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1.
Int J Mol Sci ; 24(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36614257

RESUMO

Osteoarthritis (OA) is a degenerative disease of articular cartilage that is mainly characterized by chronic and mild inflammation of the joints. Recently, many studies have reported the crucial roles of long noncoding RNAs (lncRNAs) in OA as gene transcriptional regulatory factors, diagnostic biomarkers, or therapeutic targets. However, the exact mechanisms of lncRNAs in the regulation of OA progression remain unclear. In the present study, the lncRNA WDR11 divergent transcript (lncRNA WDR11-AS1) was shown to be downregulated in osteoarthritic cartilage tissues from patients, and to promote extracellular matrix (ECM) synthesis in osteoarthritic chondrocytes with knockdown and overexpression experiments. This function of lncRNA WDR11-AS1 was linked to its ability to interact with the polyadenylate-binding protein cytoplasmic 1 (PABPC1), which was screened by RNA pulldown and mass spectrometry analyses. PABPC1 was discovered to bind ECM-related mRNAs such as SOX9, and the inhibition of PABPC1 improved the mRNA stability of SOX9 to mitigate OA progression. Our results suggest that lncRNA WDR11-AS1 has a promising inhibitory effect on inflammation-induced ECM degradation in OA by directly binding PABPC1, thereby establishing lncRNA WDR11-AS1 and PABPC1 as potential therapeutic targets in the treatment of OA.


Assuntos
Cartilagem Articular , MicroRNAs , Osteoartrite , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , MicroRNAs/genética , Osteoartrite/genética , Osteoartrite/metabolismo , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Cartilagem Articular/metabolismo , Inflamação/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo
2.
J Pharm Anal ; 12(4): 653-663, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36105166

RESUMO

MicroRNA-21 (miRNA-21) is highly expressed in various tumors. Small-molecule inhibition of miRNA-21 is considered to be an attractive novel cancer therapeutic strategy. In this study, fluoroquinolone derivatives A1-A43 were synthesized and used as miRNA-21 inhibitors. Compound A36 showed the most potent inhibitory activity and specificity for miRNA-21 in a dual-luciferase reporter assay in HeLa cells. Compound A36 significantly reduced the expression of mature miRNA-21 and increased the protein expression of miRNA-21 target genes, including programmed cell death protein 4 (PDCD4) and phosphatase and tensin homology deleted on chromosome ten (PTEN), at 10 µM in HeLa cells. The Cell Counting Kit-8 assay (CCK-8) was used to evaluate the antiproliferative activity of A36; the results showed that the IC50 value range of A36 against six tumor cell lines was between 1.76 and 13.0 µM. Meanwhile, A36 did not display cytotoxicity in BEAS-2B cells (lung epithelial cells from a healthy human donor). Furthermore, A36 significantly induced apoptosis, arrested cells at the G0/G1 phase, and inhibited cell-colony formation in HeLa cells. In addition, mRNA deep sequencing showed that treatment with A36 could generate 171 dysregulated mRNAs in HeLa cells, while the expression of miRNA-21 target gene dual-specificity phosphatase 5 (DUSP5) was significantly upregulated at both the mRNA and protein levels. Collectively, these findings demonstrated that A36 is a novel miRNA-21 inhibitor.

