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1.
Zhonghua Zhong Liu Za Zhi ; 41(10): 742-747, 2019 Oct 23.
Artigo em Chinês | MEDLINE | ID: mdl-31648495

RESUMO

Objective: To investigate the effect of thymopoietin (TMPO) gene deleted by small interfering RNA (RNAi) on the proliferation and apoptosis of lung cancer cell A549 and its mechanism. Methods: TMPO siRNA was transfected into A549 cells by lipofection. The transfected siRNA control was used as a negative control, and the parent cells were used as blank control. Forty-eight hours later, the expression of TMPO in the transfected cells was detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay, cell cycle and apoptosis were detected by flow cytometry, the protein levels of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, notch receptor 1 (Notch1) and mammalian rapamycin target protein (mTOR) were detected by Western blot analysis. Results: The results of RT-PCR and Western blot showed that the expression levels of TMPO mRNA in the blank control group, the negative control group and TMPO siRNA transfected group were (1.01±0.11), (0.99±0.10), (0.36±0.04), respectively, the protein levels were (0.27±0.02), (0.29±0.03), (0.08±0.10), respectively. The expression levels of TMPO mRNA and protein in the transfected group were significantly lower than those in the blank control and negative control group (P<0.05). The results of MTT assay showed that the OD values of the blank control group, the negative control group and the transfected group were (0.35±0.04), (0.37±0.04) and (0.34±0.03) at 24 h of transfection, respectively. The OD values at 48 h were (0.47±0.06), (0.46±0.08), (0.37±0.04), the OD values at 72 h were (0.75±0.08), (0.73±0.07), (0.49±0.05), respectively, and the OD values at 96 h were (1.09±0.07), (1.06±0.08), (0.56±0.06). The proliferation abilities of the transfected cells at 48, 72, 96 h were significantly lower than those of the blank control and the negative control group (P<0.05). The results of flow cytometry showed that the proportion of G(0)/G(1) phase cells in blank control group, negative control group and transfection group were (62.55±2.03)%, (61.24±3.15)%, (47.35±2.44)%, respectively. The proportion of cells in S phase were (17.12±1.31)%, (17.70±2.01)%, and (20.81±2.06)%, respectively. The proportion of cells in G(2)/M phase were (20.33±1.43)%, (21.06±1.52)%, (31.84±2.76)%, respectively. The proportion of cells in G(0)/G(1) phase of transfection group was significantly lower than those of blank control and negative control group (P<0.05). The proportion of cells in G(2)/M phase of transfection group was significantly higher than those of blank control and negative control group (P<0.05). The apoptosis ratio of the transfection group was (34.10±2.69)%, significantly higher than (2.96±0.03)% of the blank control and (3.01±0.04)% of the negative control group (P<0.05). Western blot analysis showed that PCNA, Notch1 and mTOR proteins were down-regulated while cleaved caspase-3 protein was up-regulated in A549 cells after deletion of TMPO. Conclusion: The inhibition of TMPO gene expression induced by small interfering RNA can significantly inhibit the proliferation and induce apoptosis of A549 cells, and the mechanism is associated with the inhibition of the activation of Notch1/mTOR signaling pathway.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Neoplasias Pulmonares/patologia , Timopoietinas/metabolismo , Animais , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Timopoietinas/genética , Transfecção
2.
Anticancer Res ; 39(10): 5297-5310, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570424

RESUMO

BACKGROUND/AIM: Low-molecular weight heparins (LMWHs) may possess putative antitumoral properties; however, the underlying mechanism(s) remains elusive. We evaluated the antiproliferative and antimigratory effects of enoxaparin (a LMWH) in lung adenocarcinoma A549 cells, and assessed the possible mechanism involved, and the effect on doxorubicin's efficacy. MATERIALS AND METHODS: Proliferation and migration were evaluated using BrdU and transwell assays, respectively. Immunoblotting was used to measure PAR-1, PAR-2, MMP-2, ERK1/2 and Akt proteins. Apoptosis and cell cycle studies examined the combined effect of enoxaparin and doxorubicin. RESULTS: Enoxaparin inhibited A549 cell proliferation and migration. Following PAR-1 gene knock down, enoxaparin's effect on A549 cell proliferation was diminished compared to scrambled siRNA. Our experiments verified that enoxaparin-mediated down-regulation of MAPK and PI3K, reduced MMP-2 expression and inhibited A549 cell migration. Additionally, enoxaparin increased doxorubicin's efficacy by enhancing apoptosis, while no effect on cell-cycle progression was observed. CONCLUSION: Results suggest that the anticancer activity of enoxaparin in A549 cells was mediated by the interference of two major PAR-1 downstream signaling pathways, MAPK/ERK and PI3K/Akt, which in turn inhibit proliferation and migration. Therefore, enoxaparin may be promising as an adjunct to traditional chemotherapy for lung cancer and warrants further investigation.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Enoxaparina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Receptor PAR-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Heparina de Baixo Peso Molecular/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo
3.
Life Sci ; 235: 116799, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31472144

