The Fuction Of E3 Ubiquitin Ligase CHIP In The Biological Behavior Of Gastric Cancer And The Regulation Of TRAF2

Objective This study is to clarify the effect of E3 ubiquitin ligase CHIP of the development and progression of gastric carcinoma and its effect on NF-?B signaling pathway and explore the internal mechanism of these effects.Methods Establish a stable transfection of CHIP-si RNA in AGS.The cell growth was measured by x CELLligence system and clonogenic assay.Ki-67 and cell viability assay was used to detect the cell proliferation.The differences of and apoptosis between AGS-si CHIP cells and the AGS-sictrl cells were detected by TUNEL assays.The experiences of cell cycle in AGS which was after CHIP blocking were dectected by using flow cytometry.The expression of cell cycle related proteins,apoptosis related proteins and proteins in extracellular signal-regulated kinase(ERK1/2)signaling pathways in the two established cell lines were detected by Western blot.The migration and invasion abilities were measured by real-time x CELLigence system,Transwell and scratch assays.The expression of Integrin ?1 was detected by Western blot.The expression of TRAF2 in AGS-si CHIP cells and the AGS-sictrl cells was detected by q RT-PCR and Western blot.Establish a stable transfection of TRAF2-si RNA in AGS.The cell growth and proliferation were measured by x CELLigence system CCK-8,cell viability and CFSE assays.The apoptosis and cell cycle of AGS-si TRAF2 and AGS-sictrl cells were detected by flow cytometry.The difference of migration and invasion abilities of AGS-si TRAF2 and AGS-sictrl cells were measured by real-time x CELLigence system and Transwell and scratch assays.The expression and activity of NF-?B signaling pathway in AGS-si TRAF2 and AGS-sictrl cells were measured by western blot and Trans AM assay.The expression of CHIP and TRAF2 in gastric cancer tissue were detected by immunohistochemistry.Chi-square or two-tailed Fisher's exact tests were used to analyze possible associations between CHIP and TRAF2 expression and clinicopathological variables.Kaplan–Meier analysis and a Cox regression model were employed to determine independent prognostic factors.Results The x CELLigence monitoring system was used on the AGS-sictrl and AGS-si CHIP cells to measure the cell growth.The cell index of AGS-si CHIP cells was significantly higher than that of AGS-sictrl cells.In clone formation assay,the number of colonies of AGS-si CHIP cells was much more than the control group.The frequencies of Ki-67 positive cells in the AGS-si CHIP group were higher than control group at 24,48 and 72 h respectively.In cell viability assay,the viability of AGS-si CHIP cells at 72 h was significantly higher than that of AGS-sictrl.The frequencies of apoptotic cells in AGS-si CHIP group were much lower than those in AGS-sictrl group.The percentages of the AGS-si CHIP cells in G0-G1 was less than the control group.The expression level of the protein of p53 in AGS-si CHIP was lower than that in AGS-sictrl.The expression level of protein of CCND1,CCND3,CDK4 and CDK6 were higher than those in AGS-sictrl.The expression level of ERK1/2 of the AGS-sictrl and AGS-si CHIP cells was close to each other while the protein expression of phosphorylated extracellular signal-regulated kinase(p-ERK1/2)in AGS-si CHIP cells was significantly higher than that in control cells.The expression of protein of Bcl-2 in AGS-si CHIP cells was higher than that in AGS-sictrl cells.In the migration experiments,the scratch healing assay indicated that AGS-si CHIP cells migrated from the edge towards the scratch center faster than AGS-sictrl cells.The number of the migrated cells of AGS-si CHIP was significantly higher than that of AGS-sictrl in Transwell assay.The migrationwas monitored for 24 h with the x CELLigence RTCA instrument.AGS-si CHIP cells migrated much faster than AGS-sictrl cells.In the invasion experiment,the results of Transwell assay showed that the number of AGS-si CHIP cells passing through Matrigel was significantly higher than that of AGS-sictrl cells cultured for 24 h.The invasion was monitored for 24 h with the x CELLigence RTCA instrument and the cell index of AGS-si CHIP cells was more than that of AGS-sictrl cells.The protein expression level of Integrin ?1 in AGS-si CHIP cells were increased than that in AGS-sictrl cells while there was no significant difference in the of expression u PA.The expression level of protein of the gene of TRAF2 markedly decreased in the AGS-h CHIP cells while there was no significant difference at m RNA level.The cell growth of AGS-si TRAF2 cells was lower than that of AGS-sictrl cells in CCK-8 assay.The x CELLigence monitoring system was used on the AGS-sictrl and AGS-si TRAF2 cells to measure the cell growth,the cell index of AGS-si TRAF2 cells was significantly lower than that of AGS-sictrl