| The carboxyl terminus of Hsc70-interacting protein(CHIP)is an E3 ubiquitin ligase,also known as STUB1(STIP1 homology and U-Box containing protein 1),plays a controversial role in different cancers,either as a tumor suppressor or a tumor promoter.To date,the exact function and underlying mechanism of CHIP in CRC is not yet clear.In the present study,the stable CHIP-overexpression and CHIP-depletion CRC cell lines were successfully transfected.The influence and underlying mechanism of CHIP on the cell growth,proliferation,migration and invasion was investigated.The results revealed that CHIP-depletion suppressed the cell proliferation and G0-G1 arrest and inhibited cell growth,migration and invasion potential of CRC cells in vitro and in vivo,accompanying with downregulation of ERK and AKT signaling activities and cyclin D1,as well as upregulation of p27 and E-cadherin.Although CHIP-overexpression exerted little influence on cell growth of CRC cells,it dramatically enhanced the migration and invasion abilities in vitro and promoted tumor metastasis in vivo,contributed to the upregulation of ERK and AKT signaling activities,and downregulation of E-cadherin.Mechanically,CHIP might downregulate E-cadherin through inactivation of GSK-3β by the upregulation of AKT pathway,subsequently increasing Slug and decreasing E-cadherin expression.Proteomic analysis confirmed that E-cadherin was decreased in CHIP overexpression cells and correlated to CRC disease and cell-cell adhesion and junction based on the Disease,GO,and KEGG pathway enrichment analysis,respectively.Furthermore,we found that there was a higher CHIP expression and lower E-cadherin expression in CRC samples compared to that of the adjacent non-neoplastic cells.CHIP and E-cadherin expression was negatively correlated.Higher CHIP,as well as lower E-cadherin expression was significantly correlated with depth of tumor invasion,lymph nodes metastasis,TNM stage and poor prognosis of CRC patients.Collectively,our findings indicated that CHIP functions as an oncogene,and may be a potential unfavorable independent predictive biomarker for CRC.Part 1 The involvement and function of CHIP in the of cellular biological behaviors of colorectal cancerObject: To elucidate the function of E3 ubiquitin ligase CHIP on CRC cellular biological behavior,including cell growth,migration and invasion.Methods: 1,The m RNA and protein expression of CHIP in CRC cell lines,including DLD-1,HT-29,COLO320 DM,and Ca CO2,were detected by q RT-PCR and Western blotting.2,CHIP RNAi sequence was designed and p Silencer3.1-H1-neo plasmid was chosed to construct the p Silencer3.1-si CHIP plasmid.CHIP overexpression plasmid,MSCV-GFP-h CHIP,was also constructed using vector MSCV-GFP.p Silencer3.1-si CHIP and vector p Silencer3.1-H1-neo,MSCV-GFP-h CHIP and vector MSCV-GFP,were transfected into DLD-1 and HT-29 cells using Lipofectamine 2000.Stably transfected CHIP overexpression and depletion DLD-1 and HT-29 cells were established.3,Cell growth of CRC cells with CHIP-overexpression and depletion were monitored by x-Celligence system.Cell proliferation of CRC transfected cells were detected using CCK-8 and Brdu proliferation assays.Cell apoptosis and cell cycle of CRC transfected cells were detected by flow cytometry analysis.4,In vitro,cell migration and invasion of DLD-1 and HT-29 transfected cells were monitored by x-Celligence system,wound healing assay and Transwell assay.In vivo intraperitoneal metastasis assay was performed to investigate the influence of CHIP on the tumor metastasis of CRC cells in nude mice.The protein expression of CHIP in metastatic tissues was detected by Western blotting and immunohistochemistry.Results: 1,The expression of CHIP m RNA and protein could be detected in CRC cell lines,including DLD-1,HT-29,COLO320 DM,and Ca CO2,but at variable levels.The CHIP m RNA expression of DLD-1 and COLO320 DM cells was at a remarkable high level compared to that of HT-29 and Ca CO2 cell lines.Western blot analysis showed that the CHIP protein expression was much higher in the HT-29 and DLD-1 cells compared to that in the COLO320 DM and Ca CO2 cells.2,CHIP RNAi plasmid p Silencer3.1-si CHIP and overexpression plasmid MSCV-GFP-h CHIP were successfully constructed and confirmed by direct sequencing.3,RNA interference of CHIP was successfully established,indicated by clear reduction of CHIP expression both at m RNA and protein level in DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells.DLD-1 and HT-29 cell lines