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1.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(3): 264-270, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32389175

RESUMO

Objective To express E6 protein of human papillomavirus (HPV) type 16 in prokaryotic expression system and prepare its polyclonal antibody. Methods HPV16 E6 gene was obtained from Siha cells by PCR and cloned into pET21a(+) vector to construct the recombinant plasmid pET21a(+)/HPV16 E6 that was confirmed by sequencing. The recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3). The HPV16 E6-His tag recombinant protein was expressed after the induction of isopropyl beta-D-1-thiogalactopyranoside (IPTG), purified by Ni-NTA affinity chromatography, and then analyzed by Western blot analysis. The purified HPV16 E6 recombinant protein was used to immunize Japanese white rabbits to prepare polyclonal antibody. The titer of the serum polyclonal antibody was determined by ELISA. The specificity of the polyclonal antibody was analyzed by Western blotting and immunofluorescence. Results The recombinant plasmid pET21a(+)/HPV16 E6 was successfully constructed and confirmed by sequencing. After the recombinant plasmid pET21a(+)/HPV16 E6 was transformed into E. coli BL21 (DE3), the recombinant HPV16 E6 protein was expressed and purified by affinity chromatography. The polyclonal antibody at a titer of 1:40 000 was obtained by immunizing Japanese big-ear white rabbit with the purified recombinant HPV16 E6 protein, and its specificity was confirmed by Western blotting and immunofluorescence assay. Conclusion HPV16 E6 recombinant protein was successfully expressed and the rabbit polyclonal antibody against HPV16 E6 recombinant protein was prepared.


Assuntos
Anticorpos/imunologia , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas Virais/imunologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/imunologia , Animais , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Plasmídeos , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia
2.
Nat Commun ; 11(1): 2666, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32471990

RESUMO

Multiple myeloma is a plasma cell blood cancer with frequent chromosomal translocations leading to gene fusions. To determine the clinical relevance of fusion events, we detect gene fusions from a cohort of 742 patients from the Multiple Myeloma Research Foundation CoMMpass Study. Patients with multiple clinic visits enable us to track tumor and fusion evolution, and cases with matching peripheral blood and bone marrow samples allow us to evaluate the concordance of fusion calls in patients with high tumor burden. We examine the joint upregulation of WHSC1 and FGFR3 in samples with t(4;14)-related fusions, and we illustrate a method for detecting fusions from single cell RNA-seq. We report fusions at MYC and a neighboring gene, PVT1, which are related to MYC translocations and associated with divergent progression-free survival patterns. Finally, we find that 4% of patients may be eligible for targeted fusion therapies, including three with an NTRK1 fusion.


Assuntos
Fusão Gênica/genética , Histona-Lisina N-Metiltransferase/genética , Mieloma Múltiplo/genética , Proteínas Proto-Oncogênicas c-myc/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Proteínas Repressoras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Variações do Número de Cópias de DNA/genética , Perfilação da Expressão Gênica/métodos , Histona-Lisina N-Metiltransferase/biossíntese , Humanos , Imunoglobulinas/genética , Pessoa de Meia-Idade , Intervalo Livre de Progressão , RNA Longo não Codificante/genética , RNA-Seq/métodos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/biossíntese , Receptor trkA/genética , Proteínas Repressoras/biossíntese
3.
J Surg Oncol ; 122(2): 243-253, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32346887

RESUMO

BACKGROUNDS AND OBJECTIVES: Recent studies have suggested that insulinoma-associated protein 1 (INSM1) is a useful marker for pathological diagnosis of pulmonary neuroendocrine tumors. In the present study, we investigated the association between INSM1 expression and prognosis in patients with pulmonary high-grade neuroendocrine carcinomas (HGNEC) and assessed the usefulness of INSM1 as a prognostic biomarker in these patients. METHODS: Seventy-five consecutive patients with HGNEC who underwent complete surgical resections from January 2000 to December 2018 were enrolled in this study. We classified these patients into two groups: the INSM1-positive group (n = 59) and INSM1-negative group (n = 16). We compared the clinicopathological characteristics, overall survival (OS), and recurrence-free survival (RFS) between the groups. In addition, we performed univariate and multivariate analyses to identify the prognostic factors associated with postoperative survival. RESULTS: Significant differences in tumor diameter and vascular invasion between the groups were found. OS and RFS were significantly poorer in the INSM1-positive group than in the INSM1-negative group. Univariate and multivariate analyses revealed that INSM1 expression was the strongest predictor of poor prognosis for OS and RFS. CONCLUSIONS: INSM1 expression had the greatest influence on the prognosis in patients with HGNEC and may be a prognostic biomarker in these patients.


