肝X受体激动剂T0901317对肝癌血管生成的作用及机制

Effect and mechanism of liver X receptor agonist T0901317 on angiogenesis phenotype of liver cancer

    摘要
  • 摘要: 目的:探讨肝脏X受体激动剂T0901317对肝细胞癌血管生成的作用及机制。
    方法:采用实验研究方法。人肝癌细胞MHCC97-H、人肝癌细胞Huh7、人脐静脉内皮细胞HUVEC各自的空白组、低浓度组、高浓度组分别加入0 、1、3 μmol/L T0901317。采用CCK-8法检测各组细胞生长情况。采用实时荧光定量聚合酶链反应(PCR)分析各组细胞肝脏X受体靶基因脂肪酸合成酶(FAS)mRNA相对表达量。测量MHCC97-H裸鼠模型(MHCC97-H对照组和MHCC97-H T0901317组)皮下肿瘤体积和体质量。处死裸鼠后获取肿瘤组织。采用免疫组织化学染色检测裸鼠肿瘤组织中肿瘤血管生成标志物CD31相对表达量。采用Transwell法检测HUVEC各组细胞迁移能力和成血管能力。观察指标：(1)T0901317对MHCC97-H、Huh7、HUVEC生长的影响。(2)T0901317对MHCC97-H、Huh7、HUVEC肝脏X受体的作用。(3)T0901317对MHCC97-H裸鼠模型皮下肿瘤生长的影响。(4)T0901317对MHCC97-H裸鼠模型皮下肿瘤组织中CD31表达的影响。(5)T0901317对HUVEC迁移能力的影响。(6)T0901317对HUVEC成血管能力的影响。正态分布的计量资料以±s表示，多组间比较采用方差分析，两组间比较采用t检验。
    结果:(1)T0901317对MHCC97-H、Huh7、HUVEC生长的影响。CCK-8法细胞生长实验结果显示：MHCC97-H空白组、MHCC97-H低浓度组、MHCC97-H高浓度组活细胞百分比分别为100.0%±1.7%、101.0%±0.7%、104.6%±1.9%，3组比较，差异无统计学意义(F=2.632，P>0.05)。Huh7空白组、Huh7低浓度组、Huh7高浓度组活细胞百分比分别为100.0%±2.7%、97.6%±2.4%、103.7%±2.8%，3组比较，差异无统计学意义(F=1.404，P>0.05)。HUVEC空白组、HUVEC低浓度组、HUVEC高浓度组活细胞百分比分别为100.0%±0.7%、100.7%±1.2%、101.3%±0.8%，3组比较，差异无统计学意义(F=0.471，P>0.05)。(2)T0901317对MHCC97-H、Huh7、HUVEC肝脏X受体的作用。实时荧光定量PCR结果显示：MHCC97-H空白组、MHCC97-H低浓度组、MHCC97-H高浓度组细胞FAS mRNA相对表达量分别为100.0%±2.2%、658.5%±7.7%、1 241.0%±106.8%，3组比较，差异有统计学意义(F=46.227，P<0.05)。其中MHCC97-H空白组分别与MHCC97-H低浓度组、MHCC97-H高浓度组比较，差异均有统计学意义(t=70.025，8.274，P<0.05)；MHCC97-H低浓度组与MHCC97-H高浓度组比较，差异有统计学意义(t=4.222，P<0.05)。