GATA6上调LOXL2促进胆管癌侵袭转移和FXR上调SOCS3抑制肝细胞癌增殖的研究

发布时间：2018-08-31 12:03

【摘要】：第一章GATA6通过上调LOXL2促进胆管癌侵袭转移的研究研究背景胆管癌(Cholangiocarcinoma, CCA)是一种常见的肝胆系统恶性肿瘤,由于早期症状不明显,晚期发现时由于侵袭转移严重影响了手术治疗的效果以及患者的预后情况。侵袭转移是包括CCA在内的多种恶性肿瘤重要的生物学特征,受多层次多因素的调控,具体过程和机理至今尚不明确。上皮间质转化(Epithelial-mesenchymal transition, EMT)是来源于上皮细胞的恶性肿瘤细胞获得迁移能力和侵袭能力的重要的过程。因此,研究EMT关键分子的调控机制、探CCA侵袭转移机理,探索有效的治疗措施、研发潜在治疗靶点具有重要意义。赖氨酰氧化酶样蛋白2(Lysyl oxidase-like 2 protein, LOXL2)是一个在维持细胞外基质稳态、细胞运动等生物学方面起着重要作用的修饰酶。GATA结合蛋白6(GATA binding protein 6, GATA6)是在胚胎发育中调控细胞增殖和分化的重要转录分子。近年研究表明LOXL2和GATA6在多种肿瘤中表达水平不一致,并且发挥的作用也不尽相同,在有的组织中被认为是肿瘤抑制因子,而在另一些肿瘤中则促进肿瘤进程,是潜在的癌基因。因此,研究LOXL2和GATA6在CCA中的表达情况、探讨二者在CCA中所扮演的角色、明确它们究竟是促进还是抑制CCA进程显得十分必要。我们前期研究发现LOXL2和GATA6在CCA组织和细胞中的表达与侵袭转移相关,并且LOXL2和GATA6在表达模式和功能上存在很多一致性,提示LOXL2和GATA6可能存在某种关联性。生物信息学分析发现在LOXL2基因启动子区存在GATA6的可能结合位点,提示GATA6可能是LOXL2的上游调控分子。另外有文献提示,LOXL2在肿瘤中表现为促癌分子参与肿瘤侵袭转移进程时,可通过介导上皮标志物E-cadherin的表达下调,从而促进EMT的发生和肿瘤的侵袭转移。因此,我们推测CCA中GATA6能调控LOXL2的表达,促进EMT发生从而增强CCA细胞的侵袭迁移能力。材料方法与研究结果1、CCA肿瘤组织和细胞中LOXL2和GATA6的表达呈显著相关性收集西南医院肝胆外科2005至2012年,手术切除的90例CCA患者的组织及24例相应癌旁组织的石蜡标本。采用免疫组化方法检测发现CCA肿瘤组织中的LOXL2和GATA6的表达水平较癌旁组织中异常增高,经统计学分析显示二者在肿瘤组织中的表达具有显著相关性。同时在CCA细胞中(QBC939和RBE)通过RT-qPCR和Western blot实验检测QBC939细胞的LOXL2的表达情况,发现LOXL2在mRNA水平与蛋白水平的表达均高于RBE细胞,进一步检测发现GATA6的表达趋势与LOXL2一致。以上结果表明LOXL2和GATA6在CCA中的表达具有相关性。2、CCA肿瘤组织和细胞中LOXL2与GATA6的高表达都与侵袭转移有关收集上述90例患者的病例资料(包括随访资料),通过统计学方法分析LOXL2和GATA6在CCA肿瘤组织中的高表达与临床病例特征的相关性,发现LOXL2和GATA6的表达水平均与患者的淋巴结转移情况显著相关。进一步运用Kaplan-Meier模型分析发现LOXL2和GATA6的高表达均导致不良预后。采用Transwell侵袭实验和损伤修复实验检测发现高表达LOXL2和GATA6的QBC939细胞的侵袭能力和迁移能力均明显强于RBE细胞。以上结果表明LOXL2和GATA6在CCA中功能具有相似性。3、CCA细胞中GATA6对LOXL2存在正向调控作用生物信息学分析发现LOXL2基因启动子区域可能存在GATA6的结合位点,提示GATA6可能是LOXL2的上游调控分子。进而通过RNA干扰和过表达策略,从正反两方面检测GATA6对LOXL2表达的影响。在QBC939细胞中干扰GATA6的表达,LOXL2的mRNA水平和蛋白水平随之降低；在RBE细胞中过表达GATA6, LOXL2的mRNA水平和蛋白水平随之升高。以上结果表明GATA6对LOXL2的表达具有正向调控作用。4、GATA6通过调控LOXL2表达影响CCA细胞的侵袭迁移采用“恢复实验”,即通过干预GATA6表达后再反向干预LOXL2的表达,通过Transwell侵袭实验和损伤修复实验检测CCA细胞侵袭迁移能力的改变。干扰GATA6表达能明显减弱QBC939细胞侵袭迁移的能力,反向恢复LOXL2表达后能够在一定程度上逆转干扰GATA6表达所导致的侵袭迁移受抑制的现象。在RBE细胞中过表达GATA6能明显增强细胞侵袭迁移的能力,而这种抑制作用能被反向干扰LOXL2表达部分削弱。