Assuntos
Carcinoma Neuroendócrino/metabolismo , Carcinoma Neuroendócrino/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Repressoras/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , Taxa de Sobrevida
4.
Genes (Basel) ; 11(1)2020 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-31940753

RESUMO

Spermatogenesis is a complex cellular-differentiation process that relies on the precise regulation of gene expression in spermatogonia, meiotic, and postmeiotic germ cells. The Ring 1 and YY1 binding protein (Rybp) is a member of the mammalian polycomb-group (PcG) protein family that plays multifunctional roles in development. Previous findings indicate that Rybp may function as an important regulator of meiosis. However, its expression in the testes and function in spermatogenesis have not been examined. In this study, we investigated Rybp expression in postnatal mouse testes using qRT-PCR and immunohistochemistry. We also examined the function of Rybp in spermatogenesis by using a conditional-knockout approach. Results showed that the relative expression of Rybp mRNA was significantly upregulated in the testes of postnatal day (PD) 6 mice. Immunofluorescent staining revealed that Rybp was enriched in the spermatocytes. Surprisingly, a conditional deletion of Rybp in fetal germ cells did not affect the fertility or normal development of spermatogenic cells. Further analysis revealed that Rybp deletion resulted in a decreased expression of meiosis-related genes, but that meiosis progression was normal. Together, these findings suggest that Rybp expression was enriched in spermatocytes, but that it was not required for spermatogenesis.


Assuntos
Regulação da Expressão Gênica , Meiose , Proteínas Repressoras/biossíntese , Espermatogênese , Testículo/metabolismo , Animais , Masculino , Camundongos , Camundongos Knockout , Proteínas Repressoras/genética
5.
Neuron ; 105(3): 464-474.e6, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31812516

RESUMO

Many neuronal types occur as pairs that are similar in most respects but differ in a key feature. In some pairs of retinal neurons, called paramorphic, one member responds to increases and the other to decreases in luminance (ON and OFF responses). Here, we focused on one such pair, starburst amacrine cells (SACs), to explore how closely related neuronal types diversify. We find that ON and OFF SACs are transcriptionally distinct prior to their segregation, dendritic outgrowth, and synapse formation. The transcriptional repressor Fezf1 is selectively expressed by postmitotic ON SACs and promotes the ON fate and gene expression program while repressing the OFF fate and program. The atypical Rho GTPase Rnd3 is selectively expressed by OFF SACs and regulates their migration but is repressed by Fezf1 in ON SACs, enabling differential positioning of the two types. These results define a transcriptional program that controls diversification of a paramorphic pair.


Assuntos
Células Amácrinas/metabolismo , Interneurônios/metabolismo , Mitose/fisiologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transcrição Genética/fisiologia , Células Amácrinas/química , Animais , Animais Recém-Nascidos , Feminino , Células HEK293 , Humanos , Interneurônios/química , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Gravidez , Proteínas Repressoras/análise
6.
Int J Mol Sci ; 21(1)2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31877944

RESUMO

HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3'UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3'UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif "AUUUA". Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3'UTR, indicating that the AU-rich elements of the 3'UTR are not contributing to RNA stability. Instead, the three "AUUUA" motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.


Assuntos
Transformação Celular Viral , Queratinócitos/metabolismo , Proteínas Oncogênicas Virais/biossíntese , Estabilidade de RNA , RNA Viral/metabolismo , Proteínas Repressoras/biossíntese , Neoplasias do Colo do Útero/metabolismo , Adenoviridae , Fusão Celular , Feminino , Vetores Genéticos , Humanos , Queratinócitos/patologia , Proteínas Oncogênicas Virais/genética , RNA Viral/genética , Proteínas Repressoras/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
7.
Chin Med Sci J ; 34(3): 184-193, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31601301

