Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 142.958
Filtrar
1.
Med Sci Monit ; 26: e921288, 2020 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-32146479

RESUMEN

BACKGROUND Leukemia is common in aging adults and has very high mortality worldwide. The present study was designed to investigate the therapeutic efficacy of miR-18a inhibitor against WEHI-3 and THP-1 leukemia cells. MATERIAL AND METHODS The changes in miR-18a inhibitor-transfected WEHI-3 and THP-1 cell proliferative potential was measured by use of the Cell Counting Kit-8 assay. Apoptotic changes were analyzed by electron microscopy, and evaluation of PI3K, AKT, mTOR, and PTEN expression was assessed by RT-qPCR assay. RESULTS Transfection of miR-18a inhibitor significantly (P<0.05) suppressed the proliferative potential of WEHI-3 and THP-1 cells. The WEHI-3 cells showed the presence of characteristic apoptotic bodies on transfection with miR-18a inhibitor at 48 h. Flow cytometry showed that miR-18a inhibitor transfection significantly (P<0.05) increased the WEHI-3 cell percentage in G1 phase. The transfection of miR-18a inhibitor significantly (P<0.05) promoted apoptosis in WEHI-3 cells. In WEHI-3 cells, miR-18a inhibitor transfection markedly suppressed the expression of PI3K, AKT, and mTOR mRNA. The expression of PTEN mRNA was significantly (P<0.05) upregulated by miR-18a inhibitor transfection in WEHI-3 cells. CONCLUSIONS The present study investigated the therapeutic efficacy of miR-18a inhibitor against WEHI-3 and THP1 leukemia cells. The study demonstrated that miR-18a inhibitor suppressed the proliferative potential of WEHI-3 and THP1 cells and activated apoptotic process through upregulation of PTEN mRNA expression. Therefore, miR-18a inhibitor can be of therapeutic importance for the treatment of leukemia.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Leucemia/patología , MicroARNs/antagonistas & inhibidores , Fosfohidrolasa PTEN/metabolismo , Apoptosis , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia/tratamiento farmacológico , Células THP-1 , Transfección , Regulación hacia Arriba
2.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 38(1): 6-10, 2020 Feb 01.
Artículo en Chino | MEDLINE | ID: mdl-32037759

RESUMEN

OBJECTIVE: To construct a PA28γ overexpression cell line and determine its effects after infecting an oral squa-mous cell carcinoma (OSCC) cell line. METHODS: The PA28γ gene was cloned into the pLOV.CMV.cherry.2A.EF1a.PuroR lentiviral vector by polymerase chain reaction (PCR), and PCR and DNA sequencing alignment analysis were used for identification. Then, 293T cells were used to package viral diseases. Infected OSCC cells were used to construct a cell line with stable PA28γ overexpression. Finally, the level of PA28γ expression in the OSCC cell line was detected through Western blot. RESULTS: The successful construction of PA28γ recombinant lentiviral vectors was confirmed by DNA sequencing. The results of immunofluorescence showed that the PA28γ overexpression lentivirus successfully infected the OSCC cells and showed cherry red fluorescence. The results of Western blot demonstrated that the constructed cells with stable PA28γ overexpression significantly increased the expression of PA28γ. CONCLUSIONS: The PA28γ overexpression lentiviral vector can significantly increase its protein expression in OSCC cells. We provide a stable OSCC cell line for further study on the effect of PA28γ in OSCC.


Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de la Boca , Autoantígenos , Línea Celular Tumoral , Vectores Genéticos , Humanos , Lentivirus , Complejo de la Endopetidasa Proteasomal , Transfección
3.
Anticancer Res ; 40(2): 767-777, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32014919

