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1.
J Ethnopharmacol ; 264: 113212, 2021 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-32768643

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Cerebral ischemia-reperfusion (CIR) injury is one of the main diseases leading to death and disability. Acanthopanax senticosus (Rupr. & Maxim.) Harms (AS), also known as Panax ginseng, has neuroprotective effects on anti-CIR injury. However, the underlying molecular mechanism of its therapeutic effects is not clear. AIM OF THE STUDY: To systematically study and explore the mechanism of Acanthopanax senticosus (Rupr. & Maxim.) Harms extract (ASE) in the treatment of CIR injury based on metabolomics and transcriptomics. MATERIALS AND METHODS: The pharmacological basis of ASE in the treatment of CIR was evaluated, and samples were used in plasma metabolomics and brain tissue transcriptomics to reveal potential biomarkers. Finally, according to online database, we analyzed biomarkers identified by the two technologies, explained reasons for the therapeutic effect of ASE, and identify therapeutic targets. RESULTS: A total of 53 differential metabolites (DMs) were identified in plasma and 3138 differentially expressed genes (DEGs) were identified in brain tissue from three groups of rats, including sham, ischemia-reperfusion (I/R), and ASE groups. Enrichment analysis showed that Nme6, Tk1, and Pold1 that are involved in the production of deoxycytidine and thymine were significantly up-regulated and Dck was significantly down-regulated by the intervention with ASE. These findings indicated that ASE participates in the pyrimidine metabolism by significantly regulating the balance between dCTP and dTTP. In addition, ASE repaired and promoted the lipid metabolism in rats, which might be due to the significant expression of Dgkz, Chat, and Gpcpd1. CONCLUSIONS: The findings of this study suggest that ASE regulates the significant changes in gene expression in metabolites pyrimidine, and lipid metabolism in CIR rats and plays an active role in the treatment of CIR injury through multiple targets and pathways.

2.
Histol Histopathol ; : 18283, 2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33215396

RESUMO

OBJECTIVE: To examine the effect of the BRD4 inhibitor JQ1 on mice with chronic obstructive pulmonary disease (COPD) via NF-κB. METHODS: COPD models constructed by exposure to cigarette smoke and intratracheal instillation of lipopolysaccharides (LPS) in mice were treated with JQ1 (15, 25 or 50 mg/kg). HE staining was performed to observe histopathological changes in the lung tissues. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of IL-10, IFN-γ, IL-17, IL-1ß, IL-6, TNF-α, MMP-2, MMP-9, MDA, SOD, T-AOC and HO-1, and gelatin zymography assays were used to examine MMP-2 and MMP-9 activity. A TransAMTM NF-κB p65 detection kit was used to test NF-κB p65/DNA binding activity. Western blotting was conducted to analyze NF-κB p65 in the nucleus and its acetylation. RESULTS: JQ1 dose-dependently improved the histopathological changes in the lung tissues and decreased the mean linear intercept (MLI), destructive index and inflammatory score of the mice with COPD. The mice with COPD showed increased levels of MMP-2, MMP-9, IFN-γ, IL-17, IL-1ß, IL-6 and TNF-α with decreased IL-10 level; these changes were reversed by JQ1 in a dose-dependent manner. In addition, JQ1 reduced the MDA level and increased the SOD, HO-1 and T-AOC levels in mice with COPD, with suppression of NF-κB p65 expression in the nucleus, NF-κB/p65 (Lys310) acetylation and NF-κB p65/DNA binding activity in the lung tissues. CONCLUSION: The BRD4 inhibitor JQ1 can downregulate MMP-2 and MMP-9 expression, reduce inflammatory responses, and alleviate oxidative stress in mice with COPD, and this mechanism might be related to the inhibition of NF-κB.

