AIM: To investigate the effects of blockade of insulin receptor substrate-1(IRS-1) on the bio-function of tube formation of human choroidal endothelial cells(HCECs).METHODS: Quantitative reverse transcriptionpolymeras...AIM: To investigate the effects of blockade of insulin receptor substrate-1(IRS-1) on the bio-function of tube formation of human choroidal endothelial cells(HCECs).METHODS: Quantitative reverse transcriptionpolymerase chain reaction(RT-PCR) and Western blot were performed to determine the expression level of IRS-1 and phospho-IRS-1 in HCECs. Tube formation of HCECs was analyzed using three dimensional in vitro Matrigel assay with or without IRS-1 blockage via IRS-1 inhibitor(GS-101) and vascular endothelial growth factor receptor 2(VEGFR2) inhibitor. In addition, cell counting kit(CCK)-8 and Transwell migration assay were exerted to analyze the effects of blockade of IRS-1 on the bio-function of proliferation and migration of HCECs, respectively. The apoptosis of HCECs was examined using flow cytometry(FCM).RESULTS: RT-PCR and Western blot revealed that IRS-1 phospho-IRS-1 were expressed in HCECs and the expression level was enhanced by stimulation of VEGF-A. The number of tube formation was decreased significantly in GS-101 treated groups compared to phosphate buffered saline(PBS) treated control groups. Furthermore, both cell proliferation and migration of HCECs were decreased in the presence of GS-101. FCM analysis showed that the apoptosis of HCECs was enhanced when the cells were treated with GS-101. Western blot also showed that the expression level of cleaved-caspase 3 in GS-101 treated group was higher than that in control group.CONCLUSION: Blockade of IRS-1 can inhibit tube formation of HCECs through reducing cell proliferation and migration and promoting cell apoptosis.展开更多
BACKGROUND Despite effective prevention and screening methods,the incidence and mortality rates associated with colorectal cancer(CRC)are still high.Insulin receptor substrate 1(IRS-1),a signaling molecule involved in...BACKGROUND Despite effective prevention and screening methods,the incidence and mortality rates associated with colorectal cancer(CRC)are still high.Insulin receptor substrate 1(IRS-1),a signaling molecule involved in cell proliferation,survival and metabolic responses has been implicated in carcinogenic processes in various cellular and animal models.However,the role of IRS-1 in CRC biology and its value as a clinical CRC biomarker has not been well defined.AIM To evaluate if and how IRS-1 expression and its associations with the apoptotic and proliferation tumor markers,Bax,Bcl-xL and Ki-67 are related to clinicopathological features in human CRC.METHODS The expression of IRS-1,Bax,Bcl-xL and Ki-67 proteins was assessed in tissue samples obtained from 127 patients with primary CRC using immunohistochemical methods.The assays were performed using specific antibodies against IRS-1,Bax,Bcl-xL,Ki-67.The associations between the expression of IRS-1,Bax,Bcl-xL,Ki-67 were analyzed in relation to clinicopathological parameters,i.e.,patient age,sex,primary localization of tumor,histopathological type,grading,staging and lymph node spread.Correlations between variables were examined by Spearman rank correlation test and Fisher exact test with a level of significance at P<0.05.RESULTS Immunohistochemical analysis of 127 CRC tissue samples revealed weak cytoplasmatic staining for IRS-1 in 66 CRC sections and strong cytoplasmatic staining in 61 cases.IRS-1 expression at any level in primary CRC was associated with tumor grade(69%in moderately differentiated tumors,G2 vs 31%in poorly differentiated tumors,G3)and with histological type(81.9%in adenocarcinoma vs 18.1%in adenocarcinoma with mucosal component cases).Strong IRS-1 positivity was observed more frequently in adenocarcinoma cases(95.1%)and in moderately differentiated tumors(85.2%).We also found statistically significant correlations between expression of IRS-1 and both Bax and Bcl-xL in all CRC cases examined.The relationships between studied proteins were related to clinicopathological parameters of CRC.No significant correlation between the expression of IRS-1 and proliferation marker Ki-67,excluding early stage tumors,where the correlation was positive and on a high level(P=0.043,r=0.723).CONCLUSION This study suggests that IRS-1 is co-expressed with both pro-and antiapoptotic markers and all these proteins are more prevalent in more differentiated CRC than in poorly differentiated CRC.展开更多
