【作者】 马瑞花；

【导师】 黄波；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 目的：饥饿状态下机体的肝细胞通过糖异生来保证重要器官的葡萄糖供给。恶性转化的肝细胞是否还具有糖异生的能力目前尚不清楚。有氧糖酵解是恶性肿瘤的重要标志，除了三步不可逆反应外，糖异生的其余七步反应均是糖酵解的逆反应，因此可以说糖异生拮抗糖酵解。本研究将探讨人和小鼠肝癌组织中糖异生的变化及相应的肝癌治疗策略。方法：(1) Real-time PCR和免疫组化分析10例肝癌病人肝癌组织中糖异生的限速酶PEPCK、G6Pase和调控糖皮质激素的两个关键酶11β-HSD1、11β-HSD2的表达，进一步免疫组化分析58例肝癌病人的肝癌组织和癌旁组织中11β-HSD1和11β-HSD2的表达，并统计分析11β-HSD1/11β-HSD2的比值与肝癌病人的生存时间和肿瘤复发率的关系。(2) RT-PCR、Western blot和免疫组化分析小鼠肝癌组织中PEPCK、G6Pas、11β-HSD1和11β-HSD2的表达，进一步建立高表达11β-HSD1或下调11β-HSD2的肝癌细胞，并将高表达11β-HSD1或下调11β-HSD2的肝癌细胞接种小鼠进行肿瘤生长实验，验证11β-HSD1和11β-HSD2对肝癌生长的影响，并采用RT-PCR检测相应高表达11β-HSD1或下调11β-HSD2的肝癌组织中PEPCK和G6Pase的表达，以探讨糖异生对肝癌生长的影响。(3)体外条件下，分别用0M、0.1M、1M、10M地塞米松处理H22细胞一周后，RT-PCR和Western blot检测PEPCK和G6Pase的表达并检测胞内葡萄糖的变化。体内条件下，皮下接种H22细胞至BALB/c右背侧，4天后随机分为四组(6只/组)，分别腹腔注射0g/g、1.25g/g、2.5g/g和5g/g地塞米松，观察并记录肿瘤生长情况，16天后处死小鼠分离肝癌组织，RT-PCR和Western blot检测PEPCK和G6Pase的表达并检测组织内葡萄糖的变化。为排除不同接种部位对地塞米松治疗效果的影响，建立小鼠原位肝癌模型，随机分为四组(6只/组)分别腹腔注射0g/g、1.25g/g、2.5g/g和5g/g地塞米松，16天后处死小鼠，观察肿瘤大小。为探讨对地塞米松对非肝脏来源的肿瘤的影响，皮下接种B16细胞至C57BL/6右背侧，4天后随机分为四组(6只/组)，分别腹腔注射0g/g、1.25g/g、2.5g/g和5g/g地塞米松，观察并记录肿瘤生长情况；为探讨地塞米松治疗肿瘤的机制，采用PEPCK抑制剂3-MPA和地塞米松联合治疗H22皮下瘤小鼠，观察并测量肿瘤生长情况；最后利用非活性形式的糖皮质激素泼尼松体外处理H22细胞，体内治疗H22皮下瘤小鼠，RT-PCR分析PEPCK和G6Pase的表达并记录肿瘤生长情况。(4)皮下接种H22细胞至BALB/c右背侧，4天后随机分为四组(6只/组)，分别腹腔注射0g/g、1.25g/g、2.5g/g和5g/g地塞米松，16天后处死小鼠分离肝癌组织，RT-PCR检测一系列代谢相关基因(HK2, PFK1, PKM2, LDHA,PDHA1, CS, SDHA, ACL, ACC, HMGCR, G6PD, GPD1)的表达，Real-time PCR和Western blot进一步检测中LDH和GPD1的表达并检测肿瘤组织内乳酸的水平。结果：(1)肝癌病人肝癌组织中糖异生限速酶PEPCK、G6Pase表达显著下调，调控糖皮质激素的关键酶11β-HSD1表达下调，而11β-HSD2表达上调，且肝癌病人中11β-HSD1和11β-HSD2在癌旁组织和肝癌组织中表达呈逆关联，即癌旁组织中11β-HSD1表达高、11β-HSD2表达低，而肝癌组织恰与此相反11β-HSD1表达低、11β-HSD2表达高，进一步统计结果显示11β-HSD1/11β-HSD2比值与肝癌病人总体生存时间与复发率相关。(2)小鼠肝癌组织中PEPCK和G6Pase表达显著下调，糖异生丧失，11β-HSD1表达下调，而11β-HSD2表达上调，恢复小鼠肝癌细胞H22中11β-HSD1和11β-HSD2的表达后，小鼠肿瘤生长变慢且肿瘤组织内PEPCK和G6Pase表达上调。(3)体外条件下，地塞米松可上调H22细胞中PEPCK和G6Pase的表达并上调胞内葡萄糖含量，体内条件下，地塞米松可抑制皮下瘤肝癌和原位肝癌的生长并上调肝癌组织内的PEPCK和G6Pase的表达和相应的葡萄糖含量，但是对小鼠黑色素瘤无影响，揭示地塞米松对肝癌治疗的特异性，同时利用PEPCK的抑制剂3-MPA可阻断地塞米松对肝癌的抑制作用，揭示地塞米松可能是通过糖异生途径抑制肝癌，且非活性形式的糖皮质激素泼尼松体外对H22的PEPCK和G6Pase表达无影响，体内对小鼠皮下瘤无抑制作用。(4)地塞米松治疗后小鼠肝癌组织中LDH和GPD1表达下调且相应组织内乳酸水平降低。结论：人和小鼠肝癌细胞中调控糖皮质激素的关键酶11β-HSD1和11β-HSD2表达逆转，导致内源性糖皮质激素的失活和糖异生的丧失。11β-HSD1/11β-HSD2的比值与肝癌病人的生存时间和肿瘤复发情况有关。糖皮质激素的活性形式地塞米松通过绕过11β-HSD1和11β-HSD2的调控上调肝癌细胞的糖异生抑制肝癌生长。这些发现揭示肝癌细胞中11β-HSD1和11β-HSD2的逆转表达可能在糖异生到糖酵解的转化中发挥重要作用，有望成为肝癌治疗的新靶点。更多还原

【Abstract】 Objective: Gluconeogenesis by which glucose is biosynthesized leading to acontinuous glucose supply to vital organs is a fundamental feature of normalhepatocytes. Whether this gluconeogenic activity is also present in malignanthepatocytes remains largely unexplored, despite that an answer may give rise to noveltherapeutic strategies to target glycolysis, one hallmark of malignant cells.Methods:(1) The expressions of PEPCK, G6Pase and11β-HSD1,11β-HSD2inhuman hepatocarcinoma were analyzed by real-time PCR and immunohistochemisty.To further clarify the exact situation of11β-HSD1/11β-HSD2in hepatocarcinomapatients,58human hepatocarcinoma specimens were immunohistochemicallyanalyzed and the relative expressions of11β-HSD1and11β-HSD2were quantified.Then, the ratios of11β-HSD1/11β-HSD2were calculated and the relationshipbetween11β-HSD1/11β-HSD2and patients’ survival time and tumor recurrence wasanalyzed by the Kaplan-Meier survival method.