Background:The chaperonin containing t-complex(CCT)proteins play an important role in cell cycle-related protein degradation in yeast and mammals.The role of the chaperonin containing t-complex 4(CCT4),one subtype of ...Background:The chaperonin containing t-complex(CCT)proteins play an important role in cell cycle-related protein degradation in yeast and mammals.The role of the chaperonin containing t-complex 4(CCT4),one subtype of CCT proteins,in the progress of hepatocellular carcinoma(HCC)was not fully elucidated.Here,we aimed to explore the mechanisms of CCT4 in HCC.Methods:In this study,we used the UALCAN platform to analyze the relationship between CCT4 and HCC,and the association of CCT4 with the overall survival(OS)of HCC patients was also analyzed.CCT4 expression in HCC tumor tissues and normal tissues was also determined by western blot(WB)assay.Lentivirus vector was used to knock down the CCT4 expression,and quantitative polymerase chain reaction and WB were used to determine the level of CCT4 in HCC cell lines.Cell counting kit-8(CCK-8)and 5-ethynyl-20-deoxyuridine(EdU)assays were used to detect the cell proliferation,and flow cytometry(FCM)was performed to evaluate the effect of CCT4 on the apoptosis of HCC cells.Co-immunoprecipitation(co-IP)assay and WB were used to explore the mechanisms of CCT4 regulating the growth of HCC.Data were calculated from at least three replicate experiments and expressed as mean±standard deviation.Student’s t test,paired t test,and Kaplan–Meier analysis were used to compare across different groups.Results:We found CCT4 was upregulated in HCC tissues compared with normal tissues,and its high expression was associated with poor prognosis(P<0.001).CCT4 was significantly increased in HCC tumor tissues compared with normal tissues(0.98±0.12 vs.0.23±0.05,t=7.73,P<0.001).After being transfected with CCT4 short-hairpin RNA(shRNA),CCT4 was decreased in mRNA level and protein level in both Huh7(mRNA level:0.41±0.07 vs.1.01±0.11,t=8.09,P=0.001;protein level:0.61±0.03 vs.0.93±0.07,t=7.19,P=0.002)and Hep3b cells(mRNA level:0.55±0.11 vs.1.04±0.15,t=4.51,P=0.011;protein level:0.64±0.10 vs.0.95±0.08,t=4.32,P=0.012).CCK8 assay indicated that CCT4 knockdown inhibited cell proliferation in both Huh7(OD value of 3 days:0.60±0.14 vs.0.97±0.16,t=3.13,P=0.036;OD value of 4 days:1.03±0.07 vs.1.50±0.12,t=5.97,P=0.004)and Hep3b(OD value of 3 days:0.69±0.14 vs.1.10±0.11,t=3.91,P=0.017;OD value of 4 days:1.12±0.12 vs.1.48±0.13,t=3.55,P=0.024)cells.EdU assay showed that CCT4 knockdown inhibited the cell proliferation in both Huh7(EdU positive rate:[31.25±3.41]%vs.[58.72±3.78]%,t=9.34,P=0.001)and Hep3b cells(EdU positive rate:[44.13±7.02]%vs.[61.79±3.96]%,t=3.79,P=0.019).FCM assay suggested that CCT4 knockdown induced apoptosis in HCC cells(apoptosis rate of Huh7:[9.10±0.80]%vs.[3.66±0.64]%,t=-9.18,P=0.001;apoptosis rate of Hep3b:[6.69±0.72]%vs.[4.20±0.86]%,t=-3.84,P=0.018).We also found that CCT4 could regulate anaphase-promoting complex(APC)Cdc20 activity via interacting with Cdc20.Furthermore,CCT4 knockdown induced securin(0.65±0.06 vs.0.44±0.05,t=-4.69,P=0.009)and B-cell lymphoma-2(Bcl-2)interacting mediator of cell death(Bim;0.96±0.06 vs.0.61±0.09,t=securin inhibited cell growth by downregulating cyclin D1(0.65±0.05 vs.1.04-±5.65,0.07,Pt==0.005)accumulation.The upregulation of 8.12,P=0.001),and the accumulation of Bim inhibited Bcl-2(0.77±0.04 vs.0.87±0.04,t=3.00,P=0.040)and activated caspase 9(caspase 9:0.77±0.04 vs.0.84±0.05,t=1.81,P=0.145;cleaved caspase 9:0.64±0.06 vs.0.16±0.07,t=1.81,P=0.001),which led to elevated apoptosis.Conclusions:Overall,these results showed that CCT4 played an important role in HCC pathogenesis through,at least partly,interacting with Cdc20.展开更多
Chromosome segregation is a tightly regulated process through which duplicated genetic materials are equally partitioned into daughter cells. During the past decades, tremendous efforts have been made to understand th...Chromosome segregation is a tightly regulated process through which duplicated genetic materials are equally partitioned into daughter cells. During the past decades, tremendous efforts have been made to understand the molecular mechanism of chromosome segregation using animals and yeasts as model systems. Recently, new insights into chromosome segregation have gradually emerged using trypanosome, an early branching parasitic protozoan, as a model organism. To uncover the unique aspects of chromosome segregation in trypanosome, which potentially could serve as new drug targets for anti-trypanosome chemotherapy, it is necessary to perform a comparative analysis of the chromosome segregation machinery between trypanosome and its human host. Here, we briefly review the current knowledge about chromosome segregation in human and Trypanosoma brucei, with a focus on the regulation of cohesin and securin degradation triggered by the activation of the anaphase promoting complex/cyclosome （APC/C）. We also include yeasts in our comparative analysis since some of the original discoveries were made using budding and fission yeasts as the model organisms and, therefore, these could provide hints about the evolution of the machinery. We highlight both common and unique features in these model systems and also provide perspectives for future research in trypanosome.展开更多
基金the Natural Science Foundation of Anhui Province(No.1908085QH325).
