RESUMEN
Previous studies have indicated that miR-146a-5p acts as an oncogene in several types of cancer, yet a tumor suppressor gene in others. In non-small cell lung cancer (NSCLC), one report showed that it was downregulated and played the role of tumor suppressor. However, another study showed that miR-146a-5p was overexpressed in the serum of NSCLC patients compared to healthy controls. Therefore, it is obvious that further study of the function of miR-146a-5p in NSCLC is necessary to fully understand its importance. Herein, we have verified that miR- 146a- 5p acts as a tumor suppressor in NSCLC. Our data revealed that the expression level of miR-146a-5p was significantly decreased in several human NSCLC cell lines, and also less abundant in human NSCLC tissues, when compared with controls. Moreover, we observed that miR-146a-5p could suppress cell proliferation, both in vitro and in vivo. Our results also showed that miR-146a-5p directly targeted the 3'-UTR of CCND1 and CCND2 mRNAs as well as decreased their expression at both mRNA and protein levels, causing cell cycle arrest at the G0/G1 phase. Furthermore, siRNA-mediated downregulation of CCND1 or CCND2 yielded the same effects on proliferation and cell cycle arrest as miR-146a-5p upregulation did in the NSCLC cell lines. We confirmed that the expression of miR-146a-5p had negative relationship with CCND1 or CCND2. Besides, we also found that miR-146a-5p could inhibit tumor growth in xengroft mouse models, and CCND1 and CCND2 were downregulated in miR-146a-5p overexpressed xengroft tumor tissues. In summary, our results demonstrated that miR-146a-5p could suppress the proliferation and cell cycle progression in NSCLC cells by inhibiting the expression of CCND1 and CCND2.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/terapia , Ciclina D1/metabolismo , Ciclina D2/metabolismo , Neoplasias Pulmonares/terapia , MicroARNs/genética , Regiones no Traducidas 3'/genética , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Ciclina D1/genética , Ciclina D2/genética , Femenino , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Desnudos , ARN Interferente Pequeño/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MicroRNAs are a class of non-coding single-stranded RNA, 20-23 nucleotide in length, which can be involved in the regulation of gene expression. Through binding with 3'-untranslated regions (3'-UTR), microRNAs can cause degradation of target mRNAs or inhibition of translation, and thus regulating the expression of genes at the post-transcriptional level. In this study, we found that miR-486-5p was significantly downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines, suggesting that miR-486-5p might function as a tumor suppressor in lung cancer. Additionally, we showed that CDK4, an oncogene that plays an important role in cell cycle G1/S phase progression, was directly targeted by miR-486-5p. Furthermore, our data reveals that knockdown of CDK4 by siRNA can inhibit cell proliferation, promote apoptosis, and impede cell-cycle progression. In epigenetics, the upstream promoter of miR-486-5p was strongly regulated by methylation in NSCLC. Collectively, our results suggest that miR-486-5p could not only inhibit NSCLC by downregulating the expression of CDK4, but also be as a promising and potent therapy in the near future.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/enzimología , Quinasa 4 Dependiente de la Ciclina/metabolismo , Neoplasias Pulmonares/enzimología , MicroARNs/metabolismo , Células A549 , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Quinasa 4 Dependiente de la Ciclina/genética , Metilación de ADN , Regulación hacia Abajo , Epigénesis Genética , Puntos de Control de la Fase G1 del Ciclo Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , MicroARNs/genética , Regiones Promotoras Genéticas , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , TransfecciónAsunto(s)
Anoicis , Autofagia , MicroARNs/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Proteína 5 Relacionada con la Autofagia/metabolismo , Western Blotting , Línea Celular Tumoral , Humanos , MicroARNs/genética , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , TransfecciónRESUMEN
Tanshinone is the liposoluble constituent of Salia miltiorrhiza, a root used in traditional herbal medicine which is known to possess certain health benefits. Although it is known that tanshinones, including tanshinone I (T1), tanshinone IIA (T2A), and cryptotanshinone (CT), can inhibit the growth of lung cancer cells in vitro, the mechanism under which they act is still unclear. AURKA, an oncogene, encodes a serine-threonine kinase which regulates mitotic processes in mammalian cells. Here, we reported that tanshinones mediate AURKA suppression partly through up-regulating the expression of miR-32. We found that tanshinones could inhibit cell proliferation, promote apoptosis, and impede cell-cycle progression, thus performing an antineoplastic function in non-small cell lung cancer (NSCLC). Additionally, we demonstrated that tanshinones attained these effects in part by down-regulating AURKA, corroborating previous reports. Our results showed that in NSCLC, similar effects were obtained with knock-down of the AURKA gene by siRNA. We also verified that AURKA was the direct target of miR-32. Collectively, our results demonstrated that tanshinones could inhibit NSCLC by suppressing AURKA via up-regulating the expressions of miR-32 and other related miRNAs.
