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1.
Nat Commun ; 12(1): 5090, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429421

RESUMO

CRISPR-Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4+ T-cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Sistemas CRISPR-Cas , Proliferação de Células/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/genética , Citocinas , Edição de Genes , Humanos , Staphylococcus aureus/genética , Linfócitos T/imunologia
2.
Nat Commun ; 12(1): 4919, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389725

RESUMO

BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of ß-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed ß-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, ß-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that ß-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Oncogenes/genética , Transcrição Genética/genética , beta Catenina/genética , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Feminino , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA-Seq/métodos , beta Catenina/metabolismo
3.
Nat Commun ; 12(1): 4821, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376672

RESUMO

Subclonal genetic heterogeneity and their diverse gene expression impose serious problems in understanding the behavior of cancers and contemplating therapeutic strategies. Here we develop and utilize a capture-based sequencing panel, which covers host hotspot genes and the full-length genome of human T-cell leukemia virus type-1 (HTLV-1), to investigate the clonal architecture of adult T-cell leukemia-lymphoma (ATL). For chronologically collected specimens from patients with ATL or pre-onset individuals, we integrate deep DNA sequencing and single-cell RNA sequencing to detect the somatic mutations and virus directly and characterize the transcriptional readouts in respective subclones. Characteristic genomic and transcriptomic patterns are associated with subclonal expansion and switches during the clinical timeline. Multistep mutations in the T-cell receptor (TCR), STAT3, and NOTCH pathways establish clone-specific transcriptomic abnormalities and further accelerate their proliferative potential to develop highly malignant clones, leading to disease onset and progression. Early detection and characterization of newly expanded subclones through the integrative analytical platform will be valuable for the development of an in-depth understanding of this disease.


Assuntos
Genoma Viral/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Leucemia-Linfoma de Células T do Adulto/genética , Análise de Célula Única/métodos , Transcriptoma/genética , Adulto , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Evolução Clonal/genética , Células Clonais/metabolismo , Células Clonais/patologia , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Mutação , RNA-Seq/métodos , Receptor Notch1/genética , Receptores de Antígenos de Linfócitos T/genética , Fator de Transcrição STAT3/genética
4.
Nat Commun ; 12(1): 5066, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417456

RESUMO

Prostate cancer (PCa) shows strong dependence on the androgen receptor (AR) pathway. Here, we show that squalene epoxidase (SQLE), an enzyme of the cholesterol biosynthesis pathway, is overexpressed in advanced PCa and its expression correlates with poor survival. SQLE expression is controlled by micro-RNA 205 (miR-205), which is significantly downregulated in advanced PCa. Restoration of miR-205 expression or competitive inhibition of SQLE led to inhibition of de novo cholesterol biosynthesis. Furthermore, SQLE was essential for proliferation of AR-positive PCa cell lines, including abiraterone or enzalutamide resistant derivatives, and blocked transactivation of the AR pathway. Inhibition of SQLE with the FDA approved antifungal drug terbinafine also efficiently blocked orthotopic tumour growth in mice. Finally, terbinafine reduced levels of prostate specific antigen (PSA) in three out of four late-stage PCa patients. These results highlight SQLE as a therapeutic target for the treatment of advanced PCa.


Assuntos
Colesterol/biossíntese , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Esqualeno Mono-Oxigenase/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular , Estudos de Coortes , Simulação por Computador , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Camundongos , Camundongos SCID , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Esqualeno Mono-Oxigenase/genética , Esqualeno Mono-Oxigenase/metabolismo , Terbinafina/farmacologia , Ativação Transcricional/genética
5.
Anim Sci J ; 92(1): e13617, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34405917

