RESUMO
OBJECTIVE: Wilms' tumor is the most common malignant tumor in children worldwide. Considering the poor therapeutic effect on Wilms' tumor, we determined the effects of microRNA-613 on cell proliferation and metastasis in vitro, providing therapeutic targets for the treatment of Wilms' tumor. PATIENTS AND METHODS: Quantitative real-time PCR (qRT-PCR) was employed to identify the expression level of miR-613. CCK8 and colony formation assays were incorporated to assess cell viability and proliferation capacity. Cell migration and invasion assays were performed to investigate the metastasis capacity of Wilms' tumor cells. Flow cytometry was used to detect cell cycle distribution and cell apoptosis. Protein levels were assessed by western blotting assay. The target gene was predicted and verified by bioinformatics analysis and luciferase assay. RESULTS: The expression of miR-613 was downregulated in Wilms' tumor tissues compared with adjacent normal tissues (n=32). Overexpression of miR-613 could attenuate Wilms' tumor cell viability, proliferation, invasion, and migration capacity, as well as induce cell cycle arrest at the G0/G1 phase. FRS2 was chosen as the target of miR-613 by bioinformatics analysis and a luciferase reporter assay. MiR-613 expression was inversely correlated with FRS2 in Wilms' tumor tissues. Moreover, restoration of FRS2 rescued the tumor suppressive role of miR-613 in Wilms' tumor cell growth and metastasis. CONCLUSIONS: MiR-613 had a tumor-suppressive effect on Wilms' tumor progression and metastasis via targeting FRS2 in vitro, which provided an innovative and candidate target for the diagnosis and treatment of Wilms' tumor.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Membrana/genética , MicroRNAs/farmacologia , Tumor de Wilms/genética , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Marcação de Genes , Humanos , Sincalida/biossíntese , Sincalida/genéticaRESUMO
Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene whose reduced expression may play an important role in the development and progression of esophageal squamous cell cancer (ESCC). The aim of this study was to investigate the clinical relevance of RUNX3 in ESCC patients and effects of overexpression on biological behaviour of Eca109 cells in vitro and in vivo. Immunohistochemistry was performed to detect the clinical relevance of RUNX3 and lymph node metastasis in 80 ESCC tissues and 40 non-cancerous tissues using the SP method. RT-PCR and Western blotting were applied to assess the RUNX3 level and verify the Eca109 cell line with stable overexpression. Localization of RUNX3 proteins was performed by cell immunofluorescence. CCK-8 and Scrape motility assays were used to determine proliferation and migration and the TUNEL assay to analyze cell apoptosis. Invasive potential was assessed in cell transwell invasion experiments. In nude mice, tumorigenesis in vivo was determined. Results showed decreased expression of RUNX3 in esophageal tissue to be significantly related to lymph node metastasis (LNM) (P<0.01). In addition, construction of a recombinant lentiviral vector and transfection into the human ESCC cell line Eca109 demonstrated that overexpression could inhibit cell proliferation, migration and invasion, and induce apoptosis. The in vivo experiments in mice showed tumorigenicity and invasiveness to be significantly reduced. Taken together, our studies indicate that underexpression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion and tumorigenesis.
