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In this study, we successfully developed a nanobody-based double antibody sandwich ELISA kit for the detection of clinical serum C-reactive protein (CRP) by using two novel CRP specific nanobodies. The developed method exhibited a linear detection range of approximately 6-200 ng/mL, with a detection limit of 1 ng/mL. Furthermore, the method demonstrated excellent specificity, as there was no cross-reactivity with interfering substances such as total bilirubin and hemoglobin and so on. To assess reproducibility, independent measurements of the samples were conducted under experimental conditions, resulting in intra- and inter-batch coefficients of variation below 10% and a recovery rate of 93%-102%. These results indicate robust reproducibility of the method. To evaluate the performance of the developed kit, we collected 90 clinical samples for correlation analysis with commercial kits. The results showed a high correlation coefficient value (R2) of 0.98, indicating accurate concordance between the developed and commercial kits. In conclusion, our study successfully developed a nanobody-based double antibody sandwich ELISA kit to detect clinical serum CRP. The utilization of nanobodies represents a significant advancement in the field of CRP immunoassay development. The developed kit demonstrates excellent performance characteristics and holds promise for clinical applications.
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Proteína C-Reativa , Ensaio de Imunoadsorção Enzimática , Anticorpos de Domínio Único , Ensaio de Imunoadsorção Enzimática/métodos , Proteína C-Reativa/análise , Humanos , Anticorpos de Domínio Único/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Limite de DetecçãoRESUMO
OBJECTIVE: The objective of this study is to investigate whether a newly introduced deep learning-based iterative reconstruction algorithm, namely, the artificial intelligence iterative reconstruction (AIIR), has a clinical value in computed tomography angiography (CTA), especially for visualizing vascular structures and related lesions, with routine dose settings. METHODS: A total of 63 patients were retrospectively collected from the triple rule-out CTA examinations, where both pulmonary and aortic data were available for each patient and were taken as the example for investigation. The images were reconstructed using the filtered back projection (FBP), hybrid iterative reconstruction (HIR), and the AIIR. The visibility of vasculature and pulmonary emboli and the general image quality were assessed. RESULTS: Artificial intelligence iterative reconstruction resulted in significantly ( P < 0.001) lower noise as well as higher signal-to-noise ratio and contrast-to-noise ratio compared with FBP and HIR. Besides, AIIR achieved the highest subjective scores on general image quality ( P < 0.05). For the vasculature visibility, AIIR offered the best vessel conspicuity, especially for the small vessels ( P < 0.05). Also, >90% of emboli on the AIIR images were graded as sharp (score 5), whereas <15% of emboli on FBP and HIR images were scored 5. CONCLUSION: As demonstrated for pulmonary and aortic CTAs, AIIR improves the image quality and offers a better depiction for vascular structures compared with FBP and HIR. The visibility of the pulmonary emboli was also increased by AIIR.
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Angiografia por Tomografia Computadorizada , Embolia Pulmonar , Humanos , Angiografia por Tomografia Computadorizada/métodos , Inteligência Artificial , Artéria Pulmonar/diagnóstico por imagem , Estudos Retrospectivos , Interpretação de Imagem Radiográfica Assistida por Computador/métodos , Aorta , Embolia Pulmonar/diagnóstico por imagem , Algoritmos , Doses de RadiaçãoRESUMO
BACKGROUND: H19 is the first long noncoding RNA (lncRNA) found to be associated with gene imprinting. It is highly expressed in the embryonic stage and may have important regulatory effects on human embryonic development. We investigated the differences between the levels of H19 promoter DNA methylation in the chorionic villi of patients who experienced spontaneous abortion (SA) following in vitro fertilization embryo transfer (IVF-ET) and those of patients with a normal early pregnancy (NEP). We also analyzed the associated DNA methyltransferase (DNMT) activity. METHODS: Chorionic villus tissue from patients with SA and NEP were collected. The DNA methylation levels of two CpG islands in the promoter region of the H19 gene in the two groups were detected by bisulfite sequencing, and the mRNA expression of DNMTs was analyzed by real-time polymerase chain reaction. RESULTS: The sample size of each group was 32, and there were no significant differences in baseline data, including age, parity, and body mass index, between the two groups. Among the 7 CpG islands measured, the methylation rates of 3 CpG islands (CpG 1, 6, and 7) were significantly lower in the SA group than in the NEP group (P < 0.01). The methylation levels of the other 4 CpG islands were not significantly different between the two groups. There were no differences in the expression of DNMT1 between the two groups (P > 0.05), but DNMT3a and DNMT3b RNA levels were significantly lower in SA group than in the NEP group (P < 0.01). CONCLUSIONS: The lower H19 promoter DNA methylation levels found in the chorionic villi of patients with SA patients following IVF-ET may be explained by decreased expression of DNMT3a and DNMT3b.
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Metilação de DNA , Fertilização in vitro , Impressão Genômica , Feminino , Humanos , Gravidez , Transferência Embrionária , Regiões Promotoras Genéticas , Aborto EspontâneoRESUMO
PURPOSE: To validate a novel deep learning-based metal artifact correction (MAC) algorithm for CT, namely, AI-MAC, in preclinical setting with comparison to conventional MAC and virtual monochromatic imaging (VMI) technique. MATERIALS AND METHODS: An experimental phantom was designed by consecutively inserting two sets of pedicle screws (size Φ 6.5 × 30-mm and Φ 7.5 × 40-mm) into a vertebral specimen to simulate the clinical scenario of metal implantation. The resulting MAC, VMI, and AI-MAC images were compared with respect to the metal-free reference image by subjective scoring, as well as by CT attenuation, image noise, signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and correction accuracy via adaptive segmentation of the paraspinal muscle and vertebral body. RESULTS: The AI-MAC and VMI images showed significantly higher subjective scores than the MAC image (all p < 0.05). The SNRs and CNRs on the AI-MAC image were comparable to the reference (all p > 0.05), whereas those on the VMI were significantly lower (all p < 0.05). The paraspinal muscle segmented on the AI-MAC image was 4.6% and 5.1% more complete to the VMI and MAC images for the Φ 6.5 × 30-mm screws, and 5.0% and 5.1% for the Φ 7.5 × 40-mm screws, respectively. The vertebral body segmented on the VMI was closest to the reference, with only 3.2% and 7.4% overestimation for Φ 6.5 × 30-mm and Φ 7.5 × 40-mm screws, respectively. CONCLUSIONS: Using metal-free reference as the ground truth for comparison, the AI-MAC outperforms VMI in characterizing soft tissue, while VMI is useful in skeletal depiction.
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Aprendizado Profundo , Tomografia Computadorizada por Raios X , Humanos , Tomografia Computadorizada por Raios X/métodos , Artefatos , Interpretação de Imagem Radiográfica Assistida por Computador/métodos , Razão Sinal-Ruído , Algoritmos , Metais , Estudos RetrospectivosRESUMO
Glioblastoma (GBM) is the most universal and devastating primary intracranial neoplasm in the central nervous system. Urolithin A (UA) possesses many pharmacological and biological activities, but its function in GBM is not clear. CCK-8 and colony formation test were used to measure the anti-proliferative potency of UA against GBM cells. Flow cytometry was applied to evaluate cell cycle arrest and apoptosis of U251 and U118 MG cells upon UA incubation. Quantitative real-time PCR and western blotting were conducted to test the regulatory effect of UA on the expression of Sirt1 and FOXO1. Immunodeficient mice were implanted with GBM cells for in vivo validation of the anti-cancer effect of UA. We found UA repressed the proliferation, migration and invasion of glioblastoma cells, while also inhibiting the induction of colony formation ability and epithelial to mesenchymal transition (EMT) in a time- or dose-dependent manner. The does-dependent relationship of UA inducing the cell cycle arrest and apoptosis of glioblastoma cells was identified. Furthermore, UA could enhance the expression levels of Sirt1 and FOXO1 and the knockdown of Sirt1 blocked the inhibitory effects of UA on the proliferation and migration of glioblastoma cells and correspondingly modified the expression level of FOXO1. Overexpression of Sirt1 restored the despaired inhibitory effect of UA induced by Sirt1 knockout on the proliferation and migration of glioblastoma cells. In animal experiments, UA decreased the tumor size and weight of glioblastoma in xenograft nude mice and promoted the expression of Sirt1 and FOXO1 in transplanted tumors. Our findings presented in this study indicate that UA exerts a repressive effect on glioblastoma cells in vivo and in vitro by regulating the Sirt1-FOXO1 axis via the ERK and AKT pathways, indicating that UA is a new novel therapeutic candidate for the treatment of glioblastoma.
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Neoplasias Encefálicas , Glioblastoma , Animais , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Cumarínicos , Transição Epitelial-Mesenquimal , Proteína Forkhead Box O1/genética , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sirtuína 1/genéticaRESUMO
Coordinated regulation of autophagy and apoptosis helps to enhance the antitumor effects of sodium selenite. However, the potential molecules that act as switch nodes in the crosstalk between autophagy and apoptosis is still elusive. Phospholipid scramblase 1 (PLSCR1) has been shown to regulate leukocyte differentiation, while its role in autophagy/apoptosis toggle switch remains unexplored. In this study, we showed that sodium selenite switched protective autophagy to apoptosis in p53-wild type NB4 cells without obvious caspase-8/apoptosis-inducing factor (AIF) axis activation, while induced autophagy-dependent caspase-8/AIF axis activation in p53-mutant Jurkat cells. Additionally, p53 was demonstrated as a positive regulator of PLSCR1. p53-dependent up-regulation of PLSCR1 accounted for the differential regulation of autophagy and apoptosis induced by sodium selenite. Furthermore, sodium selenite induced the release of AIF from mitochondria to cytosol with the facilitation of caspase-8 in Jurkat cells, while not in NB4 cells. The released AIF further enhanced autophagy flux through interacting with PLSCR1, which hereby resulting in the disassociation of PLSCR1 from Atg5-Atg12 complex. Our results indicate that PLSCR1 plays a critical role in p53-dependent regulation of autophagy and apoptosis in sodium selenite-treated leukemia cells. Manipulation of p53-PLSCR1 cascade might be beneficial to enhance the anti-tumor effects of sodium selenite.
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Apoptose , Autofagia , Leucemia/patologia , Proteínas de Transferência de Fosfolipídeos/genética , Selenito de Sódio/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Leucemia/genética , CamundongosRESUMO
To investigate the correlation between quantitative plaque parameters, the perivascular fat attenuation index, and myocardial ischaemia caused by haemodynamic impairment. Patients with stable angina who had invasive flow reserve fraction (FFR) assessment and coronary artery computed tomography (CT) angiography were retrospectively enrolled. A total of 138 patients were included in this study, which were categorized into the FFR < 0.75 group (n = 43), 0.75 ≤ FFR ≤ 0.8 group (n = 37), and FFR > 0.8 group (n = 58), depending on the range of FFR values. The perivascular FAI and CTA-derived parameters, including plaque length (PL), total plaque volume (TPV), minimum lumen area (MLA), and narrowest degree (ND), were recorded for the lesions. An FFR < 0.75 was defined as myocardial-specific ischaemia. The relationships between myocardial ischaemia and parameters such as the PL, TPV, MLA, ND, and FAI were analysed using a logistic regression model and receiver operating characteristic (ROC) curves to compare the diagnostic accuracy of various indicators for myocardial ischaemia. The PL, TPV, ND, and FAI were greater in the FFR < 0.75 group than in the grey area group and the FFR > 0.80 group (all p < 0.05). The MLA in the FFR < 0.75 group was lower than that in the grey area group and the FFR > 0.80 group (both P < 0.05). There were no significant differences in the PL, TPV, or ND between the grey area and the FFR > 0.80 group, but there was a significant difference in the FAI. The coronary artery lesions with FFRs ≤ 0.75 had the greatest FAI values. Multivariate analysis revealed that the perivascular FAI and PL density are significant predictors of myocardial ischaemia. The FAI has some predictive value for myocardial ischaemia (AUC = 0.781). After building a combination model using the FAI and plaque length, the predictive power increased (AUC, 0.781 vs. 0.918), and the change was statistically significant (P < 0.001). The combined model of PL + FAI demonstrated great diagnostic efficacy in identifying myocardial ischaemia caused by haemodynamic impairment; the lower the FFR was, the greater the FAI. Thus, the PL + FAI could be a combined measure to securely rule out myocardial ischaemia.