RESUMEN

BACKGROUND/AIM: Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) plays an important role in cancer. We examined the effect of COUP-TFII overexpression on the proliferation and invasion of the human colorectal cancer SNU-C4 cells. MATERIALS AND METHODS: SNU-C4 cells were stably transfected with COUP-TFII expression plasmid to overexpress COUP-TFII (COUP-TFII-SNU-C4 cells). Cell proliferation, colony-forming ability and transwell invasion assays were performed. To elucidate the underlying molecular mechanism of COUP-TFII action, western blot analysis, p53 shRNA transfection, and Myr-Akt transfection were performed. RESULTS: Cell proliferation and colony-forming ability were significantly inhibited in COUP-TFII-SNU-C4 cells. Western blot analyses demonstrated that while the expression of p53 and PTEN was increased, the p-Akt levels were decreased in COUP-TFII-SNU-C4 cells. Knockdown of p53 partially restored the cell proliferation, but did not reverse the inhibition of invasion. Constitutive activation of Akt via Myr-Akt transfection reversed the inhibited cell proliferation and invasion by COUP-TFII. CONCLUSION: p53 is required for the inhibition of cell proliferation, and decreased phosphorylation of Akt may mediate the inhibition of cell proliferation and invasion by COUP-TFII.


Asunto(s)
Factor de Transcripción COUP II/biosíntesis , Neoplasias Colorrectales/metabolismo , Fosfohidrolasa PTEN/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , Factor de Transcripción COUP II/metabolismo , Proliferación Celular/fisiología , Neoplasias Colorrectales/patología , Humanos , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Transfección , Proteína p53 Supresora de Tumor/metabolismo
4.
Anticancer Res ; 40(2): 813-823, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32014924

RESUMEN

BACKGROUND/AIM: Olaparib was previously shown to synergistically enhance the cytotoxicity of DNA synthesis inhibitors in oesophageal carcinoma (OC) cell lines. However, the mechanisms of this synergy are not fully understood. As P53 binding protein 1 (53BP1) expression was previously shown to potentiate the anticancer effect of olaparib, we investigated the involvement of 53BP1 in the synergetic cytotoxic effects of olaparib and anticancer drugs in KYSE70 cells. MATERIALS AND METHODS: Experiments included small interfering RNA transfection, growth inhibition assays, western blots, immunofluorescence, and flow cytometry. RESULTS: The toxicity of DNA synthesis-inhibiting agents plus olaparib was decreased when 53BP1 was depleted. Olaparib cotreatment significantly increased phosphorylated H2A histone family member X (γH2AX) foci as well as 53BP1/γH2AX co-localisation in anticancer drug-treated cells. Silencing of 53BP1 suppressed anticancer drug-induced apoptosis with or without olaparib. CONCLUSION: Olaparib potentiates the cytotoxicity of anticancer drugs through 53BP1 in OC cells.


Asunto(s)
Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteína 1 de Unión al Supresor Tumoral P53/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Humanos , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Transfección
5.
Anticancer Res ; 40(2): 653-664, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32014906

RESUMEN

BACKGROUND/AIM: Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial biogenesis and metabolism. We investigated the effect of PGC-1α knockdown in the human colorectal cancer cell line SW620, which highly expresses PGC-1α. MATERIALS AND METHODS: We established the PGC-1α shRNA-silenced SW620 stable cell line (PGC-1α shRNA-SW620 cells) and examined cell proliferation by cell counts and carboxyfluorescein succinimidyl ester (CFSE) staining, migration by wound-healing and transwell migration assay, and invasion by transwell assays. RESULTS: PGC-1α knockdown inhibited cell proliferation, migration, and invasion in SW620 cells. Western blot analysis showed that p-AKT, p-GSK-3ß, ß-catenin, N-cadherin and vimentin expression were all reduced, but E-cadherin had increased expression in PGC-1α shRNA-SW620 cells. We also examined cell proliferation, migration, invasion and the expression of p-AKT, p-GSK-3ß, ß-catenin, N-cadherin, vimentin, and E-cadherin in PGC-1α overexpressing SW480 cells (a low PGC-1α expressing line). We observed a complete reversal of the results seen in the knockdown. CONCLUSION: PGC-1α might regulate cell proliferation and invasion via AKT/GSK-3ß/ß-catenin pathway in SW620 and SW480 cells.


Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , beta Catenina/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Técnicas de Silenciamiento del Gen , Humanos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/biosíntesis , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Transducción de Señal , Transfección
6.
Anticancer Res ; 40(2): 723-731, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-32014914

RESUMEN

BACKGROUND/AIM: MicroRNAs (miRNAs) play regulatory roles in pancreatic ductal adenocarcinoma (PDAC). However, it is still required to identify the function of miRNA-301-3p in pancreatic cancer cells. MATERIALS AND METHODS: Effects of luteolin on cell growth, TRAIL cytotoxicity, and miR-301-3p levels were evaluated. The role of miRNA-301-3p in regulating cell proliferation, target gene expression, and TRAIL cytotoxicity were studied. RESULTS: The levels of miR-301-3p were down-regulated in PANC-1 cells exposed to luteolin, which inhibits the growth of PANC-1 cells and sensitizes cells to TRAIL. The knockdown of miR-301-3p attenuates cell proliferation and enhances TRAIL cytotoxicity. In addition, caspase-8 was directly targeted by miR-301-3p. CONCLUSION: Our findings unveil a critical biological function of miR-301-3p in regulating cell proliferation and elevating an antiproliferative effect of TRAIL on cancer cells. Our observation of miR-301-3p/caspase-8 relationship can also serve to clarify the role of miR-301-3p in other cancer types and related diseases.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Caspasa 8/metabolismo , Luteolina/farmacología , MicroARNs/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Carcinoma Ductal Pancreático/genética , Caspasa 8/genética , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Técnicas de Silenciamiento del Gen , Humanos , Luteolina/administración & dosificación , MicroARNs/genética , Neoplasias Pancreáticas/genética , Proteínas Recombinantes/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Transfección
7.
Chem Pharm Bull (Tokyo) ; 68(2): 129-132, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32009079

RESUMEN

Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.


Asunto(s)
ADN sin Sentido/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Aminas/química , Animales , Cationes/química , ADN sin Sentido/química , ADN sin Sentido/genética , ADN sin Sentido/farmacocinética , Disulfuros/química , Silenciador del Gen , Células HeLa , Humanos , Masculino , Ratones Endogámicos ICR , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinética , Transfección/métodos
8.
Artículo en Chino | MEDLINE | ID: mdl-32062888

RESUMEN

Objective: To investigate the effect of peroxiredoxin 2 (Prx2) overexpression on fibroblast proliferation and collagen synthesis induced by transforming growth factor-ß1 (TGF-ß1) . Methods: Fibroblasts were randomly divided into control group (DMEM medium) , TGF-ß1 group (5 µg/L TGF-ß1) , negative control group (treated with 5 µg/L TGF-ß1 and transfected with empty lentiviral vector) , and Prx2 group (treated with 5 µg/L TGF-ß1 and transfected with Prx2 overexpression lentiviral vector) . MTT assay was used to measure cell proliferation, immunofluorescence assay was used to measure the expression of 8-OHdG, and Western blot was used to measure the expression of p-JNK, p-P38, collagen type I, collagen type III, and Prx2. SPSS 18.0 was used for statistical analysis. The continuous data were expressed as mean±standard deviation; an analysis of variance was used for comparison between groups, and the least significant difference t-test was used for further comparison between two groups. Results: Lentiviral transfection was performed successfully, and the Prx2 group had a significant increase in the protein expression of Prx2 (P<0.05) . Compared with the control group, the TGF-ß1 group had a significant increase in the proliferation ability (P<0.05) , and compared with the TGF-ß1 group, the Prx2 group had a significant reduction in the proliferation ability (P<0.05) . Compared with the control group, the TGF-ß1 group had significant increases in the expression of 8-OHdG, p-JNK, p-P38, collagen type I, and collagen type III (P<0.05) ; compared with the TGF-ß1 group, the negative control group had no significant changes in the expression of 8-OHdG, p-JNK, p-P38, collagen type I, and collagen type III (P>0.05) , while the Prx2 group had significant reductions in the above parameters (P<0.05) . Conclusion: Prx2 overexpression inhibits fibroblast proliferation and collagen synthesis induced by TGF-ß1 through inhibiting reactive oxygen species and activating the JNK and P38 pathways.