3.
J Bone Miner Metab ; 40(6): 914-926, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36156740

RESUMO

INTRODUCTION: Selenium (Se) as well as selenoproteins are vital for osteochondral system development. Se deficiency (SeD) has a definite impact on the expression and activity of histone deacetylases (HDACs). Abnormal expression of some HDACs affects cartilage development. This current study aims to explore the relationship between differentially expressed HDACs and cartilage development, especially extracellular matrix (ECM) homeostasis maintenance, under SeD conditions. MATERIALS AND METHODS: Dark Agouti rats and C28/I2 cell line under SeD states were used to detect the differently expressed HDAC by RT-qPCR, western blotting and IHC staining. Meanwhile, the biological roles of the above HDAC in cartilage development and homeostasis maintenance were confirmed by siRNA transfection, western blotting, RNA sequence and inhibitor treatment experiments. RESULTS: HDAC2 exhibited lower expression at protein level in both animals and chondrocytes during SeD condition. The results of cell-level experiments indicated that forkhead box O3A (FOXO3A), which was required to maintain metabolic homeostasis of cartilage matrix, was reduced by HDAC2 knockdown. Meanwhile, induced HDAC2 was positively associated with FOXO3A in rat SeD model. Meanwhile, knockdown of HDAC2 and FOXO3A led to an increase of intracellular ROS level, which activated NF-κB pathway. Se supplementary significantly inhibited the activation of NF-κB pathway with IL-1ß treatment. CONCLUSION: Our results suggested that low expression of HDAC2 under SeD condition increased ROS content by decreasing FOXO3A in chondrocytes, which led to the activation of NF-κB pathway and ECM homeostasis imbalance.


Assuntos
Proteína Forkhead Box O3 , Histona Desacetilase 2 , Selênio , Animais , Ratos , Cartilagem , Matriz Extracelular , Histona Desacetilase 2/genética , NF-kappa B , Espécies Reativas de Oxigênio , Selênio/farmacologia , Proteína Forkhead Box O3/genética
4.
Bioorg Med Chem ; 66: 116803, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35561631

RESUMO

MicroRNA-21 is a carcinogenic microRNA, whose overexpression arises in a variety of tumor tissues. Hence, microRNA-21 a prospective target for cancer treatment, and regulation of microRNA-21 by small molecule inhibitors is deemed as a promising approach for tumor therapy. In this work, to discover potent microRNA-21 inhibitor, series of 4-(N-norfloxacin-acyl)aminobenzamides were designed and synthesized, and their inhibitory effects were appraised by utilizing dual luciferase reporter assays. The results indicated that compound A7 was the most efficient microRNA-21 small molecule inhibitor. What's more, A7 suppressed the migration of Hela cells and the colony formation of Hela and HCT-116 cells as well as promoted apoptosis of Hela cells. In the mechanism study, results of RT-qPCR certified that A7 could reduce the level of mature microRNA-21 via disrupting its expression at the transcriptional level of its primary form "pri-miR-21", which was distinct from most previous inhibitors directly binding with pre-miR-21. Noticeably, Western blotting and RT-qPCR uncovered A7 could upregulate the expression PTEN, EGR1 and SLIT2, which are the downstream functional targets of microRNA-21. These findings demonstrated that A7 was a promising microRNA-21 small molecule inhibitor and 4-(N-norfloxacin-acyl) aminobenzamide can serve as a new scaffold for discovery of potent microRNA-21 inhibitor.


Assuntos
Antineoplásicos , Benzamidas , MicroRNAs , Norfloxacino , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proliferação de Células , Células HCT116 , Células HeLa , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Norfloxacino/farmacologia
5.
Clin Immunol ; 220: 108579, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32866644

RESUMO

Endoplasmic reticulum (ER) stress associated proteins contribute to the pathogenesis of rheumatoid arthritis (RA) through affecting synoviocyte proliferation and proinflammatory cytokine production. The role of DERL3, an ER-associated degradation component, in joint inflammation of RA was explored. Synovial tissues from RA and osteoarthritis (OA) patients were collected, and in RA synovial tissue, DERL3 showed up-regulation and significantly positive correlation with the expression of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and matrix metalloproteinase (MMP)-1. Immunofluorescence result suggested DERL3 was located in fibroblast-like synoviocytes (FLS). Among different inflammatory stimuli, DERL3 could be up-regulated by TNF-α stimulation in FLS. Under TNF-α stimulation, knocking down DERL3, the expression of IL-6, IL-8, MMP-1, MMP-13 was reduced and the activation of nuclear factor kappa B (NF-κB) signaling pathway was inhibited. In pristane-induced arthritis (PIA) rat model, Derl3 was up-regulated in synovial tissue and disease was attenuated after intraarticular injection of siDerl3. Overall, we conclude that TNF-α inducing DERL3 expression promotes the inflammation of FLS through activation of NF-κB signaling pathway, suggesting DERL3 plays important roles in the pathogenesis of RA and is a promising therapeutic target.