3.
J Clin Lab Anal ; 34(9): e23362, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32463132

RESUMO

PURPOSE: To explore the effects of PAK4/LIMK1/Cofilin-1 signaling pathway on the proliferation, invasion, and migration of human osteosarcoma cells. METHODS: The expression of PAK4/LIMK1/Cofilin-1 was detected by immunohistochemistry in osteosarcoma tissues. The osteosarcoma cell line MG63 was transfected and divided into Mock, Control siRNA, si-PAK4, LIMK1, and si-PAK4+LIMK1 groups. Then, the cellular biological features of MG63 cells were detected by CCK-8, wound-healing, Transwell, and flow cytometry methods. The relationship of PAK4 and LIMK1 was performed by co-immunoprecipitation test, and the protein expression of PAK4/LIMK1/Cofilin-1 was determined by Western blotting. Finally, the effect of PAK4 on the growth of osteosarcoma was verified by subcutaneous transplantation model of osteosarcoma in nude mice. RESULTS: The expression of PAK4/LIMK1/Cofilin-1 in both osteosarcoma tissues and cells was up-regulated. Positive PAK4, LIMK1, and Cofilin-1 expressions in osteosarcoma were associated with the clinical stage, distant metastasis, and tumor grade. The MG63 cell viability, migration, and invasion, as well as the expression of PAK4, p-LIMK/LIMK, and p-Cofilin-1/Cofilin-1, were restrained by the knock down of PAK4 while it promoted apoptosis. PAK4 silencing also suppressed the growth of subcutaneous transplanted tumor in nude mice. Co-immunocoprecipitation showed that LIMK and PAK4 protein can form complex in osteosarcoma cells. Besides, LIMK1 overexpression reversed the inhibition effect of PAK4 siRNA on the growth of osteosarcoma cells. CONCLUSION: The expression of PAK4/LIMK1/Cofilin-1 pathway in osteosarcoma tissues was up-regulated. Thus, PAK4 inhibition may restrict the osteosarcoma cell proliferation, invasion, and migration but promote its apoptosis via decreasing the activity of LIMK1/Cofilin-1 pathway.

4.
Sci Prog ; 103(1): 36850419891046, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31791191

RESUMO

Breast cancer, as one of the most malignant tumors, poses a serious threat to the lives of females. Nucleotide exchange factor SIL1 is an important regulator of endoplasmic reticulum function that might have a specific role in tumor progression. In this study, we aimed to investigate the effect of SIL1 on the proliferation, apoptosis, and metastasis of human breast cancer. SIL1-specific small interfering RNA was transfected into two breast cancer cell lines, MCF7 and MDA-MB-231, to generate SIL1 knockdown cells. Clone formation and Cell Counting Kit-8 assays were performed to determine cell proliferation. Wound healing and transwell assays were used to detect the cell migration and invasion, respectively. Cell cycle and apoptosis were determined by flow cytometry. The messenger RNA and protein levels of target genes were analyzed using quantitative real-time PCR and western blot. According to the results of TCGA and GTEx database analysis, we determined that SIL1 was overexpressed in 1085 breast cancer samples compared with 291 normal samples. Knockdown of SIL1 inhibited the proliferation, migration, and invasion of MCF7 and MDA-MB-231 cells, accordingly. The cell cycle was blocked at the G1 phase following transfection of SIL1-specific small interfering RNA through the inhibition of Cyclin D1, CDK4, and CDK6. SIL1 knockdown induced apoptosis and also promoted the activity of Caspase9 and Bax. Furthermore, SIL1 was able to promote phosphorylation of ERK1/2. Based on these results, SIL1 might act as an oncogene and accelerate the progression of human breast cancer.

5.
J Phys Chem A ; 124(1): 82-89, 2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31815471

RESUMO

A new terbium (III) luminescent compound {[Tb2(PDC)2(ox)(H2O)4](H2O)2}n was synthesized by the self-assembly of Tb3+ ions with 3,5-pyridinedicarboxylate (PDC) and oxalate (ox) ligands and characterized by fluorescence spectroscopy and single-crystal X-ray diffraction. The density functional theory (DFT) and high-level correlated ab initio wave function methods with Spin-Orbit Coupling correction (CASSCF/SO and CAS-NEVPT2/SOC) were successfully applied to predict the absorption and emission spectra of this strongly correlated lanthanide system in excellent agreement with the experimental results.