RESUMO

Objective Our previous study has revealed that iASPP is elevated in human head and neck squamous cell carcinoma (HNSCC) and iASPP overexpression signifcantly correlates with tumor malignant progression and poor survival of HNSCC. This study investigated the function of iASPP playing in proliferation and invasion of HNSCC in vitro. Methods HNSCC cell line Tu686 transfected with Lentiviral vector-mediated iASPP-specific shRNA and control shRNA were named the shRNA-iASPP group and shRNA-NC group, respectively. The non-infected Tu686 cells were named the CON group. CCK-8 assay, flow cytometry, transwell invasion assay were performed to detect the effects of iASPP inhibition in vitro. Results Our results demonstrated that the proliferation of shRNA-iASPP cells at the time of 72 h (F=32.459, P=0.000), 96 h (F=51.407, P=0.000), 120 h (F=35.125, P=0.000) post-transfection, was significantly lower than that of shRNA-NC cells and CON cells. The apoptosis ratio of shRNA-iASPP cells was 9.42% ± 0.39% (F=299.490, P=0.000), which was significantly higher than that of CON cells (2.80% ± 0.42%) and shRNA-NC cells (3.18% ± 0.28%). The percentage of shRNA-iASPP cells in G0/G1 phase was 74.65% ± 1.09% (F=388.901, P=0.000), which was strikingly increased, compared with that of CON cells (55.19% ± 1.02%) and shRNA-NC cells (54.62% ± 0.88%). The number of invading cells was 56 ± 4 in the shRNA-iASPP group (F=84.965, P=0.000), which decreased significantly, compared with the CON group (111 ± 3) and the shRNA-NC group (105 ± 8). The survival rate of shRNA-iASPP cells administrated with paclitaxel was highly decreased, compared with CON cells and shRNA-NC cells (F=634.841, P=0.000). Conclusion These results suggest iASPP may play an important role in progression and aggressive behavior of HNSCC and may be an efficient chemotherapeutic target for the treatment of HNSCC.


Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas de Neoplasias/biossíntese , Paclitaxel/farmacologia , Proteínas Repressoras/biossíntese , Carcinoma de Células Escamosas de Cabeça e Pescoço , Linhagem Celular Tumoral , Humanos , Invasividade Neoplásica , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
8.
Pathol Res Pract ; 215(11): 152667, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31585814

RESUMO

MicroRNAs (miRNAs) have been found to be aberrantly expressed and exert essential roles in the tumorigenesis and progression of gastric cancer (GC). miR-301b-3p has been recognized as a cancer-related miRNA in lung cancer, bladder cancer and hepatocellular carcinoma. However, the function of miR-301b-3p in GC progression and its underlying mechanism have not been studied yet. In this study, we found that miR-301b-3p expression was up-regulated in GC tissues compared to adjacent noncancerous tissues. Furthermore, the elevated levels of miR-301b-3p were detected in GC cell lines (SGC-7901, AGS, MKN-45 and MGC-803) as compared with GES-1 cells. Interestingly, GC tissues from patients with tumor size ≥ 5 cm and advanced tumor stages showed obvious higher levels of miR-301b-3p compared to matched controls. Functionally, miR-301b-3p knockdown prominently inhibited cell proliferation, and induced cell cycle arrest at G1 phase and apoptosis in MGC-803 cells. Meanwhile, ectopic expression of miR-301b-3p conversely regulated these biological behaviors of MKN-45 cells. Next, we found that miR-301b-3p knockdown increased, whereas miR-301b-3p overexpression reduced the expression of zinc finger and BTB domain containing 4 (ZBTB4) in GC cells. Accordingly, luciferase reporter assay identified ZBTB4 as a direct target of miR-301b-3p. ZBTB4 overexpression markedly restrained the growth of MGC-803 cells. More importantly, ZBTB4 silencing partially reversed miR-301b-3p knockdown-induced tumor suppressive effects on MGC-803 cells. In conclusion, we firstly revealed that miR-301-3p was highly expressed in GC and contributed to tumor progression via attenuating ZBTB4, which might provide a novel molecular-targeted strategy for GC treatment.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Proteínas Repressoras/biossíntese , Neoplasias Gástricas/patologia , Proliferação de Células/genética , Humanos , Proteínas Repressoras/genética , Neoplasias Gástricas/genética
9.
Respir Res ; 20(1): 130, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234835