Asunto(s)
Proliferación Celular , Colágeno Tipo I/biosíntesis , Fibroblastos/citología , Peroxirredoxinas/genética , Factor de Crecimiento Transformador beta1/farmacología , Células Cultivadas , Fibroblastos/efectos de los fármacos , Humanos , Transducción de Señal , Transfección
9.
Pharm Res ; 37(2): 30, 2020 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-31915939

RESUMEN

PURPOSE: mRNA has recently emerged as a potent therapeutics and requires safe and effective delivery carriers, particularly prone to address its issues of poor stability and escape from endosomes. In this context, we designed poly(D,L-lactide) (PLA)-based micelles with N-succinimidyl (NS) ester decorated hydrophilic hairy corona to trap/couple a cationic fusogenic peptide and further complex mRNA. METHODS: Two strategies were investigated, namely (i) sequential immobilization of peptide and mRNA onto the micelles (layer-by-layer, LbL) or (ii) direct immobilization of peptide-mRNA pre-complex (PC) on the micelles. After characterization by means of size, surface charge, peptide/mRNA coupling/complexation and mRNA serum stability, carrier cytotoxicity and transfection capacity were evaluated with dendritic cells (DCs) using both GFP and luciferase mRNAs. RESULTS: Whatever the approach used, the micellar assemblies afforded full protection of mRNA in serum while the peptide-mRNA complex yielded complete mRNA degradation. In addition, the micellar assemblies allowed to significantly reduce the toxicity observed with the peptide-mRNA complex. They successfully transfected hard-to transfect DCs, with a superior efficiency for the LbL made ones (whatever mRNAs studied) showing the impact of the elaboration process on the carrier properties. CONCLUSIONS: These results show the relevance and potential of this new PLA/peptide based micelle platform to improve mRNA stability and delivery, while offering the possibility of further multifunctionality through PLA core encapsulation.


Asunto(s)
Portadores de Fármacos/química , Péptidos/química , Poliésteres/química , Povidona/análogos & derivados , ARN Mensajero/química , Animales , Línea Celular , Supervivencia Celular , Estabilidad de Medicamentos , Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Micelas , Povidona/química , ARN Mensajero/genética , Transfección
10.
Invest Ophthalmol Vis Sci ; 61(1): 1, 2020 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-31995153

RESUMEN

Purpose: Vacuolar protein sorting 35 (Vps35) mutations and protein dysfunction have been linked to the hyperphosphorylation and accumulation of tau protein in a number of central neurodegenerative disorders. The aims of the present study were to investigate the mechanism underlying the tau hyperphosphorylation caused by Vps35 deficiency. Methods: The cells used in this study were primary retinal ganglion cells (RGCs). The rat retinal glutamate excitotoxicity model was used in vivo. Fresh retinal tissues or eyeballs were collected at different time points. The expression and interactions of Vps35, Cdk5/p35, tau hyperphosphorylation, LAMP1, EEA1 and UBE1 in RGCs were studied by immunofluorescence staining, Western blotting, and immunoprecipitation. Results: The downregulation and overexpression of Vps35 increased and decreased the expression of p35 and tau hyperphosphorylation, respectively. More important, roscovitine, a Cdk5 inhibitor, could effectively decrease the hyperphosphorylated tau level induced by Vps35 deficiency. Furthermore, this study confirmed that the inhibition of Vps35 could increase the activity of Cdk5/p35 by affecting the lysosomal degradation of p35 and lead to the degeneration of RGCs. Conclusions: These findings demonstrate the possibility that Cdk5/p35 acts as a "cargo" of Vps35 and provide new insights into the pathogenesis of RGC degeneration caused by hyperphosphorylated tau protein. Vps35 is a potential target for basic research and clinical treatment of RGC degeneration in many ocular diseases such as glaucoma.


Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Fosfotransferasas/metabolismo , Células Ganglionares de la Retina/metabolismo , Proteínas de Transporte Vesicular/deficiencia , Proteínas tau/metabolismo , Animales , Western Blotting , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Regulación hacia Abajo , Técnica del Anticuerpo Fluorescente Indirecta , Ácido Glutámico/toxicidad , Glicoproteínas de la Membrana Asociadas a los Lisosomas/metabolismo , Masculino , Ratones , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/metabolismo , Roscovitina/farmacología , Transfección , Enzimas Activadoras de Ubiquitina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
11.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 64-71, 2020 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-31889183