Assuntos
Apoptose/genética , Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/patologia , Subunidade alfa 3 de Fator de Ligação ao Core/biossíntese , Neoplasias Esofágicas/patologia , Invasividade Neoplásica/patologia , Animais , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Progressão da Doença , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas do Esôfago , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/genética , RNA Mensageiro/biossíntese , Sincalida/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/genéticaRESUMO
We have reported that cholecystokinin-like immunoreactivity (CCK-LI) and its transcripts are expressed in rat pancreatic islets (Endocrinology 1998;139:389-96). The purpose of this study was to elucidate the ontogeny of CCK during pancreatic development in the rat. Fetal rats from day 13 (E13) to 20 (E20) and postnatal (P) rats from day 1 to adulthood were used in this study. Immunohistochemical studies of rat pancreas were carried out with rabbit antisera against CCK-8 and gastrin-17. The absorption studies were performed by using CCK-8 antiserum incubated with excess CCK-8 or gastrin-17. In situ hybridization was performed to demonstrate the CCK and gastrin transcripts in the pancreas. CCK and gastrin were first detected at E15, and they were distributed in the periphery of the islets in the fetal and neonatal pancreas. The mirror sections for CCK revealed positive cells with characteristics identical to those that stained positive for gastrin. The CCK-LI in early development was completely abolished by preabsorption with excess gastrin but not with CCK-8. These findings indicated that the CCK-LI in the fetal and neonatal pancreas was crossreacting gastrin rather than CCK-8. From weaning (P21) through adulthood, on the other hand, CCK-LI was expressed diffusely in pancreatic islets, but there were no gastrin-positive cells after weaning. In situ hybridization showed that CCK messenger RNA (mRNA) was present in rat pancreatic islets in adults but not in early development. Although CCK-positive cells were not detected in fetal and neonatal pancreatic islets, CCK was expressed in islets during and after weaning through adulthood, indicating that CCK in pancreatic islets might be developmentally regulated.
Assuntos
Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Sincalida/biossíntese , Animais , Animais Recém-Nascidos , DNA Complementar/metabolismo , Desenvolvimento Embrionário e Fetal , Feminino , Gastrinas/análise , Técnicas Imunoenzimáticas , Hibridização In Situ , Ilhotas Pancreáticas/metabolismo , Masculino , Gravidez , Coelhos , Ratos , Ratos Wistar , Sincalida/genéticaRESUMO
Cholecyctokinin-octapeptide (CCK-8) is one of the brain-gut peptides widely distributed in neurons of the central nervous system, especially in cerebral cortex and hippocampus. This peptide is thought as a neurotransmitter or modulator. The role of central CCK in the integration of gastric motility and food intake is still unclear. The present investigation was undertaken to see whether the gastric distension exerts some effect on the expression of CCK mRNA in cerebral cortex and hippocampus by means of in situ hybridization and semi-quantitative RT-PCR techniques. The results are as follows. (1) Discrete positive hybridization cells were seen in cerebral cortex and hippocampus in control group. (2) Significant increases of CCK mRNA expression in cerebral cortex and hippocampus produced by gastric distention observed by increased of the volume, the number, the IOD and the SA of positive cells. (3) Amplified bands of the expected size of 158 base pairs were found in cerebral cortex and hippocampus, and signals of gastric distention group were higher than those of control group. The results from RT-PCR assay are in agreement with those based on in situ hybridization. These results imply that biosynthesis of CCK-containing neurons in cerebral cortex and hippocampus is accelerated by afferent signals from gastrointestinal system and may be involved in regulation of gastric motility and food intake.
Assuntos
Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Sincalida/biossíntese , Estômago/fisiologia , Animais , Dilatação , Feminino , Mucosa Gástrica/metabolismo , Masculino , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Ratos Wistar , Sincalida/genéticaRESUMO
Two endocrine tumor cell lines from pancreas (RIN5F) and intestine (STC-1) express cholecystokinin (CCK) messenger RNA and are able to posttranslationally process pro-CCK to CCK-22 and CCK-8 amide. Both of these forms are also secreted by these cells. Because they make and secrete forms of amidated CCK larger than CCK-8, they represent a model of pro-CCK processing in the gut and allow investigation of possible mechanisms for tissue differences in prohormone processing. Both of these cells express two endoproteases convertase-1 (PC1) also known as PC3 and prohormone convertase-2 (PC2), which may be involved in pro-CCK processing. We have previously shown than inhibition of PC1 expression in these cells using stable expression of antisense messenger RNA caused a significant reduction in cellular content of amidated CCK and caused a selective depletion of CCK-8 with a comparative sparing of CCK-22. We demonstrate here that inhibition of PC2 expression in these cells also caused a large initial decrease in CCK content and produced a selective depletion of CCK-22 and a comparative sparing of CCK-8. These results support both a role for both PC1 and PC2 in pro-CCK processing in these cells and the hypothesis that tissue-specific processing of pro-CCK may be explained by differences in expression or activity of PC1 and PC2.