RESUMO

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an age-related, progressive and lethal disease, whose pathogenesis is associated with fibroblasts/myofibroblasts foci that produce excessive extracellular matrix accumulation in lung parenchyma. Hypoxia has been described as a determinant factor in its development and progression. However, the role of distinct members of this pathway is not completely described. METHODS: By western blot, quantitative PCR, Immunohistochemistry and Immunocitochemistry were evaluated, the expression HIF alpha subunit isoforms 1, 2 & 3 as well, as their role in myofibroblast differentiation in lung tissue and fibroblast cell lines derived from IPF patients. RESULTS: Hypoxia signaling pathway was found very active in lungs and fibroblasts from IPF patients, as demonstrated by the abundance of alpha subunits 1 and 2, which further correlated with the increased expression of myofibroblast marker αSMA. In contrast, HIF-3α showed reduced expression associated with its promoter hypermethylation. CONCLUSIONS: This study lends further support to the involvement of hypoxia in the pathogenesis of IPF, and poses HIF-3α expression as a potential negative regulator of these phenomena.


Assuntos
Proteínas Reguladoras de Apoptose/biossíntese , Fibrose Pulmonar Idiopática/metabolismo , Miofibroblastos/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Miofibroblastos/patologia , Proteínas Repressoras/genética
10.
Histopathology ; 75(4): 568-577, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31077609

RESUMO

AIMS: Insulinoma-associated protein 1 (INSM1) is a transcription factor that is expressed in developing and mature neuroendocrine tissue. Recent studies have shown that INSM1 is a sensitive marker for neuroendocrine tumours. The aims of this study were to evaluate INSM1 expression in primary gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) and in their known metastases, in order to assess its sensitivity as compared with chromogranin-A (CgA) and synaptophysin (SYN), and to evaluate any change in expression between primary and metastatic disease. METHODS AND RESULTS: We identified 30 patients with primary GEP-NEN. Liver metastatic tissue was available for 26 patients; two patients had two metachronous metastatic foci, yielding a total of 28 metastatic cases. An additional two and seven non-paired cases of primary and metastatic grade 3 GEP-NEN, respectively, were included. To assess specificity, we evaluated the expression of these markers in other primary tumours (colorectal adenocarcinoma, acinar cell carcinoma, solid pseudopapillary neoplasm, cholangiocarcinoma, and hepatocellular carcinoma) and metastatic tumours in the liver (adrenal cortical, breast and prostate carcinomas) that may present as differential diagnoses. In our cohort, all of the primary GEP-NENs and 94% of the metastatic GEP-NENs expressed INSM1. INSM1 showed similar sensitivity to SYN and higher sensitivity than CgA in both primary and metastatic neoplasms. INSM1 has comparable specificity to CgA, and higher specificity than SYN. CONCLUSIONS: The nuclear reactivity and the high sensitivity and specificity of INSM1 make it a preferred neuroendocrine marker. In conclusion, INSM1 can be used as a single first-line marker for primary and metastatic GEP-NEN.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Intestinais/diagnóstico , Tumores Neuroendócrinos/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Proteínas Repressoras/biossíntese , Neoplasias Gástricas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/diagnóstico , Proteínas Repressoras/análise , Estudos Retrospectivos , Sensibilidade e Especificidade
11.
J Pineal Res ; 67(2): e12587, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31090223