Asunto(s)
Abietanos/farmacología , Antineoplásicos Fitogénicos/farmacología , Aurora Quinasa A/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , MicroARNs/metabolismo , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , MicroARNs/biosíntesis , MicroARNs/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
MicroRNAs (MiRNAs) are small non-coding RNA molecules which act as important regulators of post-transcriptional gene expression by binding 3'-untranslated region (3'-UTR) of target messenger RNA (mRNA). In this study, we analyzed miRNA-34a (miR-34a) as a tumor suppressor in non-small cell lung cancer (NSCLC) H1299 cell line. The expression level of miR-34a in four different NSCLC cell lines, H1299, A549, SPCA-1, and HCC827, was significantly lower than that in the non-tumorigenic bronchial epithelium cell line BEAS-2B. In human NSCLC tissues, miR-34a expression level was also significantly decreased in pT2-4 compared with the pT1 group. Moreover, miR-34a mimic could inhibit the proliferation and triggered apoptosis in H1299 cells. Luciferase assays revealed that miR-34a inhibited TGFßR2 expression by targeting one binding site in the 3'-UTR of TGFßR2 mRNA. Quantitative real-time PCR (qRT-PCR) and Western blot assays verified that miR-34a reduced TGFßR2 expression at both mRNA and protein levels. Furthermore, downregulation of TGFßR2 by siRNA showed the same effects on the proliferation and apoptosis as miR-34a mimic in H1299 cells. Our results demonstrated that miR-34a could inhibit the proliferation and promote the apoptosis of H1299 cells partially through the downregulation of its target gene TGFßR2.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Proliferación Celular/genética , MicroARNs/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Regiones no Traducidas 3'/genética , Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genéticaRESUMEN
microRNAs (miRNAs) are a class of non-coding small RNAs that act as negative regulators of gene expression by binding to the 3'-untranslated region (3'-UTR) of target mRNAs. Tumor protein p53, a transcriptional factor, plays an important role in the progression of tumorigenesis. miR-150 was the only miRNA predicted to target 3'-UTR of p53 by Targetscan. In order to investigate the function of miR-150, p53 and relevant miRNAs in non-small cell lung cancer (NSCLC), we constructed two expression vectors of p53 (pcDNA3.1-p53 and pcDNA3.1-p53-3'-UTR) and two report vectors (pGL3-p53-3'-UTR and pGL3-p53-3'-mUTR). The activity of luciferase transfected with miR-150 mimics was lower by 30% when compared to that of the miRNA-negative control (miRNA-NC). Moreover, the p53 protein was downregulated by at least 50% when miR-150 mimics were cotransfected with pcDNA3.1-p53-3'-UTR when compared to miRNA-NC. We also determined the expression of miR-150 and p53 in NSCLC patient tissue samples. The expression of miR-150 in T2 stage tissue samples was higher than that in T1 stage tissue samples. The corresponding target gene p53 was correlated with miR-150 expression. In the present study, we further analyzed the cell cycle distribution. The cells transfected with pcDNA3.1-p53 were significantly arrested in the G1 phase when compared to the control cells. When miR-150 mimics were cotransfected with pcDNA3.1-p53-3'-UTR, the percentage of cells in the G1 phase was significantly lower by 4% when compared to miRNA-NC. To identify miRNAs that are regulated by the p53 protein, qRT-PCR was performed after pcDNA3.1-p53 transfection. miR-34a, miR-184, miR-181a and miR-148 were upregulated significantly. However, there was no distinct difference in the expression of miR-10a, miR-182 and miR-34c. Our results showed that miR-150 targets the 3'-UTR of p53, and p53 protein promotes the expression of miRNAs which affect cell cycle progression. These findings suggest that miR-150, p53 protein and relevant miRNAs are members of a regulatory network in NSCLC tumorigenesis.