Huh7空白组、Huh7低浓度组、Huh7高浓度组细胞FAS mRNA相对表达量分别为100.0%±15.8%、1 225.0%±26.7%、2 015.0%±215.1%，3组比较，差异有统计学意义(F=49.402，P<0.05)。其中Huh7空白组分别与Huh7低浓度组、Huh7高浓度组比较，差异均有统计学意义(t=39.460，8.879，P<0.05)；Huh7低浓度组与Huh7高浓度组比较，差异有统计学意义(t=2.836，P<0.05)。HUVEC空白组、HUVEC低浓度组、HUVEC高浓度组细胞FAS mRNA相对表达量分别为100.0%±19.6%、790.8%±116.5%、1 756.0%±55.0%，3组比较，差异有统计学意义(F=185.395，P<0.05)。其中HUVEC空白组分别与HUVEC低浓度组、HUVEC高浓度组比较，差异均有统计学意义(t=7.639，34.375，P<0.05)；HUVEC低浓度组与HUVEC高浓度组比较，差异有统计学意义(t=7.488，P<0.05)。(3)T0901317对MHCC97-H裸鼠模型皮下肿瘤生长的影响。MHCC97-H裸鼠模型皮下肿瘤生长实验结果显示：MHCC97-H对照组和MHCC97-H T0901317组裸鼠模型皮下肿瘤体积分别为(935±72)mm3和(552±47)mm3，两组比较，差异有统计学意义(t=4.449，P<0.05)。MHCC97-H对照组和MHCC97-H T0901317组裸鼠模型体质量分别为(23.8±0.8)g和(21.7±1.7)g，两组比较，差异无统计学意义(t=1.059，P>0.05)。(4)T0901317对MHCC97-H裸鼠模型皮下肿瘤组织中CD31表达的影响。免疫组织化学染色检测结果显示：MHCC97-H对照组和MHCC97-H T0901317组裸鼠模型皮下肿瘤组织中CD31相对表达量分别为100%±11%和35%±7%，两组比较，差异有统计学意义(t=4.919，P<0.05)。(5)T0901317对HUVEC迁移能力的影响。Transwell法细胞迁移实验结果显示：HUVEC空白组、HUVEC低浓度组、HUVEC高浓度组穿膜细胞百分比分别为100.0%±4.0%、57.3%±1.5%、32.7%±1.7%，3组比较，差异有统计学意义(F=163.944，P<0.05)。其中HUVEC空白组分别与HUVEC低浓度组、HUVEC高浓度组比较，差异均有统计学意义(t=9.998，15.434，P<0.05)；HUVEC低浓度组与HUVEC高浓度组比较，差异有统计学意义(t=10.801，P<0.05)。(6)T0901317对HUVEC成血管能力的影响。细胞成血管实验结果显示：HUVEC空白组、HUVEC低浓度组、HUVEC高浓度组成血管长度相对值分别为100.0%±3.4%、68.4%±3.5%、44.7%±0.5%，3组比较，差异有统计学意义(F=38.964，P<0.05)。其中HUVEC空白组分别与HUVEC低浓度组、HUVEC高浓度组比较，差异均有统计学意义(t=6.268，9.831，P<0.05)；HUVEC低浓度组与HUVEC高浓度组比较，差异有统计学意义(t=3.460，P<0.05)。
    结论:肝脏X受体激动剂T0901317可抑制肝细胞癌血管生成。

