目的:探讨不同浓度的二十二碳六烯酸(docosahexaenoic acid,DHA)对人肝癌细胞HepG2生长增殖抑制及促进凋亡的作用及其机制,为其应用于肝癌的药物预防和治疗提供实验及理论依据.方法:以DHA浓度0μg/mL为阴性对照,设置浓度为15、30、45、6...目的:探讨不同浓度的二十二碳六烯酸(docosahexaenoic acid,DHA)对人肝癌细胞HepG2生长增殖抑制及促进凋亡的作用及其机制,为其应用于肝癌的药物预防和治疗提供实验及理论依据.方法:以DHA浓度0μg/mL为阴性对照,设置浓度为15、30、45、60、75μg/mL的实验组.采用CCK-8和流式细胞术检测不同浓度DHA对HepG2细胞生长的相对抑制率及凋亡情况,并运用荧光定量PCR及Western blot分别从基因和蛋白质水平分析不同浓度DHA作用前后β-连环蛋白(β-catenin)及C-myc表达量的变化.结果:CCK-8实验表明DHA在体外能抑制H e p G2细胞生长,在D H A作用浓度为0-45μg/mL,作用时间24 h时,实验组与对照组细胞吸光度(A)值差异有显著的统计学意义(P<0.01),实验组内两两比较差异有统计学意义(P<0.01),细胞生长抑制率最高达90.7%.继续增加药物浓度或作用时间,差异无统计学意义.流式细胞术发现DHA可促进HepG2细胞凋亡,实验组与对照组细胞凋亡率差异有显著的统计学意义(P<0.01),实验组内两两比较差异有统计学意义(P<0.01).荧光定量PCR显示DHA可下调HepG2细胞中C-myc基因表达水平,实验组与对照组C-myc基因相对表达量的差异有显著的统计学意义(P<0.01),实验组内两两比较差异有统计学意义(P<0.01);实验组与对照组及各实验者组之间β-catenin基因的相对表达量差异无统计学意义.Western blot显示DHA可降低HepG2细胞β-catenin、C-myc蛋白质的表达量.结论:DHA对人肝癌HepG2细胞有明显的生长增殖抑制及促进凋亡的作用,与其降低β-catenin蛋白质水平进而下调C-myc基因表达水平有关.展开更多
AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We construct...AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-xL and bcl-xS were detected with western blot. RESULTS: We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved indown-regulating bcl-xL and up-regulating bcl-xS. CONCLUSION: LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.展开更多
文摘目的:探讨不同浓度的二十二碳六烯酸(docosahexaenoic acid,DHA)对人肝癌细胞HepG2生长增殖抑制及促进凋亡的作用及其机制,为其应用于肝癌的药物预防和治疗提供实验及理论依据.方法:以DHA浓度0μg/mL为阴性对照,设置浓度为15、30、45、60、75μg/mL的实验组.采用CCK-8和流式细胞术检测不同浓度DHA对HepG2细胞生长的相对抑制率及凋亡情况,并运用荧光定量PCR及Western blot分别从基因和蛋白质水平分析不同浓度DHA作用前后β-连环蛋白(β-catenin)及C-myc表达量的变化.结果:CCK-8实验表明DHA在体外能抑制H e p G2细胞生长,在D H A作用浓度为0-45μg/mL,作用时间24 h时,实验组与对照组细胞吸光度(A)值差异有显著的统计学意义(P<0.01),实验组内两两比较差异有统计学意义(P<0.01),细胞生长抑制率最高达90.7%.继续增加药物浓度或作用时间,差异无统计学意义.流式细胞术发现DHA可促进HepG2细胞凋亡,实验组与对照组细胞凋亡率差异有显著的统计学意义(P<0.01),实验组内两两比较差异有统计学意义(P<0.01).荧光定量PCR显示DHA可下调HepG2细胞中C-myc基因表达水平,实验组与对照组C-myc基因相对表达量的差异有显著的统计学意义(P<0.01),实验组内两两比较差异有统计学意义(P<0.01);实验组与对照组及各实验者组之间β-catenin基因的相对表达量差异无统计学意义.Western blot显示DHA可降低HepG2细胞β-catenin、C-myc蛋白质的表达量.结论:DHA对人肝癌HepG2细胞有明显的生长增殖抑制及促进凋亡的作用,与其降低β-catenin蛋白质水平进而下调C-myc基因表达水平有关.
文摘AIM: To investigate the effects of lentivirus vector mediated short hairpin RNA interference targeting methionine adenosyltransferase 2β gene (LV-shMAT2B) on hepatocelluar carcinoma (HCC) cells. METHODS: We constructed four plasmids of RNA interference targeting the MAT2B gene. After LV-shMAT2B was transfected with L-02 cells and two kinds of HCC cells, cell viability and proliferation were measured with MTT and [3H]thymidine assays respectively. Flow cytometry was used to assess cell apoptosis. The level of S-adenosyl methionine (SAMe) in HepG2 cells was evaluated. The expressions of cyclin D1, cyclin D2, bcl-xL and bcl-xS were detected with western blot. RESULTS: We constructed LV-shMAT2B successfully. LV-shMAT2B was safe for human normal liver cells. LV-shMAT2B caused dramatic reduction in proliferation compared with controls in HCC cells Bel-7402 (P = 0.054) and HepG2 (P = 0.031). Flow cytometry analysis showed that cell apoptosis caused by LV-shMAT2B was greater in HCC cells Bel-7402 and HepG2 than in control induced by scrambled siRNA (P = 0.047), but apoptosis rates in L-02 induced by LV-shMAT2B and scrambled siRNA respectively had no significant difference. Moreover, LV-shMAT2B significantly suppressed expression of MAT2B leading to growth-inhibition effect on HCC cells by down-regulating cyclin D1. Apoptosis induced by LV-shMAT2B was involved indown-regulating bcl-xL and up-regulating bcl-xS. CONCLUSION: LV-shMAT2B can induce cell apoptosis and growth-inhibition in HCC cells. MAT2B may be a therapy target in HCC in the future.