Asunto(s)
Carcinogénesis/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , MicroARNs/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regiones no Traducidas 3'/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Ciclo Celular/genética , Línea Celular Tumoral , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , MicroARNs/biosíntesis , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Mouse L1210 leukemia cell line is widely used as a model in the study of tumorigenesis, as well as the efficacy of chemotherapeutic drugs; however, like other suspension cell lines, the mouse L1210 cell line has lowest transfection efficiency, that many barriers exist to study about the structure, function, as well as metabolism in leukemia cells. This study was aimed to obtain higher transfection efficiency of L1210 cell line to facilitate scientific research. The transfection efficiencies of nucleofector and liposome in L1210 leukemia cells were detected by converted fluorescence microscopy and flow cytometry using EGFP (enhance green fluorescent protein); cell viability was observed by trypan blue exclusion test. The results showed that the transfection efficiency of nucleofector primarily through reporter gene pEGFP by Amaxa Nucleofector(TM) nuclear transfer apparatus was significantly higher than lipofectamine 2000 transfection, furthermore, in the same cell density (2 × 10(6)/ml) and plasmid content (10 µg), the transfection efficiency of nuclear transfer apparatus default mode A-20 was higher than that of other modes (S-18, T-20). Its survival rate was up to 50.5% after 24 hours. Cell viability of liposome transfection reached to 88% after 24 hours, but the transfection efficiency was lower (< 1%). It is concluded that the nuclear transfer apparatus A-20 transfected L1210 can reach higher transfection efficiency up to 61.6%, which is significantly higher than that of lipofectamine transfection. The survival rate is up to 50.5% well meeting the needs of scientific research. Higher transfection efficiency is helpful for in-depth research about the morphology, functions and pathogenesis in leukemia model L1210, and provides more searching space for the treatment of leukemia diseases.
Asunto(s)
Núcleo Celular/genética , Proteínas Fluorescentes Verdes/genética , Transfección/métodos , Animales , Línea Celular Tumoral , Supervivencia Celular/genética , Genes Reporteros , Liposomas , RatonesRESUMEN
OBJECTIVE: To investigate the anti-fibrogenesis property of intraportal vein injection of small interfering RNA targeting connective tissue growth factor (CTGF) in a rat model of liver fibrosis and its effect on the accumulation of extracellular matrix (ECM). METHODS: Thirty male rats were randomly divided into five groups. Some rats received CCl4 subcutaneously every three days for 6 consecutive weeks, and in the meantime they also received either siRNA targeting CTGF (preventive group), saline (model group) or siRNA (siRNA control group) by intraportal vein injections. Other rats received CCl4 by subcutaneous injection for 2 weeks, followed by CCl4 and CTGF siRNA intraportal vein injection for 4 more weeks (as treatment group). The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot respectively. Hepatic histology was evaluated by HE and Sirius red stained sections. The collagen staining areas were measured quantitatively using a computer-aided manipulator with slight modifications. Serum procollagen type III and hyaluronic acid were determined by radioimmunoassay. RESULTS: Six weeks after CCl4 injection, prominent upregulation of gene expressions of CTGF, type I and III collagen, and laminin in saline or siRNA-treated rat livers were observed. The expressions of CTGF at mRNA and protein level and type I and III collagen at mRNA level were markedly reduced in rats with CTGF siRNA treated for four or six weeks. Expressions of CTGF at mRNA and protein levels decreased by 76%+/-8%, 80%+/-3% (F = 68.630) and 95%+/-2%, 93%+/-3% (F = 21.234, P < 0.01); type I and III collagen and laminin at mRNA levels decreased by 74%+/-8%, 78%+/-8%, 31%+/-7% and 57%+/-6%, 59%+/-10%, 43%+/-9% (F = 24.219, 16.315, 9.716, P < 0.01) compared with rats in the model group at 72 h. The CTGF siRNA treatment markedly reduced serum levels of procollagen type III and hyaluronic acid and the degrees of liver fibrosis. CONCLUSION: Intraportal vein siRNA injection targeting CTGF could significantly inhibit CTGF gene expression in rats, thereby attenuating liver fibrosis by reducing ECM accumulation.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Matriz Extracelular/metabolismo , Silenciador del Gen , Cirrosis Hepática Experimental/patología , Animales , Tetracloruro de Carbono , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/metabolismo , Masculino , ARN Interferente Pequeño , Ratas , Ratas Sprague-DawleyRESUMEN
For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended beta1-beta2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNA(Trp), which could be partially rescued by adding tRNA(Trp), even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode.