RESUMO

Colorectal cancer (CRC) is one of the most common malignancies in the world. Emerging evidence has shown that dysregulation of tripartite motif (TRIM) family proteins is strongly correlated with the tumorigenesis of CRC. Here, we evaluated the biological roles of TRIM66, a member of TRIM family, in the progression of CRC. The results demonstrated that TRIM66 was markedly up-regulated in both CRC tissues and cell lines. To further investigate the functions of TRIM66 in CRC, CRC cells were infected with lentivirus expressing anti-TRIM66 shRNA (sh-TRIM66) or control lentivirus (sh-con). We found that knockdown of TRIM66 significantly inhibited cell proliferation, migration, invasion of CRC cells. TRIM66 knockdown also suppressed epithelial-mesenchymal transition (EMT), as proved by the increased E-cadherin expression and decreased expressions of N-cadherin and vimentin. Furthermore, TRIM66 knockdown markedly inhibited tumor growth in a mouse xenograft model. Knockdown of TRIM66 reduced the activation of JAK2/STAT3 signaling pathway in CRC cells. Treatment with AG490, an inhibitor of JAK2/STAT3 signaling pathway, enhanced the inhibitory effects of TRIM66 knockdown on cell proliferation, migration and invasion. These findings suggested that knockdown of TRIM66 exhibited anti-tumor activity through inhibiting the JAK2/STAT3 signaling pathway in CRC cells.


Assuntos
Movimento Celular , Proliferação de Células , Neoplasias Colorretais/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Janus Quinase 2/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Apoptose , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Janus Quinase 2/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Life Sci ; 235: 116822, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476310

RESUMO

AIMS: To investigate the effects of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) on cognitive dysfunction, the expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and amyloid ß peptide (Aß) in the hippocampus, as well as dendritic pathology in the hippocampal CA1 region in sepsis-associated encephalopathy (SAE) rats. MAIN METHODS: The rats were randomly divided into four groups: 1) control group (subjected to sham surgery), 2) control plus Mfhas1 siRNA group (rats received intracerebroventricular injection of Mfhas1 siRNA after sham surgery), 3) CLP plus control siRNA group (rats received intracerebroventricular injection of control siRNA after cecal ligation and puncture (CLP)), 4) CLP plus Mfhas1 siRNA group (rats received intracerebroventricular injection of Mfhas1 siRNA after CLP). The learning and memory capabilities of the rats were examined by means of fear conditioning and Barnes maze test. The concentration of TNF-α and IL-1ß was determined by enzyme-linked immunosorbent assay. The efficiency of siRNA transfection, MFHAS1 and Aß expression were detected by Western blotting. Total branch lengths of pyramidal dendrites of the CA1 basilar trees and spine density were determined by Golgi staining. KEY FINDINGS: We observed that MFHAS1 knock-down by Mfhas1 siRNA intracerebroventricular injection could improve cognitive impairment, reduce the expression of TNF-α, IL-1ß and Aß in the hippocampus induced by CLP, and alleviate the dendritic spinal loss of the pyramidal neurons, as well as increase the dendritic branching of the CA1 basilar trees of septic rats. SIGNIFICANCE: MFHAS1 knock-down can alleviate cognitive impairment, neuroinflammation and dendritic spinal loss in SAE rats.


