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Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a nonstochastic manner. Subsets of murine hematopoietic progenitors are privileged whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after four to five divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of â¼8 hr. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression and is increased by p53 knockdown. This ultrafast cycling population accounts for >99% of the bulk reprogramming activity in wild-type or p53 knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state and suggest that cell-cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PAPERCLIP:
Assuntos
Reprogramação Celular , Células Progenitoras de Granulócitos e Macrófagos/citologia , Células-Tronco Pluripotentes Induzidas , Animais , Células da Medula Óssea , Diferenciação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Genes p53 , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , CamundongosRESUMO
Prostate cancer is the second most common cancer in men worldwide1. Over the past decade, large-scale integrative genomics efforts have enhanced our understanding of this disease by characterizing its genetic and epigenetic landscape in thousands of patients2,3. However, most tumours profiled in these studies were obtained from patients from Western populations. Here we produced and analysed whole-genome, whole-transcriptome and DNA methylation data for 208 pairs of tumour tissue samples and matched healthy control tissue from Chinese patients with primary prostate cancer. Systematic comparison with published data from 2,554 prostate tumours revealed that the genomic alteration signatures in Chinese patients were markedly distinct from those of Western cohorts: specifically, 41% of tumours contained mutations in FOXA1 and 18% each had deletions in ZNF292 and CHD1. Alterations of the genome and epigenome were correlated and were predictive of disease phenotype and progression. Coding and noncoding mutations, as well as epimutations, converged on pathways that are important for prostate cancer, providing insights into this devastating disease. These discoveries underscore the importance of including population context in constructing comprehensive genomic maps for disease.
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Povo Asiático/genética , Epigênese Genética , Epigenômica , Genoma Humano/genética , Genômica , Mutação , Neoplasias da Próstata/classificação , Neoplasias da Próstata/genética , Proteínas de Transporte/genética , Transformação Celular Neoplásica/genética , China , Estudos de Coortes , DNA Helicases/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Neoplasias da Próstata/patologia , RNA-Seq , Transcriptoma/genéticaRESUMO
Inflammation is a beneficial host response to infection but can contribute to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases. These cells exhibit both instability (they can cease to express their signature cytokine, IL-17A) and plasticity (they can start expressing cytokines typical of other lineages) upon in vitro re-stimulation. However, technical limitations have prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as transdifferentiation. Furthermore, although Th17 cell instability/plasticity has been associated with pathogenicity, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate. Here we used two new fate-mapping mouse models to track Th17 cells during immune responses to show that CD4(+) T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a change in their signature transcriptional profile and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF-ß signalling and consequently for the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 cells transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest that Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases.
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Transdiferenciação Celular , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Células Th17/citologia , Células Th17/imunologia , Animais , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Helmintíase/imunologia , Masculino , Camundongos , Nippostrongylus/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologiaRESUMO
Zooxanthellae and coral can form an intracellular symbiotic system. Yet, little is known about the molecular mechanism underlying this symbiosis. In this study, we characterized the symbiosis based on analyses of gene expression at the single-cell level. Among 9110 single coral cells, we identified 4871 symbiotic cells based on the detection of both coral and zooxanthellae gene transcripts within a single cell. Using the bioinformatics tool Seurat, symbiotic cells were further clustered into five groups, 52 genes exhibited differential expression between groups. We proposed an index called the "symbiosis index", to indicate the degree of gene expression of both species in a single symbiotic cell. Interestingly, the index differed distinctly among the five groups. The symbiosis index was highly correlated with the expression of the coral gene gfas1.m1.6761 (ANKRD40), which encodes ankyrin repeat domain-containing protein 40 and is involved in DNA replication (r = 0.76). Two metabolism-related genes, DAGLA and betaGlu, were highly expressed in cells with a high symbiosis index. Four zooxanthellae genes, PRPF19, ATRN, aAA-ATPases and AK812-SmicGene44833, exhibited substantial changes in expression levels when zooxanthellae lived within coral. A trajectory analysis suggested that cells with a higher symbiosis index may be derived from those with a lower index during coral colony development. Taken together, our results provide evidence for zooxanthellae residing within coral, forming a symbiotic system. The symbiosis index is an effective indicator of different cell groups, with lineage relationships among groups. Additionally, we identified specific genes that exhibit expression changes in the symbiotic system.