Asunto(s)
ARN de Transferencia de Triptófano/metabolismo , Triptófano-ARNt Ligasa/química , Triptófano/metabolismo , Valina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Isoleucina/química , Datos de Secuencia Molecular , Mutación , Unión Proteica , Bases de Schiff/química , Alineación de Secuencia , Triptófano-ARNt Ligasa/genética , Triptófano-ARNt Ligasa/metabolismo , Valina/genéticaRESUMEN
Neonatal hyperbilirubinemia is a common clinical condition caused mainly by the increased production and decreased excretion of bilirubin. Current treatment is aimed at reducing the serum levels of bilirubin. Heme oxygenase-1 (HO-1) is a rate-limiting enzyme that generates bilirubin. In this study we intended to suppress HO-1 using the RNA interference technique. Small interfering RNA (siRNA)-A, -B, and -C were designed based on human HO-1 (hHO-1) mRNA sequences. siRNA was transfected into a human hepatic cell line (HL-7702). hHO-1 transcription and protein levels were then determined. In addition, the inhibitory effect of siRNA on hHO-1 was assessed in cells treated with hemin or transfected with an hHO-1 plasmid. siRNA-C showed the most potent suppressive effect on hHO-1. This inhibition is dose and time dependent. Compared with control, both hemin and hHO-1 plasmids up-regulated hHO-1 expression in HL-7702 cells. However, the up-regulation was significantly attenuated by siRNA-C. Furthermore, the decrease in hHO-1 activity was coincident with the suppression of its transcription. Finally, siRNA-C was shown to reduce hHO-1 enzymatic activity and bilirubin levels. Thus, this study provides a novel therapeutic rationale by blocking bilirubin formation via siRNA for preventing and treating neonatal hyperbilirubinemia and bilirubin encephalopathy at an early clinical stage.
Asunto(s)
Bilirrubina/metabolismo , Regulación Enzimológica de la Expresión Génica , Hemo-Oxigenasa 1/metabolismo , Hiperbilirrubinemia Neonatal/enzimología , Kernicterus/enzimología , ARN Interferente Pequeño , Línea Celular , Hemo-Oxigenasa 1/genética , Hemina/farmacología , Humanos , Hiperbilirrubinemia Neonatal/tratamiento farmacológico , Hiperbilirrubinemia Neonatal/genética , Kernicterus/tratamiento farmacológico , Kernicterus/genética , Hígado/enzimología , Oxidación-Reducción , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Regulación hacia ArribaAsunto(s)
Hepatocitos/citología , Proteínas Inmediatas-Precoces/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Proliferación Celular , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , RatasRESUMEN
OBJECTIVES: To study the inhibition of primary mouse hepatocyte apoptosis by small interfering RNA (siRNAs) against caspase-12. METHODS: The Balb/c mouse primary hepatocytes were isolated in situ with two-step liver perfusion with 0.5 g/L collagenase type IV, and apoptosis were induced with 4 micromol/L thapsigargin (TG). The three kingds of siRNAs targeting different gene sites (130, 214, 521) were synthetized chemically. The single-stranded RNAs were annealed to produce double-stranded siRNAs, then the mouse primary hepatocytes were transfected by oligofectamine package. The inhibition of caspase-12 was analyzed with RT-PCR and Western-blot. The viable hepatocytes following the induction of apoptosis were evaluated with MTT. RESULTS: All the three kinds of siRNAs could obviously inhibit normal mouse hepatocyte caspase-12 mRNA. The siRNA (214) were more effective than the other two when the concentration was 100 nmol/L. The caspase-12 mRNA expression was inhibited by 52.08%, while that of siRNA (521) was 30.73% (t=4.30, P <0.05). However when the concentration was 200 nmol/L, the inhibitions were similar (88.07%, 86.22% and 89.41% respectively). siRNA (214) could downregulate the expression of apoptotic hepatocytes procaspase-12 by 51.43% ( t=4.30, P <0.01). Contrasted with apoptotic hepatocytes, the cell activity, which was analyzed with MTT, increased by 48.76% (t=2.23, P <0.01). CONCLUSION: siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.