Assuntos
Disfunção Cognitiva/prevenção & controle , Dendritos/efeitos dos fármacos , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Proteínas Oncogênicas/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Encefalopatia Associada a Sepse/complicações , Animais , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Dendritos/metabolismo , Dendritos/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Wistar
5.
Anticancer Res ; 39(9): 4737-4742, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519573

RESUMO

BACKGROUND/AIM: There are several unresolved issues regarding the combined treatment with an immune checkpoint inhibitor and anti-angiogenic agent for renal cell carcinoma (RCC) patients. The purpose of this study was to address the inhibitory effects of programmed death-ligand 1 (PD-L1) expression on growth and sensitivity to sunitinib in the mouse RCC RenCa model. MATERIALS AND METHODS: We established RenCa/sh-PD-L1 by transfecting RenCa cells with a plasmid carrying a short hairpin RNA targeted against PD-L1. The growth pattern of RenCa/sh-PD-L1 with or without sunitinib was compared to that of RenCa cells transfected with control plasmid alone (RenCa/Co). RESULTS: No significant difference in growth or sensitivity to sinitinib was noted between RenCa/sh-PD-L1 and RenCa/Co cells in vitro. The tumor volume in mice subcutaneously injected with RenCa/sh-PD-L1 was significantly smaller than that with RenCa/Co. Treatment of mice bearing each tumor with sunitinib resulted in a significant reduction of the RenCa/sh-PD-L1 tumor compared to the RenCa/Co tumor. Moreover, infiltration by CD8+ T cells of RenCa/sh-PD-L1 tumors was significantly higher than that of RenCa/Co tumors, irrespective of treatment with sunitinib. CONCLUSION: Suppressed expression of PD-L1 could increase tumor-infiltrating CD8+ T cells and result in growth inhibition as well as enhanced sensitivity to sunitinib in the RenCa model.


Assuntos
Antineoplásicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , RNA Interferente Pequeno/genética , Sunitinibe/farmacologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Expressão Gênica , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Carga Tumoral/efeitos dos fármacos
6.
Anticancer Res ; 39(9): 4811-4816, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519583

RESUMO

BACKGROUND/AIM: γ-Glutamylcyclotransferase (GGCT) is highly expressed in many forms of cancer, and is a promising therapeutic target. The present study investigated whether inhibition of GGCT enhanced the antiproliferative effects of the drug docetaxel in prostate cancer cells. MATERIALS AND METHODS: Immunohistochemistry and western blot analysis were conducted to measure GGCT expression in prostate cancer tissue samples and cell lines. GGCT was inhibited using RNAi and a novel enzymatic inhibitor, pro-GA, and cell proliferation was evaluated with single and combination treatments of GGCT inhibitors and docetaxel. RESULTS: GGCT was highly expressed in cultured prostate cancer cells and patient samples. GGCT inhibition alone inhibited prostate cancer cell line proliferation and induced cellular senescence. GGCT inhibition in combination with apoptosis-inducing docetaxel had more potent antiproliferative effects than either drug used alone. CONCLUSION: GGCT inhibition may potentiate anticancer drug efficacy.


Assuntos
Antineoplásicos/farmacologia , Docetaxel/farmacologia , Inibidores Enzimáticos/farmacologia , gama-Glutamilciclotransferase/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , gama-Glutamilciclotransferase/genética , gama-Glutamilciclotransferase/metabolismo
7.
Anticancer Res ; 39(9): 4817-4828, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519584

RESUMO

BACKGROUND/AIM: Although epidermal growth factor receptor (EGFR) is frequently activated in lung and pancreatic cancers, the efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs) is limited. Recently, brexpiprazole, an antipsychotic drug, was reported to chemosensitize glioma cells to osimertinib, a third-generation EGFR-TKI, by suppressing survivin, an anti-apoptotic protein, but their combinational effects on lung and pancreatic cancers remain unknown. The aim of this study was to examine the combinational effects of brexpiprazole and osimertinib on lung and pancreatic cancer cells in vitro and in vivo. MATERIALS AND METHODS: YM155, a suppressor of survivin, siRNA, and immunoblot were used to examine the role of survivin in osimertinib-resistance. The effect of drugs on cell viability in vitro was examined by trypan blue staining. The in vivo effects of drugs on tumor growth were examined using a xenograft mouse model. RESULTS: Brexpiprazole exerted combinational effects with osimertinib in vitro. Pharmacological and genetic suppression of survivin chemosensitized the cells to osimertinib. Moreover, the combination of brexpiprazole and osimertinib effectively suppressed tumor growth in a mouse xenograft model. CONCLUSION: Brexpiprazole is a promising drug for lung and pancreatic cancer in combination with osimertinib.