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Antozoários/genética , Dinoflagellida/genética , Simbiose/genética , Animais , Antozoários/metabolismo , Análise por Conglomerados , Dinoflagellida/metabolismo , RNA-Seq , Análise de Célula ÚnicaRESUMO
Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population.
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Ilhas de CpG , Metilação de DNA , Epigênese Genética , Regiões Promotoras Genéticas , Análise de Célula Única/métodos , Linhagem Celular , Linhagem Celular Tumoral , Mapeamento Cromossômico , Enzimas de Restrição do DNA/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Variação Genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células K562 , Linfócitos/citologia , Linfócitos/metabolismoRESUMO
BACKGROUND/AIMS: Natural compounds are a promising resource for anti-tumor drugs. Myricetin, an abundant flavonoid found in the bark and leaves of bayberry, shows multiple promising anti-tumor functions in various cancers. METHODS: The cytotoxic, pro-apoptotic, and anti-metastatic effects of myricetin on prostate cancer cells were investigated in both in vitro and in vivo studies. Short-hairpin RNA knockdown of the proviral integration site for Moloney murine leukemia virus-1 (PIM1), pull-down and co-immunoprecipitation assays, and an intracellular Ca2+ flux assay were used to investigate the potential underlying mechanism of myricetin. ONCOMINE database data mining and immunohistochemical analysis of prostate cancer tissues were used to evaluate the expression of PIM1 and CXCR4, as well as the correlation between PIM1 and CXCR4 expression and the clinicopathologic characteristics and prognoses of prostate cancer patients. RESULTS: Myricetin exerted selective cytotoxic, pro-apoptotic, and anti-metastatic effects on prostate cancer cells by inhibiting PIM1 and disrupting the PIM1/CXCR4 interaction. Moreover, PIM1 and CXCR4 were coexpressed and associated with aggressive clinicopathologic traits and poor prognosis in prostate cancer patients. CONCLUSION: These results offer preclinical evidence for myricetin as a potential chemopreventive and therapeutic agent for precision medicine tailored to prostate cancer patients characterized by concomitant elevated expression of PIM1 and CXCR4.
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Antineoplásicos/uso terapêutico , Flavonoides/uso terapêutico , Invasividade Neoplásica/prevenção & controle , Neoplasias da Próstata/tratamento farmacológico , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Receptores CXCR4/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Flavonoides/farmacologia , Humanos , Masculino , Camundongos Nus , Invasividade Neoplásica/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidoresRESUMO
The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3' biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling.
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Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Mensageiro/genética , Análise de Célula Única/métodos , Primers do DNA/genética , Humanos , Células K562 , Reação em Cadeia da Polimerase/métodosRESUMO
Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury-induced donor hepatocyte selection: the uPA/SCID mice and Fah(-/-) Rag2(-/-)Il2rg(-/-) mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah(-/-)Rag2(-/-) mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah(-/-)Rag2(-/-) mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines.
