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1.
World J Gastroenterol ; 27(15): 1595-1615, 2021 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-33958846

RESUMEN

BACKGROUND: Expression of the full-length isoform of Abelson interactor 1 (ABI1), ABI1-p65, is increased in colorectal carcinoma (CRC) and is thought to be involved in one or more steps leading to tumor progression or metastasis. The ABI1 splice isoform-L (ABI1-SiL) has conserved WAVE2-binding and SH3 domains, lacks the homeo-domain homologous region, and is missing the majority of PxxP- and Pro-rich domains found in full-length ABI1-p65. Thus, ABI1-SiL domain structure suggests that the protein may regulate CRC cell morphology, adhesion, migration, and metastasis via interactions with the WAVE2 complex pathway. AIM: To investigate the potential role and underlying mechanisms associated with ABI1-SiL-mediated regulation of CRC. METHODS: ABI1-SiL mRNA expression in CC tissue and cell lines was measured using both qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR. A stably ABI1-SiL overexpressing SW480 cell model was constructed using Lipofectamine 2000, and cells selected with G418. Image J software, CCK8, and transwell assays were used to investigate SW480 cell surface area, proliferation, migration, and invasion. Immunoprecipitation, Western blot, and co-localization assays were performed to explore intermolecular interactions between ABI1-SiL, WAVE2, and ABI1-p65 proteins. RESULTS: ABI1-SiL was expressed in normal colon tissue and was significantly decreased in CRC cell lines and tissues. Overexpression of ABI1-SiL in SW480 cells significantly increased the cell surface area and inhibited the adhesive and migration properties of the cells, but did not alter their invasive capacity. Similar to ABI1-p65, ABI1-SiL still binds WAVE2, and the ABI1-p65 isoform in SW480 cells. Furthermore, co-localization assays confirmed these intermolecular interactions. CONCLUSION: These results support a model in which ABI1-SiL plays an anti-oncogenic role by competitively binding to WAVE2 and directly interacting with phosphorylated and non-phosphorylated ABI1-p65, functioning as a dominant-negative form of ABI1-p65.


Asunto(s)
Neoplasias Colorrectales , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/genética , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Isoformas de Proteínas
2.
Zhonghua Yi Xue Za Zhi ; 91(37): 2648-52, 2011 Oct 11.
Artículo en Chino | MEDLINE | ID: mdl-22321934

RESUMEN

OBJECTIVE: To explore the effects of receptor interacting protein (RIP) 140 gene overexpression upon the in vitro proliferation, apoptosis, invasion and migration of microglioma cells. METHODS: The BV-2 RIP140 overexpression model (BV-2-1) was constructed by Lipofection and G418 selection, then validated by real-time PCR and Western blotting. The proliferation, apoptosis, invasion and migration potencies were compared between BV-2-1 and its parents by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, flow cytometry and Transwell chamber. RESULTS: The BV-2-1 model was successfully constructed. Compared to those of the BV-2 group, the RIP140 mRNA and protein expression levels of BV-2-1 were markedly higher than those of the BV-2 group (t = 49.794, P < 0.01). MTT assay showed that the absorbance values in the BV-2 group were 1.157 ± 0.013, 1.679 ± 0.005 and 2.609 ± 0.008 at 24, 48, and 72 hours respectively. And those were 0.929 ± 0.013, 1.188 ± 0.008 and 1.528 ± 0.012 in the BV-2-1 group respectively. The proliferation at the time points of 48 and 72 hours of the BV-2-1 group were significantly lower than that of the BV-2 group (t = 6.058 and 9.245, both P < 0.01). Annexin-V staining showed that there were significant differences in the apoptosis rates between the BV-2 and BV-2-1 cells [(5.35 ± 0.23)% vs (3.46 ± 0.45)%, t = 6.619, P = 0.003)]. Transwell assay showed that the invaded cell number of the BV-2-1 group was 166 ± 43. And it was obviously higher than that of the BV-2 group (93 ± 32, t = 3.403, P = 0.007). Transwell assay also showed that the migrated cell number of BV-2 cells was 101 ± 25. And the migration potency of the BV-2-1 group (202 ± 50) was significantly stronger than that of the BV-2 group (t = 4.104, P = 0.002). CONCLUSION: RIP140 effectively inhibits the proliferation and facilitates the apoptosis of microglioma cells. And it may effectively facilitate the in vitro invasion and migration of microglioma cells.


Asunto(s)
Apoptosis , Neuroglía/citología , Neuroglía/metabolismo , Co-Represor 1 de Receptor Nuclear/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Ratones , Co-Represor 1 de Receptor Nuclear/genética
3.
Chin Med J (Engl) ; 122(3): 331-7, 2009 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-19236814

RESUMEN

BACKGROUND: Runt-related transcription factor 1 (Runx1) plays a crucial role in hematogenesis and its dysfunction may contribute to leukemogenesis. However, it is not clear whether or not abnormal expression of Runx1 will induce leukemia and how the change of Runx1 expression level could affect BCR-ABL-induced leukemogenesis. In the present study, we aimed to analyze if abnormal expression of Runx1 in BaF3 cells alone would induce leukemogenesis. And we also wanted to know if abnormal expression of Runx1 in leukemic cells would affect leukemogenesis. Furthermore, we investigated whether overexpression or knock-down of Runx1 in BaF3 cells would induce leukemogenesis. METHODS: Plasmids containing full-length Runx1 cDNA were transduced into BaF3 cells and BaF3-P185wt cells (BCR-ABL transformed BaF3 cells) by electroporation. Plasmids containing a short hairpin RNA of Runx1 were transduced into BaF3 cells and BaF3-P185wt cells by electroporation. Runx1 expression level was quantified by Western blotting and quantitative real-time PCR. The effects of overexpression or knock-down of Runx1 on proliferation, apoptosis and migration of cells were detected in vitro. Then, using MSCV-P185wt-EGFP as a control, we transplanted MSCV-P185wt-Runx1 cells or MSCV-P185wt-shRNA cells into Balb/c mice through tail vein and observed tumorgenesis of the different phenotypes. RESULTS: In vitro analysis revealed that overexpression of Runx1 in P185wt cells could inhibit cell proliferation and slow down cell migration; while knock-down of Runx1 could promote cell proliferation and speed up cell migration. In vivo analysis indicated that mice transplanted with MSCV-P185wt-Runx1 survived longer than controls. In contrast, mice transplanted with MSCV-P185wt-shRNA survived shorter than the control group. Gross pathological analysis revealed that the MSCV-P185wt-Runx1 group had less severe splenomegaly and hepatomegaly compared to the control group, and the MSCV-P185wt-shRNA group had more severe splenomegaly and hepatomegaly. No splenomegaly or hepatomegaly was detected in mice transplanted with MSCV-BaF3-Runx1 cells or MSCV-BaF3-shRNA cells. Both the mice of MSCV-BaF3-Runx1 group and MSCV-BaF3-shRNA group were healthy with no sign of leukemia for up to three months. CONCLUSIONS: Overexpression or knock-down of Runx1 gene in BaF3 cells alone could not induce leukemogenesis. However, in BaF3-P185wt cells, alteration of Runx1 expression could affect BCR-ABL-induced proliferation and migration in vitro and leukemogenesis in vivo.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Proteínas de Fusión bcr-abl/farmacología , Leucemia/metabolismo , Leucemia/patología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia/genética , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa
4.
Zhonghua Yi Xue Za Zhi ; 88(40): 2857-61, 2008 Nov 04.
Artículo en Chino | MEDLINE | ID: mdl-19080498

