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1.
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
2.
Anticancer Res ; 39(8): 4129-4136, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366497

RESUMO

BACKGROUND/AIM: 5-Aza-2-deoxycytidine (5-Aza-CdR) enhances the sensitivity to 5-fluorouracil (5-FU), but the molecular mechanism is not fully understood. The aim of this study was to investigate the molecular mechanism that enhances the sensitivity to 5-FU treated with 5-Aza-CdR via thymidine phosphorylase (TP). MATERIALS AND METHODS: The sensitivity to drugs was determined on several cancer cell lines by the MTT assay. Protein and mRNA levels were examined by immunoblot and RT-PCR, respectively. Gene silencing, binding of Sp1 to DNA and methylation of DNA was performed by siRNA, ChIP assay and sodium bisulfate genomic sequencing, respectively. RESULTS: Sp1-binding sites in the TP promoter were methylated in epidermoid carcinoma. 5-Aza-CdR demethylated Sp1-binding sites and enhanced sensitivity to 5-FU. CONCLUSION: Demethylation of Sp1-binding sites by 5-Aza-CdR was a key factor enhancing 5-FU sensitivity, which may enable more effective treatments for cancer patients with the combination of 5-Aza-CdR and 5-FU.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição Sp1/genética , Timidina Fosforilase/genética , Sítios de Ligação/efeitos dos fármacos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Decitabina/metabolismo , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , Timidina Fosforilase/química
4.
Eur J Histochem ; 63(3)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31455073

RESUMO

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.


Assuntos
Técnicas de Transferência de Genes , Lipídeos/química , Polímeros/química , RNA Interferente Pequeno/genética , Transfecção/métodos , Sequência de Bases , Carbocianinas/química , Carbocianinas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Inativação Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Metabolismo dos Lipídeos , Lipídeos/toxicidade , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/toxicidade , Neoplasias Hepáticas/genética , Polímeros/metabolismo , Polímeros/toxicidade , Interferência de RNA
5.
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
6.
Arch Endocrinol Metab ; 63(4): 438-444, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31460623

RESUMO

Pubertal timing in humans is determined by complex interactions including hormonal, metabolic, environmental, ethnic, and genetic factors. Central precocious puberty (CPP) is defined as the premature reactivation of the hypothalamic-pituitary-gonadal axis, starting before the ages of 8 and 9 years in girls and boys, respectively; familial CPP is defined by the occurrence of CPP in two or more family members. Pioneering studies have evidenced the participation of genetic factors in pubertal timing, mainly identifying genetic causes of CPP in sporadic and familial cases. In this context, rare activating mutations were identified in genes of the kisspeptin excitatory pathway (KISS1R and KISS1 mutations). More recently, loss-of-function mutations in two imprinted genes (MKRN3 and DLK1) have been identified as important causes of familial CPP, describing novel players in the modulation of the hypothalamic-pituitary-gonadal axis in physiological and pathological conditions. MKRN3 mutations are the most common cause of familial CPP, and patients with MKRN3 mutations present clinical features indistinguishable from idiopathic CPP. Meanwhile, adult patients with DLK1 mutations present high frequency of metabolic alterations (overweight/obesity, early onset type 2 diabetes and hyperlipidemia), indicating that DLK1 may be a novel link between reproduction and metabolism. Arch Endocrinol Metab. 2019;63(4):438-44.


Assuntos
Puberdade Precoce/genética , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Kisspeptinas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metilação , Mutação , Fenótipo , Puberdade Precoce/etiologia , Receptores de Kisspeptina-1/genética , Ribonucleoproteínas/genética
7.
Pharm Res ; 36(10): 142, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31376020