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Angiografia por Tomografia Computadorizada , Angiografia Coronária , Doença da Artéria Coronariana , Vasos Coronários , Reserva Fracionada de Fluxo Miocárdico , Hemodinâmica , Placa Aterosclerótica , Valor Preditivo dos Testes , Humanos , Masculino , Feminino , Estudos Retrospectivos , Pessoa de Meia-Idade , Idoso , Doença da Artéria Coronariana/fisiopatologia , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/complicações , Vasos Coronários/fisiopatologia , Vasos Coronários/diagnóstico por imagem , Curva ROC , Angina Estável/fisiopatologia , Angina Estável/diagnóstico por imagem , Tecido Adiposo/diagnóstico por imagem , Tecido Adiposo/fisiopatologia , Área Sob a Curva , Reprodutibilidade dos Testes , Modelos Logísticos , Índice de Gravidade de Doença , Tomografia Computadorizada Multidetectores , Análise Multivariada , Cateterismo Cardíaco , Prognóstico , Isquemia Miocárdica/fisiopatologia , Isquemia Miocárdica/diagnóstico por imagem , Isquemia Miocárdica/complicações , Isquemia Miocárdica/etiologiaRESUMO
BACKGROUND: Selenite at high dosage exhibits great potential in curing tumors. It has been shown that selenite inhibits tumor growth through regulation of microtubule dynamics, however, the exact underlying mechanisms remained to be fully elucidated. METHODS & RESULTS: Western blots were carried out to evaluate expression level of different molecules. Our current study discovered that selenite induced microtubule disassembly, cell cycle arrest and finally resulted in apoptosis in Jurkat leukemia cells, while during this process disassembled tubulins were re-organized after long-term exposure to selenite. Furthermore, JNK was activated in the cytoplasm of selenite-treated Jurkat cells, and inhibition of JNK activity successfully prevented the process of microtubule re-assembly. Moreover, inactivation of JNK further enhanced selenite-induced cell cycle arrest and apoptosis. According to the results from cell counting-8 assay, blockage of microtubule re-assembly by colchicine further inhibited Jurkat cell viability after exposure to selenite. Experiments in a xenograft model also proved that selenite could alter JNK activity, destroy microtubule structure and inhibit cell division in vivo. Moreover, TP53, MAPT and YWHAZ were identified to be three most confident interactors that link JNK to microtubule assembly using PPIs analysis. CONCLUSION: Our study indicated that cytosolic JNK-dependent microtubule re-organization took a protective function during selenite-induced apoptosis, while inhibition of this process would finally enhance the anti-tumor effect of selenite.
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Leucemia , Ácido Selenioso , Humanos , Ácido Selenioso/farmacologia , Células Jurkat , Apoptose , Microtúbulos/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismoRESUMO
Non-coding RNAs are responsible for oncogenesis and the development of stemness features, including multidrug resistance and metastasis, in various cancers. Expression of lncRNA MIR31HG in lung cancer tissues and peripheral sera of lung cancer patients were remarkably higher than that of healthy individuals and indicated a poor prognosis. Functional analysis showed that MIR31HG fosters stemness-associated malignant features of non-small cell lung cancer cells. Further mechanistic investigation revealed that MIR31HG modulated GLI2 expression via WDR5/MLL3/P300 complex-mediated H3K4me and H3K27Ace modification. In vivo MIR31HG repression with an antisense oligonucleotide attenuated tumor growth and distal organ metastasis, whereas MIR31HG promotion remarkably encouraged cellular invasion in lung and liver tissues. Our data suggested that MIR31HG is a potential diagnostic indicator and druggable therapeutic target to facilitate multiple strategic treatments for lung cancer patients.