RESUMO

Histone deacetylase 9 functions as an oncogene in a variety of cancers, but its role on non-small cell lung cancer (NSCLC) has not been reported. Melatonin was proven to possess anticancer actions, whereas its effect on NSCLC and underlying mechanisms remains poorly understood. In this study, 337 patients with complete clinicopathologic characteristics who underwent NSCLC surgery were recruited for the study. We found that NSCLC patients with high HDAC9 expression were correlated with worse overall survival and poor prognosis. HDAC9 knockdown significantly reduced NSCLC cell growth and induced apoptosis both in vivo and in vitro. Melatonin application also markedly inhibited cell proliferation, metastasis, and invasion and promoted apoptosis in NSCLC cells. Moreover, RNA-seq, real-time quantitative polymerase chain reaction, and western blot analyses showed that melatonin treatment decreased the HDAC9 level in NSCLC cells. A mechanistic study revealed that HDAC9 knockdown further enhanced the anticancer activities of melatonin treatment, whereas HDAC9 overexpression partially reversed the melatonin's anticancer effects. Additionally, the in vivo study found melatonin exerted anti-proliferative and pro-apoptotic effects on xenograft tumors which were also strengthened by HDAC9 knockdown. These results indicated that HDAC9 downregulation mediated the anti-NSCLC actions of melatonin, and targeting HDAC9 may be the novel therapeutic strategy for NSCLC.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Regulação para Baixo/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/biossíntese , Neoplasias Pulmonares/tratamento farmacológico , Melatonina/farmacologia , Proteínas de Neoplasias/biossíntese , Proteínas Repressoras/biossíntese , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Int J Neuropsychopharmacol ; 22(6): 383-393, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-30989210

RESUMO

BACKGROUND: There is an urgent need to identify factors that increase vulnerability to opioid addiction to help stem the opioid epidemic and develop more efficient pharmacotherapeutics. MicroRNAs are small non-coding RNAs that regulate gene expression at a posttranscriptional level and have been implicated in chronic drug-taking in humans and in rodent models. Recent evidence has shown that chronic opioid treatment regulates the microRNA miR-9. The present study was designed to test the hypothesis that miR-9 in the nucleus accumbens potentiates oxycodone addictive-like behavior. METHODS: We utilized adeno-associated virus (AAV) to overexpress miR-9 in the nucleus accumbens of male rats and tested the effects on intravenous self-administration of the highly abused prescription opioid, oxycodone, in 1-hour short-access followed by 6-h long-access sessions, the latter of which leads to escalation of drug intake. In separate rats, we assessed the effects of nucleus accumbens miR-9 overexpression on mRNA targets including RE1-silencing transcription factor (REST) and dopamine D2 receptor (DRD2), which have been shown to be regulated by drugs of abuse. RESULTS: Overexpression of miR-9 in the nucleus accumbens significantly increased oxycodone self-administration compared with rats expressing a control, scrambled microRNA. Analysis of the pattern of oxycodone intake revealed that miR-9 overexpression increased "burst" episodes of intake and decreased the inter-infusion interval. Furthermore, miR-9 overexpression decreased the expression of REST and increased DRD2 in the nucleus accumbens at time points that coincided with behavioral effects. CONCLUSIONS: These results suggest that nucleus accumbens miR-9 regulates oxycodone addictive-like behavior as well as the expression of genes that are involved in drug addiction.


Assuntos
Comportamento Aditivo/fisiopatologia , MicroRNAs/biossíntese , MicroRNAs/fisiologia , Núcleo Accumbens/metabolismo , Oxicodona/farmacologia , Animais , Comportamento Aditivo/induzido quimicamente , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Masculino , Microinjeções , Ratos , Receptores de Dopamina D2/biossíntese , Proteínas Repressoras/biossíntese , Autoadministração
13.
Exp Hematol ; 73: 38-49.e7, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30986495

RESUMO

Reduced expression of the transcription factor PU.1 is frequently associated with development of acute myeloid leukemia (AML), whereas elevated levels of CITED2 (CBP/p300-interacting-transactivator-with-an-ED-rich-tail 2) enhance maintenance of both normal and leukemic hematopoietic stem and progenitor cells (HSPCs). Recent findings indicate that PU.1 and CITED2 act in the same gene regulatory network. We therefore examined a potential synergistic effect of simultaneous PU.1 downregulation and CITED2 upregulation on stem cell biology and AML pathogenesis. We found that simultaneous PU.1/CITED2 deregulation in human CD34+ cord blood (CB) cells, as well as CITED2 upregulation in preleukemic murine PU.1-knockdown (PU.1KD/KD) bone marrow cells, significantly increased the maintenance of HSPCs compared with the respective deregulation of either factor alone. Increased replating capacity of PU.1KD/KD/CITED2 cells in in vitro assays eventually resulted in outgrowth of transformed cells, while upregulation of CITED2 in PU.1KD/KD cells enhanced their engraftment in in vivo transplantation studies without affecting leukemic transformation. Transcriptional analysis of CD34+ CB cells with combined PU.1/CITED2 alterations revealed a set of differentially expressed genes that highly correlated with gene signatures found in various AML subtypes. These findings illustrate that combined PU.1/CITED2 deregulation induces a transcriptional program that promotes HSPC maintenance, which might be a prerequisite for malignant transformation.