(2) The expressions of11β-HSD1,11β-HSD2and PEPCK, G6Pase in murine hepatocarcinoma were analyzed byRT-PCR, Western blot and immunohistochemisty. Then,11β-HSD1-overexpression or11β-HSD2-knockdown H22was constructed and these engineered H22tumor celllines were inoculated into the mice, the growth of tumor was monitored. To further investigate the effect of gluconeogenesis on hepatocarcinoma, the PEPCK andG6Pase expressions in11β-HSD1-overexpression or11β-HSD2-knockdown H22tumor were analyzed by RT-PCR.(3) In vitro, H22cells were treated with0,0.1,1or10M dexamethasone for7days. Then, the expressions of PEPCK and G6Pase weredetermined by RT-PCR and Western blot and cellular glucose was measured withglucose assay kit. In vivo, The BALB/c mice were subcutaneously injected with3×105H22cells for4days, and then treated with the intraperitoneal injections ofdifferent concentrations of dexamethasone (1.25,2.5and5g/g) or saline once perday for16days. The growth of tumor was monitored.16days later, the mice weresacrificed and hepatocarcinoma was separated for PEPCK and G6Pase expressionanalysis and tissue glucose was measured with glucose assay kit. To exclude theinfluence of different inoculation sites on dexamethasone treatment, BALB/c miceliver were inoculated with H22cells and treated with0g/g,1.25g/g,2.5g/g or5g/g dexamethasone.16days later, the mice were sacrificed and livers wereseparated and analyzed. To further investigated the influence of dexamethasone onnon-liver cancers,3×105non-liver derived melanoma B16tumor cells weresubcutaneously injected into C57BL/6mice for4days, and then treating them withdifferent concentrations of dexamethasone, the growth of tumor was monitored. Toconfirm that the antitumor effect of dexamethasone is via the gluconeogeneticpathway,3-MPA, the PEPCK-selective inhibitor was used and growth of tumor wasmonitored. To further strengthen dexamethasone as a potential agent in the treatmentof hepatocarcinoma, prednisone, a dehydrogenated inactive form of glucocorticoids,was additionally tested in H22tumor cells in vitro and H22tumor-bearing mice invivo.(4) The molecular basis of dexamethasone affecting the glucose metabolism ofhepatocarcinoma was further investigated. H22tumor-bearing mice were treated withdifferent concentrations of dexamethasone for seven days. A panel of metabolism-related genes (HK2, PFK1, PKM2, LDHA, PDHA1, CS, SDHA, ACL, ACC, HMGCR, G6PD, GPD1) in tumor tissues was analyzed by RT-PCR. Then, theexpressions of LDH and GPD1in tumor tissues were further analyzed by real-timePCR and Western blot, and lactate in tumor tissues was measured.Results:(1) Both the transcripts and proteins of PEPCK and G6Pase werestrikingly lower in the tumor tissues but much higher in the peritumoral liver tissues,as shown by real time PCR and immunohistochemical staining. In line with theseresults, it was clear that11β-HSD1was downregulated but11β-HSD2wasupregulated in hepatocarcinoma tissues. Moreover,58human hepatocarcinomaspecimens were immunohistochemically analyzed and the relative expressions of11β-HSD1and11β-HSD2were quantified, which revealed the inversely expressed11β-HSD1and11β-HSD2in hepatocarcinoma relative to normal liver tissue. Theratios of11β-HSD1/11β-HSD2were calculated and the patients’ samples were splitinto2classes (high and low) according to the median value in the whole set of58samples. The Kaplan-Meier survival analysis showed that patients with high ratio hada significant longer survival time and lesser recurrence than those with low ratio.