Asunto(s)
Apoptosis/fisiología , Caspasa 12/biosíntesis , Hepatocitos/citología , ARN Interferente Pequeño/genética , Animales , Caspasa 12/genética , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
OBJECTIVE: To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines. METHODS: Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot. RESULTS: In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis. CONCLUSION: Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteína p53 Supresora de Tumor/genética , Línea Celular Tumoral , Terapia Genética/métodos , Vectores Genéticos , Humanos , Conformación de Ácido Nucleico , Mutación Puntual , Conformación Proteica , ARN Catalítico , ARN Mensajero/síntesis química , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión , Ribonucleasa T1/farmacologíaRESUMEN
Transforming growth factorbeta1 (TGFbeta1) is considered to be the principal contributor to liver fibrosis. So in this study the ribozymes against TGFbeta1 were designed. The in vitro cleavage activities of the ribozymes were assayed through incubation of (32)p-labeled target RNAs and (32)p-labeled ribozymes in different conditions. HSC-T6 cells were transfected with the eukaryotic constructs encoding ribozyme and disable ribozyme, then the stable cell clones were used to evaluate its antifibrotic characteristic through the effect of ribozyme on biological character of activated hepatic stellate cells (HSCs). The results demonstrated that two ribozymes (Rz803 and Rz1395) could cleave target RNAs into expected products effectively, Rz803 possessed better cleavage activity in vitro. Stable transfection of Rz803 into activated HSCs reduced TGFbeta1 expression in mRNA and protein level efficiently. The further studies demonstrated that Rz803 reduced deposition of collagen I, suppressed HSC proliferation, but had no effect on HSC activation in transfected HSC-T6 cells. Therefore, it indicated that Rz803 could reverse the character of activated HSCs by down-regulating TGFbeta1 expression efficiently and diminishing TGFbeta1 signaling underlying activation of hepatic stellate cells. As the consequence, it would provide a potential therapeutic approach for liver fibrosis.
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Hepatocitos/efectos de los fármacos , Cirrosis Hepática/metabolismo , ARN Catalítico/farmacología , ARN/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Animales , Northern Blotting , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cartilla de ADN , Vectores Genéticos , Cinética , ARN/efectos de los fármacos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
AIM: To prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis. METHODS: Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neo(r)pBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with (alpha- (32)P)UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37 degrees in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg(2+)). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE(containing 8 mol/L urea). RESULTS: Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37 degrees, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%. CONCLUSION: Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis.
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Caspasas/genética , ARN Catalítico/genética , ARN Mensajero/genética , Animales , Caspasa 12 , Cartilla de ADN , ADN Complementario/genética , Cinética , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor beta subunit (PDGFR-beta) is the predominant signal transduction pathway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-beta mRNA in HSC and the effect on biological characteristics of HSC. METHODS: Expression vector of anti-PDGFR-beta ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-beta, alpha-smooth muscle actin, and typeI and type III collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy. RESULTS: The expression of PDGFR-beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49% - 57% (P < 0.05 - 0.01). The proliferation and alpha-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased (P < 0.05 - 0.01), and the type I and type III collagen synthesis were also reduced (P < 0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC (P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy. CONCLUSIONS: The anti-PDGFR-beta ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-beta expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-beta expression.
Asunto(s)
Apoptosis , Cirrosis Hepática/patología , Hígado/citología , ARN Catalítico/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Actinas/biosíntesis , Animales , Proliferación Celular , Células Cultivadas , Colágeno/biosíntesis , Cirrosis Hepática/tratamiento farmacológico , ARN Mensajero/metabolismo , RatasRESUMEN
The gynecophoral canal protein gene SjGCP of Schistosoma japonicum that is necessary for the pairing between the male and female worms is specifically expressed in the adult male worm. This protein is widely distributed in the adult female worm after pairing. Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence were employed to analyze the relationship between the RNAi effect and dsRNA dosage in the parasites. The results revealed that the inhibition of SjGCP expression by siRNA is dose-dependent. RT-PCR analysis showed that the SjGCP transcript level was reduced by 75% when 100 nM dsRNA was applied.