Assuntos
Artrite Reumatoide/imunologia , Proteínas de Membrana/imunologia , Sinoviócitos/imunologia , Idoso , Animais , Artrite Experimental/imunologia , Células Cultivadas , Citocinas/imunologia , Feminino , Humanos , Masculino , Metaloproteinase 1 da Matriz/imunologia , Metaloproteinase 13 da Matriz/imunologia , Camundongos , Pessoa de Meia-Idade , NF-kappa B/imunologia , Osteoartrite/imunologia , Ratos , Transdução de Sinais
6.
J Immunol ; 205(1): 181-192, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32503893

RESUMO

Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis. We hypothesize that Pkm2, as a key regulatory enzyme of glycolysis pathway, triggers the activation of macrophages (Mφ), which results in proinflammatory cytokine production during the arthritis progress. In this study, Pkm2 was found to be overexpressed in ED1-positive Mφ in spleens and synovial tissues from arthritic rats via immunofluorescence, Western blotting, and quantitative RT-PCR. To reveal the role of Pkm2, Dark Agouti rats were treated with either Pkm2 enzyme inhibitor shikonin or the RNA interference plasmids of Pkm2 and negative control plasmids, respectively, via i.p. injection. Pkm2 intervention could alleviate the severity of pristane-induced arthritis in aspects of the macroscopic arthritis score, perimeter changes of midpaw, and the synovitis and destruction of the bone and cartilage as well as reduce the ED1 and p-Stat1-positive cell population in rat synovial tissues. Silencing Pkm2 by RNA interference in classical activated rat and mouse Mφ resulted in less Tnf-α, Il-1ß production via Stat1 signaling. Collectively, Pkm2 is highly expressed in ED1-positive Mφ of spleens and synovial tissues from arthritic rats and promotes Mφ activation via Stat1 signaling. Pkm2 might be a promising selective metabolic target molecule for rheumatoid arthritis treatment.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Macrófagos/imunologia , Piruvato Quinase/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Artrite Experimental/diagnóstico , Artrite Experimental/patologia , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/patologia , Técnicas de Silenciamento de Genes , Humanos , Macrófagos/metabolismo , Camundongos , Naftoquinonas/administração & dosagem , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Células RAW 264.7 , RNA Interferente Pequeno/metabolismo , Ratos , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologia
7.
BMC Med Genet ; 20(1): 96, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151434

RESUMO

BACKGROUND: The highly conservative miR-15/107 family (also named as miR-15/107 gene group) including ten miRNA members is currently recognized strongly implicated in multiple human disorders. Some studies focus on the entire family rather than individual miRNA for a bigger picture, while there is also certain signature dysregulation for some of the individual miRNA implicated even in the same disorder. METHODS: Faced with the exponential growth of experimental evidence, our study tries to analyze their function and target interactions using various bioinformatics tools. RESULTS: Firstly, the evolutionary conservative "AGCAGC" sequence and possible clustered transcriptional pattern were described. Secondly, both the experimentally validated and bioinformatically predicted miRNA-target gene relationship of the entire family was analyzed to understand the mechanism of underlying collective effects for target regulation from the miR-15/107 family. Moreover, pathway analysis among miR-15/107 family was performed and displayed in detail, while its impact on cell proliferation is experimentally validated. Eventually, the dysregulation of miR-15/107 in diseases was discussed. CONCLUSIONS: In summary, our study proposes that the collective functions and implication of miR-15/107 family in various human diseases are achieved relying on the massive overlapping target genes. While the minor differences within target gene interaction among family members could also explain the signature behavior for some of the individual miRNA in aspects such as its disease-specific dysregulation and various participation in pathways.


Assuntos
Epistasia Genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes , MicroRNAs/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Análise por Conglomerados , Biologia Computacional/métodos , Predisposição Genética para Doença/genética , Humanos , Família Multigênica , Transdução de Sinais/genética
8.
Biotechnol Appl Biochem ; 66(5): 755-762, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31021480

RESUMO

The therapeutic potential of microRNA-21 (miR-21) small-molecule inhibitors has been of particular interest to medicinal chemists. Moreover, the development of more facile screening methods is lacking. In the present study, two potential screening strategies for miR-21 small-molecule inhibitor including the stem-loop reverse transcription-quantitative PCR and dual luciferase reporter assay system were demonstrated and discussed in detail. A pmirGLO-miR21cswt plasmid and its two different mutants were constructed for dual luciferase reporter assay system. In addition, the sensitivity and specificity of these two methods were validated. Our results demonstrated that both strategies are decent choices for the screening of small-molecule inhibitors for miR-21 and possibly other miRNAs. Eventually, we applied our optimized strategy to discover and characterize several promising compounds such as azobenzene derivate A, enoxacin, and norfloxacin for their potential impact on intracellular miR-21 concentration.


Assuntos
Genes Reporter/efeitos dos fármacos , Luciferases de Vaga-Lume/antagonistas & inibidores , MicroRNAs/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Bibliotecas de Moléculas Pequenas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/genética , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Células Tumorais Cultivadas
9.
Int J Mol Med ; 42(6): 3503-3512, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30272322

RESUMO

SRY­box 9 (SOX9) is the master regulator of the chondrocyte phenotype, which is essential for differentiating chondrogenic mesenchymal condensations into chondrocytes, and is involved in regulating every stage of chondrocyte differentiation. SOX9 deletion in chondrocytes at the late stages of cartilage development results in decreased chondrocyte proliferation; inhibited expression of cartilage matrix genes, including Indian hedgehog and the downstream parathyroid hormone­related protein; and premature conversion of proliferating chondrocytes into hypertrophic chondrocytes, which mineralize their matrix prematurely. Therefore, SOX9 is considered vital for the majority of phases of chondrocyte lineage, from early condensations to the differentiation of proliferating chondrocytes, leading to chondrocyte hypertrophy. It has been reported that SOX9 expression is decreased in osteoarthritis (OA) cartilage. Regeneration or repair of cartilage degradation in OA remains a challenge. Previous studies have indicated that overexpression of SOX9 can promote cartilage repair and can be used as a potential therapeutic agent at the early stages of human OA. The present study identified Scm­like with four malignant brain tumor domains 2 (SFMBT2) as a novel regulator of SOX9 expression in human chondrocytes. Our previous study revealed that SFMBT2 is negatively regulated in OA cartilage, and decreased levels of SFMBT2 contribute to the catabolic phenotype of chondrocytes. The present study detected increased expression levels of SFMBT2 in early cartilage development and during the early phases of chondrogenesis. Overexpression of SFMBT2 in C28/I2 cells upregulated SOX9 expression in a dose­dependent manner. Furthermore, SFMBT2 positively regulated C28/I2 cell proliferation and restored the decreased levels of SOX9 in chondrocytes following tumor necrosis factor­α treatment. Additional studies may reveal novel insights into the molecular mechanism involved and the potential role of SFMBT2 in cartilage repair and OA management.


Assuntos
Condrócitos/citologia , Condrócitos/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOX9/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Repressoras/genética , Fatores de Transcrição SOX9/genética , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
10.
J Cell Mol Med ; 22(11): 5753-5758, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30133133

RESUMO

The interplay between anabolic and catabolic factors regulates cartilage matrix homoeostasis. In OA, this balance is disrupted which results in cartilage degradation involving a plethora of inflammatory factors. Here, we identify a novel gene "Scm-like with four MBT domains protein 2" (SFMBT2) negatively regulated in OA cartilage. Articular cartilage from human OA patients undergoing knee arthroplasty surgery exhibited significantly decreased levels of SFMBT2 compared to the normal controls. Down-regulation of SFMBT2 by specific siRNA disturbed the metabolic homoeostasis and led to decreased expression of anabolic genes (SOX9, COL2A1) while increasing the expression of catabolic genes (MMP13 and ADAMTS4), in human chondrocytes. Finally, we revealed that SFMBT2 intervention by siRNA contributed to the catabolic phenotype of human chondrocytes mediated by NF-kB pathway.


