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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(4): 1008-1012, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31418349

RESUMO

OBJECTIVE: To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology. METHODS: shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins. RESULTS: The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G0/G1 phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex. CONCLUSION: Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Apoptose , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Células K562 , Proteínas Nucleares
2.
Anticancer Res ; 39(8): 4495-4502, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366551

RESUMO

BACKGROUND/AIM: In mice, fetal liver is the first tissue of definitive erythropoiesis for definitive erythroid expansion and maturation. ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in primitive hematopoiesis and T cell development. The aim of this study was to examine whether or not Zfat is involved in definitive erythropoiesis in the fetal liver during mammalian development. MATERIALS AND METHODS: The role of Zfat during mouse fetal erythropoiesis in the fetal liver was examined using tamoxifen-inducible CreERT2 Zfat-deficient mice. RESULTS: Zfat-deficient mice exhibit moderate anemia with small and pale fetal liver through a decreased number of erythroblasts by E12.5. Apoptosis sensitivity in fetal liver erythroid progenitors was enhanced by Zfat-deficiency ex vivo. Moreover, Zfat knockdown partially inhibited CD71-/lowTer119- to CD71highTer119- transition of fetal liver erythroid progenitors with impairment in the elevation of CD71 expression. CONCLUSION: Zfat plays a critical role for erythropoiesis in the fetal liver.


Assuntos
Antígenos CD/genética , Eritropoese/genética , Fígado/crescimento & desenvolvimento , Receptores da Transferrina/genética , Fatores de Transcrição/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Células Eritroides/metabolismo , Células Eritroides/patologia , Desenvolvimento Fetal/genética , Feto , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Fígado/metabolismo , Camundongos , Linfócitos T/citologia , Linfócitos T/metabolismo , Tireoidite Autoimune/genética , Tireoidite Autoimune/patologia
3.
Adv Exp Med Biol ; 1155: 543-553, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468430

RESUMO

Taurine transporter (TauT) has been identified as a target gene of p53 tumor suppressor. TauT is also found to be overexpressed in variety type of human cancers, such as leukemia. This study showed that expression of TauT was upregulated by c-Myc and c-Jun oncogenes. To explore whether blocking of TauT inhibits tumor development, the RNA interference (RNAi) and immune targeting approaches were tested in tumor cells in vitro and in p53 mutant mice in vivo. Knockdown of TauT expression by RNAi resulted in cell cycle G2 arrest and suppressed human breast cancer MCF-7 cells proliferation determined by colonies production and cell migration assays. Knockdown of TauT also rendered MCF-7 cells more susceptible to chemotherapeutic drug-induced apoptosis. An antibody specifically against TauT blocked taurine uptake and induced cell cycle G2 arrest leading to cell death of variety type of tumor cells without affecting the viability of normal mammalian cells. TauT peptide vaccination significantly increased median lifespan (1.5-fold) of the p53 null mice and rescued p53+/- mice by extending the median lifespan from 315 days to 621 days. Furthermore, single dose treatment of tumor-bearing (thymic lymphoma) p53 null mice with TauT peptide reduced tumor size by about 50% and significantly prolonged survival of these mice from average 7 days (after observing the thymic lymphoma) to 21 days. This finding demonstrates that a novel TauT peptide vaccine can delay, inhibit, and/or treat p53 mutation related spontaneous tumorigenesis in vivo. Therefore, TauT peptide may be used as a universal cancer vaccine to prevent and/or treat patients with p53 mutation-mediated cancers.


Assuntos
Vacinas Anticâncer , Imunoterapia , Glicoproteínas de Membrana , Proteínas de Membrana Transportadoras , Interferência de RNA , Proteína Supressora de Tumor p53/genética , Animais , Anticorpos Monoclonais/farmacologia , Pontos de Checagem do Ciclo Celular , Técnicas de Silenciamento de Genes , Genes jun , Genes myc , Humanos , Células MCF-7 , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Mutação , Taurina , Vacinas de Subunidades
4.
Vet Parasitol ; 272: 31-39, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31395202

RESUMO

The lesswright (lwr) gene and its products are essential molecules in mitosis, DNA repair, and embryo formation in many eukaryotes. In this study, immunohistochemical analysis revealed that the Lwr protein was located in the internal tissues and the surface layer of the adult Schistosoma japonicum (Sj) worms. The mRNA expression levels of SjLwr at different points were evaluated by quantitative real-time RT-PCR. The expression of SjLwr peaked at 14 days and then decreased thereafter. SjLwr expression was relatively more stable in male worms than in female worms. The functions of SjLwr were explored by siRNA-based gene silencing with a simple soaking method. The results showed that knockdown of the SjLwr gene impaired the growth and development of S. japonicum in mice, as well as survival, morphology, reproductive capacity, and egg vitality. These observations imply that SjLwr presents a novel target for the development of immuno- and/or small molecule-based therapeutics for the control and treatment of schistosome infections.


Assuntos
Proteínas de Helminto/metabolismo , Schistosoma japonicum/fisiologia , Esquistossomose Japônica/parasitologia , Animais , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Proteínas de Helminto/genética , Masculino , Camundongos , RNA Interferente Pequeno/metabolismo , Reprodução/genética , Schistosoma japonicum/genética , Schistosoma japonicum/crescimento & desenvolvimento , Esquistossomose Japônica/prevenção & controle
5.
Cytogenet Genome Res ; 158(4): 205-212, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31434093

RESUMO

EHMT2 (euchromatic histone lysine methyltransferase 2), a histone methyltransferase, has been shown to be involved in multiple human cancers. In this study, we determined mRNA and protein expression of EHMT2 in cervical cancer cells and normal cervical epithelial cells. EHMT2 was inhibited with short hairpin RNA (shEHMT2) in cervical cancer cells. Cell viability, colony proliferation, apoptosis, adhesion, and invasion assays and Western blot were performed to assess the function of EHMT2. As a result, EHMT2 was upregulated in human cervical cancer cells compared to normal cervical epithelial cells. Suppression of EHMT2 expression impairs cell proliferation and induces apoptosis. Furthermore, EHMT2 silencing inhibited cell adhesion and invasion. Finally, knockdown of EHMT2 resulted in a reduction of the expression of the tumorigenic proteins Bcl-2, Mcl-1, and Survivin and in an increase in the expression of the anti-malignant protein E-cadherin. In conclusion, our data suggest that EHMT2 plays a key role in cell proliferation and metastatic capacity in cervical cancer cells and could serve as a potential therapeutic target.


Assuntos
Inativação Gênica , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/deficiência , Histona-Lisina N-Metiltransferase/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/prevenção & controle , Neoplasias do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/patologia , Apoptose/genética , Caderinas/biossíntese , Adesão Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Neoplasias do Colo do Útero/genética
6.
Gene ; 719: 144080, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31454541

RESUMO

Trigeminal neuropathic pain is seen as a huge clinical challenge. Although numerous drugs have been developed to treat the condition, some patients have shown intolerance to the drugs and thus continue to suffer. In the present study, a rat model of trigeminal neuropathic pain was established using incorrectly positioned dental implants, which had various manifestations that were similar to human trigeminal neuropathic pain. Using this model, we investigated the differential regulation of JAK2 and PTEN. Firstly, we examined the expression of JAK2 and PTEN in the medullary dorsal horn. After inhibiting JAK2/PTEN, we evaluated nociception-related behavioral alterations. The rat models were established by replacing the left lower second molar with a mini dental implant. Immunoblot assay and immunofluorescence experiments indicated high expression of JAK2 and PTEN in medullary dorsal horn after the nerve injury, which attained plateau levels on post-operative day (POD) 5-10 and 10-20. Administration of adenovirus-shRNA-JAK2 on POD 1 reduced mechanical allodynia and downstream STAT activation. Meanwhile, the administration of adenovirus-shRNA-PTEN on POD 1 attenuated mechanical allodynia while upregulating AKT. In addition to postoperative JAK2 and PTEN activation, dexmedetomidine treatment (10 mg/kg) also modulated the downstream sensors of these signaling molecules. These data suggest that JAK2 and PTEN are pivotal to the development of trigeminal neuropathic pain, and that JAK2 and PTEN suppression alleviates the neuropathic pain.


Assuntos
Técnicas de Silenciamento de Genes , Janus Quinase 2/genética , Neuralgia/diagnóstico , PTEN Fosfo-Hidrolase/genética , Neuralgia do Trigêmeo/genética , Animais , Implantes Dentários/efeitos adversos , Dexametasona/administração & dosagem , Dexametasona/uso terapêutico , Dexmedetomidina/administração & dosagem , Dexmedetomidina/uso terapêutico , Modelos Animais de Doenças , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Janus Quinase 2/antagonistas & inibidores , Masculino , Neuralgia/genética , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Medição da Dor , Ratos , Ratos Sprague-Dawley
7.
BMC Plant Biol ; 19(1): 322, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319793

RESUMO

BACKGROUND: Besides fibers, cotton plants also produce a large amount of seeds with a high oil and protein content. The use of these seeds is restricted by their high contents of the terpenoid gossypol, which is harmful to humans and livestock. Using a genetic engineering approach, "Ultra-low gossypol cottonseed" (ULGCS) plants were produced by knocking down an enzyme that catalyzes the formation of a precursor of gossypol. This was accomplished via RNAi-mediated silencing of the target gene using a seed-specific α-globulin promotor. Since gossypol is also a crucial defense mechanism against leaf-feeding herbivores, ULGCS plants might possess lower herbivore resistance than non-engineered plants. Therefore, we tested the constitutive and inducible direct insect resistance of two ULGCS cotton lines against the African cotton leafworm, Spodoptera littoralis. RESULT: The herbivore was equally affected by both ULGCS lines and the control (Coker 312) line when feeding on fully expanded true leaves from undamaged plants and plants induced by jasmonic acid. When plants were induced by caterpillar-damage, however, S. littoralis larvae performed better on the ULGCS plants. Terpenoid analyses revealed that the ULGCS lines were equally inducible as the control plants. Levels of terpenoids were always lower in one of the two lines. In the case of cotyledons, caterpillars performed better on ULGCS cotton than on conventional cotton. This was likely caused by reduced levels of gossypol in ULGCS cotyledons. CONCLUSION: Despite those effects, the insect resistance of ULGSC cotton can be considered as largely intact and the plants may, therefore, be an interesting alternative to conventional cotton varieties.


Assuntos
Gossypium/fisiologia , Gossipol/metabolismo , Animais , Cotilédone/química , Técnicas de Silenciamento de Genes , Gossypium/genética , Gossipol/análise , Herbivoria , Larva , Folhas de Planta/química , Spodoptera
8.
Pharm Res ; 36(9): 133, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289919

RESUMO

PURPOSE: KRAS is the most frequently mutated gene in human cancers. Despite its direct involvement in malignancy and intensive effort, direct inhibition of KRAS via pharmacological inhibitors has been challenging. RNAi induced knockdown using siRNAs against mutant KRAS alleles offers a promising tool for selective therapeutic silencing in KRAS-mutant lung cancers. However, the major bottleneck for clinical translation is the lack of efficient biocompatible siRNA carrier systems. METHODS: Bovine serum albumin (BSA) nanoparticles were prepared by desolvation method to deliver siRNA targeting the KRAS G12S mutation. The BSA nanoparticles were characterized with respect to their size, zeta potential, encapsulation efficiency and nucleic acid release. Nanoparticle uptake, cellular distribution of nucleic acids, cytotoxicity and gene knock down to interfere with cancer hallmarks, uncontrolled proliferation and migration, were evaluated in KRAS G12S mutant A459 cells, a lung adenocarcinoma cell line. RESULTS: BSA nanoparticles loaded with siRNA resulted in nanoparticles smaller than 200 nm in diameter and negative zeta potentials, displaying optimal characteristics for in vivo application. Encapsulating and protecting the siRNA payload well, the nanoparticles enabled transport to A549 cells in vitro, could evade endosomal entrapment and mediated significant sequence-specific KRAS knockdown, resulting in reduced cell growth of siRNA transfected lung cancer cells. CONCLUSIONS: BSA nanoparticles loaded with mutant specific siRNA are a promising therapeutic approach for KRAS-mutant cancers.


Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno/farmacologia , Soroalbumina Bovina/química , Células A549 , Animais , Apoptose/efeitos dos fármacos , Bovinos , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Terapia Genética , Humanos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Transfecção
9.
Gene ; 714: 143997, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31348981

RESUMO

Based on Akt1 and Jak1 key roles in apoptosis and proliferation of many cancers, the aim of this study was to find a new gene therapy strategy by silencing of these main anti-apoptotic genes for HNSCC treatment. Cancerous HN5 and normal HUVEC cell lines were treated with Akt1 and Jak1 siRNAs alone or with each other combined with/without cisplatin. The MTS, flow cytometry, 4',6-diamidino-2-phenylindole staining, real-time PCR and ELISA methods were utilized in this study. The highest percentage of apoptosis was observed in the treatment of Jak1 siRNA/cisplatin group in cancerous HN5 cells (96.5%) where this treatment showed 12.84% apoptosis in normal HUVEC cell line. Cell viability reduced significantly to 64.57% after treatment with Akt1 siRNA in HN5 treated group. Knocking down Akt1 and Jak1 genes using siRNAs could increase levels of apoptosis and reduce proliferation rate in HNSCC indicating the powerful effects of these genes siRNAs with or without chemotherapeutic agents in HNSCC treatment. In conclusion, the combination of siRNA-mediated gene-silencing strategy can be considered as a valuable and safe approach for sensitizing cancer cells to chemotherapeutic agents thus proposed further studies regarding this issue to approve some siRNA based therapeutics for using in clinic.


Assuntos
Apoptose/genética , Proliferação de Células/genética , Neoplasias de Cabeça e Pescoço/genética , Janus Quinase 1/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cisplatino/farmacologia , Técnicas de Silenciamento de Genes/métodos , Inativação Gênica/efeitos dos fármacos , Inativação Gênica/fisiologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Células Endoteliais da Veia Umbilical Humana , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico
10.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 35(3): 262-267, 2019 May 28.
Artigo em Chinês | MEDLINE | ID: mdl-31257810

RESUMO

OBJECTIVE: To study the effect of exendin-4(Ex-4) on the differentiation of neural stem cells(NSCs) in adult mouse subventricular zone(SVZ)and its mechanism . METHODS: NSCs in the SVZ were derived from 5-week C57BL/6J mice and the expression of nestin was detected by immunofluorescence. The cell morphology was observed after the cells treatmed with 100 nmol/L Ex-4 for 14 days.The expressions of nestin and glucagon-like peptide-1 receptor (GLP-1R) were detected by immunofluorescence. GLP-1R was knocked down by using shRNA and the study was divided into four groups: control group, Ex-4 group, GLP-1R knockdown group, GLP-1R knockdown + Ex-4 group. After treatment with 100 nmol/L Ex-4 for 14 d, ß-tublin III and glial fibrillary acidic protein (GFAP) were labeled by immunofluorescence and then the proportion of ß-tublin III positive cells were counted. Western blot was used to detect the activation of cAMP-response element binding protein (CREB) in NSCs. In order to further study the effects of Ex-4 on mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-hydroxy kinase (PI3K) pathways, the cells were pretreated with MAPK inhibitor U0126 at a concentration of 0.07 µmol/L for 30 min or PI3K inhibitor LY294002 at 50 µmol for 2 h, respectively. The study was divided into six groups: control group, Ex-4 group, U0126 group, U0126 + Ex-4 group, LY294002 group, LY294002 + Ex-4 group. The activation of CREB in each group was detected by Western blot. The experiment was repeated three times independently. RESULTS: NSCs were successfully extracted from SVZ of C57BL/6J mice. Immunofluorescence showed that nestin and GLP-1R were positive in NSCs. Compared with the control group, the proportion of neurons differentiated from Ex-4 group was higher. The percentage of neurons in GLP-1R knockdown + Ex-4 group was basically the same as that in control group (P<0.01). The positive cells of beta-tublin III showed positive activation of GLP-1R and CREB. Western blot showed that CREB was significantly activated in the Ex-4 group, and knockdown of GLP-1R abolished its activation (P<0.01). U0126 did not affect Ex-4-mediated CERB activation, and LY294002 significantly reduced Ex-4-mediated CREB activation (P<0.01). CONCLUSION: Ex-4 promotes the differentiation of NSCs into neurons in SVZ of adult mice through GLP-1R receptor, which may be achieved through PI3K/CREB pathway.


Assuntos
Diferenciação Celular , Exenatida/farmacologia , Ventrículos Laterais/citologia , Células-Tronco Neurais/citologia , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Técnicas de Silenciamento de Genes , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases
11.
BMC Plant Biol ; 19(1): 320, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31319813

RESUMO

BACKGROUND: Plant cell walls participate in all plant-environment interactions. Maintaining cell wall integrity (CWI) during these interactions is essential. This realization led to increased interest in CWI and resulted in knowledge regarding early perception and signalling mechanisms active during CWI maintenance. By contrast, knowledge regarding processes mediating changes in cell wall metabolism upon CWI impairment is very limited. RESULTS: To identify genes involved and to investigate their contributions to the processes we selected 23 genes with altered expression in response to CWI impairment and characterized the impact of T-DNA insertions in these genes on cell wall composition using Fourier-Transform Infrared Spectroscopy (FTIR) in Arabidopsis thaliana seedlings. Insertions in 14 genes led to cell wall phenotypes detectable by FTIR. A detailed analysis of four genes found that their altered expression upon CWI impairment is dependent on THE1 activity, a key component of CWI maintenance. Phenotypic characterizations of insertion lines suggest that the four genes are required for particular aspects of CWI maintenance, cell wall composition or resistance to Plectosphaerella cucumerina infection in adult plants. CONCLUSION: Taken together, the results implicate the genes in responses to CWI impairment, cell wall metabolism and/or pathogen defence, thus identifying new molecular components and processes relevant for CWI maintenance.


Assuntos
Arabidopsis/genética , Parede Celular/metabolismo , Genes de Plantas/fisiologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Ascomicetos , Parede Celular/fisiologia , Resistência à Doença/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno , Doenças das Plantas/imunologia , Plântula/metabolismo , Plântula/fisiologia , Espectroscopia de Infravermelho com Transformada de Fourier
12.
Neoplasma ; 20192019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31307201

RESUMO

Nasopharyngeal carcinoma (NPC) is one common head and neck malignancy with leading cause of cancer-related death. Long noncoding RNAs (lncRNAs) have been reported to play essential roles in progression, prognosis and treatment of NPC. However, the exact role of lncRNA zinc finger antisense 1 (ZFAS1) in NPC progression and its potential mechanism remain largely unknown.The expressions of ZFAS1 and microRNA-135a (miR-135a) were measured in NPC tissues or cells by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between ZFAS1 and miR-135a was explored by luciferase reporter assay and RNA immunoprecipitation (RIP). Cell proliferation, apoptosis, migration and invasion were analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5 -diphenyl-2-H-tetrazolium bromide (MTT) assay, flow cytometry or trans-well assay, respectively. Our data showed the expression of ZFAS1 was up-regulated and miR-135a was down-regulated in NPC tissues and cells. miR-135a was bound to ZFAS1 in NPC cells. Moreover, knockdown of ZFAS1 or addition of miR-135a inhibited cell proliferation, migration and invasion but promoted apoptosis in NPC cells. Besides, down-regulation of miR-135a reversed abrogation of ZFAS1-mediated inhibition of proliferation, migration and invasion and increase of apoptosis in NPC cells. Our data suggested Inhibition of ZFAS1 protected against proliferation, migration and invasion but contributed to apoptosis by sponging miR-135a in NPC cells, providing a novel avenue for NPC treatment.


Assuntos
MicroRNAs , Carcinoma Nasofaríngeo , RNA Longo não Codificante , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/fisiopatologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
13.
Gene ; 716: 144010, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31352009

RESUMO

Alternative splicing can generate multiple protein messages from a single gene and has emerged as an important mechanism to regulate cancer pathways. The human SAT1 gene produces two transcript variants: one translates spermidine/spermine N-1 acetyltransferase (SSAT1), the rate-limiting enzyme in the catabolism of polyamines, and the other generates SSATX, which has largely unknown biological functions. Here, we used experimental data and analyses of several melanoma transcriptome datasets to reveal that SSATX is weakly expressed in melanoma cells. SSATX knockdown promoted the proliferation, migration, and invasion of human melanoma cells via the activation of the Wnt signaling pathway in a manner that was independent of SSAT1 expression. Based on our data, we propose that SSATX functions as a long non-coding RNA prior to its degradation in melanoma cells. Overall, our findings indicate that SSATX acts as a tumor suppressor, which may aid the future diagnosis and treatment of melanoma.


Assuntos
Melanoma/genética , RNA Longo não Codificante/fisiologia , Acetiltransferases/genética , Processamento Alternativo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Melanoma/metabolismo , Melanoma/patologia , Invasividade Neoplásica , RNA Longo não Codificante/genética , Via de Sinalização Wnt
14.
Gene ; 711: 143952, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31265880

RESUMO

Ets-1 is one of the crucial member of transcription factor family which share a unique DNA binding domain. It is predominantly expressed in various tumor subtypes and has shown its association in the regulation of various important genes which include ECM-degrading proteases. Our study aimed to understand the mechanism(s) in the pathogenesis of breast carcinogenesis by Ets-1 transcription factor and its downstream target gene MMP-9. Role of Ets-1 in MCF-7 and MDA-MB-231 breast cancer cells was studied by RNA-interference in combination with pull down and ChIP assays to identify the regulation of MMP-9 in these cell lines. Our results showed that transfection of Ets-1 siRNA in breast cancer cell lines resulted in downregulation of Ets-1 and MMP-9. Ets-1 knock down also showed reduced cell invasion and altered expression of EMT markers. Moreover, we could also predict that MMP-9 gene promoter harbors a binding site for Ets-1 transcription factor may be responsible in direct transactivation of Ets-1 along with EMT markers. Phenotypic changes and molecular alterations that may result in increased aggressiveness/invasiveness and metastatic nature of cancerous cells may lead to changes in EMT markers. Therefore, these findings may suggest a plausible role of Ets-1 dependent regulation of MMP-9 gene and may have a significant impact on breast carcinogenesis.


Assuntos
Neoplasias da Mama/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Invasividade Neoplásica , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1/genética
15.
Nat Commun ; 10(1): 2954, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273204

RESUMO

PARP-1 is rapidly recruited and activated by DNA double-strand breaks (DSBs). Upon activation, PARP-1 synthesizes a structurally complex polymer composed of ADP-ribose units that facilitates local chromatin relaxation and the recruitment of DNA repair factors. Here, we identify a function for PARP-1 in DNA DSB resection. Remarkably, inhibition of PARP-1 leads to hyperresected DNA DSBs. We show that loss of PARP-1 and hyperresection are associated with loss of Ku, 53BP1 and RIF1 resection inhibitors from the break site. DNA curtains analysis show that EXO1-mediated resection is blocked by PARP-1. Furthermore, PARP-1 abrogation leads to increased DNA resection tracks and an increase of homologous recombination in cellulo. Our results, therefore, place PARP-1 activation as a critical early event for DNA DSB repair activation and regulation of resection. Hence, our work has direct implications for the clinical use and effectiveness of PARP inhibition, which is prescribed for the treatment of various malignancies.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Cromatina/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Recombinação Homóloga/genética , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
16.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 512-517, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292055