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Hepatócitos/transplante , Terapia de Imunossupressão/métodos , Fígado/imunologia , Transplante Heterólogo , Animais , Células da Medula Óssea/imunologia , Hepatite B/imunologia , Hepatite B/metabolismo , Hepatite B/virologia , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/metabolismo , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Imuno-Histoquímica , Fígado/virologia , Camundongos , Camundongos TransgênicosRESUMO
Sorafenib is a multi-kinase blocker and one of the few suggested drug treatments for aggressive hepatocellular carcinoma (HCC) patients. However, drug resistance to sorafenib may often occur over time and cause further tumor aggression. Recently, cancer stem cells were found in HCC and were speculated to be involved in tumor progression. SOX9 is highly expressed in HCC cancer stem cells and promotes cell proliferation and self-renewal. Meanwhile, HCC patients with higher SOX9 expression show poorer prognosis. Whether SOX9 is involved in sorafenib resistance in HCC is still unclear. Here, we found that sorafenib treatment increased the proportion of SOX9 positive cells in HCC cell lines. Overexpression of exogenous SOX9 in HCC increased sorafenib resistance both in vitro and in vivo, whereas down-regulation led to inhibition of sorafenib resistance. Knock-down of SOX9 by RNA interference caused down-regulation of downstream genes, including ATP binding cassette subfamily G member 2 (ABCG2). The drug resistance to sorafenib caused by SOX9 overexpression could be ameliorated by ABCG2 inhibition in HCC cell lines. In the cohort of patients taken sorafenib, we found that patients with lower SOX9 expression had more prolonged overall survival (OS) and progression-free survival (PFS). Univariate and multivariate Cox analysis shows that SOX9 expression exerts as an independent risk factor for the OS and PFS of HCC patients with sorafenib treatment. These findings demonstrate that SOX9 enhances sorafenib resistance and may regulate this process by modulating ABCG2 expression.
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Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição SOX9/metabolismo , Sorafenibe/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição SOX9/genética , Transdução de Sinais , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Myoepithelial cells play key roles in normal mammary gland development and in limiting pre-invasive to invasive breast tumor progression, yet their differentiation and perturbation in ductal carcinoma in situ (DCIS) are poorly understood. Here, we investigated myoepithelial cells in normal breast tissues of BRCA1 and BRCA2 germline mutation carriers and in non-carrier controls, and in sporadic DCIS. We found that in the normal breast of non-carriers, myoepithelial cells frequently co-express the p63 and TCF7 transcription factors and that p63 and TCF7 show overlapping chromatin peaks associated with differentiated myoepithelium-specific genes. In contrast, in normal breast tissues of BRCA1 mutation carriers the frequency of p63+TCF7+ myoepithelial cells is significantly decreased and p63 and TCF7 chromatin peaks do not overlap. These myoepithelial perturbations in normal breast tissues of BRCA1 germline mutation carriers may play a role in their higher risk of breast cancer. The fraction of p63+TCF7+ myoepithelial cells is also significantly decreased in DCIS, which may be associated with invasive progression.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Carcinoma Ductal de Mama/metabolismo , Mutação/genética , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Ductal de Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Feminino , Imunofluorescência , Mutação em Linhagem Germinativa/genética , Humanos , Imuno-Histoquímica , Camundongos , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Induced hepatic stem cells (iHepSCs) have great potential as donors for liver cell therapy due to their self-renewal and bipotential differentiation properties. However, the efficiency of bidifferentiation and repopulation efficiency of iHepSCs is relatively low. Recent evidence shows that physiological hypoxia, a vital factor within stem cell "niche" microenvironment, plays key roles in regulating tissue stem cell biological behaviors including proliferation and differentiation. In this study, we found that physiological hypoxia (10% O2) enhanced the stemness properties and promoted the proliferation ability of iHepSCs by accelerating G1/S transition via p53-p21 signaling pathway. In addition, short-term hypoxia preconditioning improved the efficiency of hepatic differentiation of iHepSCs, and long-term hypoxia promoted cholangiocytic differentiation but inhibited hepatic differentiation of iHepSCs. These results demonstrated the potential effects of hypoxia on stemness preservation, proliferation, and bidifferentiation of iHepSCs and promising perspective to explore appropriate culture conditions for therapeutic stem cells.