RESUMO

Granulosa cells (GCs) play an important role in the development of follicles. In this study, we investigate the impact of heat stress at 41°C and 43°C on duck GCs' proliferation and steroids secretion. And, the transcriptomic responses to heat treatment were examined using RNA-sequencing analysis. Digital gene expression profiling was used to screen and identify differentially expressed genes (fold change ≥ 2 and Q value < 0.05). Further, the differential expression genes (DEGs) were classified into GO categories and KEGG pathways. The results show that duck GCs blocked in the G1 phase were increased on exposure to heat stress. Meanwhile, the expression of proliferative genes, which were essential for the transition from G1 to S phase, was inhibited. At the same time, heat stress inhibited the estradiol synthesis of GCs by decreasing CYP11A1 and CYP19A1 gene expression. A total of 241 DEGs including 181 upregulated and 60 downregulated ones were identified. Transcriptome result shows that heat shock protein and CXC chemokines gene were significantly activated during heat stress. While collagenases (MMP1 and MMP13) and strome lysins (MMP3) were downregulated. And, the hedgehog signaling pathway may be a prosurvival adaptive response under heat stress. These results offer a basis for better understanding the molecular mechanism underlying lay-eggs-less in ducks under heat stress.


Assuntos
Proliferação de Células/genética , Patos/fisiologia , Estradiol/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Expressão Gênica , Células da Granulosa/fisiologia , Resposta ao Choque Térmico/genética , Resposta ao Choque Térmico/fisiologia , Ovulação/fisiologia , Animais , Aromatase/genética , Aromatase/metabolismo , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Regulação para Baixo , Feminino , Células da Granulosa/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Hedgehog/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
6.
Theranostics ; 11(16): 7640-7657, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335955

RESUMO

Background: Since primary prostate cancer (PCa) can advance to the life-threatening metastatic PCa, exploring the molecular mechanisms underlying PCa metastasis is crucial for developing the novel targeted preventive strategies for decreasing the mortality of PCa. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism for gene expression and its specific roles in PCa progression remains elusive. Methods: Western blotting, quantitative real-time PCR and immunohistochemical analyses were used to detect target gene expression in PCa cells in vitro and prostate tissues from patients. RNA immunoprecipitation was conducted to analyze the specific binding of mRNA to the target protein. Migration and invasion assays were used to assess the migratory capacities of cancer cells. The correlation between target gene expression and survival rate of PCa patients was analyzed based the TCGA database. Results: We found that total RNA N6-methyladenosine (m6A) modification levels were markedly upregulated in human PCa tissues due to increased expression of methyltransferase like 3 (METTL3). Further studies revealed that the migratory and invasive capacities of PCa cells were markedly suppressed upon METTL3 knockdown. Mechanistically, METTL3 mediates m6A modification of USP4 mRNA at A2696, and m6A reader protein YTHDF2 binds to and induces degradation of USP4 mRNA by recruiting RNA-binding protein HNRNPD to the mRNA. Decrease of USP4 fails to remove the ubiquitin group from ELAVL1 protein, resulting in a reduction of ELAVL1 protein. Lastly, downregulation of ELAVL1 in turn increases ARHGDIA expression, promoting migration and invasion of PCa cells. Conclusions: Our findings highlight the role of METTL3 in modulating invasion and metastasis of PCa cells, providing insight into promising therapeutic strategies for hindering PCa progressing to deadly metastases.


Assuntos
Metiltransferases/genética , Neoplasias da Próstata/metabolismo , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica/fisiologia , Humanos , Masculino , Metiltransferases/metabolismo , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteases Específicas de Ubiquitina/genética
7.
Theranostics ; 11(16): 7779-7796, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335964