RESUMEN

Previous studies have shown that during severe acute pancreatitis (SAP) attacks, hydrogen sulfide (H2S) is released in the colon. However, the roles played by H2S in regulating enteric nerves remain unclear. In this study, we examined the association between SAP-induced H2S release and loss of intestinal motility, and also explored the relevant mechanism in enteric nerve cells. A rat SAP model was constructed and enteric nerve cells were prepared. Intestinal mobility was evaluated by measuring the number of bowel movements at indicated time points and by performing intestinal propulsion tests. The production of inflammatory cytokines during a SAP attack was quantified by ELISA, and the levels of cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS) were examined by immunohistochemistry and western blot analysis. In vivo studies showed that PI3K/Akt/Sp1 signaling in enteric nerve cells was blocked, confirming the mechanism of endogenous H2S formation by western blot analysis and immunofluorescence. Our results also showed that rats with SAP symptoms had reduced intestinal motility. Furthermore, PI3K/Akt/Sp1 signaling was triggered and CSE expression was up-regulated, and these changes were associated with H2S formation in the colon. In addition, propargylglycine reduced the levels of inflammatory cytokines and suppressed the release of H2S. Enteric nerve cells that were incubated with LY294002 and transfected with a Sp1-knockdown vector displayed decreased levels of CSE production, which led to a decrease in H2S production. These results suggest that SAP symptoms suppressed the intestinal motility of rats via the release of H2S in enteric nerve cells, which was dependent on the inflammation-induced PI3K/Akt/Sp1 signaling pathway.


Asunto(s)
Movimiento Celular , Sistema Nervioso Entérico/patología , Sulfuro de Hidrógeno/metabolismo , Neuronas/metabolismo , Pancreatitis/metabolismo , Animales , Cromonas/farmacología , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , Citocinas/metabolismo , Motilidad Gastrointestinal , Técnicas de Silenciamiento del Gen , Inflamación/metabolismo , Masculino , Morfolinas/farmacología , Pancreatitis/inducido químicamente , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Ácido Taurocólico/efectos adversos , Ácido Taurocólico/farmacología , Transfección
12.
Toxicol Lett ; 322: 39-49, 2020 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-31927052

RESUMEN

Exposure to the environmental pollutants organotins is of toxicological concern for the marine ecosystem and sensitive human populations, including pregnant women and their unborn children. Using a placenta cell model, we investigated whether organotins at nanomolar concentrations affect the expression and activity of 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). 11ß-HSD2 represents a placental barrier controlling access of maternal glucocorticoids to the fetus. The organotins tributyltin (TBT) and triphenyltin (TPT) induced 11ß-HSD2 expression and activity in JEG-3 placenta cells, an effect confirmed at the mRNA level in primary human trophoblast cells. Inhibition/knock-down of retinoid X receptor alpha (RXRα) in JEG-3 cells reduced the effect of organotins on 11ß-HSD2 activity, mRNA and protein levels, revealing involvement of RXRα. Experiments using RNA and protein synthesis inhibitors indicated that the effect of organotins on 11ß-HSD2 expression was direct and caused by increased transcription. Induction of placental 11ß-HSD2 activity by TBT, TPT and other endocrine disrupting chemicals acting as RXRα agonists may affect placental barrier function by altering the expression of glucocorticoid-dependent genes and resulting in decreased availability of active glucocorticoids for the fetus, disturbing development and increasing the risk for metabolic and cardiovascular complications in later life.


Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Disruptores Endocrinos/toxicidad , Expresión Génica/efectos de los fármacos , Compuestos Orgánicos de Estaño/toxicidad , Receptor alfa X Retinoide/metabolismo , Compuestos de Trialquiltina/toxicidad , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Placenta/efectos de los fármacos , Placenta/metabolismo , Embarazo , Receptor alfa X Retinoide/genética , Transfección , Regulación hacia Arriba
13.
Braz J Med Biol Res ; 53(1): e9144, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31939600

RESUMEN

Wound scarring remains a major challenge for plastic surgeons. Transforming growth factor (TGF)-ß plays a key role in the process of scar formation. Previous studies have demonstrated that truncated TGF-ß type II receptor (t-TGF-ßRII) is unable to continue signal transduction but is still capable of binding to TGF-ß, thereby blocking the TGF-ß signaling pathway. Hepatocyte growth factor (HGF) is a multifunctional growth factor that promotes tissue regeneration and wound healing. Theoretically, the combination of HGF and t-TGF-ßRII would be expected to exert a synergistic effect on promoting wound healing and reducing collagen formation. In the present study, lentivirus-mediated transfection of the two genes (t-TGF-ßRII/HGF) into fibroblasts in vitro and in a rat model in vivo was used. The results demonstrated that the expression of t-TGF-ßRII and HGF in NIH-3T3 cells was successfully induced. The expression of both molecules significantly reduced collagen I and III expression, and also inhibited fibroblast proliferation. Furthermore, histological examination and scar quantification revealed less scarring in the experimental wound in a rat model. Moreover, on macroscopic inspection, the experimental wound exhibited less visible scarring compared with the control. Therefore, the present study demonstrated that the combination gene therapy of t-TGF-ßRII and HGF promoted wound healing, with less scarring and more epithelial tissue formation, not only by suppressing the overgrowth of collagen due to its antifibrotic effect, but also by promoting tissue regeneration.