RESUMO

Objective To investigate the effect of knockdown of high mobility group protein B1 (HMGB1) on the proliferation of rat mesangial cells (GMCs) cultured in high glucose (HG) and its mechanism. Methods Rat GMCs was cultured and divided into normal group, high glucose treatment group, negative control small interfering RNA combined with high glucose treatment group (siRNA-NC-HG group) and HMGB1 small interference RNA combined with high glucose treatment group (siRNA-HMGB1-HG group). GMCs in the normal group were cultured in normal DMEM medium. GMCs in the HG treatment group were cultured with HG-DMEM medium. The GMCs in the siRNA-HMGB1-HG group, after transfected with siRNA-HMGB1 sequence for 6 hours, were cultured with high glucose medium for 24 hours. GMCs in the siRNA-NC-HG group, after transfected with siRNA-NC sequence for 6 hours, were cultured in HG medium for 24 hours. HMGB1 mRNA expression levels of GMCs were detected by real-time quantitative PCR. MTT assay was used to detect the proliferation of GMCs. Flow cytometry was performed to assess the apoptosis of GMCs. Western blot analysis was used to detect the protein levels of HMGB1, NF-κBp65 and nuclear factor kappa B inhibitor alpha (IκBα). ELISA was used to detect the levels of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor α (TNF-α) in the cell supernatants. Results Compared with the siRNA-NC-HG group or HG treatment group, HMGB1 mRNA level decreased in GMCs in the siRNA-HMGB1-HG group, and after 24-, 48-, 72- and 96-hour treatment, the proliferation activity and apoptosis rate of GMCs decreased. After knock-down of HMGB1 level of GMCs, the level of NF-κBp65 protein decreased, the level of IκBα protein increased, and the levels of IL-1ß, IL-6 and TNF-α in the supernatant decreased. Conclusion Knockdown of HMGB1 inhibits proliferation and promotes apoptosis of GMCs induced by HG, which may be related to the inhibition of NF-κB/IκB-α pathway.


Assuntos
Apoptose , Proliferação de Células , Proteína HMGB1/genética , Células Mesangiais/citologia , Animais , Meios de Cultura , Dissacarídeos , Eletrólitos , Técnicas de Silenciamento de Genes , Glucose , Glutamatos , Glutationa , Histidina , Manitol , Inibidor de NF-kappaB alfa/metabolismo , Ratos , Fator de Transcrição RelA/metabolismo
17.
Oncology ; 97(3): 180-188, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31330520

RESUMO

BACKGROUND: Hormone receptor-positive breast cancer accounts for nearly two-thirds of breast cancer cases; it ultimately acquires resistance during endocrine treatment and becomes more aggressive. This study evaluated the role of developmental endothelial locus (Del)-1 in tamoxifen-resistant (TAM-R) breast cancer. METHODS: Del-1 expression in recurrent TAM-R breast cancer tissue was evaluated and compared to that in the original tumor tissue from the same patients. Del-1 expression was also evaluated in TAM-R cells by quantitative real-time PCR, western blotting, and enzyme-linked immunosorbent assay. The effects of Del-1 knockdown on the proliferation, migration, and invasion of TAM-R cells was assessed with wound-healing and Matrigel transwell assays. RESULTS: Del-1 was more highly expressed in recurrent breast cancer as compared to the original tumor tissues before initiation of endocrine treatment. Del-1 mRNA was upregulated in TAM-R and small interfering RNA-mediated knockdown of Del-1 suppressed the migration and proliferation of TAM-R cells while partly restoring TAM sensitivity. And the TAM resistance was recovered by knockdown of Del-1. CONCLUSIONS: TAM-R breast cancer is characterized by Del-1 overexpression and tumor progression can be inhibited by Del-1 depletion, which restores TAM sensitivity. Thus, therapeutic strategies that target Del-1 may be effective for the treatment of hormone-resistant breast cancer.


Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Tamoxifeno/farmacologia , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Técnicas de Silenciamento de Genes , Humanos , Interferência de RNA , RNA Interferente Pequeno
18.
Toxicol Lett ; 314: 89-97, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31325635

RESUMO

Ethanol is a key factor in the pathogenesis of alcoholic liver disease (ALD), commonly characterized as liver inflammation. Recently, circular (circ)RNAs have emerged as important targets to cure liver diseases. However, there are no studies investigating the role of circ_1639 in reducing inflammatory responses in ALD. In this study, we found that circ_1639 was upregulated in Kupffer cells from the livers of alcohol fed mice. We hypothesized that circ_1639 inhibition is a potential novel therapy for treating ALD. To test this hypothesis, RAW 264.7 cells were treated with ethanol and transfected with circ_1639 overexpression or knockdown plasmids. We present western blotting, qRT-PCR, and ELISA data that suggest that circ_1639 is a proinflammatory factor in the liver and is involved in the activation of the NF-κB signaling pathway. Using luciferase reporter assay, we confirmed that microRNA (miR)-122 is a target gene of circ_1639. We also show that TNFRSF13C is a key regulator of RAW 264.7 cell activation, and acts as a downstream target for miR-122. In summary, our results suggest that inhibition of circ_1639 expression may reduce inflammatory responses in ALD.