Assuntos
Transformação Celular Neoplásica/metabolismo , Regulação Leucêmica da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Repressoras/biossíntese , Transativadores/biossíntese , Adulto , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Transativadores/genética
14.
Pediatr Dev Pathol ; 22(5): 440-448, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30975032

RESUMO

Insulinoma-associated protein 1 (INSM1) is a transcription factor that functions in neuroepithelial tissue development and shows expression in neuroendocrine neoplasms. Given the role of INSM1 in controlling differentiation of the sympatho-adrenal lineage, we hypothesized that INSM1 expression would define a subset of neuroblastic tumors. This study aimed to characterize the immunohistochemical profile of INSM1 in a cohort of peripheral neuroblastic tumors and compare INSM1 expression in these tumors to that seen in other embryonal neoplasms, using both tissue microarrays and whole-slide histologic sections. INSM1 showed nuclear expression in 39/50 (78%) peripheral neuroblastic tumors, including 27/32 (84%) neuroblastomas, 9/9 (100%) ganglioneuroblastomas, and 3/9 (33%) ganglioneuromas. Altogether, 70% of peripheral neuroblastic tumors showed anti-INSM1 immunoreactivity in more than 20% of tumor nuclei. Although no non-neuroblastic tumors in this study exhibited INSM1 expression in more than 20% of nuclei, focal or patchy staining was identified in 7/14 (50%) rhabdomyosarcomas, 7/22 (32%) nephroblastomas, and 4/20 (20%) Ewing sarcomas. The absence of INSM1 expression in peripheral neuroblastic tumors was restricted to undifferentiated and poorly differentiated neuroblastomas, as well as mature ganglioneuromas, mimicking the transient INSM1 expression seen in sympatho-adrenal differentiation during normal development. No significant association between MYCN amplification status and INSM1 expression was observed. We found that all 3 INSM1-negative neuroblastoma patients with available follow-up were alive at a median of 15 years, in comparison to 9 of 13 INSM1-positive neuroblastoma patients living at a median of 5 years. Additional studies are needed to determine whether INSM1 expression is indicative of a clinically significant differentiation state in neuroblastoma.


Assuntos
Neoplasias Embrionárias de Células Germinativas/patologia , Neuroblastoma/patologia , Proteínas Repressoras/biossíntese , Biomarcadores Tumorais/análise , Pré-Escolar , Feminino , Ganglioneuroma/patologia , Humanos , Masculino , Proteínas Repressoras/análise
15.
Gynecol Oncol ; 154(1): 207-217, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30979588

RESUMO

OBJECTIVE: Though metastasis-associated protein 1 (MTA1) is widely overexpressed in human cancers and is associated with advanced clinicopathological characteristics and survival in related diseases, the association between MTA1 and endometrial cancer (EC) is little known and needs to be studied. METHODS: Western blot and immunohistochemistry were used to analyze protein expression level of cells and tissues, while real-time PCR was used for RNA detection. Bioinformatics tool analysis revealed the relationship between MTA1 and clinicopathological characteristics and survival. CCK-8 assay, colony-formation assay, cell scratch assay, and Transwell assay were performed to determine cell proliferation, migration and invasion abilities, respectively. RESULTS: The expression level of MTA1 was significantly higher in human EC tissues than in normal endometrium. MTA1 expression was correlated positively with lymph nodes metastasis and poor survival rate in EC. Experimentally overexpressed MTA1 could promote cell proliferation, migration and invasion abilities of EC cell lines Ishikawa, HEC-1B, and RL-952, while reduction of MTA1 inhibited these cell biological behaviors. Moreover, MTA1 could also reverse the negative effect of miR-30c, a direct modulator of MTA1, on EC cells. Our research also revealed that overexpression of MTA1 contributed to EC tumor growth, while knockdown of MTA1 resulted in tumor growth inhibition. Additionally, the phosphorylation levels of mTOR (S2448) and 4E-BP1 (T37/46) changed significantly along with AKT (T308) under regulation of MTA1, both in vivo and vitro. CONCLUSION: Our results showed that MTA1, as a downstream target of miR-30c, might promote EC progression via AKT/mTOR/4E-BP1 pathway, which indicated the potential therapy target of MTA1 in EC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias do Endométrio/metabolismo , Histona Desacetilases/metabolismo , MicroRNAs/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Movimento Celular , Proliferação de Células , Progressão da Doença , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Xenoenxertos , Histona Desacetilases/biossíntese , Histona Desacetilases/genética , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Fosforilação , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transdução de Sinais , Células Tumorais Cultivadas
16.
J Cutan Pathol ; 46(8): 591-598, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30972814