RESUMO

BACKGROUND: With the recent approval of the first small interfering RNA (siRNA) therapeutic formulated as nanoparticles, there is increased incentive for establishing the factors of importance for the design of stable solid dosage forms of such complex nanomedicines. METHODS: The aims of this study were: (i) to identify factors of importance for the design of spray-dried siRNA-loaded lipidoid-poly(DL-lactic-co-glycolic acid) hybrid nanoparticles (LPNs), and (ii) to evaluate their influence on the resulting powders by using a quality-by-design approach. Critical formulation and process parameters were linked to critical quality attributes (CQAs) using design of experiments, and an optimal operating space (OOS) was identified. RESULTS: A series of CQAs were identified based on the quality target product profile. The loading (ratio of LPNs to the total solid content) and the feedstock concentration were determined as critical parameters, which were optimized systematically. Mannitol was chosen as stabilizing excipient due to the low water content of the resulting powders. The loading negatively affected the colloidal stability of the LPNs, whereas feedstock concentration correlated positively with the powder particle size. The optimal mannitol-based solid formulation, defined from the OOS, displayed a loading of 5% (w/w), mass median aerodynamic diameter of 3.3 ± 0.2 µm, yield of 60.6 ± 6.6%, and a size ratio of 1.15 ± 0.03. Dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to non-spray-dried LPNs. CONCLUSION: The optimal solid dosage forms represent robust formulations suitable for higher scale-up manufacturing.


Assuntos
Dessecação/métodos , Lipídeos/química , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , RNA Interferente Pequeno/química , Administração por Inalação , Animais , Composição de Medicamentos , Excipientes/química , Inativação Gênica , Técnicas de Transferência de Genes , Manitol/química , Camundongos , Nanomedicina , Tamanho da Partícula , Pós , Células RAW 264.7 , RNA Interferente Pequeno/administração & dosagem , Solubilidade , Solventes/química
8.
Dokl Biochem Biophys ; 486(1): 168-170, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367813

RESUMO

In the present study, we analyzed the uptake of radiolabeled dopamine by intact synaptosomes and purified synaptic vesicles isolated from the dorsal striatum of mice with constitutive inactivation of all three synuclein-coding genes and wild-type mice. Synuclein deficiency substantially compromised the uptake of this neurotransmitter by synaptic vesicles but had no effect on synaptosomal dopamine uptake.


Assuntos
Dopamina/metabolismo , Vesículas Sinápticas/metabolismo , Sinucleínas/deficiência , Animais , Transporte Biológico/genética , Inativação Gênica , Camundongos , Camundongos Endogâmicos C57BL , Sinucleínas/genética
9.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(4): 1013-1019, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31418350

RESUMO

OBJECTIVE: To investigate the effect of silencing LNK gene on the expression of EPO and EPOR in acute myeloid leukemia cells (THP-1). METHODS: THP-1 cells were cultured. The lentivirus was used as a vector to silence the LNK gene stably. After 72 hours of infection, GFP expression level was detected by the fluorescent inverted microscopy. The lentiviral Infection efficiencies were monitored by flow cytometry. The LNK silencing effect was confirmed. The mRNA expressions of EPO and EPOR were detected by RT-PCR. The protein levels of LNK, EPO and EPOR were detected by Western blot. RESULTS: At the time-point of 72 hours after lentivirus infection, the expression level of GFP was above 85% detected by fluorescent inverted microscopy. The infection efficiency was above 99% by flow cytometry. mRNA expressions of LNK, EPO and EPOR in LNK silencing group were signifycantly lower than those in control group (P<0.05). The protein levels of LNK, EPO and EPOR in LNK silencing group were significantly lower than those in the control group (P<0.05). CONCLUSION: THP-1 cell line of LNK gene silencing has been successfully established,the LNK gene has been silenced, the expression of EPO and EPOR decrease, indicating that LNK may participate in the regulation of EPO and EPOR.


Assuntos
Proteínas/genética , Western Blotting , Eritropoetina , Inativação Gênica , Humanos , Receptores da Eritropoetina , Células THP-1
10.
Int J Nanomedicine ; 14: 5287-5301, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31406460