Asunto(s)
Cicatriz/metabolismo , Colágeno/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Transfección , Factor de Crecimiento Transformador beta2/metabolismo , Animales , Proliferación Celular , Cicatriz/patología , Ratones , Modelos Animales , Ratas , Ratas Sprague-Dawley
14.
J Nanobiotechnology ; 18(1): 15, 2020 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-31952530

RESUMEN

BACKGROUND: The successful deliveries of siRNA depend on their stabilities under physiological conditions because greater in vivo stability enhances cellular uptake and enables endosomal escape. Viral-based systems appears as most efficient approaches for gene delivery but often compromised in terms of biocompatibility, patient safety and high cost scale up process. Here we describe a novel platform of gene delivery by elastin-like polypeptide (ELP) based targeting biopolymers. RESULTS: For better tumor targeting and membrane penetrating characteristics, we designed various chimeric ELP-based carriers containing a cell penetrating peptide (Tat), single or multiple copies of AP1 an IL-4 receptor targeting peptide along with coding sequence of ELP and referred as Tat-A1E28 or Tat-A4V48. These targeted polypeptides were further analyzed for its ability to deliver siRNA (Luciferase gene) in tumor cells in comparison with non-targeted controls (Tat-E28 or E28). The positively charged amino acids of these polypeptides enabled them to readily complex with negatively charged nucleic acids. The complexation of nucleic acid with respective polypeptides facilitated its transfection efficiency as well as stability. The targeted polypeptides (Tat-A1E28 or Tat-A4V48) selectively delivered siRNA into tumor cells in a receptor-specific fashion, achieved endosomal and lysosomal escape, and released gene into cytosol. The target specific delivery of siRNA by Tat-A1E28 or Tat-A4V48 was further validated in murine breast carcinoma 4T1 allograft mice model. CONCLUSION: The designed delivery systems efficiently delivered siRNA to the target site of action thereby inducing significant gene silencing activity. The study shows Tat and AP1 functionalized ELPs constitute a novel gene delivery system with potential therapeutic applications.


Asunto(s)
Péptidos de Penetración Celular/química , Elastina/química , Péptidos/química , ARN Interferente Pequeño/química , Animales , Biopolímeros , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Femenino , Técnicas de Transferencia de Gen , Terapia Genética , Humanos , Luciferasas/genética , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Imagen Óptica , ARN Interferente Pequeño/administración & dosificación , Receptores de Interleucina-4/metabolismo , Transfección
15.
J Nanobiotechnology ; 18(1): 16, 2020 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-31959180

RESUMEN

BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR-Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells. RESULTS: Using poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR-Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160 nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6 wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24 h and corresponded to ~ 2-3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells. CONCLUSIONS: In this work, plasmids for the CRISPR-Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR-Cas9 system.


Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Nanopartículas/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Animales , Proteína 9 Asociada a CRISPR/metabolismo , ADN/química , Colorantes Fluorescentes/química , Técnicas de Transferencia de Gen , Macrófagos/metabolismo , Ratones , Compuestos de Organosilicio/química , Plásmidos , Transfección
16.
Phys Chem Chem Phys ; 22(3): 1738-1746, 2020 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-31898698