Type 2 diabetes一associated with impaired insulin/insulin-like growth factor-1 (IGF1) signaling (IIS)一is a risk factor for cognitive impairment and dementia including Alzheimer's disease (AD). The insulin recepto...Type 2 diabetes一associated with impaired insulin/insulin-like growth factor-1 (IGF1) signaling (IIS)一is a risk factor for cognitive impairment and dementia including Alzheimer's disease (AD). The insulin receptor substrate (IRS) proteins are major components of IIS, which transmit upstream signals via the insulin receptor and/or IGF1 receptor to multiple intracellular signaling pathways, including AKT/protein kinase B and extracellular-signal-regulated kinase cascades. Of the four IRS proteins in mammals, IRS1 and IRS2 play key roles in regulating growth and survival, metabolism, and aging. Meanwhile, the roles of IRS1 and IRS2 in the central nervous system with respect to cognitive abilities remain to be clarified. In contrast to IRS2 in peripheral tissues, inactivation of neural IRS2 exerts beneficial effects, resulting in the reduction of amyloid p accumulation and premature mortality in AD mouse models. On the other hand, the increased phosphorylation of IRS 1 at several serine sites is observed in the brains from patients with AD and animal models of AD or cognitive impairment induced by type 2 diabetes. However, these serine sites are also activated in a mouse model of type 2 diabetes, in which the diabetes drug metformin improves memory impairment. Because IRS1 and IRS2 signaling pathways are regulated through complex mechanisms including positive and negative feedback loops, whether the elevated phosphorylation of IRS1 at specific serine sites found in AD brains is a primary response to cognitive dysfunction remains unknown. Here, we examine the associations between IRS 1 /1 RS2-mediated signaling in the central nervous system and cognitive decline.展开更多
Background MicroRNAs （miRNAs） contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma. Methods Ex...Background MicroRNAs （miRNAs） contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma. Methods Expression profiles of miRNAs in uveal melanoma were performed using Agilent miRNA array. Quantitative real-time polymerase chain reaction was used to screen the expression levels of miR-145 in normal uveal tissue, uveal melanoma tissue, and uveal melanoma cell lines. Lenti-virus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Cell proliferation, cell cycle, and cell apoptosis of these miR-145 overexpression cell lines were examined by MTT assay and flow cytometry respectively. The target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of insulin-like growth factor-1 receptor （IGF-1R）, insulin receptor substrate-1 （IRS-1） proteins was determined by Western blotting analysis. IRS- 1 was knocked down in OCM-1 cells. TUNEL, BrdU, and flow cytometry assay were performed in IRS-1 knocked down OCM-1 cell lines to analyze its function. Results Forty-seven miRNAs were up regulated in uveal melanoma and 61 were down regulated, miR-145 expression was significantly lower in uveal melanoma sample and the cell lines were compared with normal uveal sample. Overexpression of miR-145 suppressed cell proliferation by blocking the G1 phase entering S phase in uveal melanoma cells, and promoted uveal melanoma cell apoptosis. IRS-1 was identified as a potential target of miR-145 by dual luciferase reporter assay. Knocking down of IRS-1 had similar effect as overexpression of miR-145. Conclusion miR-145 might act as a tumor suppressor in uveal melanoma, and downregulation of the target IRS-1 might be a potential mechanism.展开更多
基金Supported by the National Natural Science Foundation in China(No.81671641 No.81970830+6 种基金 No.31600736)Suzhou Municipal Natural Science Foundation(No.SYS201745)Soochow University Doctoral Academic Talents Program(No.5832001313)Jiangsu Provincial Medical Youth Talent(No.QNRC2016718)Jiangsu Provincial Medical Innovation Team(No.CXTDA2017039)Jiangsu Provincial Natural Science Foundation(No.BK20151208)the Soochow Scholar Project of Soochow University(No.R5122001)