Assuntos
Colecistocinina/biossíntese , Fragmentos de Peptídeos/biossíntese , Sincalida/biossíntese , Subtilisinas/metabolismo , Colecistocinina/genética , Expressão Gênica , Neoplasias Intestinais/enzimologia , Neoplasias Pancreáticas/enzimologia , Fragmentos de Peptídeos/genética , Pró-Proteína Convertase 2 , RNA Antissenso/genética , RNA Mensageiro/genética , Sincalida/genética , Subtilisinas/genética , Transfecção , Células Tumorais CultivadasRESUMO
Several immortalized cell lines serve as models for procholecystokinin (pro-CCK) processing. Rin5F cells, derived from a rat insulinoma, and STC-1 cells, derived from a murine intestinal tumor, process pro-CCK mainly to amidated CCK 8. Both also make significant quantities of amidated CCK 22, a slightly larger form found in the gut. Many modifications are necessary during pro-CCK processing including cleavages performed by endoproteases, the identities of which are unknown. A candidate endoprotease is prohormone convertase 1 (PC1) also known as PC3, a Ca2+-dependent serine endoprotease of the subtilisin family. Constitutive expression of antisense PC1 message in stably transfected Rin5F cells resulted in a significant reduction of the cellular content of CCK 8 as measured by radioimmunoassay. Several affected cell lines displayed about 80% reduction in CCK content in early passages after transfection. Expression of antisense PC1 message in these cell lines resulted in a selective depletion of CCK 8 and a comparative sparing of CCK 22. The induction of antisense PC1 message within a single subclone of Rin5F cells using the Lac Switch system also resulted in a significant inhibition of CCK content. Expression of antisense PC1 message in a stably transfected STC-1 cell line also resulted in a decrease in CCK content and in PC1 protein expression, and the specific depletion of CCK 8 with comparative sparing of CCK 22. These observations support the hypothesis that PC1 is necessary for pro-CCK processing in Rin5F and STC-1 cells and suggests a role for PC1 endoprotease in the biosynthesis of CCK 8 in vivo.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Sincalida/biossíntese , Animais , Ácido Aspártico Endopeptidases/genética , Colecistocinina/metabolismo , Cromatografia em Gel , Insulinoma/metabolismo , Neoplasias Intestinais/metabolismo , Modelos Químicos , Oligonucleotídeos Antissenso/metabolismo , Neoplasias Pancreáticas/metabolismo , Fragmentos de Peptídeos/metabolismo , Pró-Proteína Convertases , Ratos , Células Tumorais CultivadasRESUMO
To investigate cholecystokinin (CCK) mRNA changes induced by social isolation rats were isolated in single cages soon after weaning for 30 days. They were then sacrificed and their brains removed for in situ hybridization (ISH) study. Control animals were housed in groups of 6 per cage for the same period. ISH was performed using a 32P-labelled oligonucleotide probe complementary to CCK-8 mRNA and the results analysed by computerized densitometry. They showed a significant increase (from 59.5-152.3%) in CCK mRNA expression in the basolateral amygdala, cortex, CA1, dorsal raphe nucleus, geniculate body and ventral tegmental area of isolated rats. These results suggest that social isolation may influence CCK gene expression.
