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BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most common malignancies with the highest morbidity and mortality worldwide. Long non-coding RNAs (lncRNAs) are recently recognized as noteworthy regulators of different tumors, counting NSCLC. However, the biological functions and regulatory mechanism of lncRNA WT1-AS in NSCLC progression still stay uninvestigated. METHODS: WT1-AS and miR-494-3p levels in NSCLC cell lines were detected by real-time quantitative polymerase chain reaction (RT-qPCR). In the current study, the regulatory effects of WT1-AS/miR-494-3p axis on cellular behaviors of NSCLC cell lines (A549 and NCI-H1975) were evaluated by a variety of methods. Cell counting kit-8 (CCK-8) and EDU assays were adopted to assess NSCLC cell proliferation. Tunnel staining and flow cytometry assay were applied to determine cell apoptosis and cell cycle distribution. Besides, cell migration and invasion abilities were analyzed by performing wound healing and transwell assays. Meanwhile, the levels of key proteins related to NSCLC cell apoptosis and PTEN/PI3K/AKT pathway were examined using Western blot assay. In addition, luciferase reporter assays were used to determine the interaction between WT1-AS and miR-494-3p or miR-494-3p and PTEN. RESULTS: Visibly downregulated WT1-AS in NSCLC cell lines was obtained from Broad Institute Cancer Cell Line Encyclopedia (CCLE) database and further verified by performing RT-qPCR. Besides, miR-494-3p was the downstream target gene of WT1-AS and obviously upregulated miR-494-3p in NSCLC cell lines was confirmed. WT1-AS overexpression suppressed cell proliferation, migration and invasion abilities while enhanced cell apoptosis of A549 and NCI-H1975 cells. Furthermore, upregulation of miR-494-3p distinctly reversed these inhibitory effects of WT1-AS overexpression on the tumorigenesis and progression of NSCLC. In addition, WT1-AS promoted PTEN expression and thereby inhibited activation of PI3K/AKT pathway by sponging miR-494-3p. CONCLUSION: To conclude, lncRNA WT1-AS impeded cell proliferation, migration, invasion but accelerated cell apoptosis via negatively regulating miR-494-3p to mediate PTEN/PI3K/AKT pathway in NSCLC.
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Background: Cervical cancer (CC) is the second frequent cancer of women in developing countries. Plentiful studies proved that long noncoding RNA antisense of the tumor suppressor gene WT1 (WT1-AS) participated in the progression of CC. However, the role of WT1-AS remains unclear. This study investigated the potential mechanisms of WT1-AS in CC. Methods: The expression of WT1-AS and miR-205 were determined by quantitative real-time polymerase chain reaction. The cellular localization of WT1-AS in CC cells was detected by subcellular fractionation assay. The level of epithelial-mesenchymal transition (EMT)-related proteins of N-cadherin, E-cadherin, MMP9, and MMP2 were measured by Western blot. Moreover, cell cycle, apoptosis, migration, and invasion were detected by flow cytometry and transwell assay, respectively. The interrelation between WT1-AS and miR-205 was verified by dual-luciferase reporter and RNA immunoprecipitation assays. The role of WT1-AS in modifying CC growth was identified using xenograft tumor model. Results: WT1-AS was downregulated in cervical tissues and cell lines. WT1-AS was predominantly located in the cytoplasm of CC cells. Upregulation of WT1-AS promoted cell apoptosis, blocked cell cycle, migration, invasion, and EMT in vitro. Moreover, miR-205, as a target gene of WT1-AS, was increased in cervical tissues and cell lines. Besides, miR-205 mimic reversed the effect of WT1-AS upregulation on cell cycle, apoptosis, migration, invasion, and EMT. Also, WT1-AS caused the curb of xenograft tumor growth in vivo. Conclusion: Upregulation of WT1-AS suppressed CC development through sponging miR-205, providing experimental basis for clinical targeted treatment of CC.