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Hipóxia Celular/fisiologia , Fígado/citologia , Fígado/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , CamundongosRESUMO
PURPOSE: Liver cancer stem cells (CSCs) are known to be associated with the development, survival, proliferation, metastasis, and recurrence of liver tumors. The aim of this study was to investigate the association of liver-enriched activator protein 1 (LAP1) with hepatocellular carcinoma (HCC) and liver CSCs (LCSCs) and explore the impact of LAP1 on LCSCs. MATERIALS AND METHODS: Differences in LAP1 expression in liver cancer tissues versus matched para-tumoral liver tissues and LCSCs versus non-CSCs were analyzed by Western blotting, real-time polymerase chain reaction, immunohistochemistry, and flow cytometry. The effect of LAP1 on liver cancer cells was evaluated by the expression of CSC markers, oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle distribution and the number of apoptotic cells were analyzed to assess cell cycle and cell apoptosis. Furthermore, a mouse subcutaneous tumor implant model was established to explore the role of LAP1 in the development of HCC in vivo. Finally, the expression of CSC markers in paraffin-embedded sections was evaluated by immunofluorescence. RESULTS: LAP1 was weakly expressed in HCC tumors and cell lines and even weaker in LCSCs. LAP1 inhibited the expression of stem cell-associated genes and reduced the abilities of oncosphere formation, proliferation, migration, and invasion in vitro. Cell cycle assay revealed that LAP1 induced G1/G0 arrest. Furthermore, LAP1 decreased subcutaneous tumor-formation ability and the expression of CSC markers and Ki67 in vivo. CONCLUSION: LAP1 suppressed the stem cell features of HCC, indicating that it possessed an antitumor effect in liver cancer, both in vitro and in vivo; therefore, LAP1 may prove to be a potential target in liver CSC-targeted therapy.
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AIM: There is little knowledge about the expression profile and function of circular RNAs (circRNAs) in prostate cancer (PCa). METHODS: The expression profiles of circRNAs in RWPE-1, 22RV1 and PC3 cells were explored via high-throughput circRNAs sequencing and validated by real-time qPCR. The roles of differentially expressed circRNAs were evaluated by bioinformatics analyses. RESULTS: Altogether 9545 circRNAs were identified and hundreds of differentially expressed circRNAs were recognized. CircRNA-miRNA networks analysis showed that many circRNAs, including circSLC7A6, circGUCY1A2 and circZFP57 could cross-talk with tumor-related miRNAs such as miR-21, miR-143 and miR-200 family. CONCLUSION: The results of our bioinformatics analyses suggested that circRNAs should play critical roles in the development and progression of PCa.
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The prognosis of bladder urothelial carcinoma (BLCA) varies greatly even for patients with similar pathological characteristics. We conducted transcriptome sequencing on ten pairs of BLCA samples and adjacent normal tissues to identify differentially expressed genes. Anillin (ANLN) was identified as a transcript that was significantly up-regulated in BLCA samples compared with normal tissues. Prognostic power of candidate gene was studied using qRT-PCR and immunohistochemistry on 40 and 209 patients, respectively. Patients with elevated ANLN expression level was correlated with poorer cancer-specific (median, 22.4 vs. 37.3 months, p = 0.001), progression-free (median, 19.7 vs. 27.9 months, p = 0.001) and recurrence-free survival (median, 17.1 vs. 25.2 months, p = 0.011) compared with low ANLN expression. Public datasets TCGA and NCBI-GEO were analyzed for external validation. Knockdown of ANLN in J82 and 5637 cells using small interfering RNA significantly inhibited cell proliferation, migration, and invasion ability. Moreover, knockdown of ANLN resulted in G2/M phase arrest and decreased expression of cyclin B1 and D1. Microarray analysis suggested that ANLN played a major role in cell migration and was closely associated with several cancer-related signaling pathways. In conclusion, ANLN was identified as a promising prognostic biomarker which could be used to stratify different risks of BLCA.