Assuntos
Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases/farmacologia , Quinolonas/farmacologia , Survivina/genética , Tiofenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Humanos , Masculino , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Anticancer Res ; 39(9): 4853-4864, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31519588

RESUMO

BACKGROUND/AIM: Colorectal cancer (CRC) is the leading cause of cancer mortality worldwide. Its poor prognosis can be ascribed primarily to high recurrence rates. Accordingly, the aim of this study was to identify novel prognostic biomarkers and therapeutic targets for management of CRC. MATERIALS AND METHODS: To develop prognostic biomarkers, we performed RNA-seq analysis and real-time RT-PCR in primary cancer tissues with or without systemic recurrence. To characterize the molecular functions of the encoded proteins, CRC cells underexpressing or overexpressing the candidate genes were established and appropriate cell-based assays were applied. RESULTS: ITGB1 and RHOC mRNA levels were up-regulated in the recurrence group of CRC patients. Overexpression of ITGB1 or RHOC stimulated CRC cell proliferation, invasion and migration, whereas the opposite effects were observed in cells underexpressing either protein. Five-year recurrence-free survival rates were significantly higher in the ITGB1- and RHOC-underexpression groups than those in the overexpression. CONCLUSION: ITGB1 and RHOC are potential predictors of recurrence and therapeutic targets for CRC, possibly predicting a high-risk group of stage II patients.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Integrina beta1/metabolismo , Proteína de Ligação a GTP rhoC/metabolismo , Idoso , Biomarcadores Tumorais , Proliferação de Células , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Interferente Pequeno/genética , Recidiva , Análise de Sobrevida
9.
J Cancer Res Clin Oncol ; 145(10): 2433-2444, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485767

RESUMO

PURPOSE: The clinical importance of cancer stem cells (CSCs) in head and neck squamous cell carcinoma (HNSCC) is well recognized. However, a reliable method for the detection of functioning CSC has not yet been established. We hypothesized that YAP1, a transcriptional coactivator, and SOX2, a master transcription factor of SCC, may cooperatively induce stemness through transcriptional reprogramming. METHODS: We immunohistochemically examined the expression of SOX2 and YAP1 in the CD44 variant 9 (CD44v9)-positive invasion front. A CSC-inducible module was identified through a combination of siRNAs and sphere formation assays. YAP1 and SOX2 interactions were analyzed in vitro. RESULTS: The triple overexpression of SOX2, YAP1, and CD44v9 was significantly associated with poor prognosis. TCGA data revealed that the CSC-inducible module, which was related to EMT and angiogenesis, was significantly correlated with poor prognosis. The KLF7 expression, representatively chosen from the module, also correlated with poor prognosis and was essential for sphere formation and CSC propagation. Sphere stress-activated YAP1 enhanced SOX2 activity. CONCLUSIONS: The stress-triggered activation of YAP1/SOX2 transcriptionally reprograms HNSCC for the acquisition of stemness. Triple SOX2, YAP1, and CD44v9 immunostaining assays may be useful for the selection of high-risk patients with functioning CSCs, and YAP1 targeting may lead to the development of a CSC-targeting therapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fosfoproteínas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Estresse Fisiológico , Ativação Transcricional , Biomarcadores , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Estresse Fisiológico/genética
10.
Chem Commun (Camb) ; 55(77): 11619-11622, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31501844

RESUMO

Mesoporous organosilica nanoparticles (PHT-PMO) have been prepared from an octa-triethoxysilylated Zn phthalocyanine precursor. These PHT-PMO nanoparticles had no dark toxicity but high phototoxicity when irradiated at 650 nm, and remarkable near-infrared phototoxicity when excited at 760 and 810 nm. The PHT-PMO were then aminated to promote electrostatic complexation with siRNA. Transfection experiments were performed upon NIR irradiation and photochemical internalization was very efficient, leading to 65% luciferase extinction in MCF-7 cancer cells expressing stable luciferase.