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Glicoproteínas/biosíntesis , Proteínas del Helminto/biosíntesis , Interferencia de ARN , ARN Bicatenario/farmacología , Schistosoma japonicum/metabolismo , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schistosoma japonicum/genética , Conducta Sexual AnimalRESUMEN
To study the inhibitory effects of plasmid-derived small interfering RNA (siRNA) and synthetic siRNA on the expression of the hepatitis B virus surface (HBs) gene, three plasmid-derived siRNAs and one synthetic siRNA that complement the coding region of the HBs gene were prepared. The HBs expression plasmid pHBs-EGFP was also constructed. HeLa cells were co-transfected with pHBs-EGFP and the above siRNAs. The HBs mRNA quantities were measured by reverse-transcription PCR, and the level of HBs-EGFP fusion protein was quantified by fluorescent microscope. The concentrations of the hepatitis B virus surface antigen (HBsAg) derived from the culture supernatant of transfected HepG2.2.15 cells were measured by an enzyme-linked immunosorbent assay (ELISA) kit. The results showed that the three plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the quantities of HBs mRNA and protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBs expression. It can inhibit HBs-EGFP expression by 63.3% and suppress HBs mRNA by 75.6%. To further substantiate the above observations, psiRNA1 was transfected into HepG2.2.15 cells (an HBV secreting cell line). The transfections resulted in almost complete blockage of HBsAg production, whereas control vector-transfected cells secreted high levels of HBsAg 7 days post-transfection. In conclusion, our data suggests that RNA interference (RNAi) is an efficient approach for reducing the level of HBs transcripts and proteins and for suppressing HBsAg production.
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Silenciador del Gen , Marcación de Gen/métodos , Ingeniería Genética/métodos , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , ARN Interferente Pequeño/genética , Análisis de Secuencia de ARN/métodos , Secuencia de Bases , Estudios de Factibilidad , Regulación Viral de la Expresión Génica , Terapia Genética/métodos , Células HeLa , Hepatitis B/genética , Hepatitis B/terapia , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia/métodosRESUMEN
RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNA-mediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression. Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0 approximately 60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5'end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.
Asunto(s)
Interferencia de ARN , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Proteínas Estructurales Virales/biosíntesis , Animales , Chlorocebus aethiops , Clonación Molecular , Proteínas M de Coronavirus , Proteínas de la Nucleocápside de Coronavirus , Regulación Viral de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Nucleocápside/antagonistas & inhibidores , Proteínas de la Nucleocápside/biosíntesis , Proteínas de la Nucleocápside/genética , ARN Interferente Pequeño/farmacología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/efectos de los fármacos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Células Vero , Proteínas del Envoltorio Viral/antagonistas & inhibidores , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Proteínas de la Matriz Viral/antagonistas & inhibidores , Proteínas de la Matriz Viral/biosíntesis , Proteínas de la Matriz Viral/genética , Proteínas Estructurales Virales/antagonistas & inhibidores , Proteínas Estructurales Virales/genética , Proteínas ViroporinasRESUMEN
BACKGROUND/AIMS: Transforming growth factor beta (TGFbeta1) is considered the key mediator in the process of liver fibrosis. The purpose of this investigation was to evaluate the activity of ribozymes against TGFbeta1 in a cell-free system and activated hepatic stellate cells (HSCs), and antifibrotic effect in activated HSCs in vitro and in rats. METHODS: Three ribozymes targeting against TGFbeta1 mRNA were designed, and then cloned into the U1 snRNA expression cassette. The chimeric ribozymes were selected for the analysis of their performances in activated HSCs through the detection of their cleavage activities in a cell-free system. After ribozyme-encoding plasmids had been transfected into HSC-T6 cells, the effects of ribozymes on activated HSCs were evaluated through the analysis of proliferation, activation and collagen deposition of HSC-T6. The adenoviral vector expressing the ribozymes was constructed, and then delivered into rat models of hepatic fibrosis induced by carbon tetrachloride. RESULTS: TGFbeta1 expression was efficiently down-regulated in activated HSCs by U1 snRNA chimeric ribozymes which possessed perfect cleavage activity in a cell-free system. Further studies demonstrated that U1 snRNA chimeric ribozymes inhibited the synthesis of collagen I, reduced deposition of collagen I, suppressed BrdU incorporation, but had no effect on desmin and alpha-SMA expression in transfected HSC-T6 cells. Histological analysis demonstrated that the adenoviral vector expressing ribozyme (Rz803) could alleviate fibrotic pathology in rats treated with carbon tetrachloride. CONCLUSIONS: The anti-TGFbeta1 ribozymes could reverse the character of activated HSCs in vitro and improve fibrotic pathology in vivo. It indicated that TGFbeta1 could be considered as a novel candidate for a therapeutic agent against hepatic fibrosis.