RESUMEN

Cationic liposomes, a type of non-viral vectors, often play the important biological function of delivering nucleic acids during cell transfection. Variations in the molecular architecture of di-alkyl dihydroxy ethyl ammonium chloride-based cationic lipids involving hydrophobic tails have been found to influence their biological function in terms of cell transfection efficiency. For example, liposomes based on a cationic lipid (Lip1814) with asymmetry in the hydrophobic chains were found to display higher transfection efficacy in cultured mammalian cell lines than those comprising of symmetric Lip1818 or asymmetric Lip1810. The effect of variations in the molecular architecture of the cationic lipids on the biological activity of liposomes has been explored here via the photophysical studies of 8-anilino-1-naphthalenesulphonate (ANS) and Nile Red (NR) in three cationic liposomes, namely Lip1810, Lip1814 and Lip1818. Time-resolved fluorescence of ANS revealed reduced hydration at the lipid-water interface and enhanced relaxation dynamics of surface water (lipid headgroup bound water molecules) in Lip1810- and Lip1814-based liposomes in the presence of cholesterol. As the probe ANS failed to be incorporated into the lipid-water interface of Lip1818 due to the significantly high rigidity of these liposomes, no information concerning the extent of hydration of the lipid-water interface or the interfacial water dynamics could be obtained. Time-resolved polarization-gated anisotropy measurements of NR in the presence of cholesterol revealed the rigidity of the cationic liposomes to be increasing in the order of Lip1810 < Lip1814 < Lip1818. In the presence of cholesterol, moderately higher rigidity, reduced membrane hydration and enhanced relaxation dynamics of the interfacial water molecules gave rise to the superior cell transfection efficacy of Lip1814-based cationic liposomes than those of the highly flexible Lip1810 or the highly rigid Lip1818.


Asunto(s)
Lípidos/química , Línea Celular , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación Molecular , Transfección
17.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(1): 17-20, 2020 Jan 10.
Artículo en Chino | MEDLINE | ID: mdl-31922588

RESUMEN

OBJECTIVE: To explore the genetic basis of a pedigree affected with hereditary spherocytosis. METHODS: Peripheral blood samples were collected from 17 members of the pedigree. Genomic DNA of the proband was subjected to next generation sequencing. Candidate variant was validated by co-segregation analysis. pCAS2(c.5798+1G) and pCAS2(c.5798+1A) plasmids were constructed by homologous recombination and transfected into 293T cells. Reverse transcription PCR, TA cloning and Sanger sequencing were used to analyze the effect of candidate variant on splicing. Meanwhile, peripheral blood RNAs were extracted to analyze the effect of candidate variant on splicing in vivo. RESULTS: The proband was found to carry a c.5798+1G>A variant of the SPTB gene. The variant has co-segregated with the phenotype in the pedigree. In vitro and in vivo splicing experiments confirmed that the mutation has significantly affected the splicing, resulting in shift of reading frame and produced a premature termination codon. CONCLUSION: The novel c.5798+1G>A variant of the SPTB gene probably underlies the pathogenesis of hereditary spherocytosis in this pedigree.


Asunto(s)
Espectrina , Esferocitosis Hereditaria , Codón sin Sentido/genética , Variación Genética , Células HEK293 , Humanos , Mutación/genética , Linaje , Plásmidos , Empalme del ARN , Espectrina/genética , Esferocitosis Hereditaria/genética , Transfección
18.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(1): 24-29, 2020 Jan.
Artículo en Chino | MEDLINE | ID: mdl-31950785

