhIL-2 and rhIL-12 combined with rhIL-18. When the dose of antibodies against CD3/CD28 was 1 µg/mL, RetroNectinr was 20 µg/mL, and MOI was 3, the virus infection efficiency was optimal. The positive rate of CAR-T cells was 34% under the optimal condition. Conclusion The study achieved the optimal cell culture system of CD19 CAR-T cells in vitro and the conditions of lentivirus infection on primary T cells.
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Lentivirus , Antígenos CD19 , Feminino , Humanos , Lentivirus/genética , Leucócitos Mononucleares , Gravidez , Receptores de Antígenos Quiméricos , Linfócitos T/imunologiaRESUMO
Long noncoding RNAs (lncRNAs) are a class of functional non-coding transcripts that are longer than 200 nt and regulate gene expression via diverse mechanisms in eukaryotes. In fact, they have emerged as critical epigenetic and transcriptional regulators of autophagy in mammals in response to various stressors. Autophagy not only plays a crucial role in maintaining cellular homeostasis, but it is also essential to immunity, targets intracellular pathogens for degradation, modulates inflammation, and participates in adaptive immune responses. However, the expression profile of lncRNA and its role in regulating autophagy in macrophages have been poorly defined. Here, we used transcriptomic and bioinformatics to analysis LncRNA expression profile during autophagy and functional studies to evaluate the function of the metastasis-associated lung adenocarcinoma transcript-1 (Malat1) lncRNA in macrophages. A total of 1112 putative lncRNAs (240 novel lncRNAs) were identified, including 831 large intergenic, 129 intronic, and 152 anti-sense lncRNA, of which 59 differentially expressed transcripts exhibited a greater than 1.5-fold change under different conditions. The interaction of Malat1 lncRNA with microRNA (mir)-23-3p and lysosomal-associated membrane protein 1 (Lamp1) was found, Malat1 releases inhibition of Lamp1 expression in macrophages through competitive adsorption of mir-23-3p. The results of this study provide a better understanding of lncRNA function in macrophages and a basis for further investigation into the roles and mechanisms of ncRNA in immunology, particularly the functions of Malat1 and mir-23-3p in the pathogenesis of macrophages.
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Autofagia/genética , Proteínas de Membrana Lisossomal/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Biologia Computacional , Camundongos , MicroRNAs/genética , Células RAW 264.7 , RNA-SeqRESUMO
Autophagy is crucial for immune defense against Mycobacterium tuberculosis (Mtb) infection. Mtb can evade host immune attack and survival within macrophages by manipulating the autophagic process. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in regulating vital genes during Mtb infection. The precise role of miRNAs in autophagy with the exits of Mtb remains largely unknown. In this study, we found miR-1958, a new miRNA that could regulate autophagy by interacting with 3'UTR of autophagy-related gene 5 (Atg5). In addition, Mtb infection triggered miR-1958 expression in RAW264.7 cells. What's more, miR- 1958 overexpression blocked autophagic flux by impairing the fusion of autophagosomes and lysosomes. Overexpression of miR-1958 reduced Atg5 expression and LC3 puncta while inhibition of miR-1958 brought an increase of Atg5 and LC3 puncta; the opposite results were observed in detection of p62. The survival of Mtb in RAW264.7 cells transfected with mimic of miR-1958 was enhanced. Taken together, our research demonstrated that a novel miR-1958 could inhibit autophagy by interacting with Atg5 and favored intracellular Mtb survival in RAW264.7 cells.
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Proteína 5 Relacionada à Autofagia/genética , Autofagia/fisiologia , MicroRNAs/metabolismo , Viabilidade Microbiana/genética , Mycobacterium tuberculosis/fisiologia , Regiões 3' não Traduzidas , Animais , Autofagossomos/metabolismo , Expressão Gênica , Evasão da Resposta Imune , Lisossomos/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , MicroRNAs/genética , Mycobacterium tuberculosis/imunologia , Células RAW 264.7RESUMO
Autophagy plays an important role in the fight against Mycobacterium tuberculosis infection. Massive researches proved that miRNAs could be the regulators of autophagy, which implied miRNAs could favor MTB invasion or latent infection. In our study, multiple bioinformatics databases and software were used to seek and lock the miRNAs associating with regulation of autophagy. Notably, a novel miR-129-3p was found and its target gene Atg4b showed grand potential in mediation of autophagy. Moreover, BCG infection triggered miR-129-3p overexpression in RAW264.7 cells. Up-regulation of miR-129-3p decreased mRNA or protein level of Atg4b and resulted in the inhibition of autophagy. The antagomir of miR-129-3p had the opposite impact. The LC3 puncta formation in RAW264.7 cells were also affected after transfection of miR-129-3p mimic or antagomir. The mRFP-GFP-LC3 analysis indicated that mimic of miR-129-3p impaired autophagic flux while antagomir improved autophagy. The CFU assay results showed that miR-129-3p promoted the intracellular survival of BCG in macrophages. Consequently, these data suggested that miR-129-3p could favor MTB survival by inhibiting autophagy via Atg4b.