RESUMO

Rationale: The progression of prostate cancer (PCa) to castration-resistant PCa (CRPC) despite continuous androgen deprivation therapy is a major clinical challenge. Over 90% of patients with CRPC exhibit sustained androgen receptor (AR) signaling. KDM4B that removes the repressive mark H3K9me3/2 is a transcriptional activator of AR and has been implicated in the development of CRPC. However, the mechanisms of KDM4B involvement in CRPC remain largely unknown. Here, we sought to demonstrate the molecular pathway mediated by KDM4B in CRPC and to provide proof-of-concept evidence that KDM4B is a potential CRPC target. Methods: CRPC cells (C4-2B or CWR22Rv1) depleted with KDM4B followed by cell proliferation (in vitro and xenograft), microarray, qRT-PCR, Seahorse Flux, and metabolomic analyses were employed to identify the expression and metabolic profiles mediated by KDM4B. Immunoprecipitation was used to determine the KDM4B-c-Myc interaction region. Reporter activity assay and ChIP analysis were used to characterize the KDM4B-c-Myc complex-mediated mechanistic actions. The clinical relevance between KDM4B and c-Myc was determined using UCSC Xena analysis and immunohistochemistry. Results: We showed that KDM4B knockdown impaired CRPC proliferation, switched Warburg to OXPHOS metabolism, and suppressed gene expressions including those targeted by c-Myc. We further demonstrated that KDM4B physically interacted with c-Myc and they were co-recruited to the c-Myc-binding sequence on the promoters of metabolic genes (LDHA, ENO1, and PFK). Importantly, KDM4B and c-Myc synergistically promoted the transactivation of the LDHA promoter in a demethylase-dependent manner. We also provided evidence that KDM4B and c-Myc are co-expressed in PCa tissue and that high expression of both is associated with worse clinical outcome. Conclusions: KDM4B partners with c-Myc and serves as a coactivator of c-Myc to directly enhance c-Myc-mediated metabolism, hence promoting CRPC progression. Targeting KDM4B is thus an alternative therapeutic strategy for advanced prostate cancers driven by c-Myc and AR.


Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Antagonistas de Androgênios , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Histona Desmetilases com o Domínio Jumonji/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo
8.
Theranostics ; 11(16): 8112-8128, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335983

RESUMO

The coiled-coil domain containing protein members have been well documented for their roles in many diseases including cancers. However, the function of the coiled-coil domain containing 65 (CCDC65) remains unknown in tumorigenesis including gastric cancer. Methods: CCDC65 expression and its correlation with clinical features and prognosis of gastric cancer were analyzed in tissue. The biological role and molecular basis of CCDC65 were performed via in vitro and in vivo assays and a various of experimental methods including co-immunoprecipitation (Co-IP), GST-pull down and ubiquitination analysis et al. Finally, whether metformin affects the pathogenesis of gastric cancer by regulating CCDC65 and its-mediated signaling was investigated. Results: Here, we found that downregulated CCDC65 level was showed as an unfavourable factor in gastric cancer patients. Subsequently, CCDC65 or its domain (a.a. 130-484) was identified as a significant suppressor in GC growth and metastasis in vitro and in vivo. Molecular basis showed that CCDC65 bound to ENO1, an oncogenic factor has been widely reported to promote the tumor pathogenesis, by its domain (a.a. 130-484) and further promoted ubiquitylation and degradation of ENO1 by recruiting E3 ubiquitin ligase FBXW7. The downregulated ENO1 decreased the binding with AKT1 and further inactivated AKT1, which led to the loss of cell proliferation and EMT signal. Finally, we observed that metformin, a new anti-cancer drug, can significantly induce CCDC65 to suppress ENO1-AKT1 complex-mediated cell proliferation and EMT signals and finally suppresses the malignant phenotypes of gastric cancer cells. Conclusion: These results firstly highlight a critical role of CCDC65 in suppressing ENO1-AKT1 pathway to reduce the progression of gastric cancer and reveals a new molecular mechanism for metformin in suppressing gastric cancer. Our present study provides a new insight into the mechanism and therapy for gastric cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , China , Feminino , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Glicoproteínas/genética , Humanos , Masculino , Metformina/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oncogenes , Prognóstico , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
9.
FASEB J ; 35(9): e21827, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383980