RESUMEN

Objective: To investigate the effect of antisense oligodeoxynucleotides (ASODN) of Homeobox A1 gene ( HOXA1) on proliferation, apoptosis, invasion and migration of esophageal carcinoma cells. Methods: The expression of HOXA1 protein in normal esophageal epithelial cells Het-1A and esophageal cancer TE-1, EC9706 and Eca109 cells was detected by Western blot. Screening of highly expressed of HOXA1 protein esophageal squamous cell carcinoma cells for follow-up experiments. HOXA1 antisense oligonucleotide (ASODN) chains, sense oligodeoxynucleotides (SODN) chain, and nonsense oligodeoxy nucleotides (N-ODN) chain were designed. The screened esophageal squamous cell carcinoma cells with high expression were divided into HOXA1 ASODN group (5, 10, 15 µmol/L HOXA1 ASODN transfected Eca109 cells), control group (conventional culture medium, no cell transfection), SODN group (cells transfected with 15 µmol/L of SODN) and N-ODN group (cells transfected with 15 µmol/L N-ODN). Cell viability, apoptosis rate and invasion and migration ability were detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method, flow cytometry, transwell chamber respectively; The expression of HOXA1, phosphorylation serine/threonine kinase (p-AKT), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP-2) and B-cell lymphoma2 (Bcl-2) associated X protein (Bax) protein was detected by Western blot. Results: Compared with normal esophageal epithelial cells Het-1A, the expression of HOXA1 protein in human esophageal squamous cell carcinoma cells TE-1, EC9706 and Eca109 was significantly higher ( P<0.05). The expression of HOXA1 protein was the highest in Eca109 cells, therefore, Eca109 cells were selected for follow-up experiments. The expression of HOXA1 protein in Eca109 cells transfected with HOXA1 ASODN was significantly decreased ( P<0.05). After transfection of Eca109 cells with HOXA1 ASODN, the viability of Eca109 cells decreased with the increase of concentration and time, the difference was significant compared with the control, SODN and N-ODN groups ( P<0.05). 15 µmol/L HOXA1 ASODN significantly inhibited cell viability. After 15 µmol/L HOXA1 ASODN was transfected into Eca109 cells, the invasion and migration abilities of cells were significantly decreased, the apoptosis rate was increased, the expressions of p-AKT, PCNA and MMP-2 were significantly decreased, and the expression of Bax was significantly increased ( P<0.05). Conclusion: Antisense oligodeoxynucleotides of HOXA1 gene can inhibit the proliferation, invasion and migration of esophageal cancer cells, and induce apoptosis. The mechanism is related to the inhibition of PI3K/AKT signaling pathway.


Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Oligonucleótidos Antisentido , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Esofágicas/fisiopatología , Carcinoma de Células Escamosas de Esófago/fisiopatología , Humanos , Oligonucleótidos Antisentido/farmacología , Transfección
19.
Gene ; 730: 144318, 2020 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-31917231

RESUMEN

Although the chicken embryo has been a classical model for developmental studies, the lack of straightforward technologies for chicken transgenesis limited the usefulness of this animal model. Here, we assessed electroporation and lipofection approaches for in ovo transfection of Sleeping Beauty transposon system in stage X-XII chicken embryos. Electroporation of chicken embryos could transfect the trophectodermal cells. Then, a mixture of transposon lipoplexes and high concentrated carboxymethylcellulose (HCC) solution was injected into the subgerminal cavity of day 0 embryos. The lipoplex-HCC mixture substantially increased the number of trophectodermal cells expressing the reporter. Importantly, the fluorescent reporter was detected in cells inside of the embryos as well as circulation cells in the bloodstream during days 3-4 of incubation. This study provided evidence for direct in ovo transfection of early chicken embryos, though the long-term outcome of this approach warrants further studies.


Asunto(s)
Electroporación/métodos , Transfección/métodos , Transposasas/genética , Animales , Animales Modificados Genéticamente , Carboximetilcelulosa de Sodio , Embrión de Pollo , Pollos/genética , Elementos Transponibles de ADN/genética , Embrión de Mamíferos/embriología , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas de Transferencia de Gen
20.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 58-63, 2020 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-31681945

RESUMEN

Cardiac hypertrophy is considered to be a leading factor in heart function-related deaths. In this study, we explored the potential mechanism underlying cardiac hypertrophy induced by isoproterenol. Our results showed that isoproterenol induced cardiac hypertrophy in AC16 cells, as reflected by the increased cell surface area and increased hypertrophic markers, which was accompanied by increased ubiquitin-protein ligase E3a (UBE3A) expression. Moreover, UBE3A knockdown by siRNAs accelerated cardiac hypertrophy, suggesting that increased UBE3A expression induced by isoproterenol might be a protective response and UBE3A might be a protective factor against cardiac hypertrophy. Our study also revealed that UBE3A knockdown increased the protein expression of the TLR4/MMP-9 pathway that has been shown to be associated with cardiac hypertrophy, which suggested that UBE3A-mediated protection is likely to be associated with the blockade of the TLR4/MMP-9 signaling pathway. UBE3A might be thus a potential target gene for the treatment of cardiac hypertrophy.


Asunto(s)
Cardiomegalia/inducido químicamente , Cardiomegalia/metabolismo , Isoproterenol/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Receptor Toll-Like 4/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Isoproterenol/efectos adversos , Miocitos Cardíacos/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Transfección , Ubiquitina-Proteína Ligasas/genética
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA