AIM To determine the role of G0/G1 switch gene 2(G0 S2) and its transcriptional regulation in palmitate-induced hepatic lipid accumulation.METHODS Hep G2 cells were treated with palmitate,or palmitate in combination w...AIM To determine the role of G0/G1 switch gene 2(G0 S2) and its transcriptional regulation in palmitate-induced hepatic lipid accumulation.METHODS Hep G2 cells were treated with palmitate,or palmitate in combination with CCAAT/enhancer binding protein(C/EBP)β si RNA or G0 S2 si RNA. The m RNA expression of C/EBPβ,peroxisome proliferator-activated receptor(PPAR)γ and PPARγ target genes(G0 S2,GPR81,GPR109 A and Adipoq) was examined by q PCR. The protein expression of C/EBPβ,PPARγ,and G0 S2 was determined by Western blotting. Lipid accumulation was detected with Oil Red O staining and quantified by absorbance value of the extracted Oil Red O dye. Lipolysis was evaluated by measuring the amount of glycerol released into the medium. RESULTS Palmitate caused a dose-dependent increase in lipid accumulation and a dose-dependent decrease in lipolysis in Hep G2 cells. In addition,palmitate increased the m RNA expression of C/EBPβ,PPARγ,and PPARγ target genes(G0 S2,GPR81,GPR109 A,and Adipoq) and the protein expression of C/EBPβ,PPARγ,and G0 S2 in a dose-dependent manner. Knockdown of C/EBPβ decreased palmitate-induced PPARγ and its target genes(G0 S2,GPR81,GPR109 A,and Adipoq) m RNA expression and palmitate-induced PPARγ and G0 S2 protein expression in Hep G2 cells. Knockdown of C/EBPβ also attenuated lipid accumulation and augmented lipolysis in palmitate-treated Hep G2 cells. G0 S2 knockdown attenuated lipid accumulation and augmented lipolysis,while G0 S2 knockdown had no effects on the m RNA expression of C/EBPβ,PPARγ,and PPARγ target genes(GPR81,GPR109 A and Adipoq) in palmitate-treated Hep G2 cells. CONCLUSION Palmitate can induce lipid accumulation in Hep G2 cells by activating C/EBPβ-mediated G0 S2 expression.展开更多
Tetrahydroisoquinolines are known to have various biological effects, including antitumor activity. This study investigated the effect of 1-chloromethyl-6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-sulfonic acid amid...Tetrahydroisoquinolines are known to have various biological effects, including antitumor activity. This study investigated the effect of 1-chloromethyl-6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-sulfonic acid amide (CDST), a newly synthesized anticancer agent, on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells were determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide staining, western blot analysis and immunoprecipitation, respectively. CDST induced the differentiation of HL-60, as shown by increased expression of differentiation surface antigen CD11b (but no significant change in CD14 expression) and increased NBT-reducing functional activity. DNA flow cytometry analysis indicated that CDST markedly induced a G0/G1 phase arrest of HL-60 cells. Subsequently, we examined the expre-ssion of G0/G1 phase cell cycle-related proteins, including cyclin-dependent kinases (CDKs), cyclins and cyclin dependent kinase inhibitors (CKIs), during the differentiation of HL-60. The levels of CDK2, CDK6, cyclin E and cyclin A were decreased, whereas steady-state levels of CDK4 and cyclin D1 were unaffected. The expression of the p27Kip1 was markedly increased by CDST, but not p21WAF1/Cip1. Moreover, CDST markedly enhanced the binding of p27Kip1 with CDK2 and CDK6, resulting in the reduced activity of both kinases. Taken together, these results demonstrate that CDST is capable of inducing cellular differentiation and growth inhibition through p27Kip1 protein-related G0/G1 phase arrest in HL-60 cells.展开更多
Short-chain fatty acids(SCFAs)butyrate promote the postnatal rumen epithelial development and maturation in ruminants.However,molecular mechanisms of effects of butyrate on the bovine rumen epithelial cells(BRECs)prol...Short-chain fatty acids(SCFAs)butyrate promote the postnatal rumen epithelial development and maturation in ruminants.However,molecular mechanisms of effects of butyrate on the bovine rumen epithelial cells(BRECs)proliferation remain elusive.Therefore,purpose of this study was to investigate the effects of butyrate on the expression of genes and proteins at G0/G1 and S phase of BRECs cycle.Our results showed that BRECs treated with butyrate inhibited(P<0.05)the proliferation of BRECs,relatively to control.Flow cytometric assays revealed that butyrate triggers the BRECs cycle arrest at the G0/G1 phase.qRT-PCR analyses of mRNA level of genes involved in the G0/G1 phase of cell cycle showed that butyrate significantly upregulated(P<0.001)the expression of mRNA encoding p21^(Cip1)compared with control group,but it decreased(P<0.05)the mRNA levels of cyclin D1 and CDK4 genes at G0/G1 phase checkpoint compared with control.Moreover,Western blot also revealed that butyrate downregulated the expression of cyclin D3,CDK6,p-Rb,and E2F1 proteins involved in the modulation of G0/G1 phase of cell cycle.In conclusion,our results demonstrated that butyrate inhibits the proliferation of BRECs via downregulation of positive regulators at G0/G1 phase checkpoint.展开更多