RESUMO

Neuron-derived orphan receptor 1, NR4A3 (Nor1)/NR4A3 is an orphan nuclear receptor involved in the transcriptional control of developmental and neurological functions. Oxidative stress-induced conditions are primarily associated with neurological defects in humans, yet the impact on Nor1-mediated transcription of neuronal genes remains with unknown mechanism. Here, we demonstrate that Nor1 is a non-conventional target of SUMO2/3 conjugation at Lys-137 contained in an atypic ψKxSP motif referred to as the pSuM. Nor1 pSuM SUMOylation differs from the canonical process with the obligate phosphorylation of Ser-139 by Ras signaling to create the required negatively charged interface for SUMOylation. Additional phosphorylation at sites flanking the pSuM is also mediated by the coordinated action of protein kinase casein kinase 2 to function as a small ubiquitin-like modifier enhancer, regulating Nor1-mediated transcription and proteasomal degradation. Nor1 responsive genes involved in cell proliferation and metabolism, such as activating transcription factor 3, cyclin D1, CASP8 and FADD-like apoptosis regulator, and enolase 3 were upregulated in response to pSuM disruption in mouse HT-22 hippocampal neuronal cells and human neuroblastoma SH-SY5Y cells. We also identified critical antioxidant genes, such as catalase, superoxide dismutase 1, and microsomal glutathione S-transferase 2, as responsive targets of Nor1 under pSuM regulation. Nor1 SUMOylation impaired gene transcription through less effective Nor1 chromatin binding and reduced enrichment of histone H3K27ac marks to gene promoters. These effects resulted in decreased neuronal cell growth, increased apoptosis, and reduced survival to oxidative stress damage, underlying the role of pSuM-modified Nor1 in redox homeostasis. Our findings uncover a hierarchical post-translational mechanism that dictates Nor1 non-canonical SUMOylation, disrupting Nor1 transcriptional competence, and neuroprotective redox sensitivity.


Assuntos
Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Sumoilação/genética , Animais , Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinase do Ponto de Checagem 2/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Hipocampo/metabolismo , Homeostase/genética , Humanos , Camundongos , Neuroblastoma/genética , Neurônios/metabolismo , Oxirredução , Estresse Oxidativo/genética , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Regulação para Cima/genética
10.
Nat Commun ; 12(1): 4923, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389713

RESUMO

With increasing age of the population, countries across the globe are facing a substantial increase in osteoporotic fractures. Genetic association signals for fractures have been reported at the RSPO3 locus, but the causal gene and the underlying mechanism are unknown. Here we show that the fracture reducing allele at the RSPO3 locus associate with increased RSPO3 expression both at the mRNA and protein levels, increased trabecular bone mineral density and reduced risk mainly of distal forearm fractures in humans. We also demonstrate that RSPO3 is expressed in osteoprogenitor cells and osteoblasts and that osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and an important regulator of vertebral trabecular bone mass and bone strength in adult mice. Mechanistic studies revealed that RSPO3 in a cell-autonomous manner increases osteoblast proliferation and differentiation. In conclusion, RSPO3 regulates vertebral trabecular bone mass and bone strength in mice and fracture risk in humans.


Assuntos
Osso Esponjoso/metabolismo , Fraturas Ósseas/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Trombospondinas/genética , Animais , Densidade Óssea , Osso Esponjoso/lesões , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Humanos , Análise da Randomização Mendeliana/métodos , Camundongos Knockout , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Fatores de Risco , Trombospondinas/deficiência
11.
Clin Lab ; 67(8)2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34383427

RESUMO

BACKGROUND: Gastric cancer (GC) remains the fourth-leading malignancy worldwide and has a high mortality rate. Accumulating evidence reveals that long noncoding RNAs (lncRNAs) play essential roles in tumorigenesis and metastasis and can be used as potential biomarkers for diagnosis and prognosis. METHODS: We downloaded gene expression profiles from the National Center of Biotechnology Information Gene Expression Omnibus (GEO), screened lncRNAs differentially expressed in gastric cancer tissues and adjacent tissues, and then constructed a lncRNA-miRNA-mRNA network. Seventy patients with gastric cancer were divided into two groups according to different clinical characteristics. The expression of lncRNA LUCAT1 in gastric cancer was detected by reverse transcription polymerase chain reaction (RT-PCR). The AGS and SGC-7901 cell lines were used in CCK8 assay, apoptosis, cell cycle test, transwell assay, and wound healing assay. RESULTS: The expression level of LUCAT1 was associated with tumor diameter (p < 0.001), tissue differentiation grade (p = 0.026), and LNM status (p = 0.020) in GC. The results showed that the lncRNA LUCAT1 could promote the proliferation, invasion, and migration of GC cells, inhibit the apoptosis of GC cells, and affect the process of cell cycles. CONCLUSIONS: The lncRNA LUCAT1 may be used as a potential biomarker for early signs of LNM in GC and may play a crucial role in the development of GC.