文摘AIM: To investigate the effects of blockade of insulin receptor substrate-1(IRS-1) on the bio-function of tube formation of human choroidal endothelial cells(HCECs).METHODS: Quantitative reverse transcriptionpolymerase chain reaction(RT-PCR) and Western blot were performed to determine the expression level of IRS-1 and phospho-IRS-1 in HCECs. Tube formation of HCECs was analyzed using three dimensional in vitro Matrigel assay with or without IRS-1 blockage via IRS-1 inhibitor(GS-101) and vascular endothelial growth factor receptor 2(VEGFR2) inhibitor. In addition, cell counting kit(CCK)-8 and Transwell migration assay were exerted to analyze the effects of blockade of IRS-1 on the bio-function of proliferation and migration of HCECs, respectively. The apoptosis of HCECs was examined using flow cytometry(FCM).RESULTS: RT-PCR and Western blot revealed that IRS-1 phospho-IRS-1 were expressed in HCECs and the expression level was enhanced by stimulation of VEGF-A. The number of tube formation was decreased significantly in GS-101 treated groups compared to phosphate buffered saline(PBS) treated control groups. Furthermore, both cell proliferation and migration of HCECs were decreased in the presence of GS-101. FCM analysis showed that the apoptosis of HCECs was enhanced when the cells were treated with GS-101. Western blot also showed that the expression level of cleaved-caspase 3 in GS-101 treated group was higher than that in control group.CONCLUSION: Blockade of IRS-1 can inhibit tube formation of HCECs through reducing cell proliferation and migration and promoting cell apoptosis.
文摘BACKGROUND Despite effective prevention and screening methods,the incidence and mortality rates associated with colorectal cancer(CRC)are still high.Insulin receptor substrate 1(IRS-1),a signaling molecule involved in cell proliferation,survival and metabolic responses has been implicated in carcinogenic processes in various cellular and animal models.However,the role of IRS-1 in CRC biology and its value as a clinical CRC biomarker has not been well defined.AIM To evaluate if and how IRS-1 expression and its associations with the apoptotic and proliferation tumor markers,Bax,Bcl-xL and Ki-67 are related to clinicopathological features in human CRC.METHODS The expression of IRS-1,Bax,Bcl-xL and Ki-67 proteins was assessed in tissue samples obtained from 127 patients with primary CRC using immunohistochemical methods.The assays were performed using specific antibodies against IRS-1,Bax,Bcl-xL,Ki-67.The associations between the expression of IRS-1,Bax,Bcl-xL,Ki-67 were analyzed in relation to clinicopathological parameters,i.e.,patient age,sex,primary localization of tumor,histopathological type,grading,staging and lymph node spread.Correlations between variables were examined by Spearman rank correlation test and Fisher exact test with a level of significance at P<0.05.RESULTS Immunohistochemical analysis of 127 CRC tissue samples revealed weak cytoplasmatic staining for IRS-1 in 66 CRC sections and strong cytoplasmatic staining in 61 cases.IRS-1 expression at any level in primary CRC was associated with tumor grade(69%in moderately differentiated tumors,G2 vs 31%in poorly differentiated tumors,G3)and with histological type(81.9%in adenocarcinoma vs 18.1%in adenocarcinoma with mucosal component cases).Strong IRS-1 positivity was observed more frequently in adenocarcinoma cases(95.1%)and in moderately differentiated tumors(85.2%).We also found statistically significant correlations between expression of IRS-1 and both Bax and Bcl-xL in all CRC cases examined.The relationships between studied proteins were related to clinicopathological parameters of CRC.No significant correlation between the expression of IRS-1 and proliferation marker Ki-67,excluding early stage tumors,where the correlation was positive and on a high level(P=0.043,r=0.723).CONCLUSION This study suggests that IRS-1 is co-expressed with both pro-and antiapoptotic markers and all these proteins are more prevalent in more differentiated CRC than in poorly differentiated CRC.