RESUMO

BACKGROUND: Several studies have reported the oncogenic role of human papillomavirus (HPV) in non-melanoma skin cancer (NMSC) carcinogenesis. Considering that HPV could affect tumor protein 53 (TP53) degradation via E6 oncoprotein, we evaluated the expression of TP53 according to HPV infection and E6 expression. METHODS: Biopsy specimens from 79 NMSCs (28 squamous cell carcinomas, 21 keratoacanthomas and 30 basal cell carcinomas) were enrolled. Nested PCR was used to detect mucosal HPV (mHPV) DNA. Genotyping was performed by reverse line hybridization. Expression of TP53 and E6 was evaluated by immunohistochemistry. RESULTS: mHPVs were detected in 34.2% (27/79) of NMSC, with 92.6% (25/27) of high-risk HPV (HR-HPV) types. HPV16-E6-positive expression was observed in all HPV16-positive samples. TP53 high expression was found in 51.4% (37/72) of specimens. In this group, 78.4% were HPV-negative (P = 0.014). TP53 expression was negative in 8/10 of HPV E6-positive specimens. Multivariate analysis showed that TP53 was associated with HPV infection independently of histopathologic type (P = 0.005). CONCLUSION: This study showed a high prevalence of mHPV in NMSC. Active infections assessed by E6 expression are associated with loss of p53 function, highlighting the involvement of mHPV in NMSC carcinogenesis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/biossíntese , Infecções por Papillomavirus , Proteínas Repressoras/biossíntese , Neoplasias Cutâneas , Proteína Supressora de Tumor p53/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/patologia , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Tunísia/epidemiologia
17.
J Biol Chem ; 294(21): 8640-8652, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-30962285

RESUMO

Histone deacetylase 5 (HDAC5) and HDAC9 are class IIa HDACs that function as signal-responsive repressors of the epigenetic program for pathological cardiomyocyte hypertrophy. The conserved deacetylase domains of HDAC5 and HDAC9 are not required for inhibition of cardiac hypertrophy. Thus, the biological function of class IIa HDAC catalytic activity in the heart remains unknown. Here we demonstrate that catalytic activity of HDAC5, but not HDAC9, suppresses mitochondrial reactive oxygen species generation and subsequent induction of NF-E2-related factor 2 (NRF2)-dependent antioxidant gene expression in cardiomyocytes. Treatment of cardiomyocytes with TMP195 or TMP269, which are selective class IIa HDAC inhibitors, or shRNA-mediated knockdown of HDAC5 but not HDAC9 leads to stimulation of NRF2-mediated transcription in a reactive oxygen species-dependent manner. Conversely, ectopic expression of catalytically active HDAC5 decreases cardiomyocyte oxidative stress and represses NRF2 activation. These findings establish a role of the catalytic domain of HDAC5 in the control of cardiomyocyte redox homeostasis and define TMP195 and TMP269 as a novel class of NRF2 activators that function by suppressing the enzymatic activity of an epigenetic regulator.