RESUMO

Purpose: Nanoparticle (NP)-mediated targeted delivery of therapeutic genes or siRNAs to tumors has potential advantages. In this study, hyaluronic acid (HA)-modified chitosan nanoparticles (CS NPs-HA) loaded with cyanine 3 (Cy3)-labeled siRNA (sCS NPs-HA) were prepared and characterized. Methods: Human non-small cell lung cancer (NSCLC) A549 cells expressing receptor CD44 and tumor-bearing mice were used to evaluate the cytotoxic and antitumor effects of sCS NPs-HA in vitro and in vivo. Results: The results showed that noncytotoxic CS NPs-HA of small size (100-200 nm) effectively delivered the Cy3-labeled siRNA to A549 cells via receptor CD44 and inhibited cell proliferation by downregulating the target gene BCL2. In vivo experiment results revealed that sCS NPs-HA directly delivered greater amounts of Cy3-labeled siRNA to the tumor sites, resulting in the inhibition of tumor growth by downregulating BCL2, as compared to unmodified NPs loaded with siRNA (sCS NPs) and to naked Cy3-labeled siRNA. Conclusion: The HA-modified NPs based on chitosan could serve as a promising carrier for siRNA delivery and targeted therapy for NSCLC expressing CD44.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/terapia , Quitosana/química , Ácido Hialurônico/química , Neoplasias Pulmonares/terapia , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Células A549 , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Fluorescência , Inativação Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem
11.
Cancer Sci ; 110(10): 3157-3172, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432600

RESUMO

The underlying mechanisms of breast cancer cells metastasizing to distant sites are complex and multifactorial. Bone sialoprotein (BSP) and αvß3 integrin were reported to promote the metastatic progress of breast cancer cells, particularly metastasis to bone. Most theories presume that BSP promotes breast cancer metastasis by binding to αvß3 integrin. Interestingly, we found the αvß3 integrin decreased in BSP silenced cells (BSPi), which have weak ability to form bone metastases. However, the relevance of their expression in primary tumor and the way they participate in metastasis are not clear. In this study, we evaluated the relationship between BSP, αvß3 integrin levels, and the bone metastatic ability of breast cancer cells in patient tissues, and the data indicated that the αvß3 integrin level is closely correlated to BSP level and metastatic potential. Overexpression of αvß3 integrin in cancer cells could reverse the effect of BSPi in vitro and promote bone metastasis in a mouse model, whereas knockdown of αvß3 integrin have effects just like BSPi. Moreover, The Cancer Genome Atlas data and RT-PCR analysis have also shown that SPP1, KCNK2, and PTK2B might be involved in this process. Thus, we propose that αvß3 integrin is one of the downstream factors regulated by BSP in the breast cancer-bone metastatic cascade.


Assuntos
Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Integrina alfaVbeta3/metabolismo , Sialoproteína de Ligação à Integrina/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Quinase 2 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Inativação Gênica , Humanos , Sialoproteína de Ligação à Integrina/genética , Células MCF-7 , Camundongos , Transplante de Neoplasias , Osteopontina/genética , Canais de Potássio de Domínios Poros em Tandem/genética
12.
Nat Commun ; 10(1): 2958, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273195

RESUMO

RNAi-based bone anabolic gene therapy has demonstrated initial success, but many practical challenges are still unmet. Here, we demonstrate that a recombinant adeno-associated virus 9 (rAAV9) is highly effective for transducing osteoblast lineage cells in the bone. The adaptor protein Schnurri-3 (SHN3) is a promising therapeutic target for osteoporosis, as deletion of shn3 prevents bone loss in osteoporotic mice and short-term inhibition of shn3 in adult mice increases bone mass. Accordingly, systemic and direct joint administration of an rAAV9 vector carrying an artificial-microRNA that targets shn3 (rAAV9-amiR-shn3) in mice markedly enhanced bone formation via augmented osteoblast activity. Additionally, systemic delivery of rAAV9-amiR-shn3 in osteoporotic mice counteracted bone loss and enhanced bone mechanical properties. Finally, we rationally designed a capsid that exhibits improved specificity to bone by grafting the bone-targeting peptide motif (AspSerSer)6 onto the AAV9-VP2 capsid protein. Collectively, our results identify a bone-targeting rAAV-mediated gene therapy for osteoporosis.


Assuntos
Reabsorção Óssea/complicações , Reabsorção Óssea/prevenção & controle , Osso e Ossos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dependovirus/metabolismo , Inativação Gênica , Osteoporose/complicações , Animais , Reabsorção Óssea/patologia , Osso e Ossos/virologia , Capsídeo/metabolismo , Cartilagem/virologia , Modelos Animais de Doenças , Deleção de Genes , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Sorogrupo
13.
Yonsei Med J ; 60(8): 751-759, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31347330

