Gankyrin在CCl4诱导的肝纤维化—肝癌发生中的作用及机制研究
发布时间：2018-08-10 18:18
【摘要】：肝癌发生一个是慢性的多因素、多步骤的复杂过程。HCC的发生通常与多种因素相关：肝炎病毒的持续感染、长期酒精摄取、长期接触致癌剂黄曲霉素等。此外,代谢疾病如糖尿病、肥胖也是肝癌发病的危险因素。多项临床流行病学研究证实,大部分肝癌都是在慢性炎症背景下发展而来,通常经由慢性肝病发展到肝纤维化、肝硬化,最后恶化形成肝癌。肝硬化病理背景为肿瘤形成提供了合适的细胞转化环境。目前,肝癌发生特别是从慢性肝损伤恶性转化导致的肿瘤形成的确切分子机制尚不完全清楚。Gankyrin是最早发现在肝癌中特异性高表达的癌基因。近年来的研究显示,gankyrin还在其他包括胆管癌、肠癌、乳腺癌以及肺癌等多种恶性肿瘤中表达上调。Gankyrin在肿瘤相关的信号转导中具有非常重要的调控作用。Gankyrin被证明可促进RB蛋白的磷酸化降解,导致肿瘤细胞周期紊乱,促进增殖；通过与MDM2相互作用,增加P53的泛素化降解,从而抑制肿瘤细胞凋亡。本实验室前期研究发现gankyrin直接结合RelA,促进NF-κB出核,抑制其转录活性；通过激活PI3K/AKT信号以促进肝癌的侵袭和转移；通过抑制OCT4的降解进而促进肿瘤前体细胞的扩增。虽然gankyrin在肝癌中已经被广泛研究,但大部分集中在肝癌的发展阶段的功能,至于其对于肝癌发生的具体调控或者说在肝癌发生时期是起始因素还是伴随现象,这一点仍不十分清楚。结合以往的研究数据,gankyrin不仅仅在肝癌阶段高表达,且在DEN诱导的大鼠肝癌发生过程的肝硬化阶段出现表达升高,提示我们gankyrin在这一阶段可能发挥某种调控作用。所以本研究课题将注意力集中在肝癌发生之前的肝损伤、肝纤维化、肝硬化阶段,通过在肝脏特异性过表达gankyrin的转基因小鼠基础上构建经由慢性损伤、肝纤维化、肝硬化到肝癌的动物模型,尝试从体内水平探索gankyrin对肝癌发生有无调控功能以及其内在机制。实验方法1.临床样本检测肝硬化临床样本中gankyrin的蛋白表达水平。2.转基因小鼠构建：构建由Albumin启动子连接gankyrin全长cDNA以实现gankyrin的肝脏特异性过表达。3.DEN以及DEN联合CCl4肝癌动物模型构建：小鼠出生后15天单次注射DEN后9个月后观察肝癌形成情况；DEN处理联合每周一次CCl4给药,不同时间后观察两组之间肿瘤形成情况差异。4.CCl4单独处理诱导纤维化及恶性转化模型：每周3次CCl4注射持续16周,收取中间时间点样品检测肝损伤及纤维化相关指标。5.机制探索：(1)不同时间点CCl4处理的野生对照和gankyrinhep小鼠肝脏样品中检测JNK及其他相关通路变化情况；CCl4处理后分选小鼠原代肝细胞检测相关通路活化情况。(2)CCl4处理的小鼠肝脏组织样品进行Rac1激酶活性检测；免疫共沉淀检测gankyrin对RhoGDIa与Rac1抑制性相互作用的影响。(3)CCl4处理的小鼠肝脏组织样品进项ROCK激酶活性检测；免疫共沉淀检测gankyrin对RhoGDIa与RhoA相互作用的影响。(4)对CCl4处理小鼠同时给予Rac1抑制剂处理小鼠,检测对JNK活性的调节。(5)对CCl4处理小鼠同时给予JNK抑制剂或Rac1抑制剂,检测凋亡相关分子、肝纤维化以及炎症因子分泌情况。(6)CCl4处理小鼠16周期间同时给予JNK抑制剂3个月,观察肝脏肿瘤形成。实验结果1.肝硬化临床样本中gankyrin表达升高(P0.05)。2. DEN+CC14诱导肝癌形成过程中gankyrinhep组的肿瘤数目和肿瘤大小均明显高于野生对照组；同时肝纤维化在gankyrinhep组明显加重；而DEN诱癌模型中,gankyrinhep组与野生对照组肿瘤形成情况无明显差异。3.CCl4单独处理16周后在gankyrinhep组有50%小鼠形成肝癌,而野生组未发生。4.CCl4单独处理2周、4周和8周时,gankyrinhep组的肝纤维化以及星状细胞活性化均较野生组增强；gankyrinhep组的肝脏损伤及炎症反应加重,同时代偿性增殖明显高于野生对照。5.短时间CCl4处理的gankyrinhep组肝细胞坏死和凋亡较野生对照加重。6.CCl4处理的gankyrinhep组呈现JNK通路相对野生对照组持续增强活化。7.CCl4处理的gankyrinhep组的Rac1激酶持续活化；而RhoA、ROCK活性下降；gankyrinhep组的RhoGDIl与RhoA结合减弱；Rac1抑制剂处理逆转gankyrin对JNK持续激活。8.JNK抑制剂和Rac1抑制剂均能缓解CCl4引起的gankyrinhep组的细胞凋亡和炎症因子分泌。9.JNK抑制剂可完全抑制长期CCl4诱导的gankyrinhep组肝癌形成。结论本研究主要通过构建不同类型的肝癌模型,从动物体内水平证明gankyrin明显促进肝损伤剂CCl4诱导的肝纤维化以及肝癌发生；而对单独DNA损伤剂DEN诱导的遗传突变导致的肝癌发生没有影响。这一点证明gankyrin促进的肝癌发生与持续的损伤和炎症微环境相关。Gangkyrin通过调节RhoGDI与RhoA的相互作用,下调RhoA的活性进而增强Rac1活性,导致CCl4处理过程中JNK通路的持续活化,促进肝损伤加重,炎症反应加强,肝细胞代偿性增殖加强,肝纤维化加重并恶性转化,最终导致肝癌形成。
[Abstract]:Hepatocellular carcinoma (HCC) is a chronic, multifactorial, multistep, complex process. The occurrence of HCC is usually associated with a variety of factors: persistent hepatitis virus infection, long-term alcohol intake, long-term exposure to carcinogens such as aflatoxin. In addition, metabolic diseases such as diabetes, obesity is also a risk factor for liver cancer. Several clinical epidemiological studies have confirmed that Most hepatocellular carcinomas develop in the context of chronic inflammation, usually through chronic liver disease to liver fibrosis, cirrhosis, and eventually deterioration to form liver cancer. Gankyrin is the earliest oncogene to be found in hepatocellular carcinoma. Recent studies have shown that Gankyrin is up-regulated in many other malignant tumors, including cholangiocarcinoma, intestinal cancer, breast cancer and lung cancer. Gankyrin plays an important role in tumor-related signal transduction. Gankyrin has been shown to promote the phosphorylation and degradation of RB protein, lead to cell cycle disorder and promote proliferation; increase the ubiquitination and degradation of P53 by interacting with MDM2, thereby inhibiting tumor cell apoptosis. I3K/AKT signaling promotes invasion and metastasis of hepatocellular carcinoma and promotes the proliferation of precursor cells by inhibiting the degradation of OCT4. Although Gankyrin has been extensively studied in hepatocellular carcinoma, most of it focuses on the function of hepatocellular carcinoma at the stage of development, and the specific regulation of hepatocellular carcinoma or the initiation of hepatocellular carcinoma at the stage of development. It is still unclear whether Gankyrin is a concomitant phenomenon. In combination with previous data, Gankyrin is not only overexpressed in the stage of hepatocellular carcinoma, but also elevated in the stage of cirrhosis in the process of hepatocarcinogenesis induced by DEN in rats, suggesting that Gankyrin may play a regulatory role in this stage. Focusing on liver injury, liver fibrosis and cirrhosis before hepatocarcinogenesis, the animal models from chronic injury, liver fibrosis, cirrhosis to hepatocarcinogenesis were constructed on the basis of transgenic mice with liver-specific overexpression of gankyrin. Methods 1. Detection of Gankyrin protein expression in clinical samples of cirrhosis. 2. Construction of transgenic mice: construction of a full-length Gankyrin cDNA linked by an Albumin promoter to achieve Gankyrin liver-specific overexpression. 3. Establishment of animal models of DEN and DEN combined with CCl4 liver cancer: 9 mice after a single injection of DEN 15 days after birth Hepatocellular carcinoma formation was observed after 6 months; DEN treatment combined with weekly CCl4 administration was used to observe the difference of tumor formation between the two groups after different time. 4. CCl4 treatment alone induced fibrosis and malignant transformation model: CCl4 injection three times a week for 16 weeks, collected samples at intermediate time points to detect liver injury and fibrosis-related indicators. 5. Mechanism exploration (1) The changes of JNK and other related pathways were detected in the liver samples of wild control and gankyrinhep mice treated with CCl4 at different time points, and the activation of related pathways was detected in the primary hepatocytes of mice treated with CCl4. (2) Rac1 kinase activity was detected in the liver samples of mice treated with CCl4, and gankyrinhep was detected by immunoprecipitation. (3) The activity of ROCK kinase was detected in CCl4 treated mice liver tissue samples; the effect of Gankyrin on the interaction between RhoGDIa and RhoA was detected by immunoprecipitation. (4) The mice treated with CCl4 were also treated with Rac1 inhibitor to detect the regulation of JNK activity. (5) The mice treated with CCl4 were treated with CCl4. At the same time, JNK inhibitor or Rac1 inhibitor were given to detect apoptosis-related molecules, hepatic fibrosis and inflammatory factor secretion. (6) CCl4 treated mice were given JNK inhibitor for 3 months at the same time for 16 weeks to observe liver tumor formation. Experimental results 1. Gankyrin expression in clinical samples of liver cirrhosis increased (P 0.05). 2. DEN + CC14 induced hepatocarcinogenesis. The number and size of tumors in the middle gankyrinhep group were significantly higher than those in the wild control group, and liver fibrosis was significantly aggravated in the gankyrinhep group, while there was no significant difference in tumor formation between the gankyrinhep group and the wild control group in the DEN induced cancer model. CCl4 treatment alone for 2 weeks, 4 weeks and 8 weeks, gankyrinhep group of liver fibrosis and stellate cell activity were stronger than wild group; gankyrinhep group of liver injury and inflammation aggravated, while compensatory proliferation was significantly higher than wild control. 5. short-term CCl4 treatment of gankyrinhep group of liver cell necrosis and apoptosis than wild control. CCl4-treated gankyrinhep group showed sustained enhanced activation of the JNK pathway compared with the wild control group. 