Assuntos
Mediadores da Inflamação/metabolismo , Hepatopatias Alcoólicas/metabolismo , Fígado/metabolismo , MicroRNAs/metabolismo , RNA/metabolismo , Animais , Receptor do Fator Ativador de Células B/genética , Receptor do Fator Ativador de Células B/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Macrófagos do Fígado/metabolismo , Fígado/patologia , Hepatopatias Alcoólicas/genética , Hepatopatias Alcoólicas/patologia , Hepatopatias Alcoólicas/prevenção & controle , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Células RAW 264.7 , RNA/genética , Transdução de Sinais
19.
Life Sci ; 232: 116637, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31288014

RESUMO

Keloid is characterized by overactive fibroblasts. Forkhead box M1 (FOXM1) is transcription factor that plays important roles in the progression of fibrosis. However, the role of FOXM1 in keloid has not been elucidated. In the present study, we examined the expression levels of FOXM1 in clinical keloid tissue specimens and primary keloid fibroblasts (KFs). The results showed that FOXM1 levels were significantly increased in both keloid tissues and KFs. To further investigate the biological functions of FOXM1, FOXM1 was knocked down in KFs by transfection with small interfering RNA targeting FOXM1 (si-FOXM1). Knockdown of FOXM1 inhibited transforming growth factor-ß1 (TGF-ß1)-induced cell proliferation and migration of KFs. Besides, the increased expressions of collagen (coll I), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) in TGF-ß1-induced KFs were suppressed by si-FOXM1 transfection. Furthermore, TGF-ß1-induced increase in p-Smad2 and p-Smad3 expressions was attenuated by FOXM1 knockdown. These data indicated that knockdown of FOXM1 inhibited TGF-ß1-induced KFs activation and extracellular matrix (ECM) accumulation, which was attributed to the inhibition of TGF-ß1/Smad pathway.


Assuntos
Proteína Forkhead Box M1/deficiência , Queloide/metabolismo , Actinas/metabolismo , Adolescente , Adulto , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Técnicas de Silenciamento de Genes/métodos , Humanos , Queloide/genética , Masculino , Fosforilação , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteína Smad2/antagonistas & inibidores , Proteína Smad2/metabolismo , Proteína Smad3/antagonistas & inibidores , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo
20.
Cancer Sci ; 110(9): 2734-2747, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31278886

RESUMO

Lung cancer has the highest morbidity and mortality among all cancers. Discovery of early diagnostic and prognostic biomarkers of lung cancer can greatly facilitate the survival rate and reduce its mortality. In our study, by analyzing Gene Expression Omnibus and Oncomine databases, we found a novel potential oncogene uridine-cytidine kinase 2 (UCK2), which was overexpressed in lung tumor tissues compared to adjacent nontumor tissues or normal lung. Then we confirmed this finding in clinical samples. Specifically, UCK2 was identified as highly expressed in stage IA lung cancer with a high diagnostic accuracy (area under the receiver operating characteristic curve > 0.9). We also found that high UCK2 expression was related to poorer clinicopathological features, such as higher T stage and N stage and higher probability of early recurrence. Furthermore, we found that patients with high UCK2 expression had poorer first progression survival and overall survival than patients with low UCK2 expression. Univariate and multivariate Cox regression analyses showed that UCK2 was an independent risk factor related with worse DFS and OS. By gene set enrichment analysis, tumor-associated biological processes and signaling pathways were enriched in the UCK2 overexpression group, which indicated that UCK2 might play a vital role in lung cancer. Furthermore, in cytology experiments, we found that knockdown of UCK2 could suppress the proliferation and migration of lung cancer cells. In conclusion, our study indicated that UCK2 might be a potential early diagnostic and prognostic biomarker for lung cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Pulmonares/patologia , Recidiva Local de Neoplasia/diagnóstico , Uridina Quinase/metabolismo , Idoso , Linhagem Celular Tumoral , Proliferação de Células , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Pulmão/patologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Prognóstico , Análise de Sobrevida , Uridina Quinase/genética
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