RESUMO

PURPOSE: This study aimed to explore the effects and mechanisms of long non-coding RNA (lncRNA) anti-differentiation non-coding RNA (ANCR) on the osteogenesis of osteoblast cells in postmenopausal osteoporosis (PMOP). MATERIALS AND METHODS: Mice models of PMOP were established. ANCR expression and intracellular calcium ions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and laser confocal microscopy, respectively. ANCR was silenced in osteoblast cells from PMOP mice by the transfection of siRNA-ANCR (si-ANCR). The proliferation and apoptosis of osteoblast cells was analyzed by MTT and flow cytometry, respectively. Alkaline phosphatase (ALP) activity and calcium nodules were examined by ALP and alizarin red staining assay, respectively. The expression of enhancer of zeste homolog 2 (EZH2), runt related transcription factor 2 (RUNX2), and OSTERIX was detected by qRT-PCR and Western blot. Furthermore, an osteogenesis model was constructed in mice, and osteoid formation was observed by hematoxylin-eosin (HE) staining. The interaction between lncRNA-ANCR and EZH2 was further identified by RNA pull-down assay. RESULTS: ANCR expression and intracellular calcium ions were increased in PMOP mice. Si-ANCR significantly increased the proliferation, ALP activity, calcium deposition of osteoblast cells and decreased apoptosis. ANCR and EZH2 were down-regulated by si-ANCR, while RUNX2 and OSTERIX were upregulated. Si-ANCR also promoted osteoid formation in mice treated with hydroxyapatite-tricalcium phosphate. In addition, ANCR specifically bound to EZH2. CONCLUSION: Silencing ANCR promotes the osteogenesis of PMOP osteoblast cells. The specific binding of ANCR with EZH2 suppressed RUNX2, thereby inhibiting osteogenesis.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inativação Gênica , Osteoblastos/metabolismo , Osteogênese , Osteoporose Pós-Menopausa/genética , Osteoporose Pós-Menopausa/patologia , RNA Longo não Codificante/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Osteoblastos/patologia , Osteogênese/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Fator de Transcrição Sp7/metabolismo
14.
J Agric Food Chem ; 67(28): 7898-7907, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31282664

RESUMO

This study aimed to explore the effects of silencing HB12 and TT8 genes on protein utilization characteristics of alfalfa. Ground samples of 11 HB12-silenced (HB12i), 5 TT8-silenced (TT8i) and 4 wild type (WT) were incubated in a Daisy II incubator with N15 labeled ammonium sulfate for 0, 4, 8, 12, and 24 h. CP degradation and degradational kinetics, microbial nitrogen fractions, and protein metabolic profiles were determined. Moreover, relationships between protein profiles and FTIR spectral parameters were estimated. Results showed that transgenic alfalfa had lower CP degradation, microbial protein, and total available protein compared with WT, especially for HB12i. In addition, CP degradation and protein metabolic profiles were closely correlated with FTIR spectral parameters and thereby could be predicted from spectral parameters. In conclusion, silencing of HB12 and TT8 genes in alfalfa decreased protein degradational and metabolic profiles, which were predictable with FTIR spectral parameters.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Inativação Gênica , Proteínas de Homeodomínio/genética , Medicago sativa/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rúmen/metabolismo , Ração Animal/análise , Animais , Bactérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bovinos , Digestão , Proteínas de Homeodomínio/metabolismo , Cinética , Medicago sativa/química , Medicago sativa/metabolismo , Proteínas de Plantas/química , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteólise , Rúmen/química , Rúmen/microbiologia
15.
BMC Plant Biol ; 19(1): 317, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307384