7. CCl4-treated gankyrinhep group showed sustained activation of Rac1 kinase; RhoA and ROCK activity decreased; Gankyrinhep-treated RhoGDIl and RhoA binding decreased; Rac1 inhibitor treatment reversed Gankyrin's sustained activation of JNK. 8. JNK inhibitors and Rac1 inhibitors. JNK inhibitor can completely inhibit the formation of hepatocellular carcinoma in gankyrinhep group induced by long-term CCl4. Conclusion Gankyrinhep can significantly promote the hepatic fibrosis induced by CCl4 in vivo by constructing different types of hepatocellular carcinoma models. Gankyrin promotes hepatocarcinogenesis by modulating the interaction between RhoGDI and RhoA, thereby increasing the activity of Rac1 and leading to CCl4. The continuous activation of JNK pathway promotes the aggravation of liver injury, inflammation, compensatory proliferation of hepatocytes, liver fibrosis and malignant transformation, leading to the formation of liver cancer.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
本文编号：2175812
[Abstract]:Hepatocellular carcinoma (HCC) is a chronic, multifactorial, multistep, complex process. The occurrence of HCC is usually associated with a variety of factors: persistent hepatitis virus infection, long-term alcohol intake, long-term exposure to carcinogens such as aflatoxin. In addition, metabolic diseases such as diabetes, obesity is also a risk factor for liver cancer. Several clinical epidemiological studies have confirmed that Most hepatocellular carcinomas develop in the context of chronic inflammation, usually through chronic liver disease to liver fibrosis, cirrhosis, and eventually deterioration to form liver cancer. Gankyrin is the earliest oncogene to be found in hepatocellular carcinoma. Recent studies have shown that Gankyrin is up-regulated in many other malignant tumors, including cholangiocarcinoma, intestinal cancer, breast cancer and lung cancer. Gankyrin plays an important role in tumor-related signal transduction. Gankyrin has been shown to promote the phosphorylation and degradation of RB protein, lead to cell cycle disorder and promote proliferation; increase the ubiquitination and degradation of P53 by interacting with MDM2, thereby inhibiting tumor cell apoptosis. I3K/AKT signaling promotes invasion and metastasis of hepatocellular carcinoma and promotes the proliferation of precursor cells by inhibiting the degradation of OCT4. Although Gankyrin has been extensively studied in hepatocellular carcinoma, most of it focuses on the function of hepatocellular carcinoma at the stage of development, and the specific regulation of hepatocellular carcinoma or the initiation of hepatocellular carcinoma at the stage of development. It is still unclear whether Gankyrin is a concomitant phenomenon. In combination with previous data, Gankyrin is not only overexpressed in the stage of hepatocellular carcinoma, but also elevated in the stage of cirrhosis in the process of hepatocarcinogenesis induced by DEN in rats, suggesting that Gankyrin may play a regulatory role in this stage. Focusing on liver injury, liver fibrosis and cirrhosis before hepatocarcinogenesis, the animal models from chronic injury, liver fibrosis, cirrhosis to hepatocarcinogenesis were constructed on the basis of transgenic mice with liver-specific overexpression of gankyrin. Methods 1. Detection of Gankyrin protein expression in clinical samples of cirrhosis. 2. Construction of transgenic mice: construction of a full-length Gankyrin cDNA linked by an Albumin promoter to achieve Gankyrin liver-specific overexpression. 