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Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Cisteína Endopeptidases/metabolismo , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Animais , Proteínas Relacionadas à Autofagia/genética , Vacina BCG/uso terapêutico , Biologia Computacional/métodos , Cisteína Endopeptidases/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , MicroRNAs/metabolismo , Mycobacterium tuberculosis/patogenicidade , Fagossomos/metabolismo , Células RAW 264.7 , RNA Mensageiro/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/genética , Tuberculose/prevenção & controleRESUMO
Hepatocelluar carcinoma (HCC) is one of the most frequently diagnosed cancers worldwide and among the leading causes of cancer-related death. Although deregulation of microRNAs has been frequently described in HCC, imperfection is known about the precise molecular mechanisms by which microRNAs modulate the process of tumorogenesis and behavior of cancer cells. In this study, we demonstrated that miR-758-3p could suppress cell proliferation, migration and invasion in hepatocellular carcinoma cells. We screened and identified two novel miR-758-3p targets, MDM2 and mTOR. Up-regulation of miR-758-3p could specifically and markedly down-regulate the expression of MDM2 and mTOR. Additionally, miR-758-3p over-expression displayed significant suppression in HCC development. To identify the mechanisms, we further investigated the P53 and mTOR pathway and found that p-p70S6 kinase(Ser371), p-p70 S6 kinase(Thr389) and p-4E-BP1were dramatically down-regulated after miR-758-3p transfection, while an enhanced expression of P53, AKT and PRAS40 were visualized, thus suggesting that the role of miR-758-3p in HCC progression may be associated with MDM2-p53 and mTOR signaling pathways. Collectively, our results indicate that miR-758-3pserves as a tumor suppressor and plays a crucial role in inhibiting the proliferation, migration and invasion of HCC via targeting MDM2 and mTOR and implicate its potential application in cancer therapy.
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Carcinoma Hepatocelular/metabolismo , Proliferação de Células/fisiologia , Neoplasias Hepáticas/metabolismo , MicroRNAs/biossíntese , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adulto , Carcinoma Hepatocelular/patologia , Movimento Celular/fisiologia , Feminino , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
Autophagy plays important roles in the host immune response against mycobacterial infection. Mycobacterium tuberculosis (M. tuberculosis) can live in macrophages owing to its ability to evade attacks by regulating autophagic response. MicroRNAs (miRNAs) are small noncoding, endogenously encoded RNA which plays critical roles in precise regulation of macrophage functions. Whether miRNAs specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that BCG infection of macrophages resulted in enhanced expression of miRNA-20a, which inhibits autophagic process by targeting ATG7 and ATG16L1 and promotes BCG survival in macrophages. Forced overexpression of miR-20a decreased the expression levels of LC3-II and the number of LC3 puncta in macrophages, and promoted BCG survival in macrophages, while transfection with miR-20a inhibitor had the opposite effect. Moreover, the inhibitory effect of miR-20a on autophagy was further confirmed by transmission electron microscopy (TEM) analysis. Quantification of autophagosomes per cellular cross-section revealed a significant reduction upon transfection with miR-20a mimic, but transfection with miR-20a inhibitor increased the number of autophagosomes per cellular cross-section. Moreover, silencing of ATG7 significantly inhibited autophagic response, and transfection with ATG7 siRNA plus miR-20a mimic could further decrease autophagic response. Collectively, our data reveal that miR-20a inhibits autophagic response and promotes BCG survival in macrophages by targeting ATG7 and ATG16L1, which may have implications for a better understanding of pathogenesis of M. tuberculosis infection.
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Proteína 7 Relacionada à Autofagia/antagonistas & inibidores , Autofagia , Proteínas de Transporte/antagonistas & inibidores , Evasão da Resposta Imune , Macrófagos/microbiologia , MicroRNAs/metabolismo , Mycobacterium bovis/patogenicidade , Animais , Proteínas Relacionadas à Autofagia , Macrófagos/imunologia , Camundongos , Viabilidade Microbiana , Microscopia Eletrônica de Transmissão , Mycobacterium bovis/fisiologia , Células RAW 264.7RESUMO
De novo root organogenesis is the process in which adventitious roots regenerate from detached or wounded plant tissues or organs. In tissue culture, appropriate types and concentrations of plant hormones in the medium are critical for inducing adventitious roots. However, in natural conditions, regeneration from detached organs is likely to rely on endogenous hormones. To investigate the actions of endogenous hormones and the molecular mechanisms guiding de novo root organogenesis, we developed a simple method to imitate natural conditions for adventitious root formation by culturing Arabidopsis thaliana leaf explants on B5 medium without additive hormones. Here we show that the ability of the leaf explants to regenerate roots depends on the age of the leaf and on certain nutrients in the medium. Based on these observations, we provide examples of how this method can be used in different situations, and how it can be optimized. This simple method could be used to investigate the effects of various physiological and molecular changes on the regeneration of adventitious roots. It is also useful for tracing cell lineage during the regeneration process by differential interference contrast observation of ß-glucuronidase staining, and by live imaging of proteins labeled with fluorescent tags.