cells.The fluorescence of CFSE in AGS-si TRAF2 cells was higher than that of AGS-sicrtl cells after 72 h.In the cell viability assay,the viability of AGS-si TRAF2 cells was significantly lower than that of AGS-sictrl cells.The percentage of apoptosis of AGS-si TRAF2 cells was significantly higher than that of the control group.In the migration experiments,the scratch healing assay indicated that AGS-si TRAF2 cells migrated from the edge towards the scratch center slower than AGS-sictrl cells.The number of the migrated cells of AGS-si TRAF2 was significantly lower than that of AGS-sictrl in Transwell assay.The difference was statistically significant.The migration was monitored with the x CELLigence RTCA instrument.AGS-si TRAF2 cells migrated much slower than AGS-sictrl cells.In the invasion experiment,the results of Transwell assay showed that the number of AGS-si TRAF2 cells passing through Matrigel was significantly less than that of AGS-sictrl cells.The invasion was monitored with the x CELLigence RTCA instrument.The cell index of AGS-si TRAF2 cells was less than that of AGS-sictrl cells.The expression of all subunits in NF-?B pathway were clearly decreased in both the cytoplasmic(CE)and nuclear fraction(NE)in the AGS-si TRAF2 cells compared to that in the AGS-control cells.The NF-k B activity in AGS-si TRAF2 was lower than that in control group in the Trans AM assay.The expression of CHIP in gastric cancer tissues was lower than that in paracancerous tissues;the expression of TRAF2 in gastric cancer tissues was higher than that in adjacent tissues.There was a negative correlation between the expression of CHIP and TRAF2 in gastric cancer.The median overall survival of patients with low CHIP expression and higt TRAF2 expression were shorter.TRAF2 expression and tumor diameter are independent prognostic factors of gastric cancer.Conclusions 1.Knockdown of CHIP enhanced AGS cell growth,migration and invasion,likely due to the suppressed phosphorylation of ERK1/2 signaling and the promotion Integrin?-1 2.The expression level of protein of the gene of TRAF2 markedly decreased in the AGS-h CHIP cells.3.Knockdown of TRAF2 inhibited AGS cell growth,migration and invasion.4.The expression of CHIP was decreased in tumor tissue.The expression of TRAF2 was increased in tumor tissue.5.TRAF2 expression is independent prognostic factors of gastric cancer.Part1 The role of E3 ubiquitin ligase CHIP in the biological behavior in gastric cancerObjective This study is to clarify the effect of E3 ubiquitin ligase CHIP on the biological behavior of gastric cancer cells.Methods Establish a stable transfection of CHIP-si RNA in AGS.The cell growth was measured by x CELLligence system and clonogenic assay.Ki-67 and cell viability assay was used to detect the cell proliferation.The differences of and apoptosis between AGS-si CHIP cells and the AGS-sictrl cells were detected by TUNEL assays.The experiences of cell cycle in AGS which was after CHIP blocking were dectected by using flow cytometry.The expression of cell cycle related proteins,apoptosis related proteins and proteins in extracellular signal-regulated kinase(ERK1/2)signaling pathways in the two established cell lines were detected by Western blot.The migration and invasion abilities were measured by real-time x CELLigence system,Transwell and scratch assays.The expression of Integrin ?1 was detected by Western blot.The expression of TRAF2 in AGS-si CHIP cells and the AGS-sictrl cells was detected by q RT-PCR and Western blot.Results The x CELLigence monitoring system was used on the AGS-sictrl and AGS-si CHIP cells to measure the cell growth.The cell index of AGS-si CHIP cells was significantly higher than that of AGS-sictrl cells.In clone formation assay,the number of colonies of AGS-si CHIP cells was much more than the control group.The frequencies of Ki-67 positive cells in the AGS-si CHIP group were higher than control group at 24,48 and 72 h respectively.In cell viability assay,the viability of AGS-si CHIP cells at 72 h was significantly higher than that of AGS-sictrl.The frequencies of apoptotic cells in AGS-si CHIP group were much lower than those in AGS-sictrl group.The percentages of the AGS-si CHIP cells in G0-G1 was less than the control group.The expression level of the protein of p53 in AGS-si CHIP was lower than that in AGS-sictrl.The expression level of protein of CCND1,CCND3,CDK4 and CDK6 were higher than those in AGS-sictrl.The expression level of ERK1/2 of the AGS-sictrl and AGS-si CHIP cells was close to each other while the protein expression of phosphorylated extracellular signal-regulated kinase(p-ERK1/2)in AGS-si CHIP cells was significantly higher than that in control cells.The expression of protein of Bcl-2 in AGS-si CHIP cells was higher than that in AGS-sictrl cells.In the migration experiments,the scratch healing assay indicated