overexpressing the human CHIP c DNA was successfully established.CHIP was remarkably induced in DLD-1-h CHIP-1,DLD-1-h CHIP-3 and HT-29-h CHIP-3 and HT-29-h CHIP-5 cells both at m RNA and protein levels,compared to that in the control cells.4,Cell growth was monitored continuously using a real-time x-Celligence system for 72 h.The DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells grew much slower than DLD-1-sictrl cells,with statistical significances at all the indicated time points.There was no obvious difference between the DLD-1-h CHIP-1,DLD-1-h CHIP-3 and DLD-1-ctrl cells,as well as HT-29-h CHIP-3,HT-29-h CHIP-5 and HT-29-ctrl cells for all the indicated time points.5,Cell proliferation of DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells was decreased compared to that of the DLD-1-sictrl cells,revealed by CCK-8 and Brdu proliferation assay.There was no obvious difference for the cell proliferation between the DLD-1-h CHIP-1,DLD-1-h CHIP-3 and DLD-1-ctrl cells,as well as HT-29-h CHIP-3,HT-29-h CHIP-5 and HT-29-ctrl cells.6,The spontaneous apoptosis rarely occurred in DLD-1-si CHIP-3,DLD-1-si CHIP-9 and DLD-1-sictrl cells,as well as DLD-1-h CHIP-1,DLD-1-h CHIP-3 and HT-29-ctrl cells,and had no statistical significances at 24,48,and 72 h time points.The CHIP-silencing significantly induced a strong accumulation of the DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells in G0-G1 phase compared to that in the DLD-1-sictrl cells,while CHIP overexpression exerted no effect on the cell cycle of DLD-1 cells.7,Knockdown of CHIP expression significantly impaired the migratory potential of DLD-1 cells in a time-dependent manner,and had statistically significant difference from 16 to 24 h(P<0.001).In the contrast,CHIP-overexpressing dramatically increased the migration ability of DLD-1 cells in a time-dependent manner,and had statistically significant difference from 12 h to 24 h(P<0.001).Consistently,Transwell assay revealed that,the numbers of migrated and invaded DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells were significantly lesser than those of the DLD-1-sictrl cells(P<0.001),while the numbers of migrated and invaded DLD-1-h CHIP-1 and DLD-1-h CHIP-3 cells were notably greater than those of the DLD-1-ctrl cells(P<0.001).Wound healing assay showed that,the CHIP-silencing significantly hampered the wound closure of DLD-1 cells,while the CHIP-overexpression evidently promoted the wound closure as compared to the ctrl group,indicating the enhanced migration poteintial in DLD-1-h CHIP-1 and DLD-1-h CHIP-3 cells.The migration and invasion abilities of HT-29-h CHIP-3 and HT-29-h CHIP-5 cells were much higher than those of HT-29-ctrl cells in 24 h.In vivo,more metastatic nodules on the mensentery were formed in mice injected with the DLD-1-h CHIP-1 cells compared with those injected with the DLD-1-ctrl cells(P<0.001).On the contrary,less metastasis nodules were formed on the mensentery in mice injected with the DLD-1-si CHIP-3 cells than those injected with the DLD-1-sictrl cells(P<0.001).Western blotting and IHC analysis of the metastasis nodules revealed that CHIP protein was substantially downregulated in the DLD-1-si CHIP-3 group compared with that in the DLD-1-sictrl group.Whereas tumor tissues from the mice injected with the DLD-1-h CHIP-1 cells had an upregulation of CHIP expression compared with that from the DLD-1-ctrl group.Conclusions: CHIP-silencing in CRC cells inhibited cell growth and cell proliferation and resulted in G0-G1 arrest,CHIP-overexpression exerted no obvious influence on the cell growth,apoptosis,proliferation or cell cycle of CRC cells.CHIP-silencing attenuated the CRC cell migratory and invasive potential in vitro and metastasis in vivo.Conversely,cell migratory and invasive potential in vitro and metastasis in vivo were enhanced dramatically in CHIP-overexpressing CRC cells.Part 2 The underlying mechanism of CHIP in the cellular biological behaviors of colorectal cancerObject: To investigate the influence and function of CHIP on the protein expression of CRC cells and to elucidate the underlying mechanism of E3 ubiquitin ligase CHIP in the cellular biological behaviors of colorectal cancer.Methods: 1,Whole cell extracts from DLD-1-h CHIP-3 and DLD-1-ctrl were extracted,quantified with BCA assay and detected by SDS-PAGE electrophoresis.Total protein was selected and isobaric tags for relative and absolute quantification(i TRAQ)labels were performed and loaded on the C18 column.The LC-MS/MS