6.
J Asian Nat Prod Res ; 22(4): 303-315, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30843729

RESUMO

Three new saponins (1-3), a new natural product (4) and six other known compounds (5-10) were isolated from the whole Reineckia carnea plant. Their structures were established by comparison of their NMR spectra and MS data with literature data. In addition, all the isolated compounds were evaluated in vitro for anti-inflammatory activities against LPS-stimulated nitric oxide (NO) production in RAW 264.7 macrophages. Compounds 1-4 exhibited anti-inflammatory activities with IC50 values of 37.5 µM, 31.4 µM, 34.6 µM, and 56.1 µM, respectively. Furthermore, compounds 5-10 showed anti-inflammatory activities with IC50 values ranging from 20.3 to 42.9 µM.


Assuntos
Anti-Inflamatórios , Saponinas , Lipopolissacarídeos , Macrófagos , Estrutura Molecular , Óxido Nítrico , Extratos Vegetais
7.
J Cell Mol Med ; 23(8): 4991-5005, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31207106

RESUMO

Melanoma is one of the most malignant skin tumours with constantly increasing incidence worldwide. Previous studies have demonstrated that microRNA-374 (miR-374) is a novel biomarker for cancer therapy. Therefore, this study explores whether miR-374 targeting tyrosinase (TYR) affects melanoma and its underlying mechanism. We constructed subcutaneous melanoma models to carry out the following experiments. The cells were transfected with a series of miR-374 mimics, miR-374 inhibitors or siRNA against TYR. Dual luciferase reporter gene assay was used for the verification of the targeting relationship between miR-374 and TYR. Reverse transcription quantitative polymerase chain reaction and western blot analysis were conducted to determine the expression of miR-374, TYR, ß-catenin, B-cell leukaemia 2 (Bcl-2), Bcl-2 associated X protein (Bax), Low-density lipoprotein receptor-related protein 6 (LRP6), Leucine-rich repeat G protein-coupled receptor 5 (LGR5) and CyclinD1. Cell proliferation, migration, invasion, cell cycle distribution and apoptosis were evaluated using cell counting kit-8 assay, scratch test, transwell assay and flow cytometry respectively. TYR was proved as a putative target of miR-374 as the evidenced by the result. It was observed that up-regulated miR-374 or down-regulated TYR increased expression of Bax and decreased expressions of TYR, ß-catenin, LRP6, Bcl-2, CyclinD1 and LGR5, along with diminished cell proliferation, migration, invasion and enhanced apoptosis. Meanwhile, cells with miR-374 inhibitors showed an opposite trend. These findings indicated that up-regulated miR-374 could inhibit the expression of TYR to suppress cell proliferation, migration, invasion and promote cell apoptosis in melanoma cells by inhibiting the Wnt signalling pathway.

8.
Cells ; 8(6)2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31163575

RESUMO

Two unrecognizable strains of the same bacterial species form a distinct colony boundary. During growth as colonies, Myxococcus xanthus uses multiple factors to establish cooperation between recognized strains and prevent interactions with unrecognized strains of the same species. Here, ΔMXAN_0049 is a mutant strain deficient in immunity for the paired nuclease gene, MXAN_0050, that has a function in the colony-merger incompatibility of Myxococcus xanthus DK1622. With the aim to investigate the factors involved in boundary formation, a proteome and metabolome study was employed. Visualization of the boundary between DK1622 and ΔMXAN_0049 was done scanning electron microscope (SEM), which displayed the presence of many damaged cells in the boundary. Proteome analysis of the DK1622- boundary disclosed many possible proteins, such as cold shock proteins, cell shape-determining protein MreC, along with a few pathways, such as RNA degradation, phenylalanine, tyrosine and tryptophan biosynthesis, and Type VI secretion system (T6SS), which may play major roles in the boundary formation. Metabolomics studies revealed various secondary metabolites that were significantly produced during boundary formation. Overall, the results concluded that multiple factors participated in the boundary formation in M. xanthus, leading to cellular damage that is helpful in solving the mystery of the boundary formation mechanism.


Assuntos
Metabolômica/métodos , Myxococcus xanthus/crescimento & desenvolvimento , Myxococcus xanthus/metabolismo , Proteômica/métodos , Sistemas de Secreção Bacterianos , Contagem de Colônia Microbiana , Regulação para Baixo , Viabilidade Microbiana , Myxococcus xanthus/ultraestrutura , Mapas de Interação de Proteínas , Proteoma/metabolismo , Metabolismo Secundário , Regulação para Cima
9.
Microb Biotechnol ; 12(4): 763-774, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31069998

RESUMO

Glycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti-tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC-subbranch with 160 GT sequences. We demonstrated that the YjiC-subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC-subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high-active GTs (Q66 and P77) but changed in two low-efficient GTs. Site-directed residues swapping at the two positions in the two low-active GTs (BssGT and BamGT) and the high-active GT BsGT-1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.