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Selenium is an essential trace element. High dosage of selenite exhibits a great potential in treating leukemia. Previous study discovered selenite could promote leukemia cells apoptosis through inducing DNA damage and cell cycle arrest, while the switch mechanisms of these events and autophagy were still unclear. Current study discovered selenite promoted autophagy and apoptosis of leukemia Jurkat cells. In this process, DNA damage related ATM/IKK alpha axis was activated. This axis could stabilize pro-apoptotic P73, and promote autophagy through regulating NF-kappaB signaling pathway. Moreover, survivin-2B was also confirmed to be necessary for the ATM-induced nuclear location of IKK alpha, and therefore stood at the node position of apoptosis and autophagy cascades inside Jurkat cells. Finally, our in vivo experiments proved that selenite exhibited some anti-tumor effects on Jurkat cells-bearing mice. Moreover, alterations of ATM and IKK alpha expression observed in vivo were similar to that identified in vitro. Therefore, our findings had fully confirmed survivin-2B dependent activation of ATM/IKK alpha axis might be another crosstalk between autophagy and apoptosis of selenite-treated leukemia cells.
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Leucemia , Selênio , Oligoelementos , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Autofagia , Humanos , Quinase I-kappa B/metabolismo , Células Jurkat , Leucemia/patologia , Camundongos , NF-kappa B/metabolismo , Ácido Selenioso/metabolismo , Ácido Selenioso/farmacologia , Selênio/farmacologia , Survivina/metabolismo , Oligoelementos/metabolismoRESUMO
Methylotrophic bacteria are widespread microbes which can use one carbon compound as their only carbon and energy sources. Here we report the finished, annotated genome sequence of the methylotrophic bacterium Methylovorus sp. strain MP688, which was isolated from soil for high-level production of pyrroloquinolone quinone (PQQ) in our lab.
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Genoma Bacteriano , Methylophilaceae/genética , Methylophilaceae/metabolismo , Cofator PQQ/metabolismo , Methylophilaceae/isolamento & purificação , Dados de Sequência Molecular , Microbiologia do SoloRESUMO
[This retracts the article DOI: 10.7150/thno.32738.].
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[This corrects the article DOI: 10.1155/2019/8607859.].
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BACKGROUND: Increasing numbers of studies have demonstrated that circulating tumor cells (CTCs) undergo a phenotypic change termed epithelial-mesenchymal transition (EMT), and researchers have proposed that EMT might provide CTCs with increased potential to survive in the different microenvironments encountered during metastasis through various ways, such as by increasing cell survival and early colonization. However, the exact role of EMT in CTCs remains unclear. METHODS: In this study, we identified CTCs of 41 patients with gastric cancer using Cyttel-CTC and im-FISH (immune-fluorescence in situ hybridization) methods, and tested the expression of EMT markers and ULBP1 (a major member of the NKG2D-natural killer [NK] group 2 member D-ligand family) on CTCs. Moreover, we investigated the relationship between the expression of EMT markers and ULBP1 on CTCs and gastric cancer cell lines. RESULTS: Our results showed that the CTCs of gastric cancer patients exhibited three EMT marker subtypes, and that the expression of ULBP1 was significantly lower on mesenchymal phenotypic CTCs (M+ CTCs) than on epithelial phenotypic CTCs (E+ CTCs). EMT induced by TGF-ß in vitro produced a similar phenomenon, and we therefore proposed that EMT might be involved in the immune evasion of CTCs from NK cells by altering the expression of ULBP1. CONCLUSIONS: Our study indicated that EMT might play a vital role in the immune invasion of CTCs by regulating the expression of ULBP1 on CTCs. These findings could provide potential strategies for targeting the immune evasion capacity of CTCs.
