果糖-1,6-二磷酸酶在肝癌中的表达及其调控机制

发布时间：2018-04-27 20:29

本文选题：肝癌 + 果糖-1　；参考：《苏州大学》2016年博士论文

【摘要】：目的:检测果糖-1,6-二磷酸酶(FBP1)在肝癌中的表达,分析其与患者预后的关系。探讨组蛋白去乙酰化酶(HDAC)对FBP1缺失的调控机制,及FBP1对肝脏肿瘤细胞生物学行为的影响。方法:通过免疫组织化学染色法检测90例肝癌患者肿瘤组织及相应的癌旁组织中FBP1的表达。采用配对秩和检验比较肝癌组织与癌旁组织中FBP1的表达水平,Kaplan-Meier法和Log-rank检验分析FBP1表达水平与患者生存期的关系。为进一步探讨肝癌中FBP1的调控机制采用HDAC抑制剂NaBu、SAHA、LBH按浓度梯度处理HepG2及SK-Hep1细胞,western blot及real-time PCR检测FBP1蛋白及mRNA水平的变化。利用siRNA转染实验敲除肝癌细胞株中HDAC1及HDAC2,并检测其对FBP1表达的影响。染色质免疫共沉淀实验检测HDAC与FBP1的结合位点。90例临床组织标本同时经免疫组化检测FBP1、HDAC1及HDAC2,并分析其相关性。HepG2及SK-Hep1细胞经病毒感染,分别构建稳转pcDNA3.1,pLenti6.3/FBP1,NS shRNA,FBP1 shRNA的稳转细胞株。葡萄糖检测试剂盒检测转染后细胞对葡萄糖消耗的变化。MTS检测肝癌细胞株过表达FBP1对细胞增殖的影响。免疫缺陷小鼠荷瘤实验检测FBP1过表达对肿瘤增殖的影响。结果:FBP1在肝癌组织中的表达明显低于相应的癌旁组织,差异有统计学意义(Z=7.952,P0.001)。小肝癌中FBP1的表达水平高于非小肝癌(P=0.005)。AJCC分期越高,FBP1表达越低(P=0.001)。FBP1高表达组患者平均生存期为50.17个月,低表达组患者平均生存期为34.88个月,差异有统计学意义(P=0.034)。肝癌细胞株经HDAC抑制剂NaBu、SAHA、LBH处理后,FBP1蛋白及mRNA水平明显升高,并且具有药物浓度依赖性。单独干扰肝癌细胞株中HDAC1及HDAC2后可上调FBP1的蛋白及mRNA水平,共同敲除HDAC1及HDAC2后FBP1上调更显著。HDAC通过与FBP1增强子区AcH3K27位点结合,影响FBP1的转录。90例临床标本免疫组化检测结果也提示FBP1与HDAC1及HDAC2的表达均呈负相关(r=-0.64,P0.05;r=-0.61,P0.05)。恢复肝癌细胞中FBP1的表达,可减低肿瘤细胞对葡萄糖的消耗,抑制肿瘤细胞增殖,抑制肿瘤细胞在免疫缺陷小鼠皮下成瘤。结论:FBP1在肝癌中功能缺失,是判断患者预后的因素。HDAC1及HDAC2协同抑制FBP1增强子区H3K27位点的乙酰化,调控FBP1的表达。FBP1干扰肝脏肿瘤细胞糖酵解过程,降低葡萄糖消耗,减低肿瘤细胞供能,抑制肿瘤细胞增殖。
[Abstract]:Aim: to detect the expression of fructose 6 diphosphatase (FBP1) in hepatocellular carcinoma (HCC) and to analyze the relationship between FBP1 and prognosis. To investigate the regulatory mechanism of histone deacetylase (HDAC) on FBP1 deletion and the effect of FBP1 on the biological behavior of liver tumor cells. Methods: immunohistochemical staining was used to detect the expression of FBP1 in tumor tissues and adjacent tissues of 90 patients with liver cancer. The expression level of FBP1 in HCC and para-cancerous tissues was compared by paired rank sum test. Kaplan-Meier method and Log-rank test were used to analyze the relationship between FBP1 expression and survival. In order to further investigate the regulatory mechanism of FBP1 in hepatocellular carcinoma, the changes of FBP1 protein and mRNA levels in HepG2 and SK-Hep1 cells were detected by HDAC inhibitor NaBU SAHAA LBH and SK-Hep1 cells treated with western blot and real-time PCR according to the concentration gradient. SiRNA transfection assay was used to knockout the expression of HDAC1 and HDAC2 in hepatoma cells and the effect of HDAC2 on the expression of FBP1 was detected. The binding sites of HDAC and FBP1 were detected by chromatin coprecipitation assay. FBP1HDAC1 and HDAC2 were detected by immunohistochemistry, and the correlation between HDAC1 and HDAC2 was analyzed. HepG2 and SK-Hep1 cells were infected with virus to construct the stable transformed cell line of pcDNA3.1pLenti6.3 / FBP1NSshRNAFBP1 shRNA. Glucose detection kit was used to detect the change of glucose consumption after transfection. MTS was used to detect the effect of overexpression of FBP1 on cell proliferation. The effect of overexpression of FBP1 on tumor proliferation in immunodeficient mice was detected by tumor-bearing assay. Results the expression of FBP1 in HCC was significantly lower than that in adjacent tissues, and the difference was statistically significant (P 0.001). The expression of FBP1 in small hepatocellular carcinoma was significantly higher than that in P0. 005. AJCC stage. The lower the expression of FBP1 was, the lower the expression of FBP1 was. The average survival time was 50. 17 months in the high expression group and 34. 88 months in the low expression group. The difference was statistically significant (P 0. 034 4). The levels of FBP1 protein and mRNA in hepatoma cells treated with HDAC inhibitor NaBu-SAHAL LBH were significantly increased in a concentration-dependent manner. Interfering with HDAC1 and HDAC2 alone could up-regulate the level of FBP1 protein and mRNA in hepatoma cell lines, and after knockout HDAC1 and HDAC2, FBP1 upregulated more significantly. HDAC could bind to AcH3K27 site of FBP1 enhancer region. The expression of FBP1 and HDAC1 and HDAC2 were negatively correlated with the expression of HDAC1 and HDAC2 in 90 clinical specimens. The results showed that the expression of FBP1 was negatively correlated with the expression of HDAC1 and HDAC2. Restoring the expression of FBP1 in hepatoma cells can reduce the glucose consumption of tumor cells, inhibit the proliferation of tumor cells, and inhibit tumor cells subcutaneous tumorigenesis in immunodeficient mice. Conclusion the loss of function of FBP1 in hepatocellular carcinoma is a prognostic factor. HDAC1 and HDAC2 synergistically inhibit the acetylation of H3K27 site in FBP1 enhancer region, and regulate the expression of FBP1. FBP1 interferes with the glycolysis process of liver tumor cells and reduces glucose consumption. Reduce the energy supply of tumor cells and inhibit the proliferation of tumor cells.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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