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma in Situ/diagnóstico , Regulação Neoplásica da Expressão Gênica , Proteínas dos Microfilamentos/genética , Recidiva Local de Neoplasia/diagnóstico , Transcriptoma , Neoplasias da Bexiga Urinária/diagnóstico , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma in Situ/genética , Carcinoma in Situ/mortalidade , Carcinoma in Situ/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Conjuntos de Dados como Assunto , Pontos de Checagem da Fase G2 do Ciclo Celular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Análise em Microsséries , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/metabolismo , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Prognóstico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Análise de Sobrevida , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To investigate immune escape during breast tumor progression, we analyzed the composition of leukocytes in normal breast tissues, ductal carcinoma in situ (DCIS), and invasive ductal carcinomas (IDC). We found significant tissue and tumor subtype-specific differences in multiple cell types including T cells and neutrophils. Gene expression profiling of CD45+CD3+ T cells demonstrated a decrease in CD8+ signatures in IDCs. Immunofluorescence analysis showed fewer activated GZMB+CD8+ T cells in IDC than in DCIS, including in matched DCIS and recurrent IDC. T-cell receptor clonotype diversity was significantly higher in DCIS than in IDCs. Immune checkpoint protein TIGIT-expressing T cells were more frequent in DCIS, whereas high PD-L1 expression and amplification of CD274 (encoding PD-L1) was only detected in triple-negative IDCs. Coamplification of a 17q12 chemokine cluster with ERBB2 subdivided HER2+ breast tumors into immunologically and clinically distinct subtypes. Our results show coevolution of cancer cells and the immune microenvironment during tumor progression.Significance: The design of effective cancer immunotherapies requires the understanding of mechanisms underlying immune escape during tumor progression. Here we demonstrate a switch to a less active tumor immune environment during the in situ to invasive breast carcinoma transition, and identify immune regulators and genomic alterations that shape tumor evolution. Cancer Discov; 7(10); 1098-115. ©2017 AACR.See related commentary by Speiser and Verdeil, p. 1062This article is highlighted in the In This Issue feature, p. 1047.
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Neoplasias da Mama/imunologia , Carcinoma Ductal de Mama/imunologia , Carcinoma Intraductal não Infiltrante/imunologia , Perfilação da Expressão Gênica/métodos , Linfócitos T/imunologia , Antígeno B7-H1/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Complexo CD3/genética , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Antígenos Comuns de Leucócito/genética , Receptor ErbB-2/genética , Microambiente TumoralRESUMO
Identification of the cellular origin of primary liver cancer remains challenging. Some data point toward liver stem cells (LSCs) or liver progenitor cells (LPCs) not only as propagators of liver regeneration, but also as initiators of liver cancer. LSCs exhibit a long lifespan and strong duplicative potential upon activation and are inclined to accumulate more mutations that can be passed down to the next generations. Recent evidence shows that dysregulation of signaling pathways associated with self-renewal of LSCs can drive their aberrant proliferation and even malignant transformation. If LSCs could be proved to be an initiator of liver carcinogenesis, they would be promising for ultra-early diagnosis and targeting therapy of liver cancer. This review mainly summarizes the potential role of LSCs in the carcinogenesis of primary liver cancer.