Assuntos
Encéfalo/metabolismo , Colecistocinina/biossíntese , Sincalida/biossíntese , Isolamento Social , Transcrição Gênica , Animais , Hibridização In Situ , Masculino , Sondas de Oligonucleotídeos , Especificidade de Órgãos , RNA Mensageiro/biossíntese , Ratos , Ratos WistarRESUMO
Cholecystokinin-octapeptide (CCK-8S) is widely distributed in neurones of the central nervous system, where it is thought to act as a transmitter or modulator. CCK-8S has been shown to exert anti-convulsant activity in animal seizure models and changes in cortical and hippocampal CCK-immunoreactivity and preproCCK messenger RNA (mRNA) have been reported following electrically- and chemically-induced seizures. In the present study, the spatiotemporal effect of amygdaloid-kindled seizures on levels of preproCCK messenger RNA in rat brain were determined using quantitative in situ hybridization histochemistry. Stimulation-evoked seizures produced bilateral increases (45-70%) in preproCCK mRNA throughout layers II-III of the cerebral cortex. These increases were rapidly induced, occurring 30-60 min after the last stage 5 seizure, but transient, as no significant changes were detected after 2 h, or subsequently at 24 or 72 h, or 2-8 weeks, post-stimulation. Rapid changes in the relative levels of preproCCK mRNA, post-seizure, suggest a possible stabilization of preproCCK transcripts and increased production of CCK-8S peptide, which may be involved in anticonvulsant mechanisms in response to the acute seizures.
Assuntos
Tonsila do Cerebelo/metabolismo , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Colecistocinina/biossíntese , Excitação Neurológica , Precursores de Proteínas/biossíntese , Convulsões/fisiopatologia , Transcrição Gênica , Animais , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Cinética , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Convulsões/metabolismo , Sincalida/análogos & derivados , Sincalida/biossíntese , Fatores de TempoRESUMO
The subtilisin-like endoprotease PC1 (PC3) has been implicated in the processing of a number of prohormones. To evaluate whether PC1 may be important for the processing of pro CCK to CCK 8, stable cell lines expressing a portion of the PC1 cDNA in the antisense orientation were established from RIN5F cells. These cells express CCK mRNA, produce and display regulated secretion of CCK 8. One of the clones, R1E8, expresses antisense PC1 mRNA as determined by reverse transcriptase-PCR (RT-PCR) and contains a significantly reduced level of PC1 protein. As compared to both RIN5F and RIN5F control cells (transfected with the expression plasmid containing no antisense message), R1E8 contains only about 30% cell content of CCK 8. These results suggest that PC1 may be important for the processing of CCK 8 from pro CCK.
Assuntos
Ácido Aspártico Endopeptidases/biossíntese , RNA Antissenso/farmacologia , RNA Mensageiro/efeitos dos fármacos , Sincalida/biossíntese , Transfecção , Animais , Células Clonais/efeitos dos fármacos , Reação em Cadeia da Polimerase , Pró-Proteína Convertases , RNA Mensageiro/biossíntese , Ratos , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Immunocytochemistry and in situ hybridization techniques were used for investigating changes in cholecystokinin immunoreactivity and mRNA in the cerebral cortex and hippocampus after kainic acid-induced limbic seizures in the rat. Marked increases in cholecystokinin mRNA concentrations were observed in layers II/III and V/VI of the cerebral cortex, in CA1 pyramidal neurons of the hippocampus, and in presumptive basket cells of the dentate gyrus 1 and 2 days after the acute seizures. Whereas cholecystokinin mRNA contents returned to normal in the cerebral cortex and the CA1 sector at later intervals, high concentrations were observed in basket cells even 2 months after the initial seizures. Accordingly, cholecystokinin-like immunoreactivity was intensified in the cerebral cortex, CA1 sector and in presumed basket cells of the hippocampus 30 days after kainic acid. Besides its high content in basket cells, cholecystokinin-like immunoreactivity was primarily present in neuronal fibers or diffusely distributed in the respective brain area. In the hippocampus, strongly enhanced staining for cholecystokinin was also observed in the alveus, the stratum lacunosum moleculare, and in the inner molecular layer, suggesting increased concentrations of the peptide in afferent and efferent fibers of the hippocampus. The present experiments suggest a strong activation of cholecystokinin systems in the brain after kainic acid-induced limbic seizures in the rat. This is indicated by pronounced increases in cholecystokinin mRNA in the cortex and individual cell types of the hippocampus (basket cells, granule cells, and CA1 pyramidal neurons). The subsequent increases in cholecystokin immunoreactivity even surpass those in mRNA. The observed changes may be part of the self-defense mechanisms that protect the animals during subsequent epileptic episodes.