Assuntos
Indóis/química , Nanopartículas/química , Compostos Organometálicos/química , Fotoquimioterapia/métodos , RNA Interferente Pequeno/química , Silanos/química , Sobrevivência Celular , Cetrimônio/química , Humanos , Raios Infravermelhos , Luciferases/genética , Células MCF-7 , Processos Fotoquímicos , Porosidade , RNA Interferente Pequeno/metabolismo , Eletricidade Estática , Propriedades de Superfície
11.
Rev Assoc Med Bras (1992) ; 65(8): 1067-1073, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531603

RESUMO

OBJECTIVE: Diabetes is a risk factor for acute kidney injury (AKI). However, its mechanism of pathogenesis has not been elucidated. The aim of the study was to investigate the role of inflammation and the toll-like receptor 7 (TLR7) in ischemic AKI for diabetes. METHODS: A high glucose hypoxia-reoxygenation model of human renal tubular epithelial (HK-2) cells was used to generate AKI induced by ischemia-reperfusion in diabetes. The activity of cells was measured by CCK-8 assay and LDH activity. Inflammatory cytokines were assessed by ELISA. TLR7, MyD88, and NF-κB expressions were examined by western blotting. Apoptosis was evaluated by flow cytometry. RESULTS: The high glucose group and low glucose group were subjected to hypoxia-reoxygenation. The low glucose group developed only mild cell damage, apoptosis, and inflammatory response. In contrast, an equivalent hypoxia-reoxygenation injury provoked severe cell damage, apoptosis, and inflammatory response in the high glucose group. Expression of TLR7 and its related proteins were measured in the high glucose group before and after hypoxia-reoxygenation. The high glucose group exhibited more significant increases in TLR7 expression following hypoxia-reoxygenation than the low glucose group. In addition, the expression of TLR7 and its related proteins after hypoxia-reoxygenation were higher in the high glucose group than in the low glucose group. Inhibition of TLR7 provides significant protection against ischemic injury in diabetes. CONCLUSION: Our results suggest that diabetes increases the vulnerability to ischemia-induced renal injury. This increased vulnerability originates from a heightened inflammatory response involving the TLR7 signal transduction pathway.


Assuntos
Lesão Renal Aguda/metabolismo , Diabetes Mellitus/metabolismo , Isquemia/metabolismo , Receptor 7 Toll-Like/metabolismo , Lesão Renal Aguda/fisiopatologia , Células Cultivadas , Diabetes Mellitus/fisiopatologia , Citometria de Fluxo , Humanos , Isquemia/fisiopatologia , RNA Interferente Pequeno , Transdução de Sinais , Receptor 7 Toll-Like/fisiologia , Transfecção
12.
Genes Dev ; 33(17-18): 1208-1220, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31416967

RESUMO

The PIWI-interacting RNA (piRNA) pathway is a conserved small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilization. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use noncanonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by nuclear export factor 1 (Nxf1). Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila nuclear export factor family protein Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We found that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Precursores de RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Elementos de DNA Transponíveis/genética , Drosophila/genética , Proteínas de Drosophila/genética
13.
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
14.
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
15.
BMC Bioinformatics ; 20(1): 434, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438847

RESUMO

BACKGROUND: The epidermal growth factor receptor (EGFR) is a major regulator of proliferation in tumor cells. Elevated expression levels of EGFR are associated with prognosis and clinical outcomes of patients in a variety of tumor types. There are at least four splice variants of the mRNA encoding four protein isoforms of EGFR in humans, named I through IV. EGFR isoform I is the full-length protein, whereas isoforms II-IV are shorter protein isoforms. Nevertheless, all EGFR isoforms bind the epidermal growth factor (EGF). Although EGFR is an essential target of long-established and successful tumor therapeutics, the exact function and biomarker potential of alternative EGFR isoforms II-IV are unclear, motivating more in-depth analyses. Hence, we analyzed transcriptome data from glioblastoma cell line SF767 to predict target genes regulated by EGFR isoforms II-IV, but not by EGFR isoform I nor other receptors such as HER2, HER3, or HER4. RESULTS: We analyzed the differential expression of potential target genes in a glioblastoma cell line in two nested RNAi experimental conditions and one negative control, contrasting expression with EGF stimulation against expression without EGF stimulation. In one RNAi experiment, we selectively knocked down EGFR splice variant I, while in the other we knocked down all four EGFR splice variants, so the associated effects of EGFR II-IV knock-down can only be inferred indirectly. For this type of nested experimental design, we developed a two-step bioinformatics approach based on the Bayesian Information Criterion for predicting putative target genes of EGFR isoforms II-IV. Finally, we experimentally validated a set of six putative target genes, and we found that qPCR validations confirmed the predictions in all cases. CONCLUSIONS: By performing RNAi experiments for three poorly investigated EGFR isoforms, we were able to successfully predict 1140 putative target genes specifically regulated by EGFR isoforms II-IV using the developed Bayesian Gene Selection Criterion (BGSC) approach. This approach is easily utilizable for the analysis of data of other nested experimental designs, and we provide an implementation in R that is easily adaptable to similar data or experimental designs together with all raw datasets used in this study in the BGSC repository, https://github.com/GrosseLab/BGSC .