Assuntos
Regulação Enzimológica da Expressão Gênica , Histona Desacetilases/biossíntese , Miócitos Cardíacos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Animais , Benzamidas/farmacologia , Células HEK293 , Histona Desacetilases/genética , Humanos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/citologia , Fator 2 Relacionado a NF-E2/genética , Oxidiazóis/farmacologia , Domínios Proteicos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética
18.
J Biol Chem ; 294(21): 8617-8629, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-30967472

RESUMO

We previously reported that the cell cycle-related cyclin-dependent kinase 4-retinoblastoma (RB) transcriptional corepressor pathway is essential for stroke-induced cell death both in vitro and in vivo However, how this signaling pathway induces cell death is unclear. Previously, we found that the cyclin-dependent kinase 4 pathway activates the pro-apoptotic transcriptional co-regulator Cited2 in vitro after DNA damage. In the present study, we report that Cited2 protein expression is also dramatically increased following stroke/ischemic insult. Critically, utilizing conditional knockout mice, we show that Cited2 is required for neuronal cell death, both in culture and in mice after ischemic insult. Importantly, determining the mechanism by which Cited2 levels are regulated, we found that E2F transcription factor (E2F) family members participate in Cited2 regulation. First, E2F1 expression induced Cited2 transcription, and E2F1 deficiency reduced Cited2 expression. Moreover, determining the potential E2F-binding regions on the Cited2 gene regulatory sequence by ChIP analysis, we provide evidence that E2F1/4 proteins bind to this DNA region. A luciferase reporter assay to probe the functional outcomes of this interaction revealed that E2F1 activates and E2F4 inhibits Cited2 transcription. Moreover, we identified the functional binding motif for E2F1 in the Cited2 gene promoter by demonstrating that mutation of this site dramatically reduces E2F1-mediated Cited2 transcription. Finally, E2F1 and E2F4 regulated Cited2 expression in neurons after stroke-related insults. Taken together, these results indicate that the E2F-Cited2 regulatory pathway is critically involved in stroke injury.


Assuntos
Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F4/metabolismo , Regulação da Expressão Gênica , Neurônios/metabolismo , Proteínas Repressoras/biossíntese , Acidente Vascular Cerebral/metabolismo , Transativadores/biossíntese , Motivos de Aminoácidos , Animais , Morte Celular , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F4/genética , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Proteínas Repressoras/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Transativadores/genética
19.
Gene ; 701: 1-8, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30898696

RESUMO

Myocardial infarction (MI) is a severe heart disease caused by acute, persistent ischemia or hypoxia and finally leads to heart failure and sudden death. However, the intrinsic molecular mechanisms of MI remain largely unknown. lncRNAs have also been implicated in the process of ischemic heart diseases. However, the role of lncRNA TTTY15 in MI is not elucidated. We evaluated the expression of TTTY15 in MI and human cardiomyocyte under hypoxia. We explored the role of TTTY15 in cell injury under hypoxia. We searched for potential target of TTTY15. Up-regulation of TTTY15 was associated with hypoxia. Silencing TTTY15 prevented hypoxia-induced cell apoptosis and rescued the cell migration and invasion. TTTY15 targeted miR-455-5p, which regulated the Jun dimerization protein 2 (JDP2) expression. Knocking down miR-455-5p abolished effects of TTTY-15 silencing on cell injury. Suppression of long noncoding RNA TTTY15 attenuates hypoxia-induced cardiomyocytes injury by targeting miR-455-5p.


Assuntos
Apoptose , Movimento Celular , MicroRNAs/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/metabolismo , Hipóxia Celular/genética , Linhagem Celular , Inativação Gênica , Humanos , MicroRNAs/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , RNA Longo não Codificante/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética
20.
Elife ; 82019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874502

RESUMO

In the unfolded protein response (UPR), stress in the endoplasmic reticulum (ER) activates a large transcriptional program to increase ER folding capacity. During the budding yeast UPR, Ire1 excises an intron from the HAC1 mRNA and the exon products of cleavage are ligated, and the translated protein induces hundreds of stress-response genes. Using cells with mutations in RNA repair and decay enzymes, we show that phosphorylation of two different HAC1 splicing intermediates is required for their degradation by the 5'→3' exonuclease Xrn1 to enact opposing effects on the UPR. We also found that ligated but 2'-phosphorylated HAC1 mRNA is cleaved, yielding a decay intermediate with both 5'- and 2'-phosphates at its 5'-end that inhibit 5'→3' decay and suggesting that Ire1 degrades incompletely processed HAC1. These decay events expand the scope of RNA-based regulation in the budding yeast UPR and have implications for the control of the metazoan UPR.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Regulação Fúngica da Expressão Gênica , Processamento de RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Repressoras/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/fisiologia , Resposta a Proteínas não Dobradas , Exorribonucleases/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
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