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MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , Neoplasias del Cuello Uterino/genética , Proteínas WT1/metabolismo , Animales , Femenino , Humanos , Ratones , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Objective To investigate the effect of LncRNA WT1-AS on the invasion and migration of triple-negative breast cancer MDA-MB-231 cells. Methods qRT-PCR was used to detect the expression of WT1-AS and the efficiency of gene silencing in MDA-MB-231 cells. Transwell invasion test and scratch test were used to detect the invasion and migration of MDA-MB-231 cells; Western blot was used to detect the expression of E-cadherin, N-cadherin, Vimentin and Snail in MDA-MB-231 cells. Results Compared with normal human mammary epithelial cells, the expression of WT1-AS in MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells were significantly up-regulated. Knockdown of WT1-AS significantly reduced the invasion and migration abilities of MDA-MB-231 cells, the expression of E-cadherin was increased but N-cadherin, Vimentin and Snail expression were decreased. Conclusion WT1-AS could promote the invasion and migration of triple-negative breast cancer cells MDA-MB-231 via regulating epithelial-mesenchymal transition.
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OBJECTIVE: To study the effect of lncRNA WT1-AS on oxidative stress injury (OSI) and apoptosis of neurons in Alzheimer's disease (AD) and its specific mechanisms related to the microRNA-375 (miR-375)/SIX4 axis and WT1 expression. RESULTS: After bioinformatic prediction, WT1-AS was found to be downregulated in Aß25-35treated SH-SY5Y cells, and WT1-AS overexpression inhibited WT1 expression. WT1 could target miR-375 to promote its expression. miR-375 bound to SIX4, and miR-375 overexpression inhibited SIX4 expression. WT1-AS inhibited OSI and apoptosis, while WT1 and miR-375 overexpression or SIX4 silencing reversed the WT1-AS effect on OSI and apoptosis. In vivo experiments revealed that WT1-AS improved learning/memory abilities and inhibited OSI and apoptosis in AD mice. CONCLUSION: Overexpression of WT1-AS can inhibit the miR-375/SIX4 axis, OSI and neuronal apoptosis in AD by inhibiting WT1 expression. METHODS: Related lncRNAs were identified, and miR-375 downstream targets were predicted. WT1-AS, WT1, miR-375 and SIX4 expression was detected in a cell model induced by Aß25-35. The binding of WT1 with miR-375 and that of miR-375 with SIX4 were further confirmed. Adenosine triphosphate (ATP), reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and lactate dehydrogenase (LDH) activities, and apoptosis levels were tested after mitochondrial membrane potential observation. Learning/memory abilities and neuronal apoptosis were tested in a mouse model.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , MicroARNs/metabolismo , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , ARN Largo no Codificante/metabolismo , Transactivadores/metabolismo , Proteínas WT1/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/psicología , Animales , Conducta Animal , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Prueba del Laberinto Acuático de Morris , Neuronas/metabolismo , Neuronas/patología , ARN Largo no Codificante/genética , Transducción de Señal , Transactivadores/genética , Proteínas WT1/genéticaRESUMEN
BACKGROUND: LncRNA WT1-AS is a recently identified potential tumor suppressor in gastric cancer. This study mainly explored the role of WT1-AS in non-small cell lung cancer (NSCLC). METHODS: WT1-AS and TGF-ß1 mRNA in two types of tissues of 74 NSCLC patients were detected by performing RT-qPCR experiments. WT1-AS and TGF-ß1 expression vectors were established using the pcDNA3.1 vector. Protein concentration was measured by BCA assay. Mean values in this study were calculated using the data of three biological replicates of each experiment. RESULTS: We found that WT1-AS was down-regulated, while TGF-ß1 was upregulated in NSCLC tissues. Survival analysis showed that low levels of WT1-AS expression predicted poor survival of NSCLC patients. WT1-AS and TGF-ß1 were inversely correlated in NSCLC tissues. Over-expression experiments revealed down-regulated TGF-ß1 after WT1-AS over-expression, while TGF-ß1 over-expression failed to affect WT1-AS. WT1-AS over-expression resulted in inhibited cancer cell stemness. TGF-ß1 over-expression played an opposite role and attenuated the effects of TGF-ß1 over-expression. CONCLUSION: Therefore, WT1-AS over-expression may inhibit non-small cell lung cancer cell stemness by down-regulating TGF-ß1. TRIAL REGISTRATION: The First Affiliated Hospital of Anhui Medical University Ethics committee approved this study (AHMU20101009).