analysis was carried out in Capital Bio Technology using a Q Exactive mass spectrometer.The high confident peptides with a global false discovery rate(FDR)<1% were used as filter parameters.Proteins that contained at least two unique peptides were used to quantify proteins.The results were calculated,differentially expressed proteins were screened and undergone Gene ontology(GO)enrichment analysis,kyoto encyclopedia of gene and genomes(KEGG)pathway analysis and Disease analysis.2,The expression of ERK,AKT,NF-кB signaling related proteins,cell cycle-related proteins and the expression of Integrin β1,u PA and u PAR after CHIP overexpression and depletion in CRC cells were detected by Western blotting.3,The expression of E-cadherin,Ep CAM and CK8/18 in DLD-1 transfected cells were detected using IHC analysis.The m RNA and protein expression of EMT related proteins in DLD-1 transfected cells were further detected by q RT-PCR and Western blotting.The AKT signaling pathway related protein GSK-3β,p-GSK-3β and Slug after CHIP-depletion and overexpression were also detected by Western blotting.Results: 1,Compared with DLD-1-ctrl group,789 different proteins were detected in DLD-1-h CHIP-3 group,including 414 upregulated proteins and 375 downregulated proteins.E-cadherin is one of the downregulated proteins.The Cellular component of GO analysis revealed that,the 789 differentiated proteins were predominantly intrinsic component of membrane,integral component of membrane,cell projection,extracellular space,and so on.Biological process of GO analysis revealed that,the 789 differentiated proteins were predominantly involved in regulation of cell communication,signal transduction,regulation of molecular function,movement of cell or subcellular component,and so on.Molecular Function of GO analysis revealed that,the 789 differentiated proteins were predominantly molecular function regulator,structural constituent of ribosome,receptor activity,enzyme inhibitor activity,and so on.KEGG analysis revealed that,the 789 differentiated proteins were predominantly involved in 28 signalling pathway(P≤0.05),including Metabotropic glutamate receptor group III pathway,Nicotine pharmacodynamics pathway,Generic Transcription Pathway,Glutamatergic synapse,and so on.Disease analysis revealed that,the 789 differentiated proteins were predominantly involved in Response to methotrexate in juvenile idiopathic arthritis,Tourette’s syndrome or obsessive-compulsive disorder,Chronic myeloid leukemia,Crohn’s disease,and so on.GO enrichment analysis also revealed that the downregulation of E-cadherin was also correlated to the cell-cell adhesion and cell-cell adhesion via plasma-membrane adhesion molecules pathway.KEGG enrichment analysis also revealed that the downregulation of E-cadherin was also correlated to the Extracellular matrix organization and Cell-cell junction organization,and Integrin cell surface interactions.According to the Disease Enrichment analysis,9 out of the 790 differentially expressed proteins were correlated to the CRC disease,including E-cadherin.2,The phosphorylation of ERK1/2(p-ERK1/2)was markedly deduced in the DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells,while increased in DLD-1-h CHIP-1 and DLD-1-h CHIP-3 cells.The phosphorylation of AKT was also obviously decreased in DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells.While,the expression of p-AKT was markedly induced in DLD-1-h CHIP-1 and DLD-1-h CHIP-3 cells.The expression of NF-κB subunits was similar in DLD-1-si CHIP-3,DLD-1-si CHIP-9 and DLD-1-sictrl cells,as well as DLD-1-h CHIP-1,DLD-1-h CHIP-3 and DLD-1-ctrl cells.In addition,cyclin D1 was downregulated,while p27 was upregulated in DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells,when compared to those of DLD-1-sictrl cells.The expression of cyclin D1 and p27 remained unchanged in DLD-1-h CHIP-1,DLD-1-h CHIP-3 and DLD-1-ctrl cells.The expression of Integrin β1 and u PAR was downregulated in DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells,while upregulated in DLD-1-h CHIP-1 and DLD-1-h CHIP-3 cells,when compared to the control cells.3,IHC analysis revealed that,CHIP was predominantly expressed on the membrane and in the cytoplasm of DLD-1 cells.The expression of CHIP was much lower in DLD-1-si CHIP-3 cells,while higher in DLD-1-h CHIP-1 cells when compared to those of control cells.E-cadherin,Ep CAM and CK8/18 were predominantly expressed on the membrane and in the cytoplasm of DLD-1 cells.The expression