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Biomarcadores Tumorais/metabolismo , Transição Epitelial-Mesenquimal , Evasão da Resposta Imune , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Neoplásicas Circulantes/imunologia , Neoplasias Gástricas/imunologia , Microambiente Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Prognóstico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
An amendment to this paper has been published and can be accessed via the original article.
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OBJECTIVES: Cisplatin (DDP) is one of the most commonly used chemotherapeutic drugs for several cancers, including non-small-cell lung cancer (NSCLC). However, resistance to DDP eventually develops, limiting its further application. New therapy targets are urgently needed to reverse DDP resistance. METHODS: The mRNA expression of UBE2C, ZEB1/2, ABCG2, and ERCC1 was analyzed by reverse transcription-polymerase chain reaction. The protein levels of these molecules were analyzed by Western blotting and immunofluorescent staining. Cell proliferation was detected by CCK8 and MTT assays. Cell migration and invasion were analyzed by wound healing assay and Transwell assays. Promoter activities and gene transcription were analyzed by luciferase reporter assay. RESULTS: In this study, we examined the effect of UBE2C and ZEB1/2 expression levels in DDP-resistant cells of NSCLC. We confirmed that aberrant expression of UBE2C and ZEB1/2 plays a critical role in repressing the DDP sensitivity to NSCLC cells. Additionally, knockdown of UBE2C significantly sensitized resistant cells to DDP by repressing the expression of ZEB1/2. Mechanistic investigations indicated that UBE2C transcriptionally regulated ZEB1/2 by accelerating promoter activity. This study revealed that ZEB1/2 promotes the epithelial mesenchymal transition and expression of ABCG2 and ERCC1 to participate in UBE2C-mediated NSCLC DDP-resistant cell progression, metastasis, and invasion. CONCLUSION: UBE2C may be a novel therapy target for NSCLC for sensitizing cells to the chemotherapeutic agent DDP.
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Background: Recent evidence indicates that UBE2C participates in carcinogenesis by regulating the cell cycle, apoptosis, metastasis, and transcriptional processes. Additionally, miR-548e-5p dysregulation plays a vital role in tumor progression. However, the molecular mechanism via which UBE2C is directly targeted by miR-548-5p, resulting in increase in cellular growth and invasiveness of cancer cells, and its interactions with the epithelial-mesenchymal transition (EMT) marker protein ZEB1/2 in non-small cell lung cancer (NSCLC) is not understood. Methods: Expression of UBE2C and miR-548e-5p was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein level of UBE2C and ZEB1/2 was analyzed using western blotting and immunofluorescence staining. Cellular proliferation was detected using the cell counting kit 8 (CCK8) and 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell migration, invasion, and growth were analyzed using the wound healing and transwell assay. Promoter activity and transcription was analyzed using the luciferase reporter assay. Chromatin immunoprecipitation was used to detect binding of UBE2C to 5'UTR-ZEB1/2. Results: We observed that 4,5-ubiquitin-conjugating enzyme E2C (UBE2C) expression was higher in NSCLC tissue than in the adjacent normal tissue and was associated with increased cell proliferation and invasion. UBE2C enhanced NSCLC progression and metastasis by affecting the cell cycle and inhibiting apoptosis. We also observed that miR-548e-5p was significantly downregulated in lung cancer tissue specimens, which decreased the expression of its direct substrate, UBE2C. Moreover, miR-548e-5p overexpression and UBE2C under-expression significantly suppressed lung cancer cell proliferation, migration, and invasion. Luciferase reporter and chromatin immunoprecipitation assays indicated that miR-548e-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. Furthermore, UBE2C knockdown downregulated the mesenchymal marker vimentin and upregulated the epithelial marker E-cadherin. Bioinformatics assays, coupled with western blotting and luciferase assays, revealed that UBE2C directly binds to the 5'-untranslated region (UTR) of the transcript of the E-cadherin repressor ZEB1/2 and promotes EMT in lung cancer cells. Conclusion: miR-548e-5p directly binds to the 3'-UTR of UBE2C and decreases UBE2C mRNA expression. UBE2C is an oncogene that promotes EMT in lung cancer cells by directly targeting the 5'-UTR of the transcript encoding the E-cadherin repressor ZEB1/2. miR-548e-5p, UBE2C, and ZEB1/2 constitute the miR-548e-5p-UBE2C-ZEB1/2 signal axis, which enhances cancer cell invasiveness by directly interacting with e EMT marker proteins. We believe that the miR-548e-5p-UBE2C-ZEB1/2 signal axis may be a suitable diagnostic marker and a potential target for lung cancer therapy.