Assuntos
Epotilonas/metabolismo , Glicosiltransferases/metabolismo , Moduladores de Tubulina/metabolismo , Biotransformação , Domínio Catalítico , Sequência Conservada , Glicosilação , Glicosiltransferases/classificação , Glicosiltransferases/genética , Cinética , Simulação de Acoplamento Molecular , Filogenia , Alinhamento de Sequência
10.
Sci Rep ; 8(1): 15070, 2018 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-30305664

RESUMO

Polarization imaging detection has its unique advantage in discriminating the man-made objects from natural objects. Grating integrated super pixel for polarization imaging detection can simultaneously obtain the first three elements of the Stokes vector, which is the trend of infrared polarization imaging detection in recent years. Here, we demonstrate the first super pixel for long wavelength infrared polarization imaging detection with the extinction ratio of its four polarization directions more than 100. The measured highest polarization extinction ratio is as high as 136, which is the highest reported value of long wavelength infrared polarization imaging detection super pixel. The mechanism is attributed to the excellent mode selectivity of plasmonic microcavity according to the results of three-dimensional theoretical simulation. The experimental responses of the super pixel with four polarization directions are in good agreement with the Malus' Law. In addition, the super pixel can accurately resolve the Stokes parameters at the same time. It is expected to develop the super pixel into a new generation of practical high-polarization-discriminating long wavelength infrared focal plane array.

11.
Nanomaterials (Basel) ; 8(9)2018 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-30200542

RESUMO

MP2 (Second order approximation of Møller⁻Plesset perturbation theory) and DFT/TD-DFT (Density functional theory/Time-dependent_density_functional_theory) investigations have been performed on metallophilic nanomaterials of host clusters [Au(NHC)2]⁺⋅⋅⋅[M(CN)2]-⋅⋅⋅[Au(NHC)2]⁺ (NHC = N-heterocyclic carbene, M = Au, Ag) with high phosphorescence. The phosphorescence quantum yield order of clusters in the experiments was evidenced by their order of µS1/ΔES1-T1 values ( µ S 1 : S0 → S1 transition dipole, ∆ E S 1 - T 1 : splitting energy between the lowest-lying singlet S1 and the triplet excited state T1 states). The systematic variation of the guest solvents (S1: CH3OH, S2: CH3CH2OH, S3: H2O) are employed not only to illuminate their effect on the metallophilic interaction and phosphorescence but also as the probes to investigate the recognized capacity of the hosts. The simulations revealed that the metallophilic interactions are mainly electrostatic and the guests can subtly modulate the geometries, especially metallophilic Au⋅⋅⋅M distances of the hosts through mutual hydrogen bond interactions. The phosphorescence spectra of hosts are predicted to be blue-shifted under polar solvent and the excitation from HOMO (highest occupied molecular orbital) to LUMO (lowest unoccupied molecular orbital) was found to be responsible for the ³MLCT (triplet metal-to-ligand charge transfer) characters in the hosts and host-guest complexes. The results of investigation can be introduced as the clues for the design of promising blue-emitting phosphorescent and functional materials.

12.
Appl Microbiol Biotechnol ; 102(13): 5599-5610, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29705958

RESUMO

Promoter optimization is an economical and effective approach to overexpress heterologous genes and improve the biosynthesis of valuable products. In this study, we swapped the original promoter of the epothilone biosynthetic gene cluster in Myxococcus xanthus with two endogenous strong promoters P pilA and P groEL1 , respectively, which, however, decreased the epothilone production ability. The transcriptional abilities by the two promoters were found to be bloomed in the growth stage but markedly decreased after the growth, whereas the original promoter P epo functioned majorly after the exponential growth stage. Tandem repeat engineering on the original promoter P epo remarkably increased epothilone production. The tandem promoter exerted similar expressional pattern as P epo did in M. xanthus. We demonstrated that differential transcriptional modes markedly affected the efficiency of promoters in controlling the gene expressions for the production of the secondary metabolite epothilones. Our study provides an insight into exploiting powerful promoters to produce valuable secondary metabolites, especially in host with limited known promoters.


Assuntos
Epotilonas/biossíntese , Microbiologia Industrial , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Regiões Promotoras Genéticas/genética , Transcrição Genética , Família Multigênica/genética
13.
J Pharm Biomed Anal ; 154: 236-244, 2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-29558724

RESUMO

The Chimonanthus nitens Oliv. leaf (CNOL). is a widely used traditional Chinese medicine (TCM) used for treating colds and influenza. In the present study, a comprehensive strategy integrating multiple chromatographic analysis and chemometric methods was firstly proposed for structural characterization and discrimination of CNOL from different geographical origins. It consists of three steps: Firstly, the ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) method was applied for comprehensive profiling of characterization constituents in CNOL by high-resolution diagnostic product ions/neutral loss filtering, and a total of 40 constituents were identified. Secondly, chemical fingerprints were established by HPLC coupled with photodiode array detector (PDA), and similarity analyses were calculated based on nineteen common characteristic peaks. Subsequently, the nine major constituents, including coumarins, flavonoids, and phenolic acids were quantified, and the quantitative data further analyzed by principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). Thirdly, a hot map visualization was conducted for clarifying the distribution of major compounds among different geographical origins. Also, nine constituents detected could be used as chemical markers for discrimination of CNOL from different provinces. Collectively, these results indicated that our proposed platform was a powerful tool for chemical profiling and discrimination of herbs with multiple botanical origins, providing promising perspectives in tracking the formulation process of TCMs products.


Assuntos
Calycanthaceae/química , Medicamentos de Ervas Chinesas/química , Folhas de Planta/química , Cromatografia Líquida de Alta Pressão/métodos , Cumarínicos/química , Análise Discriminante , Flavonoides/química , Hidroxibenzoatos/química , Medicina Tradicional Chinesa/métodos , Análise de Componente Principal/métodos , Espectrometria de Massas em Tandem/métodos
14.
Molecules ; 23(3)2018 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-29495641

RESUMO

Ziyuglycoside I (ZGS1) is a promising drug candidate for the treatment of leucopenia. Currently, information on ZGS1 and its in vivo metabolite ziyuglycoside II (ZGS2) is limited. The objective of this study was to investigate the pharmacokinetics, tissue distribution, and excretion of ziyuglycoside I (ZGS1) and its metabolite ziyuglycoside II (ZGS2) in rats. In our study, a simple and sensitive high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method was established for simultaneous determination of ZGS1 and its metabolite for Sprague-Dawley rat pharmacokinetics studies. The method was validated following internationally-approved guidelines. The results presented in this study indicated that subcutaneous administration of ZGS1 prolonged its extension time and increased the area under the curve (AUC0-t) of ZGS2 during 0 to t minutes. In summary, in this study, the pharmacokinetic characteristics of ZGS1 and its metabolite ZGS2 were defined and its tissue distribution, and excretion in rats were described. Our finding may be beneficial for leucopenia drug that focus on ZGS1.


Assuntos
Cromatografia Líquida de Alta Pressão , Saponinas/farmacocinética , Espectrometria de Massas em Tandem , Animais , Estrutura Molecular , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise Espectral , Distribuição Tecidual
15.
Microb Cell Fact ; 17(1): 15, 2018 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-29378572

RESUMO

BACKGROUND: The CRISPR/dCas9 system is a powerful tool to activate the transcription of target genes in eukaryotic or prokaryotic cells, but lacks assays in complex conditions, such as the biosynthesis of secondary metabolites. RESULTS: In this study, to improve the transcription of the heterologously expressed biosynthetic genes for the production of epothilones, we established the CRISPR/dCas9-mediated activation technique in Myxococcus xanthus and analyzed some key factors involving in the CRISPR/dCas9 activation. We firstly optimized the cas9 codon to fit the M. xanthus cells, mutated the gene to inactivate the nuclease activity, and constructed the dCas9-activator system in an epothilone producer. We compared the improvement efficiency of different sgRNAs on the production of epothilones and the expression of the biosynthetic genes. We also compared the improvement effects of different activator proteins, the ω and α subunits of RNA polymerase, and the sigma factors σ54 and CarQ. By using a copper-inducible promoter, we determined that higher expressions of dCas9-activator improved the activation effects. CONCLUSIONS: Our results showed that the CRISPR/dCas-mediated transcription activation is a simple and broadly applicable technique to improve the transcriptional efficiency for the production of secondary metabolites in microorganisms. This is the first time to construct the CRISPR/dCas9 activation system in myxobacteria and the first time to assay the CRISPR/dCas9 activations for the biosynthesis of microbial secondary metabolites.