of E-cadherin was much higher in DLD-1-si CHIP-3 cells,while lower in DLD-1-h CHIP-1 cells when compared to those of control cells.The expression of Ep CAM and CK8/18 were comparable in all transfected cells.4,The m RNA and protein expression of E-cadherin were upregulated in DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells cells,while downregulated in DLD-1-h CHIP-1 and DLD-1-h CHIP-3 cells,according to the q RT-PCR and Western blotting analysis.The m RNA and protein expression of Ep CAM and CK8/18 were comparable in all the transfected cells.In addition,the p-GSK-3β and Slug were markedly deduced in the DLD-1-si CHIP-3 and DLD-1-si CHIP-9 cells,while increased in DLD-1-h CHIP-1 and DLD-1-h CHIP-3 cells.Conclusions: CHIP-silencing inhibited AKT and ERK signaling activities,led to the downregulated cyclin D1 and upregulated p27.While,ERK and AKT signaling activities were upregulated in CHIP overexpression CRC cells.CHIP overexpression didn’t affect the expression of cyclin D1 and p27.E-cadherin was upregulated in DLD-1 CHIP-depletion cells while downregulated in CHIP-overexpression cells.CHIP probably activated the AKT signaling,which inactivated GSK-3β.The GSK-3β inactivation,in turn,upregulated Slug,leading to the downregulation of E-cadherin and epithelial-mesenchymal transition(EMT)initiation.Part 3 The expression and the clinical significance of CHIP and E-cadherin in colorectal cancer tissuesObject: To investigate the expression of CHIP and E-cadherin in CRC tissues and the paired adjacent counterparts and the clinical significance.Methods: The Commercial CRC tissue microarray was provided by Shanghai Outdo Biotech Company from the National Human Genetic Resources Sharing Service Platform(2005DKA21300).The expression of CHIP and E-cadherin in CRC tissues and the paired adjacent counterparts were evaluated by IHC.The χ2 test was used to exam the differences in the distributions between the clinical characteristics.The association between CHIP and E-cadherin protein expression was analyzed by Spearman’s test(r;P-value).The survival curves were plotted using Kaplan-Meier analysis and compared by log-rank test.Univariate and Multivariate analyses were performed using the Cox proportional hazards model.Results: Significantly increased CHIP expression(CHIP-high)was found in 72.04%(67/93)of CRC samples,while higher CHIP expression was only observed in 42.53%(37/87)of the paired adjacent non-neoplastic tissues.CHIP expression was much higher in cancer tissues when compared with that in adjacent counterparts(P<0.001).Significantly increased E-cadherin expression(E-cadherin-low)was found in 61.29%(57/93)of CRC samples,while decreased E-cadherin expression was only observed in 25.29%(22/87)of the paired adjacent non-neoplastic tissues.E-cadherin expression was much lower in cancer tissues when compared with that in adjacent counterparts(P<0.001).Spearman rank correlation analysis showed that the expression of CHIP and E-cadherin expression was negatively correlated(r=-0.243,P=0.019).The expression of CHIP was positively correlated with depth of tumor invasion(P=0.039),lymph nodes invasion(P=0.003),and TNM stage(P=0.003).E-cadherin expression was negatively correlated with depth of tumor invasion(P=0.048),lymph nodes invasion(P=0.043),and TNM stage(P=0.020).Kaplan-Meier analysis revealed that CRC patients with high CHIP expression were significantly correlated with a poorer overall survivalthan those with low CHIP expression(P=0.001).High E-cadherin expression was evidently correlated with a better overall survival of CRC patients than low E-cadherin expression(P=0.010).Tumor differentiation,lymph node metastasis,TNM stage,CHIP and E-cadherin expression showed statistical significances in the univariate survival analysis,respectively(P<0.01).Multivariate Cox regression analysis indicated that CHIP expression and tumor differentiation had statistical significances with the overall survival,with hazard ratio of 7.163 for CHIP(95%CI 2.188 to 23.453,P=0.001)and 3.030 for tumor differentiation(95%CI 1.485 to 6.180,P=0.002).Conclusions: CHIP expression was much higher in cancer tissues when compared with that in adjacent counterparts,while E-cadherin expression was much lower in cancer tissues when compared with that in adjacent counterparts.The expression of CHIP and E-cadherin expression was negatively correlated.CHIP expression was an independent prognostic factor in the development of CRC. |
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