MIF对人结肠癌细胞血管生成因子表达及PI3K信号通路的影响
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摘要
目的:
研究巨噬细胞移动抑制因子(MIF)在体外对人结肠癌细胞HT-29增殖及VEGF和IL-8表达的影响,并进一步探讨MIF通过PI3K/Akt通路调控结肠癌细胞生长及血管生成因子表达的信号转导机制。
方法:
HT-29细胞饥饿24h达同步化后,实验分成对照组、不同浓度的人重组MIF(rhMIF 25、50、100μg/L)组和50μg/L rhMIF联合LY294002预处理组,分别作用于HT-29细胞不同时间,应用:
(1)MTT(四甲基偶氮唑蓝比色法)法分别于0、12、24、48h检测rhMIF对HT-29细胞生长增殖的影响及LY294002预处理组细胞增殖状态。
(2)RT-PCR法检测不同浓度rhMIF对HT-29细胞作用0、12、24、48h后及LY294002预处组24h VEGF、IL-8 mRNA的表达。
(3)ELISA法分别于0、12、24、48h检测rhMIF组细胞培养上清液中VEGF和IL-8蛋白水平及LY294002预处理组24h上清液中VEGF、IL-8蛋白水平。
(4)Western-blot法检测rhMIF作用于HT-29细胞0、15、30、60min时Akt蛋白的磷酸化水平及LY294002预处理组30min时p-Akt水平。
结果:
(1)不同浓度rhMIF作用12,24,48h后,能够促进HT-29细胞生长,增殖活性随浓度增加、时间延长逐渐上升(P<0.05),呈现量-效、时-效关系;LY294002预处理组对HT-29细胞的生长具有抑制作用,与rhMIF组比较差异有显著性意义(P<0.01),而与对照组相比未见明显变化(P>0.05)。
(2)不同浓度rhMIF处理HT-29细胞24h,50μg/L rhMIF作用12,24,48h,VEGF、IL-8 mRNA表达均明显高于对照组,存在时间和浓度依赖性(P<0.05),而LY294002预处理能明显下调VEGF、IL-8 mRNA的表达(P<0.01)。
(3)正常对照组HT-29细胞培养上清液中VEGF、IL-8蛋白含量低, rhMIF处理细胞12h,24h,48h显著诱导VEGF、IL-8蛋白分泌,存在时间和浓度依赖性(P<0.05);上清中VEGF最高含量为对照组的6倍,IL-8最高含量为对照组的30倍。LY294002对rhMIF诱导的VEGF和IL-8蛋白分泌均产生抑制(P<0.01),与对照组相比无明显变化(P>0.05)。
(4)Western-blot结果显示:50μg/L rhMIF作用于HT-29细胞0、15min、30min、60min,与对照组相比,p-Akt蛋白表达在30min时达高峰。25、50、100μg/L rhMIF作用于HT-29细胞30min,与对照组比较,p-Akt表达明显增加(P <0.01);rhMIF浓度越高,p-Akt蛋白水平上升趋势越明显,呈剂量依赖性。PI3K抑制剂LY294002预处理后再施加rhMIF,磷酸化Akt蛋白水平明显降低,与rhMIF组相比差异有显著性意义(P<0.05)。
结论:
(1)MIF能够促进结肠癌细胞生长和增殖,MIF参与结肠癌血管形成可能是通过上调促血管生成因子VEGF和IL-8的表达发挥作用。
(2)MIF诱导人结肠癌细胞增殖和血管生成因子表达可能部分是通过活化PI3K/Akt信号转导通路实现的。
【Objectives】
To investigate the effects of Macrophage migration inhibitory factor on the proliferation and expression of vascular endothelial growth factor (VEGF) and Interleukin-8 (IL-8) in colon carcinoma cell line HT-29,and to further analyze the signal transduction mechanism of MIF in regulating the growth of HT-29 cells and angiogenesis via PI3K/Akt pathway.
【Methods】
The cultured HT-29 cells were treated with different concentrations of recombinant human Macrophage migration inhibitory factor ( rhMIF ) (0 , 25, 50,100μg/L) and 50μg/L rhMIF combined LY294002(25μmol/L) for different time,and the cells were harvested. Using:
(1) MTT assay were used to measure the proliferation of HT-29 in 0,12,24,48 hours respectively.
(2) The expression of VEGF and IL-8 mRNA in HT-29 cells were examined by real-time quantitative reverse-transcription-polymerase chain reaction (RT-PCR).
(3) The levels of VEGF and IL-8 protein in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA).
(4) Western blotting was performed to detect the expressions of p-Akt after the rhMIF treatment for 0,15,30,60 minutes,and the inhibition of PI3K/Akt by LY294002 for 30 minutes.
【Results】
(1) RhMIF promoted the proliferation of HT-29 cells in a concentration- and time-dependent manner (P <0.05), and the proliferating activity was raised with the increase of concentration and prolonging of action time. LY294002 suppressed the proliferation of HT-29 cells (P < 0.01),LY294002 combined with rhMIF couldn’t promote the proliferation of HT-29 cells(P>0.05).
(2) The expression of VEGF and IL-8 was significantly increased at mRNA levels in the cells treated by rhMIF in a concentration- and time-dependent manner(P<0.05), but the up-regulation of VEGF and IL-8 was obviously inhibited by LY294002 pre-treatment (P<0.01).
(3) The levels of VEGF and IL-8 protein in supernatants were raised gradually after rhMIF treatment with the increase of concentration or action time (P<0.05),the highest levels of VEGF and IL-8 was six times and thirty times than control group, respectively. But that in LY294002 pre-treatment groups was lower than the rhMIF-induced (P< 0.01).
(4) Western-blot analysis demonstrated that:50μg/L rhMIF significantly induced the activity of Akt at 15,30,60minutes.The maximal activation of Akt took place at 30min,with increased doses of rhMIF,the activation of Akt increased gradually.PI3K inhibitor, LY294002, could significantly inhibited the activation of Akt induced by rhMIF in HT-29.
【Conclusions】
(1) MIF promotes the growth and proliferation of colon carcinoma cells,MIF plays a crucial role in the angiogenesis of colon cancer and the roles of MIF may be realized via increasing the expression of angiogenic factors such as VEGF and IL-8.
(2) MIF can promote the proliferation and regulate the expression of VEGF and IL-8 of colon carcinoma cells via PI3K/Akt pathway.
研究巨噬细胞移动抑制因子(MIF)在体外对人结肠癌细胞HT-29增殖及VEGF和IL-8表达的影响,并进一步探讨MIF通过PI3K/Akt通路调控结肠癌细胞生长及血管生成因子表达的信号转导机制。
方法:
HT-29细胞饥饿24h达同步化后,实验分成对照组、不同浓度的人重组MIF(rhMIF 25、50、100μg/L)组和50μg/L rhMIF联合LY294002预处理组,分别作用于HT-29细胞不同时间,应用:
(1)MTT(四甲基偶氮唑蓝比色法)法分别于0、12、24、48h检测rhMIF对HT-29细胞生长增殖的影响及LY294002预处理组细胞增殖状态。
(2)RT-PCR法检测不同浓度rhMIF对HT-29细胞作用0、12、24、48h后及LY294002预处组24h VEGF、IL-8 mRNA的表达。
(3)ELISA法分别于0、12、24、48h检测rhMIF组细胞培养上清液中VEGF和IL-8蛋白水平及LY294002预处理组24h上清液中VEGF、IL-8蛋白水平。
(4)Western-blot法检测rhMIF作用于HT-29细胞0、15、30、60min时Akt蛋白的磷酸化水平及LY294002预处理组30min时p-Akt水平。
结果:
(1)不同浓度rhMIF作用12,24,48h后,能够促进HT-29细胞生长,增殖活性随浓度增加、时间延长逐渐上升(P<0.05),呈现量-效、时-效关系;LY294002预处理组对HT-29细胞的生长具有抑制作用,与rhMIF组比较差异有显著性意义(P<0.01),而与对照组相比未见明显变化(P>0.05)。
(2)不同浓度rhMIF处理HT-29细胞24h,50μg/L rhMIF作用12,24,48h,VEGF、IL-8 mRNA表达均明显高于对照组,存在时间和浓度依赖性(P<0.05),而LY294002预处理能明显下调VEGF、IL-8 mRNA的表达(P<0.01)。
(3)正常对照组HT-29细胞培养上清液中VEGF、IL-8蛋白含量低, rhMIF处理细胞12h,24h,48h显著诱导VEGF、IL-8蛋白分泌,存在时间和浓度依赖性(P<0.05);上清中VEGF最高含量为对照组的6倍,IL-8最高含量为对照组的30倍。LY294002对rhMIF诱导的VEGF和IL-8蛋白分泌均产生抑制(P<0.01),与对照组相比无明显变化(P>0.05)。
(4)Western-blot结果显示:50μg/L rhMIF作用于HT-29细胞0、15min、30min、60min,与对照组相比,p-Akt蛋白表达在30min时达高峰。25、50、100μg/L rhMIF作用于HT-29细胞30min,与对照组比较,p-Akt表达明显增加(P <0.01);rhMIF浓度越高,p-Akt蛋白水平上升趋势越明显,呈剂量依赖性。PI3K抑制剂LY294002预处理后再施加rhMIF,磷酸化Akt蛋白水平明显降低,与rhMIF组相比差异有显著性意义(P<0.05)。
结论:
(1)MIF能够促进结肠癌细胞生长和增殖,MIF参与结肠癌血管形成可能是通过上调促血管生成因子VEGF和IL-8的表达发挥作用。
(2)MIF诱导人结肠癌细胞增殖和血管生成因子表达可能部分是通过活化PI3K/Akt信号转导通路实现的。
【Objectives】
To investigate the effects of Macrophage migration inhibitory factor on the proliferation and expression of vascular endothelial growth factor (VEGF) and Interleukin-8 (IL-8) in colon carcinoma cell line HT-29,and to further analyze the signal transduction mechanism of MIF in regulating the growth of HT-29 cells and angiogenesis via PI3K/Akt pathway.