     

    Abstract: Objective:To explore the effect and mechanism of liver X receptor agonist T0901317 on angiogenesis phenotype of liver cancer.
    Methods:The experimental study was conducted. Hepatocellular carcinoma MHCC97-H and Huh7 cells and human umbilical vein endothelial cells (HUVEC) were cultured in vitro. Each cell line was divided into 3 groups: control group (non-treated), low concentration group (treated using 1 μmol/L T0901317) and high concentration group (treated using 3 μmol/L T0901317). Cell proliferation was counted with a CCK-8 assay. Quantitative real-time polymerase chain reaction (PCR) was applied to confirm the relative mRNA expression of fatty acid synthetase (FAS) of liver X receptor target genes in 3 groups. Subcutaneous xenograft tumor volume and body mass were measured in MHCC97-H nude mice model. Then mice were sacrificed and tumor tissues were analyzed for CD31 relative expression by immunohistochemistry (IHC) staining. Migration and vessel angiogenesis of HUVEC were determined by Transwell method. Observation indicators: (1) effects of T0901317 on MHCC97-H, Huh7 and HUVEC cells proliferation, (2) effects of T0901317 on liver X receptor with MHCC97-H, Huh7 and HUVEC cells, (3) effects of T0901317 on subcutaneous xenograft tumor growth in MHCC97-H nude mice model, (4) effects of T0901317 on CD31 relative expression in subcutaneous xenograft tumor tissues of MHCC97-H nude mice model, (5) effects of T0901317 on migration of HUVEC, (6) effects of T0901317 on vessel angiogenesis of HUVEC. Measurement data with normal distribution were represented as ±s, and comparisons between groups were analyzed by the t test.
    Results:(1) Effects of T0901317 on MHCC97-H, Huh7 and HUVEC cells proliferation: results of CCK-8 assay showed that percentage of living cells was respectively 100.0%±1.7%, 101.0%±0.7% and 104.6%±1.9% in MHCC97-H control, low concentration and high concentration groups, with no statistically significant difference (F=2.632, P>0.05). Percentage of living cells was respectively 100.0%±2.7%, 97.6%±2.4% and 103.7%±2.8% in Huh7 control, low concentration and high concentration groups, with no statistically significant difference (F=1.404, P>0.05). Percentage of living cells was respectively 100.0%±0.7%, 100.7%±1.2% and 101.3%±0.8% in HUVEC control, low concentration and high concentration groups, with no statistically significant difference (F=0.471, P>0.05). (2) Effects of T0901317 on liver X receptor with MHCC97-H, Huh7 and HUVEC cells: results of quantitative real-time PCR showed that relative mRNA expressions of FAS in MHCC97-H control, low concentration and high concentration groups were respectively 100.0 %±2.2%, 658.5%±7.7% and 1 241.0%±106.8%, with a statistically significant difference among groups (F=46.227, P<0.05), and with a statistically significant difference between MHCC97-H control group and MHCC97-H low concentration and high concentration groups (t=70.025，8.274, P<0.05) and between MHCC97-H low concentration and high concentration groups (t=4.222, P<0.05). Relative mRNA expressions of FAS in Huh7 control, low concentration and high concentration groups were respectively 100.0%±15.8%, 1 225.0%±26.7% and 2 015.0%±215.1%, with a statistically significant difference among groups (F=49.402, P<0.05), and with a statistically significant difference between Huh7 control group and Huh7 low concentration and high concentration groups (t=39.460，8.879, P<0.05) and between Huh7 low concentration and high concentration groups (t=2.836, P<0.05). Relative mRNA expressions of FAS in HUVEC control, low concentration and high concentration groups were respectively 100.0%±19.6%, 790.8%±116.5% and 1 756.0%±55.0%, with a statistically significant difference among groups (F=185.395, P<0.05), and with a statistically significant difference between HUVEC control group and HUVEC low concentration and high concentration groups (t=7.639，34.375, P<0.05) and between HUVEC low concentration and high concentration groups (t=7.488, P<0.05). (3) Effects of T0901317 on subcutaneous xenograft tumor growth in MHCC97-H nude mice model: results of assay showed that subcutaneous xenograft tumor volume in MHCC97-H control group and MHCC97-H T0901317 group were respectively (935±72)mm3 and (552±47)mm3, with a statistically significant difference between groups (t=4.449, P<0.05). Body masses of nude mice model in MHCC97-H control group and MHCC97-H T0901317 group were respectively (23.8±0.8)g and (21.7±1.7)g, with no statistically significant difference between groups (t=1.059, P>0.05). (4) Effects of T0901317 on CD31 relative expression in subcutaneous xenograft tumor tissues of MHCC97-H nude mice model: results of IHC staining showed that CD31 relative expression in subcutaneous xenograft tumor tissues of MHCC97-H nude mice model was 100%±11% and 35%±7% in MHCC97-H control group and MHCC97-H T0901317 group, with a statistically significant difference between groups (t=4.919, P<0.05). (5) Effects of T0901317 on migration of HUVEC: results of Transwell method showed that percentages of membrane cells in HUVEC control, low concentration and high concentration groups were respectively 100.0%±4.0%, 57.3%±1.5% and 32.7%±1.7%, with a statistically significant difference among groups (F=163.944, P<0.05), and with statistically significant differences between HUVEC control group and HUVEC low concentration and high concentration groups (t=9.998，15.434, P<0.05) and between HUVEC low concentration and high concentration groups (t=10.801, P<0.05). (6) Effects of T0901317 on vessel angiogenesis of HUVEC: results of vessel angiogenesis assay showed that length of vessel angiogenesis in HUVEC control, low concentration and high concentration groups were respectively 100.0%±3.4%, 68.4%±3.5% and 44.7%±0.5%, with a statistically significant difference among groups (F=38.964, P<0.05), and with statistically significant differences between HUVEC control group and HUVEC low concentration and high concentration groups (t=6.268，9.831, P<0.05) and between HUVEC low concentration and high concentration groups (t=3.460, P<0.05).
    Conclusions:Liver X receptor agonist T0901317 can inhibit vessel angiogenesis of liver cancer.

     

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