基金supported by a MEXTGrant-in-Aid for Scientific Research on Innovative Areas(brain environment)(JP24111536 to AT)+3 种基金JSPS KAKENHI(JP24650201,JP26282026,JP17K19951,JP17H02188 to AT)grants from the Mitsubishi Foundation(to AT)NOVARTIS Foundation Japan for the Promotion of Science(to AT)
文摘Type 2 diabetes一associated with impaired insulin/insulin-like growth factor-1 (IGF1) signaling (IIS)一is a risk factor for cognitive impairment and dementia including Alzheimer's disease (AD). The insulin receptor substrate (IRS) proteins are major components of IIS, which transmit upstream signals via the insulin receptor and/or IGF1 receptor to multiple intracellular signaling pathways, including AKT/protein kinase B and extracellular-signal-regulated kinase cascades. Of the four IRS proteins in mammals, IRS1 and IRS2 play key roles in regulating growth and survival, metabolism, and aging. Meanwhile, the roles of IRS1 and IRS2 in the central nervous system with respect to cognitive abilities remain to be clarified. In contrast to IRS2 in peripheral tissues, inactivation of neural IRS2 exerts beneficial effects, resulting in the reduction of amyloid p accumulation and premature mortality in AD mouse models. On the other hand, the increased phosphorylation of IRS 1 at several serine sites is observed in the brains from patients with AD and animal models of AD or cognitive impairment induced by type 2 diabetes. However, these serine sites are also activated in a mouse model of type 2 diabetes, in which the diabetes drug metformin improves memory impairment. Because IRS1 and IRS2 signaling pathways are regulated through complex mechanisms including positive and negative feedback loops, whether the elevated phosphorylation of IRS1 at specific serine sites found in AD brains is a primary response to cognitive dysfunction remains unknown. Here, we examine the associations between IRS 1 /1 RS2-mediated signaling in the central nervous system and cognitive decline.
基金This study was supported by grants from the National Natural Science Foundation of China （No. 81272981） and Natural Sciences Fundation of Beijing, China （No. 7112031）.
文摘Background MicroRNAs （miRNAs） contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma. Methods Expression profiles of miRNAs in uveal melanoma were performed using Agilent miRNA array. Quantitative real-time polymerase chain reaction was used to screen the expression levels of miR-145 in normal uveal tissue, uveal melanoma tissue, and uveal melanoma cell lines. Lenti-virus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Cell proliferation, cell cycle, and cell apoptosis of these miR-145 overexpression cell lines were examined by MTT assay and flow cytometry respectively. The target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of insulin-like growth factor-1 receptor （IGF-1R）, insulin receptor substrate-1 （IRS-1） proteins was determined by Western blotting analysis. IRS- 1 was knocked down in OCM-1 cells. TUNEL, BrdU, and flow cytometry assay were performed in IRS-1 knocked down OCM-1 cell lines to analyze its function. Results Forty-seven miRNAs were up regulated in uveal melanoma and 61 were down regulated, miR-145 expression was significantly lower in uveal melanoma sample and the cell lines were compared with normal uveal sample. Overexpression of miR-145 suppressed cell proliferation by blocking the G1 phase entering S phase in uveal melanoma cells, and promoted uveal melanoma cell apoptosis. IRS-1 was identified as a potential target of miR-145 by dual luciferase reporter assay. Knocking down of IRS-1 had similar effect as overexpression of miR-145. Conclusion miR-145 might act as a tumor suppressor in uveal melanoma, and downregulation of the target IRS-1 might be a potential mechanism.