RESUMO

BACKGROUND: Anthocyanins, which are colored pigments, have long been used as food and pharmaceutical ingredients due to their potential health benefits, but the intermediate signals through which environmental or developmental cues regulate anthocyanin biosynthesis remains poorly understood. Fleshy fruits have become a good system for studying the regulation of anthocyanin biosynthesis, and exploring the mechanism underlying pigment metabolism is valuable for controlling fruit ripening. RESULTS: The present study revealed that ABA accumulated during Lycium fruit ripening, and this accumulation was positively correlated with the anthocyanin contents and the LbNCED1 transcript levels. The application of exogenous ABA and of the ABA biosynthesis inhibitor fluridon increased and decreased the content of anthocyanins in Lycium fruit, respectively. This is the first report to show that ABA promotes the accumulation of anthocyanins in Lycium fruits. The variations in the anthocyanin content were consistent with the variations in the expression of the genes encoding the MYB-bHLH-WD40 transcription factor complex or anthocyanin biosynthesis-related enzymes. Virus-induced LbNCED1 gene silencing significantly slowed fruit coloration and decreased both anthocyanin and ABA accumulation during Lycium fruit ripening. An qRT-PCR analysis showed that LbNCED1 gene silencing clearly reduced the transcript levels of both structural and regulatory genes in the flavonoid biosynthetic pathway. CONCLUSIONS: Based on the results, a model of ABA-mediated development-dependent anthocyanin biosynthesis and fruit coloration during Lycium fruit maturation was proposed. In this model, the developmental cues transcriptionally activates LbNCED1 and thus enhances accumulation of the phytohormone ABA, and the accumulated ABA stimulates transcription of the MYB-bHLH-WD40 transcription factor complex to upregulate the expression of structural genes in the flavonoid biosynthetic pathway and thereby promoting anthocyanin production and fruit coloration. Our results provide a valuable strategy that could be used in practice to regulate the ripening and quality of fresh fruit in medicinal and edible plants by modifying the phytohormone ABA.


Assuntos
Ácido Abscísico/metabolismo , Antocianinas/biossíntese , Frutas/metabolismo , Lycium/metabolismo , Pigmentação , Reguladores de Crescimento de Planta/metabolismo , Dioxigenases/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Lycium/genética , Lycium/crescimento & desenvolvimento , Proteínas de Plantas/genética , Transdução de Sinais
16.
Life Sci ; 232: 116665, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31323273

RESUMO

AIMS: Overexpression of the mechanistic target of rapamycin (mTOR), a member of the PIKK (phosphoinositide kinase-related kinase) family, protects cardiomyocytes from cell death induced by pathological stimuli such as ischemia. We previously reported that posttranslational modification of mTOR plays an important role in regulating cardiac mTOR expression. The aim of this study was to see if Tel2 (telomere maintenance 2), a protein that regulates the abundance of PIKKs, confers similar cardioprotective effects as mTOR. Tel2 is not well-characterized in cardiomyocytes, therefore we examined the effects of Tel2 on cardiomyocyte viability under ischemic stress conditions. MATERIALS AND METHODS: We overexpressed Tel2 or silenced Tel2 with siRNA in the HL-1 cardiomyocyte cell line to survey the effects of Tel2 overexpression and downregulation on cell survival during hypoxia. Adult mouse cardiomyocytes transfected with Tel2 adenoviruses were used to test whether Tel2 sufficiently prevented cardiomyocyte cell death against hydrogen peroxide (H2O2). KEY FINDINGS: Overexpressing Tel2 increased mTOR expression with a concomitant increase in mTOR Complex 1 (mTORC1) and mTORC2 activity in HL-1 cells. Tel2 deletion decreased mTOR expression, and mTORC1 and mTORC2 activity accordingly. In both HL-1 cells and adult mouse cardiomyocytes, Tel2 overexpression protected cardiomyocytes under ischemic stress. These effects were mTOR-dependent, as mTOR inhibitors blunted the effects of Tel2. While gene silencing of Tel2 did not affect cell survival under normoxia, Tel2 silencing made cardiomyocytes more vulnerable to cell death under hypoxia. SIGNIFICANCE: Upregulating Tel2 expression increases mTOR-mediated cardiomyocyte survival and targeting Tel2 could be another therapeutic strategy against ischemic heart disease.


Assuntos
Sobrevivência Celular/fisiologia , Miócitos Cardíacos/citologia , Proteínas de Ligação a Telômeros/fisiologia , Adenoviridae/genética , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Inativação Gênica , Peróxido de Hidrogênio/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , Transdução de Sinais , Proteínas de Ligação a Telômeros/genética , Transfecção
17.
Plant Sci ; 286: 28-36, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300139

RESUMO

MYB family genes act as important regulators modulating the response to abiotic stress in plants. However, much less is known about MYB proteins in cotton. Here, we found that a cotton MYB gene, GhMYB73, was induced by NaCl and abscisic acid (ABA). Silencing GhMYB73 expression in cotton increased sensitivity to salt stress. The cotyledon greening rate of Arabidopsis thaliana over-expressing GhMYB73 under NaCl or mannitol treatment was significantly enhanced during the seedling germination stage. What's more, several osmotic stress-induced genes, such as AtNHX1, AtSOS3 and AtP5CS1, were more highly induced in the over-expression lines than in wild type under salt treatment, supporting the hypothesis that GhMYB73 contributes to salinity tolerance by improving osmotic stress resistance. Arabidopsis lines over-expressing GhMYB73 had superior germination and cotyledon greening under ABA treatment, and some abiotic stress-induced genes involved in ABA pathways (AtPYL8, AtABF3, AtRD29B and AtABI5), had increased transcription levels under salt-stress conditions in these lines. Furthermore, we found that GhMYB73 physically interacts with GhPYL8 and AtPYL8, suggesting that GhMYB73 regulates ABA signaling during salinity stress response. Taken together, over-expression of GhMYB73 significantly increases tolerance to salt and ABA stress, indicating that it can potentially be used in transgenic technology approaches to improve cotton salt tolerance.


Assuntos
Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Gossypium/fisiologia , Proteínas de Plantas/genética , Estresse Salino/genética , Fatores de Transcrição/genética , Arabidopsis/genética , Inativação Gênica , Genes myb , Gossypium/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tolerância ao Sal/genética , Fatores de Transcrição/metabolismo
18.
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
19.
Zhongguo Ying Yong Sheng Li Xue Za Zhi ; 35(3): 273-278, 2019 May 28.
Artigo em Chinês | MEDLINE | ID: mdl-31257812

RESUMO

OBJECTIVE: To investigate the effects of myeloid differentiation-2 (MD2) gene silencing on high glucose-induced proliferation inhibition, apoptosis and inflammation in rat cardiomyocytes. METHODS: The immortalized rat cardiomyocyte cell line H9C2 were transfected with MD2 small interfering RNA (si-MD2) and negative control for 24 h, then stimulated with high glucose (HG) for 48 h. RT-qPCR was performed to detect the mRNA levels of MD2 and inflammatory factors TNF-α, IL-1ß and IL-6. MTS and flow cytometry were used to evaluate cell proliferation, cell cycle and apoptosis rate. Western blot was used to detect protein expression levels and phosphorylation levels. RESULTS: The mRNA and protein levels of MD2 in H9C2 cells were dramatically decreased after transfected with si-MD2 (P<0.01). After stimulation of high glucose, the mRNA levels of inflammatory factors, the cells in G0/G1 phase , the cell apoptosis rate and the protein level of cleaved Caspase-3 were significantly increased, while the cell proliferation ability was decreased (P<0.01). MD2 gene silencing antagonized the effects of high glucose on cell proliferation, cell cycle, cell apoptosis and the mRNA levels of TNF-α, IL-1ß , IL-6(P<0.05). Western blot analysis showed that the phosphorylation levels of extracellular signal-regulated kinase(ERK1/2), P38 mitogen-activated protein kinase(P38 MAPK) and C-Jun N-terminal kinase(JNK) protein were increased significantly in H9C2 cells treated with high glucose, which could be reversed by silencing of MD2 (P<0.01). CONCLUSION: This study demonstrates that MD2 gene silencing reverses high glucose-induced myocardial inflammation, apoptosis and proliferation inhibition via the mechanisms involving suppression of ERK, P38 MAPK, JNK signaling pathway.


Assuntos
Apoptose , Proliferação de Células , Inativação Gênica , Antígeno 96 de Linfócito/genética , Miócitos Cardíacos/citologia , Animais , Células Cultivadas , Citocinas/metabolismo , Glucose , Inflamação , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
Chem Commun (Camb) ; 55(56): 8170-8173, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31241120

RESUMO

Lipid-complexed small interfering RNA (siRNA) nanoparticles are promising gene regulation materials with excellent genetic, but little cellular, selectivity. Herein, we report a chemical strategy to enhance the gene silencing selectivity of these nanoparticles against cancer cells through the covalent integration of a reactive oxygen species (ROS)-degradable thioketal into the lipid nanoparticles. These lipid nanoparticles can efficiently deliver siRNA into cells, and selectively silence cancer cell gene expression in response to the high levels of intracellular ROS in cancer cells.


Assuntos
Inativação Gênica , Lipídeos/química , Nanopartículas/química , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética
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