that AGS-si CHIP cells migrated from the edge towards the scratch center faster than AGS-sictrl cells.The number of the migrated cells of AGS-si CHIP was significantly higher than that of AGS-sictrl in Transwell assay.The migrationwas monitored for 24 h with the x CELLigence RTCA instrument.AGS-si CHIP cells migrated much faster than AGS-sictrl cells.In the invasion experiment,the results of Transwell assay showed that the number of AGS-si CHIP cells passing through Matrigel was significantly higher than that of AGS-sictrl cells cultured for 24 h.The invasion was monitored for 24 h with the x CELLigence RTCA instrument and the cell index of AGS-si CHIP cells was more than that of AGS-sictrl cells.The protein expression level of Integrin ?1 in AGS-si CHIP cells were increased than that in AGS-sictrl cells while there was no significant difference in the of expression u PA.The expression level of protein of the gene of TRAF2 markedly decreased in the AGS-h CHIP cells while there was no significant difference at m RNA level.Conclusions 1.Knockdown of CHIP enhanced AGS cell growth,migration and invasion,likely due to the suppressed phosphorylation of ERK1/2 signaling and the promotion Integrin?-1 2.The expression level of protein of the gene of TRAF2 markedly decreased in the AGS-h CHIP cells.Part2 CHIP regulates NF-?B signaling through TRAF2 in gastric cancerObjective This study is to clarify the effect of E3 ubiquitin ligase CHIP on regulating of TRAF2 and explore the internal mechanism of these effects.Methods Establish a stable transfection of TRAF2-si RNA in AGS.The cell growth and proliferation were measured by x CELLigence system CCK-8,cell viability and CFSE assays.The apoptosis and cell cycle of AGS-si TRAF2 and AGS-sictrl cells were detected by flow cytometry.The difference of migration and invasion abilities of AGS-si TRAF2 and AGS-sictrl cells were measured by real-time x CELLigence system and Transwell and scratch assays.The expression and activity of NF-?B signaling pathway in AGS-si TRAF2 and AGS-sictrl cells were measured by western blot and Trans AM assay.The expression of CHIP and TRAF2 in gastric cancer tissue were detected by immunohistochemistry.Chi-square or two-tailed Fisher's exact tests were used to analyze possible associations between CHIP and TRAF2 expression and clinicopathological variables.Kaplan–Meier analysis and a Cox regression model were employed to determine independent prognostic factors.Results The cell growth of AGS-si TRAF2 cells was lower than that of AGS-sictrl cells in CCK-8 assay.The x CELLigence monitoring system was used on the AGS-sictrl and AGS-si TRAF2 cells to measure the cell growth,the cell index of AGS-si TRAF2 cells was significantly lower than that of AGS-sictrl cells.The fluorescence of CFSE in AGS-si TRAF2 cells was higher than that of AGS-sicrtl cells after 72 h.In the cell viability assay,the viability of AGS-si TRAF2 cells was significantly lower than that of AGS-sictrl cells.The percentage of apoptosis of AGS-si TRAF2 cells was significantly higher than that of the control group.In the migration experiments,the scratch healing assay indicated that AGS-si TRAF2 cells migrated from the edge towards the scratch center slower than AGS-sictrl cells.The number of the migrated cells of AGS-si TRAF2 was significantly lower than that of AGS-sictrl in Transwell assay.The difference was statistically significant.The migration was monitored with the x CELLigence RTCA instrument.AGS-si TRAF2 cells migrated much slower than AGS-sictrl cells.In the invasion experiment,the results of Transwell assay showed that the number of AGS-si TRAF2 cells passing through Matrigel was significantly less than that of AGS-sictrl cells.The invasion was monitored with the x CELLigence RTCA instrument.The cell index of AGS-si TRAF2 cells was less than that of AGS-sictrl cells.The expression of all subunits in NF-?B pathway were clearly decreased in both the cytoplasmic(CE)and nuclear fraction(NE)in the AGS-si TRAF2 cells compared to that in the AGS-control cells.The NF-k B activity in AGS-si TRAF2 was lower than that in control group in the Trans AM assay.The expression of CHIP in gastric cancer tissues was lower than that in paracancerous tissues;the expression of TRAF2 in gastric cancer tissues was higher than that in adjacent tissues.There was a negative correlation between the expression of CHIP and TRAF2 in gastric cancer.The median overall survival of patients with low CHIP expression and higt TRAF2 expression were shorter.TRAF2 expression and tumor diameter are independent prognostic factors of gastric cancer.Conclusions 1.Knockdown of TRAF2 inhibited AGS cell growth,migration and invasion.2.The expression of CHIP was decreased in tumor tissue.The expression of TRAF2 was increased in tumor tissue.3.TRAF2 expression is independent prognostic factors of gastric cancer.