【Methods】
The cultured HT-29 cells were treated with different concentrations of recombinant human Macrophage migration inhibitory factor ( rhMIF ) (0 , 25, 50,100μg/L) and 50μg/L rhMIF combined LY294002(25μmol/L) for different time,and the cells were harvested. Using:
(1) MTT assay were used to measure the proliferation of HT-29 in 0,12,24,48 hours respectively.
(2) The expression of VEGF and IL-8 mRNA in HT-29 cells were examined by real-time quantitative reverse-transcription-polymerase chain reaction (RT-PCR).
(3) The levels of VEGF and IL-8 protein in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA).
(4) Western blotting was performed to detect the expressions of p-Akt after the rhMIF treatment for 0,15,30,60 minutes,and the inhibition of PI3K/Akt by LY294002 for 30 minutes.
【Results】
(1) RhMIF promoted the proliferation of HT-29 cells in a concentration- and time-dependent manner (P <0.05), and the proliferating activity was raised with the increase of concentration and prolonging of action time. LY294002 suppressed the proliferation of HT-29 cells (P < 0.01),LY294002 combined with rhMIF couldn’t promote the proliferation of HT-29 cells(P>0.05).
(2) The expression of VEGF and IL-8 was significantly increased at mRNA levels in the cells treated by rhMIF in a concentration- and time-dependent manner(P<0.05), but the up-regulation of VEGF and IL-8 was obviously inhibited by LY294002 pre-treatment (P<0.01).
(3) The levels of VEGF and IL-8 protein in supernatants were raised gradually after rhMIF treatment with the increase of concentration or action time (P<0.05),the highest levels of VEGF and IL-8 was six times and thirty times than control group, respectively. But that in LY294002 pre-treatment groups was lower than the rhMIF-induced (P< 0.01).
(4) Western-blot analysis demonstrated that:50μg/L rhMIF significantly induced the activity of Akt at 15,30,60minutes.The maximal activation of Akt took place at 30min,with increased doses of rhMIF,the activation of Akt increased gradually.PI3K inhibitor, LY294002, could significantly inhibited the activation of Akt induced by rhMIF in HT-29.
【Conclusions】
(1) MIF promotes the growth and proliferation of colon carcinoma cells,MIF plays a crucial role in the angiogenesis of colon cancer and the roles of MIF may be realized via increasing the expression of angiogenic factors such as VEGF and IL-8.
(2) MIF can promote the proliferation and regulate the expression of VEGF and IL-8 of colon carcinoma cells via PI3K/Akt pathway.
引文
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[2] Maxime V, Fitting C, Annane D,et al.Corticoids normalize leukocyte production of macrophage migration inhibitory factor in septic shock.J Infect Dis. 2005 Jan 1;191(1):138-44. Epub 2004 Nov 30.
[3] Ren Y, Law S, Huang X,et al.Macrophage migration inhibitory factor stimulates angiogenic factor expression and correlates with differentiation and lymph node status in patients with esophageal squamous cell carcinoma.Ann Surg. 2005 Jul;242(1):55-63.
[4] Lue H, Kapurniotu A, Fingerle-Rowson G, et al.Rapid and transient activation of the ERK MAPK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on JAB1/CSN5 and Src kinase activity.Cell Signal. 2006 May;18(5):688-703. Epub 2005 Aug 24.
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