Assuntos
Sistemas CRISPR-Cas/genética , Epotilonas/biossíntese , Família Multigênica , Myxococcus xanthus/genética , Proteínas Recombinantes/genética , Transcrição Genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Epotilonas/genética , Myxococcus xanthus/metabolismo , Regiões Promotoras Genéticas , RNA Guia/genética , Metabolismo Secundário , Ativação Transcricional
16.
J Biomater Sci Polym Ed ; 29(6): 663-682, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29375018

RESUMO

Abdominal wall defects are a common medical problem, and inadequate repair methods can lead to serious complications. Abdominal wall reconstruction using autologous tissue, or non-biological, biological, or composite patches is often performed to repair defective areas. In particular, composite patches containing both polymeric and biological materials have gained increasing attention due to their good mechanical properties and biocompatibility. However, it is still unclear whether the quality of repairs using composite patches is superior to that of a biological patch. Based on the limitations of previous studies, we compared small intestinal submucosa (SIS) patches with SIS + polypropylene mesh (PPM) patches for repairing abdominal wall defects in adult beagle dogs. Forty-five female dogs were subjected to surgical resection to produce abdominal wall defects. SIS or SIS + PPM was used as patch for the defects. Morphology, biomechanics, and histological evaluations were performed to evaluate the efficacy and safety of such therapies. Our findings demonstrated that SIS had advantages over SIS + PPM considering biological activity and histocompatibility without increasing the risk of repair failure.


Assuntos
Parede Abdominal/cirurgia , Intestino Delgado/citologia , Polipropilenos/farmacologia , Telas Cirúrgicas , Adesividade , Animais , Materiais Biocompatíveis/farmacologia , Cães , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Resistência à Tração
17.
Oncol Lett ; 14(4): 4106-4114, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28943917

RESUMO

Malignant melanoma (MM) is a highly malignant skin tumor. The mechanism of MM pathogenesis and its signaling pathways are not well characterized. C-X-C chemokine receptor type 7 (CXCR7) has been reported to regulate cancer cell invasion. The present study sought to investigate the effects of CXCR7 on MM development. First, CXCR7 expression levels were assessed in the skin tumor tissue of patients with MM. Then, CXCR7 small hairpin RNA was used in M14 melanoma cells in a Transwell culture model and in a transplanted mouse model to test the effects of CXCR7. In addition, immunohistochemistry staining, reverse transcription-quantitative polymerase chain reaction and western blotting were used. The results revealed that CXCR7 expression levels were significantly higher in MM tissue compared with squamous cell carcinoma or basal cell carcinoma tissue. Knocking down CXCR7 in M14 cells significantly inhibited cell migration and invasion in the Transwell culture model. Furthermore, CXCR7 knockdown also significantly reduced the transplanted tumor size, weight and vascular number in the mouse model. It was concluded that CXCR7 interacts with C-X-C motif chemokine ligand 12 to activate the chemokine receptor signaling pathway, and to increase melanoma cell migration, invasion and development.

18.
Microb Cell Fact ; 16(1): 142, 2017 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-28814300

RESUMO

BACKGROUND: The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. RESULTS: In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA-sgRNA-tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. CONCLUSIONS: By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.