基金Supported by the grants from the Natural Science Foundation of Shanxi Province,China,No.2014011043-1the Social Development Project of Jinzhong,Shanxi Province,No.S1601
文摘AIM To determine the role of G0/G1 switch gene 2(G0 S2) and its transcriptional regulation in palmitate-induced hepatic lipid accumulation.METHODS Hep G2 cells were treated with palmitate,or palmitate in combination with CCAAT/enhancer binding protein(C/EBP)β si RNA or G0 S2 si RNA. The m RNA expression of C/EBPβ,peroxisome proliferator-activated receptor(PPAR)γ and PPARγ target genes(G0 S2,GPR81,GPR109 A and Adipoq) was examined by q PCR. The protein expression of C/EBPβ,PPARγ,and G0 S2 was determined by Western blotting. Lipid accumulation was detected with Oil Red O staining and quantified by absorbance value of the extracted Oil Red O dye. Lipolysis was evaluated by measuring the amount of glycerol released into the medium. RESULTS Palmitate caused a dose-dependent increase in lipid accumulation and a dose-dependent decrease in lipolysis in Hep G2 cells. In addition,palmitate increased the m RNA expression of C/EBPβ,PPARγ,and PPARγ target genes(G0 S2,GPR81,GPR109 A,and Adipoq) and the protein expression of C/EBPβ,PPARγ,and G0 S2 in a dose-dependent manner. Knockdown of C/EBPβ decreased palmitate-induced PPARγ and its target genes(G0 S2,GPR81,GPR109 A,and Adipoq) m RNA expression and palmitate-induced PPARγ and G0 S2 protein expression in Hep G2 cells. Knockdown of C/EBPβ also attenuated lipid accumulation and augmented lipolysis in palmitate-treated Hep G2 cells. G0 S2 knockdown attenuated lipid accumulation and augmented lipolysis,while G0 S2 knockdown had no effects on the m RNA expression of C/EBPβ,PPARγ,and PPARγ target genes(GPR81,GPR109 A and Adipoq) in palmitate-treated Hep G2 cells. CONCLUSION Palmitate can induce lipid accumulation in Hep G2 cells by activating C/EBPβ-mediated G0 S2 expression.
文摘Tetrahydroisoquinolines are known to have various biological effects, including antitumor activity. This study investigated the effect of 1-chloromethyl-6, 7-dimethoxy-3, 4-dihydro-1H-isoquinoline-2-sulfonic acid amide (CDST), a newly synthesized anticancer agent, on cellular differentiation and proliferation in HL-60 cells. Differentiation and proliferation of HL-60 cells were determined through expression of CD11b and CD14 surface antigens using flow cytometry and nitroblue tetrazolium (NBT) assay, and through analysis of cell cycle using propidium iodide staining, western blot analysis and immunoprecipitation, respectively. CDST induced the differentiation of HL-60, as shown by increased expression of differentiation surface antigen CD11b (but no significant change in CD14 expression) and increased NBT-reducing functional activity. DNA flow cytometry analysis indicated that CDST markedly induced a G0/G1 phase arrest of HL-60 cells. Subsequently, we examined the expre-ssion of G0/G1 phase cell cycle-related proteins, including cyclin-dependent kinases (CDKs), cyclins and cyclin dependent kinase inhibitors (CKIs), during the differentiation of HL-60. The levels of CDK2, CDK6, cyclin E and cyclin A were decreased, whereas steady-state levels of CDK4 and cyclin D1 were unaffected. The expression of the p27Kip1 was markedly increased by CDST, but not p21WAF1/Cip1. Moreover, CDST markedly enhanced the binding of p27Kip1 with CDK2 and CDK6, resulting in the reduced activity of both kinases. Taken together, these results demonstrate that CDST is capable of inducing cellular differentiation and growth inhibition through p27Kip1 protein-related G0/G1 phase arrest in HL-60 cells.
基金This study was supported by the National Natural Science Foundation of China(No.32002200)the Research Project of Natural Science Foundation of Jiangsu Province(BK20190898)China Agriculture Research System of MOF and MARA.
文摘Short-chain fatty acids(SCFAs)butyrate promote the postnatal rumen epithelial development and maturation in ruminants.However,molecular mechanisms of effects of butyrate on the bovine rumen epithelial cells(BRECs)proliferation remain elusive.Therefore,purpose of this study was to investigate the effects of butyrate on the expression of genes and proteins at G0/G1 and S phase of BRECs cycle.Our results showed that BRECs treated with butyrate inhibited(P<0.05)the proliferation of BRECs,relatively to control.Flow cytometric assays revealed that butyrate triggers the BRECs cycle arrest at the G0/G1 phase.qRT-PCR analyses of mRNA level of genes involved in the G0/G1 phase of cell cycle showed that butyrate significantly upregulated(P<0.001)the expression of mRNA encoding p21^(Cip1)compared with control group,but it decreased(P<0.05)the mRNA levels of cyclin D1 and CDK4 genes at G0/G1 phase checkpoint compared with control.Moreover,Western blot also revealed that butyrate downregulated the expression of cyclin D3,CDK6,p-Rb,and E2F1 proteins involved in the modulation of G0/G1 phase of cell cycle.In conclusion,our results demonstrated that butyrate inhibits the proliferation of BRECs via downregulation of positive regulators at G0/G1 phase checkpoint.