Assuntos
Artroplastia do Joelho , Cartilagem Articular/metabolismo , Osteoartrite/genética , Proteínas Repressoras/genética , Adulto , Idoso , Cartilagem Articular/patologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/metabolismo , Osteoartrite/fisiopatologia , Osteoartrite/cirurgia , RNA Interferente Pequeno/genética , Fatores de Transcrição SOX9/genética
11.
Cell Death Dis ; 9(6): 699, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899528

RESUMO

Osteoarthritis (OA) is the most common form of arthritis involving major structural changes of peripheral joints and local or systemic inflammation and in lack of therapeutic approaches because of complexity of underlying molecular basis. Our previous work showed that HS6ST2, an enzyme involved in the transfer of sulfate, is downregulated in cartilage tissues of OA patients compared with normal donors, but little is known about its regulatory mechanism. In this study, we demonstrated that the expression of HS6ST2 was lower in OA-damaged cartilage than smooth cartilage from the same patient. In chondrocytes, HS6ST2 could be targeted by miR-23b-3p, which was higher expressed in OA-damaged cartilage. Under TNF-α stimulation, the expression of HS6ST2 was found inversely correlated with the expression of miR-23b-3p. Downregulation of HS6ST2 regulated by overexpression of miR-23b-3p and siRNAs against HS6ST2 could enhance the protein level of MMP13 and aggravate the matrix degradation in chondrocytes. Increased expression of MMP13 depended on activity of p38 MAPK rather than total p38 MAPK level and was abrogated by HS6ST2 overexpression. Together, the results indicated that downregulated HS6ST2 targeted by miR-23b-3p promotes matrix degradation by activating p38 MAPK in chondrocytes and OA cartilage.


Assuntos
Regulação para Baixo , Matriz Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , Osteoartrite/enzimologia , Osteoartrite/genética , Sulfotransferases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Idoso , Cartilagem Articular/enzimologia , Cartilagem Articular/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Metaloproteinase 13 da Matriz/metabolismo , MicroRNAs/genética , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfotransferases/genética , Fator de Necrose Tumoral alfa/farmacologia
12.
Acta Biochim Biophys Sin (Shanghai) ; 49(2): 110-118, 2017 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-28039148

RESUMO

Glutathione peroxidase 1 (GPx1) is a selenium (Se)-containing protein and is induced in cartilage formation. GPx1 eliminates reactive oxygen species (ROS), which are required for chondrogenic induction. The physiological properties of GPx1 in cartilage and the redox mechanisms involved are not known. The effects of GPx1 on chondrogenic differentiation of ATDC5 cells were examined through short hairpin RNA-mediated gene silencing. The results demonstrated that GPx1 knockdown impaired gene expression of sex determining region Y-box 9, collagen II (Col II), and aggrecan. GPx1 knockdown suppressed the accumulation of cartilage glycosaminoglycans (GAGs) and the proliferation of chondrocyte. GPx1 knockdown also induced cell apoptosis. However, cell sensitivity toward exogenous oxidative stress was not increased after GPx1 knockdown. Unexpectedly, GPx1 knockdown not only induced oxidative stress characterized by the increased production of ROS but also caused reductive stress indicated by an elevation of glutathione (GSH)/oxidized GSH (GSSG) ratio. Furthermore, GPx1 knockdown-mediated reductive and oxidative stress could be antagonized by a thiol-oxidizing agent diamide and a thiol-containing compound N-acetylcysteine (NAC), respectively. Moreover, NAC attenuated GPx1 knockdown-induced cell apoptosis, while diamide prevented GPx1 knockdown-suppressed chondrocyte proliferation. Finally, diamide but not NAC could rescue GPx1 knockdown-mediated impaired chondrogenic differentiation. In summary, GPx1 is essential for chondrogenic induction in ATDC5 cells mainly through modulation of intracellular GSH/GSSG ratio, rather than an antioxidant enzyme to detoxify ROS. In addition, GPx1 knockdown-induced impaired chondrogenesis may participate in the pathogenesis of the endemic osteoarthropathy due to Se deficiency. These observations offer novel insights for the development of therapeutic target during cartilage degeneration.


Assuntos
Diferenciação Celular/genética , Condrócitos/metabolismo , Glutationa Peroxidase/genética , Estresse Oxidativo , Interferência de RNA , Agrecanas/genética , Agrecanas/metabolismo , Animais , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/genética , Condrogênese/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Expressão Gênica , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Glutationa Peroxidase GPX1
13.
Oncol Lett ; 9(6): 2603-2608, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26137114

RESUMO

In glioblastomas, the surface glycoprotein CD133 (prominin-1) indicates the presence of cancer stem cells (CSCs), which are able to initiate tumor growth and are highly resistant to conventional chemo/radiotherapy. However, a number of studies have reported that certain CD133- glioma cells are able to self-renew and retain tumorigenic potential. In addition, the reliability of CD133 as a CSC marker is controversial due to inconsistent findings with regard to the prognostic values and distribution of CD133. Such controversies may be due to the detection limits using currently available anti-CD133 antibodies. In the present study, novel anti-human CD133 monoclonal antibodies (mAbs) were generated using two recombinant extracellular domains of human CD133: CD133 ectodomain 1 (amino acids 171-420) and CD133 ectodomain 2 (amino acids 507-716). One of the antibodies produced against CD133 ectodomain 2, C2E1, detected high expression levels of CD133 protein in glioblastoma U87 cells, in contrast to previous studies which did not detect CD133 expression in these cells. The cells exhibited a cytoplasmic distribution pattern of CD133 and produced a 95 kDa band following western blot analysis. In addition, C2E1 was able to bind the full-length glycosylated CD133 on the cell surface and inhibit the proliferation of tumor cells. Therefore, this antibody may be a valuable tool to study CD133 as a CSC marker and may be significant in future cancer treatments.

14.
PLoS One ; 7(9): e45169, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23028823

RESUMO

ZFN technology is a powerful research tool and has been used for genome editing in cells lines, animals and plants. The generation of functional ZFNs for particular targets in mammalian genome is still challenging for an average research group. The modular-assembly method is relatively fast, easy-to-practice but has a high failure rate. Some recent studies suggested that a ZFP with low binding activity might be able to form a working ZFN pair with another binding active half-ZFP. In order to unveil the potential ZFP candidates among those with low binding activities, this paper established a highly sensitive mammalian cell-based transcriptional reporter system to assess the DNA binding activities of ZFPs by inserting multiple copies of ZFN target sequence fragment (TSF) of an interested gene (e. g., hPGRN or hVEGF). Our results showed that this system increased the screening sensitivity up to 50-fold and markedly amplified the differences in the binding activities between different ZFPs. We also found that the targeted chromosomal gene repair efficiency of each hPGRN or hVEGF ZFN pair was in proportion with the combination of the binding activities of the ZFL (Left zinc finger) and ZFR (Right zinc finger). A hPGRN ZFR with low binding ability was able to form a biological active ZFN if combined with a hPGRN ZFL with relatively high binding ability. Lastly, site-specific genome editing by hPGRN ZFNs generated by this system was confirmed by sequencing, and the PGRN knock-out cell line showed significantly decreased cell growth compared with the control. Our system will provide a valuable tool for further optimizing the nucleases with regard to specificity and cytotoxicity.


Assuntos
Endonucleases/genética , Engenharia Genética/métodos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fator A de Crescimento do Endotélio Vascular/genética , Dedos de Zinco/genética , Sequência de Bases , Endonucleases/metabolismo , Genes Reporter , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Luciferases , Dados de Sequência Molecular , Plasmídeos , Ligação Proteica , Análise de Sequência de DNA , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/metabolismo
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