3. Establishment of animal models of DEN and DEN combined with CCl4 liver cancer: 9 mice after a single injection of DEN 15 days after birth Hepatocellular carcinoma formation was observed after 6 months; DEN treatment combined with weekly CCl4 administration was used to observe the difference of tumor formation between the two groups after different time. 4. CCl4 treatment alone induced fibrosis and malignant transformation model: CCl4 injection three times a week for 16 weeks, collected samples at intermediate time points to detect liver injury and fibrosis-related indicators. 5. Mechanism exploration (1) The changes of JNK and other related pathways were detected in the liver samples of wild control and gankyrinhep mice treated with CCl4 at different time points, and the activation of related pathways was detected in the primary hepatocytes of mice treated with CCl4. (2) Rac1 kinase activity was detected in the liver samples of mice treated with CCl4, and gankyrinhep was detected by immunoprecipitation. (3) The activity of ROCK kinase was detected in CCl4 treated mice liver tissue samples; the effect of Gankyrin on the interaction between RhoGDIa and RhoA was detected by immunoprecipitation. (4) The mice treated with CCl4 were also treated with Rac1 inhibitor to detect the regulation of JNK activity. (5) The mice treated with CCl4 were treated with CCl4. At the same time, JNK inhibitor or Rac1 inhibitor were given to detect apoptosis-related molecules, hepatic fibrosis and inflammatory factor secretion. (6) CCl4 treated mice were given JNK inhibitor for 3 months at the same time for 16 weeks to observe liver tumor formation. Experimental results 1. Gankyrin expression in clinical samples of liver cirrhosis increased (P 0.05). 2. DEN + CC14 induced hepatocarcinogenesis. The number and size of tumors in the middle gankyrinhep group were significantly higher than those in the wild control group, and liver fibrosis was significantly aggravated in the gankyrinhep group, while there was no significant difference in tumor formation between the gankyrinhep group and the wild control group in the DEN induced cancer model. CCl4 treatment alone for 2 weeks, 4 weeks and 8 weeks, gankyrinhep group of liver fibrosis and stellate cell activity were stronger than wild group; gankyrinhep group of liver injury and inflammation aggravated, while compensatory proliferation was significantly higher than wild control. 5. short-term CCl4 treatment of gankyrinhep group of liver cell necrosis and apoptosis than wild control. CCl4-treated gankyrinhep group showed sustained enhanced activation of the JNK pathway compared with the wild control group. 7. CCl4-treated gankyrinhep group showed sustained activation of Rac1 kinase; RhoA and ROCK activity decreased; Gankyrinhep-treated RhoGDIl and RhoA binding decreased; Rac1 inhibitor treatment reversed Gankyrin's sustained activation of JNK. 8. JNK inhibitors and Rac1 inhibitors. JNK inhibitor can completely inhibit the formation of hepatocellular carcinoma in gankyrinhep group induced by long-term CCl4. Conclusion Gankyrinhep can significantly promote the hepatic fibrosis induced by CCl4 in vivo by constructing different types of hepatocellular carcinoma models. Gankyrin promotes hepatocarcinogenesis by modulating the interaction between RhoGDI and RhoA, thereby increasing the activity of Rac1 and leading to CCl4. The continuous activation of JNK pathway promotes the aggravation of liver injury, inflammation, compensatory proliferation of hepatocytes, liver fibrosis and malignant transformation, leading to the formation of liver cancer.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7
【参考文献】
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1 ;Roles of Chemokine Receptor 4 (CXCR4) and Chemokine Ligand 12 (CXCL12) in Metastasis of Hepatocellular Carcinoma Cells[J];Cellular & Molecular Immunology;2008年05期
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