Gankyrin在CCl4誘導的肝纖維化—肝癌發(fā)生中的作用及機制研究
發(fā)布時間：2018-08-10 18:18
【摘要】：肝癌發(fā)生一個是慢性的多因素、多步驟的復雜過程。HCC的發(fā)生通常與多種因素相關：肝炎病毒的持續(xù)感染、長期酒精攝取、長期接觸致癌劑黃曲霉素等。此外,代謝疾病如糖尿病、肥胖也是肝癌發(fā)病的危險因素。多項臨床流行病學研究證實,大部分肝癌都是在慢性炎癥背景下發(fā)展而來,通常經(jīng)由慢性肝病發(fā)展到肝纖維化、肝硬化,最后惡化形成肝癌。肝硬化病理背景為腫瘤形成提供了合適的細胞轉(zhuǎn)化環(huán)境。目前,肝癌發(fā)生特別是從慢性肝損傷惡性轉(zhuǎn)化導致的腫瘤形成的確切分子機制尚不完全清楚。Gankyrin是最早發(fā)現(xiàn)在肝癌中特異性高表達的癌基因。近年來的研究顯示,gankyrin還在其他包括膽管癌、腸癌、乳腺癌以及肺癌等多種惡性腫瘤中表達上調(diào)。Gankyrin在腫瘤相關的信號轉(zhuǎn)導中具有非常重要的調(diào)控作用。Gankyrin被證明可促進RB蛋白的磷酸化降解,導致腫瘤細胞周期紊亂,促進增殖；通過與MDM2相互作用,增加P53的泛素化降解,從而抑制腫瘤細胞凋亡。本實驗室前期研究發(fā)現(xiàn)gankyrin直接結合RelA,促進NF-κB出核,抑制其轉(zhuǎn)錄活性；通過激活PI3K/AKT信號以促進肝癌的侵襲和轉(zhuǎn)移；通過抑制OCT4的降解進而促進腫瘤前體細胞的擴增。雖然gankyrin在肝癌中已經(jīng)被廣泛研究,但大部分集中在肝癌的發(fā)展階段的功能,至于其對于肝癌發(fā)生的具體調(diào)控或者說在肝癌發(fā)生時期是起始因素還是伴隨現(xiàn)象,這一點仍不十分清楚。結合以往的研究數(shù)據(jù),gankyrin不僅僅在肝癌階段高表達,且在DEN誘導的大鼠肝癌發(fā)生過程的肝硬化階段出現(xiàn)表達升高,提示我們gankyrin在這一階段可能發(fā)揮某種調(diào)控作用。所以本研究課題將注意力集中在肝癌發(fā)生之前的肝損傷、肝纖維化、肝硬化階段,通過在肝臟特異性過表達gankyrin的轉(zhuǎn)基因小鼠基礎上構建經(jīng)由慢性損傷、肝纖維化、肝硬化到肝癌的動物模型,嘗試從體內(nèi)水平探索gankyrin對肝癌發(fā)生有無調(diào)控功能以及其內(nèi)在機制。實驗方法1.臨床樣本檢測肝硬化臨床樣本中gankyrin的蛋白表達水平。2.轉(zhuǎn)基因小鼠構建：構建由Albumin啟動子連接gankyrin全長cDNA以實現(xiàn)gankyrin的肝臟特異性過表達。3.DEN以及DEN聯(lián)合CCl4肝癌動物模型構建：小鼠出生后15天單次注射DEN后9個月后觀察肝癌形成情況；DEN處理聯(lián)合每周一次CCl4給藥,不同時間后觀察兩組之間腫瘤形成情況差異。4.CCl4單獨處理誘導纖維化及惡性轉(zhuǎn)化模型：每周3次CCl4注射持續(xù)16周,收取中間時間點樣品檢測肝損傷及纖維化相關指標。5.機制探索：(1)不同時間點CCl4處理的野生對照和gankyrinhep小鼠肝臟樣品中檢測JNK及其他相關通路變化情況；CCl4處理后分選小鼠原代肝細胞檢測相關通路活化情況。(2)CCl4處理的小鼠肝臟組織樣品進行Rac1激酶活性檢測；免疫共沉淀檢測gankyrin對RhoGDIa與Rac1抑制性相互作用的影響。(3)CCl4處理的小鼠肝臟組織樣品進項ROCK激酶活性檢測；免疫共沉淀檢測gankyrin對RhoGDIa與RhoA相互作用的影響。(4)對CCl4處理小鼠同時給予Rac1抑制劑處理小鼠,檢測對JNK活性的調(diào)節(jié)。(5)對CCl4處理小鼠同時給予JNK抑制劑或Rac1抑制劑,檢測凋亡相關分子、肝纖維化以及炎癥因子分泌情況。(6)CCl4處理小鼠16周期間同時給予JNK抑制劑3個月,觀察肝臟腫瘤形成。實驗結果1.肝硬化臨床樣本中gankyrin表達升高(P0.05)。2. DEN+CC14誘導肝癌形成過程中gankyrinhep組的腫瘤數(shù)目和腫瘤大小均明顯高于野生對照組；同時肝纖維化在gankyrinhep組明顯加重；而DEN誘癌模型中,gankyrinhep組與野生對照組腫瘤形成情況無明顯差異。3.CCl4單獨處理16周后在gankyrinhep組有50%小鼠形成肝癌,而野生組未發(fā)生。4.CCl4單獨處理2周、4周和8周時,gankyrinhep組的肝纖維化以及星狀細胞活性化均較野生組增強；gankyrinhep組的肝臟損傷及炎癥反應加重,同時代償性增殖明顯高于野生對照。5.短時間CCl4處理的gankyrinhep組肝細胞壞死和凋亡較野生對照加重。6.CCl4處理的gankyrinhep組呈現(xiàn)JNK通路相對野生對照組持續(xù)增強活化。7.CCl4處理的gankyrinhep組的Rac1激酶持續(xù)活化；而RhoA、ROCK活性下降；gankyrinhep組的RhoGDIl與RhoA結合減弱；Rac1抑制劑處理逆轉(zhuǎn)gankyrin對JNK持續(xù)激活。8.JNK抑制劑和Rac1抑制劑均能緩解CCl4引起的gankyrinhep組的細胞凋亡和炎癥因子分泌。9.JNK抑制劑可完全抑制長期CCl4誘導的gankyrinhep組肝癌形成。結論本研究主要通過構建不同類型的肝癌模型,從動物體內(nèi)水平證明gankyrin明顯促進肝損傷劑CCl4誘導的肝纖維化以及肝癌發(fā)生；而對單獨DNA損傷劑DEN誘導的遺傳突變導致的肝癌發(fā)生沒有影響。這一點證明gankyrin促進的肝癌發(fā)生與持續(xù)的損傷和炎癥微環(huán)境相關。Gangkyrin通過調(diào)節(jié)RhoGDI與RhoA的相互作用,下調(diào)RhoA的活性進而增強Rac1活性,導致CCl4處理過程中JNK通路的持續(xù)活化,促進肝損傷加重,炎癥反應加強,肝細胞代償性增殖加強,肝纖維化加重并惡性轉(zhuǎn)化,最終導致肝癌形成。
[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.
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7
本文編號：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.
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7
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相關期刊論文 前1條
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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