Assuntos
MicroRNAs , RNA Longo não Codificante , Neoplasias Gástricas , Biomarcadores , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética
12.
BMC Genomics ; 22(1): 613, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34384362

RESUMO

BACKGROUND: Hypertrophic scar (HTS) is a fibroproliferative skin disorder characterized by excessive cell proliferation, migration, and extracellular matrix (ECM) deposition. The CUB and Sushi multiple domains 1 (CSMD1) has previously been identified as the key regulatory gene of hypertrophic scar by a large sample GWAS study. However, further research has not yet been conducted to verify this finding in other HTS patients and to determine the underlying mechanism. RESULTS: In this study, we verified that CSMD1 was downregulated in both HTS tissue and HTS-derived fibroblasts. The knockdown of CSMD1 resulted in enhanced migration and fibronectin1 (FN1) secretion in fibroblasts in vitro. In addition, the upstream and downstream regulatory mechanisms of CSMD1 were also investigated through microRNA (miRNA) databases screening and RNA-sequencing (RNA-seq) respectively. The screening of four common microRNA (miRNA) databases suggested that miR-190a-3p binds to the CSMD1 and may regulate its expression. We confirmed that miR-190a-3p directly targeted the CSMD1-3'-UTR using luciferase reporter assays. Furthermore, the overexpression of miR-190a-3p showed promotion of migratory activity and FN1 secretion in fibroblasts, resembling the effect of CSMD1 knockdown; whereas the knockdown of miR-190a-3p exerted the opposite effect. Finally, transcriptomic analysis showed activation of Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway in the CSMD1 knockdown fibroblasts. CONCLUSIONS: This study has validated the conclusions of the previous GWAS study conducted in Chinese population. In vitro experiments have provided further evidence on the function of CSMD1 in the development of HTS, and have also revealed the underlying upstream and downstream regulating mechanisms. Additionally, the JAK/STAT signaling pathway identified using RNA-seq might provide a potential treatment approach, especially for HTS.


Assuntos
Cicatriz Hipertrófica , MicroRNAs , Movimento Celular/genética , Proliferação de Células/genética , Cicatriz Hipertrófica/genética , Cicatriz Hipertrófica/patologia , Fibroblastos , Humanos , Proteínas de Membrana , MicroRNAs/genética , Proteínas Supressoras de Tumor
13.
Mol Biol (Mosk) ; 55(4): 667-675, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34432784

RESUMO

MicroRNAs epigenetically regulate physiological and pathological processes. Previously, we found that miR-204-5p is expressed at low levels in melanoma cells, and an increase in its level leads to a change in proliferation, migration, and invasion of these cancer cells. Now, using bioinformatics analysis, it has been shown that the target of miR-204-5p is FOXC1 transcription factor, which is implicated in carcinogenesis. Using the luciferase reporter assay, it was found that miR-204-5p suppresses expression of the FOXC1 gene by binding to its 3' non-coding region. Transfection of small interfering RNA (siRNA) targeting FOXC1 into melanoma cells caused a decrease in miR-204-5p levels, which is consistent with the generally accepted concept of feedback regulation of miRNA expression by target genes. According to the results of the MTT test and fluorescence microscopy, the proliferation level of melanoma cells under the influence of siRNA to FOXC1 decreased 72 h after transfection. Changes in the ratio of cells by cell cycle phase were analyzed using flow cytometry. Regulatory relationships between FOXC1 and miR-204-5p, and an inhibitory effect of FOXC1 knockdown on melanoma cell proliferation were revealed. Based on the results, it can be assumed that miR-204-5p regulates proliferation of melanoma cells by affecting FOXC1 expression.


Assuntos
Melanoma , MicroRNAs , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Fatores de Transcrição Forkhead , Humanos , Melanoma/genética , MicroRNAs/genética
14.
Ann Palliat Med ; 10(7): 7960-7969, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34353082