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Carcinoma Pulmonar de Células não Pequenas/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Enzimas de Conjugação de Ubiquitina/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Regiões 3' não Traduzidas/genética , Células A549 , Apoptose/genética , Biomarcadores Tumorais/genética , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Invasividade Neoplásica/genética , RNA Mensageiro/genéticaRESUMO
BACKGROUND: METTL3 is an RNA methyltransferase that mediates m6A modification and is implicated in mRNA biogenesis, decay, and translation. However, the biomechanism through which METTL3 regulates MALAT1-miR-1914-3p-YAP axis activity to induce NSCLC drug resistance and metastasis is not very clear. METHODS: The expression of mRNA was analyzed by qPCR assays. Protein levels were analyzed by western blotting and immunofluorescent staining. Cellular proliferation was detected by CCK8 assays. Cell migration and invasion were analyzed by wound healing and transwell assays, respectively. Promoter activities and gene transcription were analyzed by luciferase reporter assays. Finally, m6A modification was analyzed by MeRIP. RESULTS: METTL3 increased the m6A modification of YAP. METTL3, YTHDF3, YTHDF1, and eIF3b directly promoted YAP translation through an interaction with the translation initiation machinery. Moreover, the RNA level of MALAT1 was increased due to a higher level of m6A modification mediated by METTL3. Meanwhile, the stability of MALAT1 was increased by METTL3/YTHDF3 complex. Additionally, MALAT1 functions as a competing endogenous RNA that sponges miR-1914-3p to promote the invasion and metastasis of NSCLC via YAP. Furthermore, the reduction of YAP m6A modification by METTL3 knockdown inhibits tumor growth and enhances sensitivity to DDP in vivo. CONCLUSION: Results indicated that the m6A mRNA methylation initiated by METTL3 promotes YAP mRNA translation via recruiting YTHDF1/3 and eIF3b to the translation initiation complex and increases YAP mRNA stability through regulating the MALAT1-miR-1914-3p-YAP axis. The increased YAP expression and activity induce NSCLC drug resistance and metastasis.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Pulmonar de Células não Pequenas/secundário , Resistencia a Medicamentos Antineoplásicos/genética , Metiltransferases/genética , MicroRNAs/genética , Biossíntese de Proteínas , RNA Longo não Codificante/genética , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina/análogos & derivados , Adenosina/química , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Taxa de Sobrevida , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
PURPOSE: Sodium selenite (SS) has been widely reported to induce apoptosis in various cancer cell types. However, the underlying molecular mechanisms governing SS-mediated repression of lung cancer stem cells remain largely undefined. METHODS: In vitro assays of cell proliferation, clonal formation, apoptosis, migration and cancer stemness cell sphere formation were performed to examine the inhibitory effects of SS on lung adenocarcinoma (LAD) cells with or without the overexpression of SRY-related high-mobility-group box 2 (SOX2). RESULTS: SS significantly inhibited cell growth and induced apoptosis in LAD cells in a dose-dependent manner with marginal effects on normal epithelial cell HBEC. SS dramatically repressed expression of SOX2 and its upstream regulator GLI1 and strongly decreased stemness sphere formation in LAD cells at 10 µM. Forced expression of SOX2 significantly buffered anti-cancer effects of SS. CONCLUSIONS: Our results demonstrate that SS attenuates lung adenocarcinoma progression by repressing SOX2 and its upstream regulator GLI1, which suggests that SS may be a potential therapeutic drug candidate for lung cancer patients.