Assuntos
Processamento Alternativo/genética , Biologia Computacional/métodos , Receptores ErbB/genética , Glioblastoma/genética , Teorema de Bayes , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Probabilidade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
17.
Nat Biotechnol ; 37(8): 884-894, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31375812

RESUMO

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.


Assuntos
Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Huntingtina/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , Animais , Proteína Huntingtina/genética , Camundongos , Mutação , RNA Mensageiro , RNA Interferente Pequeno/metabolismo
18.
Cell Biochem Funct ; 37(7): 504-515, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31368195

RESUMO

The treatment of neural deficiency after cerebral infarction is challenging, with limited therapeutic options. The transplantation of mesenchymal stem cells (MSCs) to the ischemic penumbra is a potential therapeutic approach. In the present study, a cerebral infarction model was generated by performing middle cerebral artery occlusion (MCAO) in SD rats. The expression of CXCR4 increased, and the number of MSCs migrating to the peri-infarct area was higher in rats transplanted with preconditioned MSCs than in rats transplanted with untreated MSCs. The rate of apoptosis, as evaluated by TUNEL staining and immunoblotting assays, was reduced in rats receiving preconditioned MSCs. A significant amelioration of neural regeneration and improved neurological function were observed in rats injected with preconditioned MSCs compared with those injected with untreated MSCs. However, the application of an siRNA targeting CXCL12 significantly inhibited the protective role of preconditioned MSCs against apoptosis and promoted the migration of MSCs to the ischemic area, leading to impaired neuronal regeneration and limited recovery of neuronal function. Hypoxic preconditioning of MSCs prior to transplantation suppressed apoptosis and increased their migration abilities, leading to the promotion of neuronal regeneration and improvement in neural function after transplantation. This preconditioning strategy may be considered as a potential approach for the modification of MSCs prior to cell transplantation therapy in patients with cerebral infarction. SIGNIFICANCE OF THE STUDY: We found that hypoxic preconditioning of MSCs improved their ability to promote neuronal regeneration and the recovery of neuronal function. Moreover, we showed that CXCR4 inhibited apoptosis, improved cell homing, and promoted neuronal differentiation, without influencing angiogenesis. Our study provides a relatively safe preconditioning method for potential use for cell transplantation therapy in ischemic cerebral infarction. The results presented here will facilitate the development of novel strategies and techniques to improve the tolerance and migration ability of transplanted cells for the treatment of cerebral infarction sequelae.


Assuntos
Infarto Cerebral/metabolismo , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , Hipóxia , Células-Tronco Mesenquimais/metabolismo , Receptores CXCR4/metabolismo , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular , Infarto Cerebral/terapia , Quimiocina CXCL12/antagonistas & inibidores , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Neurônios , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores CXCR4/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos
19.
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
20.
Anticancer Res ; 39(8): 4073-4077, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366490

RESUMO

BACKGROUND/AIM: ANRIL is a long noncoding RNA located on INK4 locus, which encodes p15 and p16 that cause G1 phase arrest in the cell cycle. ANRIL positively regulates proliferation of several kinds of cancer cells such as lung and gastric cancers. This study, examined the effect of ANRIL in head and neck squamous cell carcinoma cells. MATERIALS AND METHODS: Cells were transfected with siRNA oligonucleotides targeting ANRIL. Transfected cells were subjected to cell-cycle and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. RESULTS: Depletion of ANRIL increased p15 mRNA in FaDu cells, and p15 and p16 mRNA in CAL27 cells and inhibited proliferation of these cells. Cell cycle analysis showed that depletion of ANRIL caused arrest at the G1 phase of the cell cycle. CONCLUSION: ANRIL promotes G1 phase progression by repressing p15 and p16, and thus promotes FaDu and CAL27 cell proliferation.


Assuntos
Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , RNA Longo não Codificante/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
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