RESUMO

BACKGROUND: Rheumatoid arthritis (RA) is a chronic joint disease. The study aimed to explore the effects of microRNA (miR)-449 and histone deacetylase 1 (HDAC1) on the proliferation, invasion, and apoptosis of synovial fibroblasts in rheumatoid arthritis. METHODS: Synovial tissue was collected from 20 patients with RA and 20 patients with osteoarthritis (OA) who underwent joint replacement surgery. RA synovial fibroblasts (RASFs) and OA synovial fibroblasts (OASFs) were isolated and cultured. Real-time quantitative PCR was used to detect the expression levels of miR-449 and HDAC1 in synovial tissues and cells. Western blot was performed to detect the cellular expression levels of HDAC1 protein, and apoptosis and invasion-related proteins. The proliferation, invasion, and apoptosis of RASFs were detected by MTT assay, Transwell assay, and flow cytometry. The dual-luciferase reporter gene was used to test the targeting relationship between inflammatory miR-449 and HDAC1. RESULTS: Compared with normal synovial tissue and OASFs, the levels of HDAC1 messenger RNA in RA synovial tissue and RASF cells were significantly increased (P<0.01), while the expression levels of miR-449 were significantly decreased (P<0.01). The dual-luciferase reporter gene experiment confirmed that miR-449 could specifically bind to the 3' untranslated region of HDAC1 to inhibit its luciferase activity (P<0.05). HDAC1 inhibition or miR-449 overexpression significantly inhibited the proliferation and invasion of RASFs (P<0.001), while inducing their apoptosis (P<0.001). HDAC1 overexpression reversed the biological effects of miR-449 on RASFs (P<0.001). CONCLUSIONS: miR-449 inhibits the proliferation and invasion of RASFs and induces their apoptosis by targeting HDAC1, thereby exerting a protective effect against RA.


Assuntos
Artrite Reumatoide , MicroRNAs , Apoptose/genética , Artrite Reumatoide/genética , Proliferação de Células/genética , Fibroblastos , Histona Desacetilase 1/genética , Humanos , MicroRNAs/genética
15.
Medicina (Kaunas) ; 57(7)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34356991

RESUMO

Background and Objectives: Matrix metalloproteinases (MMP) have been implicated as major determinants of tumour growth and metastasis, which are considered two of the main hallmarks of cancer. The interaction of MMP8 and other signalling molecules within and adjacent tumoral tissues, including immune cells, are rather elusive, particularly of adenocarcinoma cell type. In this study, we aimed to investigate the role of MMP8 in non-small cell lung cancer proliferation and invasiveness potential. Materials and Methods: We individually lipofected with two different single guide RNA (sgRNAs) that specifically targeted on MMP8, with CRISPR-Cas 9 protein into the cells. Results: Our results clearly indicated that the lipofection of these complexes could lead to reduced ability of A549 cells to survive and proliferate to form colonies. In addition, when compared to non-transfected cells, the experimental cell groups receiving sgRNAs demonstrated relatively decreased migration rate, hence, wider wound gaps in scratch assay. The quantitative real time-polymerase chain reaction (qRT-PCR) demonstrated significant reduction in the MAP-K, survivin and PI3-K gene expression. MMP8 might have protective roles over tumour growth and spread in our body. Conclusions: The delivery of sgRNAs targeting on the MMP8 gene could induce tumour cell death and arrest cell migratory activity.


Assuntos
Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Neoplasias Pulmonares/genética , Metaloproteinase 8 da Matriz , Invasividade Neoplásica , RNA Guia
16.
Cancer Sci ; 112(9): 3585-3597, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34252986

RESUMO

Diffuse large B cell lymphoma (DLBCL) heterogeneity promotes recurrence and anti-CD20-based therapeutic resistance. Previous studies have shown that downregulation of MS4A1/CD20 expression after chemoimmunotherapy with rituximab leads to rituximab resistance. However, the mechanisms of CD20 loss remain unknown. We identified that pyruvate dehydrogenase kinase 4 (PDK4) is markedly elevated in DLBCL cells derived from both patients and cell lines with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) resistance. We found that overexpression of PDK4 in DLBCL cells resulted in cell proliferation and resistance to rituximab in vitro and in vivo. Furthermore, loss of PDK4 expression or treatment with the PDK4 inhibitor dichloroacetate was able to significantly increase rituximab-induced cell apoptosis in DLBCL cells. Further studies suggested PDK4 mediates a metabolic shift, in that the main energy source was changed from oxidative phosphorylation to glycolysis, and the metabolic changes could play an important role in rituximab resistance. Importantly, by knocking down or overexpressing PDK4 in DLBCL cells, we showed that PDK4 has a negative regulation effect on MS4A1/CD20 expression. Collectively, this is the first study showing that targeting PDK4 has the potential to overcome rituximab resistance in DLBCL.