文摘Background:The chaperonin containing t-complex(CCT)proteins play an important role in cell cycle-related protein degradation in yeast and mammals.The role of the chaperonin containing t-complex 4(CCT4),one subtype of CCT proteins,in the progress of hepatocellular carcinoma(HCC)was not fully elucidated.Here,we aimed to explore the mechanisms of CCT4 in HCC.Methods:In this study,we used the UALCAN platform to analyze the relationship between CCT4 and HCC,and the association of CCT4 with the overall survival(OS)of HCC patients was also analyzed.CCT4 expression in HCC tumor tissues and normal tissues was also determined by western blot(WB)assay.Lentivirus vector was used to knock down the CCT4 expression,and quantitative polymerase chain reaction and WB were used to determine the level of CCT4 in HCC cell lines.Cell counting kit-8(CCK-8)and 5-ethynyl-20-deoxyuridine(EdU)assays were used to detect the cell proliferation,and flow cytometry(FCM)was performed to evaluate the effect of CCT4 on the apoptosis of HCC cells.Co-immunoprecipitation(co-IP)assay and WB were used to explore the mechanisms of CCT4 regulating the growth of HCC.Data were calculated from at least three replicate experiments and expressed as mean±standard deviation.Student’s t test,paired t test,and Kaplan–Meier analysis were used to compare across different groups.Results:We found CCT4 was upregulated in HCC tissues compared with normal tissues,and its high expression was associated with poor prognosis(P<0.001).CCT4 was significantly increased in HCC tumor tissues compared with normal tissues(0.98±0.12 vs.0.23±0.05,t=7.73,P<0.001).After being transfected with CCT4 short-hairpin RNA(shRNA),CCT4 was decreased in mRNA level and protein level in both Huh7(mRNA level:0.41±0.07 vs.1.01±0.11,t=8.09,P=0.001;protein level:0.61±0.03 vs.0.93±0.07,t=7.19,P=0.002)and Hep3b cells(mRNA level:0.55±0.11 vs.1.04±0.15,t=4.51,P=0.011;protein level:0.64±0.10 vs.0.95±0.08,t=4.32,P=0.012).CCK8 assay indicated that CCT4 knockdown inhibited cell proliferation in both Huh7(OD value of 3 days:0.60±0.14 vs.0.97±0.16,t=3.13,P=0.036;OD value of 4 days:1.03±0.07 vs.1.50±0.12,t=5.97,P=0.004)and Hep3b(OD value of 3 days:0.69±0.14 vs.1.10±0.11,t=3.91,P=0.017;OD value of 4 days:1.12±0.12 vs.1.48±0.13,t=3.55,P=0.024)cells.EdU assay showed that CCT4 knockdown inhibited the cell proliferation in both Huh7(EdU positive rate:[31.25±3.41]%vs.[58.72±3.78]%,t=9.34,P=0.001)and Hep3b cells(EdU positive rate:[44.13±7.02]%vs.[61.79±3.96]%,t=3.79,P=0.019).FCM assay suggested that CCT4 knockdown induced apoptosis in HCC cells(apoptosis rate of Huh7:[9.10±0.80]%vs.[3.66±0.64]%,t=-9.18,P=0.001;apoptosis rate of Hep3b:[6.69±0.72]%vs.[4.20±0.86]%,t=-3.84,P=0.018).We also found that CCT4 could regulate anaphase-promoting complex(APC)Cdc20 activity via interacting with Cdc20.Furthermore,CCT4 knockdown induced securin(0.65±0.06 vs.0.44±0.05,t=-4.69,P=0.009)and B-cell lymphoma-2(Bcl-2)interacting mediator of cell death(Bim;0.96±0.06 vs.0.61±0.09,t=securin inhibited cell growth by downregulating cyclin D1(0.65±0.05 vs.1.04-±5.65,0.07,Pt==0.005)accumulation.The upregulation of 8.12,P=0.001),and the accumulation of Bim inhibited Bcl-2(0.77±0.04 vs.0.87±0.04,t=3.00,P=0.040)and activated caspase 9(caspase 9:0.77±0.04 vs.0.84±0.05,t=1.81,P=0.145;cleaved caspase 9:0.64±0.06 vs.0.16±0.07,t=1.81,P=0.001),which led to elevated apoptosis.Conclusions:Overall,these results showed that CCT4 played an important role in HCC pathogenesis through,at least partly,interacting with Cdc20.
文摘Chromosome segregation is a tightly regulated process through which duplicated genetic materials are equally partitioned into daughter cells. During the past decades, tremendous efforts have been made to understand the molecular mechanism of chromosome segregation using animals and yeasts as model systems. Recently, new insights into chromosome segregation have gradually emerged using trypanosome, an early branching parasitic protozoan, as a model organism. To uncover the unique aspects of chromosome segregation in trypanosome, which potentially could serve as new drug targets for anti-trypanosome chemotherapy, it is necessary to perform a comparative analysis of the chromosome segregation machinery between trypanosome and its human host. Here, we briefly review the current knowledge about chromosome segregation in human and Trypanosoma brucei, with a focus on the regulation of cohesin and securin degradation triggered by the activation of the anaphase promoting complex/cyclosome （APC/C）. We also include yeasts in our comparative analysis since some of the original discoveries were made using budding and fission yeasts as the model organisms and, therefore, these could provide hints about the evolution of the machinery. We highlight both common and unique features in these model systems and also provide perspectives for future research in trypanosome.