Assuntos
Transformação Celular Neoplásica/patologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/patologia , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transdução de SinaisRESUMO
@#[摘 要] 单细胞测序技术是指以单个细胞为单位,通过高通量测序技术全方位分析单个细胞内的遗传信息,其已成为现代生物学深度研究的一种强有力工具,使生物医药领域研究和临床应用转化研究达到了传统测序方法难以实现的极高精确度和灵敏度。目前,单细胞转录组测序(又称“单细胞RNA测序”,scRNA-seq)已成为最广泛应用的单细胞测序技术。简要介绍scRNA-seq技术的发展历程、技术原理和方法,系统阐述其应用在肿瘤异质性、肿瘤转移、肿瘤免疫、肿瘤耐药等基础领域的研究成果及其在肿瘤精准医疗的临床转化与应用中的研究进展,单细胞测序技术的发展和应用必将推动肿瘤精准医疗迈向更高水平。
RESUMO
AIM: To establish a mice model harboring hepatitis B virus x gene (adr subtype) for studying the function of hepatitis B virus X protein, a transactivator of viral and cellular promoter/enhancer elements. METHODS: Expression vector pcDNA3-HBx, containing CMV promoter and hepatitis B virus x gene open reading fragment, was constructed by recombination DNA technique. Hela cells were cultured in DMEM and transfected with pcDNA3-HBx or control pcDNA3 plasmids using FuGENE6 Transfection Reagent. Expression of pcDNA3-HBx vectors in the transfected Hela cells was confirmed by Western blotting. After restriction endonuclease digestion, the coding elements were microinjected into male pronuclei of mice zygotes. The pups were evaluated by multiplex polymerase chain reaction (PCR) at genomic DNA level. The x gene transgenic mice founders were confirmed at protein level by Western blotting, immunohistochemistry and immunogold transmission electron microscopy. RESULTS: Expression vector pcDNA3-HBx was constructed by recombination DNA technique and identified right by restriction endonuclease digestion and DNA direct sequencing. With Western blotting, hepatitis X protein was detected in Hela cells transfected with pcDNA3-HBx plasmids, suggesting pcDNA3-HBx plasmids could express in eukaryotic cells. Following microinjection of coding sequence of pcDNA3-HBx, the embryos were transferred to oviducts of pseudopregnant females. Four pups were born and survived. Two of them were verified to have the HBx gene integrated in their genomic DNA by multiplex PCR assay, and named C57-TgN(HBx)SMMU1 and C57-TgN(HBx)SMMU3 respectively. They expressed 17KD X protein in liver tissue by Western blotting assay. With the immunohistochemistry, X protein was detected mainly in hepatocytes cytoplasm of transgenic mice, which was furthermore confirmed by immunogold transmission electon microscopy. CONCLUSION: We have constructed the expression vector pcDNA3-HBx that can be used to study the function of HBx gene in eukaryotic cells in vitro. We also established HBx gene (adr subtype) transgenic mice named C57-TgN (HBx)SMMU harboring HBx gene in their genome and express X protein in hepatocytes, Which might be a valuable animal system for studying the roles of HBx gene in hepatitis B virus life cycle and development of hepatocellular carcinoma in vivo.
Assuntos
Antígenos da Hepatite B/metabolismo , Transativadores/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Regulação Viral da Expressão Gênica , Células HeLa , Hepatite B/genética , Antígenos da Hepatite B/genética , Humanos , Fígado/ultraestrutura , Fígado/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transativadores/genética , Proteínas Virais Reguladoras e AcessóriasRESUMO
Despite the recent advance of single-cell gene expression analyses, co-measurement of both genomic and transcriptional signatures at the single-cell level has not been realized. However such analysis is necessary in order to accurately delineate how genetic information is transcribed, expressed, and regulated to give rise to an enormously diverse range of cell phenotypes. Here we report on a microfluidics-facilitated approach that allows for controlled separation of cytoplasmic and nuclear contents of a single cell followed by on-chip amplification of genomic DNA and cytoplasmic mRNA. When coupled with off-chip polymerase chain reaction, gel electrophoresis and Sanger sequencing, a panel of genes and transcripts from the same single cell can be co-detected and sequenced. This platform is potentially an enabling tool to permit multiple genomic measurements performed on the same single cells and opens new opportunities to tackle a range of fundamental biology questions including non-genetic cell-to-cell variability, epigenetic regulation, and stem cell fate control. It also helps address clinical challenges such as diagnosing intra-tumor heterogeneity and dissecting complex cellular immune responses.
Assuntos
Epigênese Genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Microfluídica , Análise de Célula Única , Linhagem Celular Tumoral , DNA/genética , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , RNA Mensageiro/biossínteseRESUMO
Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1ß and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.