Assuntos
Antígenos CD20/metabolismo , Antineoplásicos Imunológicos/administração & dosagem , Reprogramação Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Glicoproteínas/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Rituximab/administração & dosagem , Transdução de Sinais/genética , Adolescente , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Transfecção , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
17.
Cancer Sci ; 112(9): 3846-3855, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34286904

RESUMO

Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial-mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from -273 to -10 base pairs upstream of the transcription start site identified by 5'-RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB-downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.


Assuntos
Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Sítios de Ligação , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Progressão da Doença , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HT29 , Humanos , Metástase Neoplásica , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/genética , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Transcrição Genética/genética , Transfecção
18.
Cancer Sci ; 112(9): 3856-3870, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34288281

RESUMO

Patients with BRAF-mutated colorectal cancer (CRC) have a poor prognosis despite recent therapeutic advances such as combination therapy with BRAF, MEK, and epidermal growth factor receptor (EGFR) inhibitors. To identify microRNAs (miRNAs) that can improve the efficacy of BRAF inhibitor dabrafenib (DAB) and MEK inhibitor trametinib (TRA), we screened 240 miRNAs in BRAF-mutated CRC cells and identified five candidate miRNAs. Overexpression of miR-193a-3p, one of the five screened miRNAs, in CRC cells inhibited cell proliferation by inducing apoptosis. Reverse-phase protein array analysis revealed that proteins with altered phosphorylation induced by miR-193a-3p were involved in several oncogenic pathways including MAPK-related pathways. Furthermore, overexpression of miR-193a-3p in BRAF-mutated cells enhanced the efficacy of DAB and TRA through inhibiting reactivation of MAPK signaling and inducing inhibition of Mcl1. Inhibition of Mcl1 by siRNA or by Mcl1 inhibitor increased the antiproliferative effect of combination therapy with DAB, TRA, and anti-EGFR antibody cetuximab. Collectively, our study demonstrated the possibility that miR-193a-3p acts as a tumor suppressor through regulating multiple proteins involved in oncogenesis and affects cellular sensitivity to MAPK-related pathway inhibitors such as BRAF inhibitors, MEK inhibitors, and/or anti-EGFR antibodies. Addition of miR-193a-3p and/or modulation of proteins involved in the miR-193a-3p-mediated pathway, such as Mcl1, to EGFR/BRAF/MEK inhibition may be a potential therapeutic strategy against BRAF-mutated CRC.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/metabolismo , Genes Supressores de Tumor , Imidazóis/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Oximas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Piridonas/farmacologia , Pirimidinonas/farmacologia , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Cetuximab/farmacologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Quimioterapia Combinada/métodos , Receptores ErbB/antagonistas & inibidores , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção
19.
Nat Commun ; 12(1): 4640, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330896

RESUMO

Cranial sutures are major growth centers for the calvarial vault, and their premature fusion leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/- mouse model of coronal craniosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co-transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/- mice. Our study reveals that decrease and/or imbalance of skeletal stem/progenitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis.


Assuntos
Suturas Cranianas/metabolismo , Craniossinostoses/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Células-Tronco/metabolismo , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Suturas Cranianas/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Sistema Musculoesquelético/citologia , Sistema Musculoesquelético/metabolismo , Células-Tronco/citologia , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Via de Sinalização Wnt/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo
20.
Nat Commun ; 12(1): 4626, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330913

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development.Statement of significancePDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors.


Assuntos
Carcinoma Ductal Pancreático/genética , Dano ao DNA , Neoplasias Pancreáticas/genética , Proteína-Arginina N-Metiltransferases/genética , RNA/genética , Proteínas Repressoras/genética , Animais , Biocatálise/efeitos dos fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/prevenção & controle , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/prevenção & controle , Proteína-Arginina N-Metiltransferases/metabolismo , RNA/metabolismo , Interferência de RNA , Proteínas Repressoras/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
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