RESUMEN

OBJECTIVE: To investigate the effects of receptor-interacting protein (RIP)140 gene knockdown on the proliferation of microglioma cells. METHODS: Mouse microglioma cells of the line BV-2 were cultured and transfected with 2 kinds of recombinant RIP140-shRNA plasmids (V2MM-71674 and V2MM-71080) or blank plasmid MSCV-EGFP. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of RIP140; and the cell proliferation was detected by MTT assay. RESULTS: There were not significant differences in the RIP140 mRNA and protein expression between the BV-2 and BV-2-MGCV-EGFP groups. Compared to those of the BV-2 group, the RIP140 mRNA expression levels of the BV-2-71674 and BV-2-71080 groups were lower by 73% and 75% respectively. The protein expression levels of the BV-2-71674 and BV-2-71080 groups were remarkably lower than those of the BV-2 and BV-MSGV-EGFP groups. MTT assay showed that there were not significant differences in the proliferation rates at different time points between the BV-2 and BV-2-MSCV-EGFP groups, however, the proliferation rates at the time points of 24, 48, 72, and 96 h of the BV-2-71674 and BV-2-71080 groups were significantly lower than those of the BV-2 group (all P<0.01). CONCLUSION: RIP140 gene knockdown effectively inhibits the proliferation of microglioma cells.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proliferación Celular , Proteínas Nucleares/genética , ARN Interferente Pequeño , Animales , Apoptosis , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Microglía , Proteína de Interacción con Receptores Nucleares 1 , ARN Mensajero/genética , Ratas , Transfección
5.
Reprod Toxicol ; 24(1): 89-96, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17561372

RESUMEN

Our previous study demonstrated that cystathionine beta synthase (CBS) is highly expressed in the cumulus-oocyte complex during ovulation. However, the role of CBS during oocyte maturation remains uncertain. In this study, a small-interfering (si) RNA interference (siRNA) approach was used to investigate the potential role of CBS during oocyte maturation. Accompanied with a gradual increase of homocysteine, the introduction of CBS-siRNA into murine granulosa cells selectively depleted the corresponding target mRNA and protein for CBS as assessed by semi-quantitative reverse-transcriptase PCR (RT-PCR) and immunofluorescence staining. When fully grown, germinal vesicle (GV) oocytes matured in vitro for 16 h using medium from transfected granulosa cells, the functional suppression of CBS resulted in a significant increase in the rate of GV-arrested oocytes. The results of this study provide evidence that CBS participates in the process of oocyte maturation. Furthermore, this effect may be fulfilled by conditioning the level of homocysteine in the microenvironment of the oocyte.


Asunto(s)
Cistationina betasintasa/metabolismo , Células de la Granulosa/metabolismo , Homocisteína/metabolismo , Oocitos/metabolismo , Oogénesis , Comunicación Paracrina , Animales , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Cistationina betasintasa/genética , Femenino , Células de la Granulosa/enzimología , Metionina/metabolismo , Ratones , Ratones Endogámicos ICR , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Transfección
6.
Chin Med J (Engl) ; 119(22): 1877-83, 2006 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-17134586

RESUMEN

BACKGROUND: In vitro fertilization (IVF) researches have suggested that cystathionine beta synthase (CBS) is involved in oocyte development. However, little is known about the regional and cellular expression patterns of CBS in the ovary. The purpose of this study was to analyze the localization of CBS in mice ovaries and to investigate the expression profile during follicular development. METHODS: We used in situ hybridization and immunohistochemical analysis to determine CBS expression in the ovaries of female Balb/c mice. Then the follicles were collected from F1 (C57BL x Balb/c) mice and cultured in vitro. With the method of semi-quantitative RT-PCR, we also investigated the expression profile of CBS during follicular development. RESULTS: CBS was absent in the oocytes, although it was ubiquitously expressed in the ovary with the strongest expression in follicular cells at all stages. In late antral follicles, CBS expression was markedly higher in granulosa cells located close to the antrum and in cumulus cells around the oocyte. The semi-quantitative RT-PCR showed that CBS mRNA was detected in follicles at all stages in vitro. In cumulus-oocyte complexes superovulated, CBS expression also increased rapidly. CONCLUSIONS: CBS was located mainly in the follicular cells in the ovaries. The level of CBS expression is high in follicles during folliculogenesis in mice. Differences in the CBS expression profile between oocyte and follicular cells suggest a role for CBS as a mediator in interactions between oocyte and granulosa cells.


Asunto(s)
Cistationina betasintasa/genética , Perfilación de la Expresión Génica , Folículo Ovárico/fisiología , Ovario/enzimología , Animales , Cistationina betasintasa/análisis , Femenino , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Mensajero/análisis
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