Assuntos
Sistemas CRISPR-Cas/genética , Genes Bacterianos , Myxococcus xanthus/genética , Sequência de Bases , Família Multigênica , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , RNA de Transferência/genética , Deleção de Sequência
19.
Biosci Rep ; 37(5)2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-28724603

RESUMO

The study aims to evaluate the effects of miR-136 on the proliferation, apoptosis, and epithelial-mesenchymal transition (EMT) of melanoma cells by targetting premelanosome protein (PMEL) through the Wnt signaling pathway. After establishment of melanoma mouse models, melanoma (model group) and normal tissues (normal group) were collected. Immunohistochemistry was performed to determine PMEL protein concentration. Mouse melanoma cells were assigned into control, blank, negative control (NC), miR-136 mimics, miR-136 inhibitors, siRNA-PMEL, and miR-136 inhibitors + siRNA-PMEL, LiC1 (Wnt signaling pathway activator), and siRNA-PMEL+ LiCl groups. MTT, Scratch test, Transwell assay, and flow cytometry were performed to measure cell proliferation, migration, invasion, and apoptosis. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to evaluate miR-136, PMEL, ß-catenin, Wnt3a, Bcl-2, Bax, Caspase, E-cadherin, and N-cadherin expressions. PMEL is highly expressed in melanoma tissues. MiR-136, Bax, Caspase, and E-cadherin expressions decreased in the model group, whereas PMEL, ß-catenin, Bcl-2, Wnt3a, and N-cadherin expressions increased. Bax, Caspase, and E-cadherin expressions increased in the miR-136 mimics and siRNA-PMEL groups, whereas the expressions decreased in the miR-136 inhibitors group and LiC1 group. PMEL, ß-catenin, Bcl-2, Wnt3a, and N-cadherin expressions, cell proliferation, migration, and invasion decreased, and the apoptosis rate inceased in the miR-136 mimics and siRNA-PMEL groups; whereas the tendencies were opposite to those in the miR-136 inhibitors group and LiC1 group. In the siRNA-PMEL+ LiCl group, PMEL expression decreased. These findings indicated that overexpression of miR-136 inhibits melanoma cell EMT, proliferation, migration, invasion, and promotes apoptosis by targetting PMEL through down-regulation of the Wnt signaling pathway.


Assuntos
Transição Epitelial-Mesenquimal , Melanoma/genética , MicroRNAs/genética , Neoplasias Cutâneas/genética , Via de Sinalização Wnt , Antígeno gp100 de Melanoma/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Masculino , Melanoma/metabolismo , Melanoma/patologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , MicroRNAs/metabolismo , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Antígeno gp100 de Melanoma/metabolismo
20.
Biosci Rep ; 37(4)2017 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-28679648

RESUMO

The present study investigated the effects of microRNA-374 (miR-374) on human squamous cell carcinoma (SCC) cell proliferation, migration, invasion, and apoptosis through P53 signaling pathway by targeting growth arrest and DNA-damage-inducible protein 45 α (Gadd45a). Skin samples were collected from patients with skin SCC and normal skin samples. Expression of miR-374, Gadd45a, P53, P73, P16, c-myc, bcl-2, Bax, caspase-3, and caspase-9 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. A431 and SCL-1 cells were divided into blank, negative control (NC), miR-374 mimics, miR374 inhibitors, siRNA-Gadd45a, and miR-374 inhibitors + siRNA-Gadd45a groups. Their proliferation, migration, invasion, cell cycle, and apoptosis were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, scratch test, Transwell assay, and flow cytometry. SCC skin tissues exhibited decreased expression of miR-374, P73, P16, Bax caspase-3 and caspase-9, and increased levels of Gadd45a, P53, c-myc, and Bcl-2 compared with the normal skin tissues. The miR-374 inhibitors group exhibited decreased expression of miR-374, P73, P16, Bax caspase-3 and caspase-9, and increased expression of Gadd45a, P53, c-myc, and Bcl-2, enhanced cell proliferation, migration, and invasion, and reduced apoptosis compared with the blank and NC groups; the miR-374 mimics group followed opposite trends. Compared with the blank and NC groups, the miR-374 inhibitors + siRNA-Gadd45a group showed decreased miR-374 level; the siRNA-Gadd45a group showed elevated levels of P73, P16, Bax, caspase-3 and caspase-9, decreased levels of Gadd45a, P53, c-myc, and Bcl-2, reduced cell proliferation, migration, and invasion, and accelerated apoptosis. miR-374 induces apoptosis and inhibits proliferation, migration, and invasion of SCC cells through P53 signaling pathway by down-regulating Gadd45a.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Ciclo Celular/metabolismo , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas Nucleares/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteína Supressora de Tumor p53/genética
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