(2)When we used murine hepatocarcinoma tumor cell line H22to generatehepatocarcinoma, the expressions of PEPCK and G6Pase were found to be strikinglydecreased and were not affected by fasting, as shown by RT-PCR, Western blot andimmunohistochemical staining. Surprisingly,11β-HSD1was markedly downregulatedand11β-HSD2was markedly upregulated in hepatocarcinoma tissues, compared tonormal liver tissues. In addition, the primary hepatocarcinoma cells and normalhepatocytes, isolated from tumor-bearing mice, also showed downregulation of11β-HSD1and upregulation of11β-HSD2. Not therefore unexpectedly,11β-HSD1-overexpression or11β-HSD2-knockdown both restored gluconeogenesiswith upregulations of PEPCK and G6Pase in H22tumor tissues. As a result, theinoculation of these engineered H22tumor cell lines to the mice resulted in theinhibition of tumor growth and the corresponding prolonged survival of the mice.(3) In vitro, dexamethasone-treated H22tumor cells showed the upregulation of PEPCKand G6Pase expressions under the concentrations of1and10M. Consistently, thelevels of intracellular glucose were also found to be increased. In line with these invitro results the in vivo dexamethasone treatments resulted in increases in PEPCK andG6Pase expressions and tissue glucose in all the mice, compared to the saline control.Moreover, dexamethasone treatment showed significant inhibition of the ectopic H22tumor growths and orthotopic H22tumor growths. But dexamethasone did notsignificantly suppress tumor growth and only produced marginal effects, suggesting arelative selectivity of dexamethasone for hepatocarcinoma but not non-liver cancers.It was found that the intragastric administration of3-MPA the PEPCK-selectiveinhibitor effectively counteracted the inhibitory effects of dexamethasone on H22tumor. Unlike dexamethasone and its efficacy, prednisone a dehydrogenated inactiveform of glucocorticoids did not show the upregulation of PEPCK and G6Pase or anyantitumor effect.(4) LDHA and GPD1were found to be downregulated afterdexamethasone treatment and the lactate level was also downregulated.Conclusions: We show here that gluconeogenesis was not present in human ormouse malignant hepatocytes. Two critical enzymes11β-HSD1and11β-HSD2thatregulate glucocorticoid activities were found to be expressed inversely in malignanthepatocytes, resulting in the inactivation of endogenous glucocorticoids and the lossof gluconeogenesis. In patients’ hepatocarcinoma, the expressions of11β-HSD1and11β-HSD2are strikingly linked to patients’ prognosis and survival. Dexamethasone,the active form of synthesized glucocorticoids, is capable of restoringgluconeogenesis in malignant cells by bypassing the abnormal regulation of11β-HSDenzymes, leading to therapeutic efficacy against hepatocarcinoma. These findingsreveal that the reversed expressions of11β-HSD1and11β-HSD2may play animportant role in the switch gluconeogenesis to glycolysis in hepatocarcinoma,begging it to be a new hepatocarcinoma treatment target.更多还原