以上结果表明GATA6可通过调控LOXL2的表达影响CCA细胞的侵袭迁移。5、GATA6-LOXL2通路通过促进EMT增强CCA细胞的侵袭迁移采用免疫组化方法检测发现高表达GATA6和LOXL2的CCA肿瘤组织中,上皮标志物E-cadherin低表达,而间质标志物Vimentin高表达,且经统计学分析显示E-cadherin和Vimentin的表达水平分别与GATA6和LOXL2的表达水平显著相关。进一步在CCA细胞中通过“恢复实验”,即干预GATA6表达后再反向干预LOXL2的表达,检测EMT标志物E-cadherin表达水平的变化,结果显示QBC939细胞中GATA6介导的LOXL2上调抑制CCA细胞中E-cadherin的表达,而RBE细胞中GATA6介导的LOXL2下调促进CCA细胞中E-cadherin的表达。以上结果表明GATA6通过调控LOXL2表达影响EMT,进而促进EMT发生和侵袭转移。研究结论本研究明确了GATA6和LOXL2表达水平具有正向相关性。通过统计学分析发现二者在CCA中的高表达均与患者的淋巴结转移情况、不良预后密切相关。进一步在CCA细胞模型上,通过RNA干扰和过表达策略,从正反两方面证实了GATA6对LOXL2表达具有正向调控作用,并通过“恢复实验”进一步证实了GATA6通过上调LOXL2表达促进EMT,从而促进CCA细胞的侵袭迁移。以上实验结果不仅可能发现LOXL2在CCA侵袭转移中的自身表达调控机制,更重要的是可能为将来基于GATA6-LOXL2为靶标的CCA临床治疗提供理论及实验依据。第二章核受体FXR通过上调SOCS3抑制肝细胞癌增殖的研究研究背景肝细胞癌(Hepatocellular carcinoma, HCC)是一种全世界范围高发的恶性肿瘤,死亡率极高。致癌转录因子STAT3信号通路在其发生发展中发挥着重要促进作用。细胞信号转导抑制蛋白3(Suppressors of cytokine signaling 3, SOCS3),作为STAT3通路重要的内源性负反馈调节分子,在多种癌症中低表达,导致STAT3的异常活化。而越来越多的证据也显示SOCS3的恢复表达(即功能回复)在HCC、炎症性结肠癌、恶性间皮胸膜瘤等肿瘤中具有显著的抑癌活性。SOCS3目前被认为是一个重要的内源性抗肿瘤分子,上调其表达无疑将有助于HCC等多种肿瘤的防治。核受体法尼酯X受体(Farnesoid X receptor, FXR)除了密切参与物质代谢与抗炎外,在HCC等多种恶性肿瘤中能发挥显著的抑癌保护作用,但总体来说具体机制还有待深入探讨。鉴于FXR和SOCS3在HCC中的重要作用,那么FXR能否通过调控SOCS3表达而发挥抗癌作用呢?因此我们以HCC细胞为模型,通过解析FXR在SOCS3基因启动子区的具体结合位点,阐明FXR通过上调SOCS3抑制HCC的新机制,从而为探索以FXR-SOCS3为靶标的HCC治疗提供新的实验依据和理论基础。材料方法与研究结果一、FXR通过上调SOCS3抑制肝细胞癌增殖生长通过MTS细胞增殖实验、流式细胞技术、RT-qPCR和Western blot等实验证实了FXR激动剂GW4064可以抑制人HCC细胞(HepG2和Huh7)的增殖,诱导细胞周期阻滞在G1期,诱导细胞周期关键分子p21的表达、抑制STAT3的磷酸化活化。并通过RNA干扰策略证实GW4064的上述抑增殖效应是通过上调SOCS3表达实现的。进一步进行在体实验证实GW4064能抑制裸鼠HCC移植瘤的生长。二、阐明GW4064上调SOCS3表达的具体分子机制采用针对FXR的siRNA证实GW4064上调SOCS3是通过活化FXR实现的。生物信息学分析SOCS3基因启动子区可能存在FXR反应元件。进而通过报告基因检测验(结合点突变策略)、EMSA实验、ChIP实验证实FXR通过与人SOCS3基因启动子区-1878～-1858区域的FXR反应元件(IR9)结合,促进SOCS3的转录活性,从而上调其表达。三、肝细胞癌组织中FXR与SOCS3表达明显降低,而STAT3异常活化收集西南医院肝胆外科2013至2014年间手术切除的66例HCC临床标本,采用免疫组化方法检测发现,与相应癌旁组织相比,HCC肿瘤组织中的FXR和SOCS3的表达水平显著降低(统计学分析表明二者的表达水平具有显著相关性),同时STAT3的活化水平(即p-STAT3的表达水平)在HCC肿瘤组织中明显增高。临床标本上的结果与细胞水平实验的结果相符。研究结论我们以肝细胞癌(HCC)细胞为模型,详细解析了FXR在SOCS3基因启动子区的具体结合位点,阐明了FXR上调SOCS3的分子机制。通过RNA干扰策略证实了FXR通过上调SOCS3表达抑制HCC细胞的增殖、诱导细胞周期阻滞、上调细胞周期关键分子p21的表达,抑制STAT3的磷酸化活化；在裸鼠HCC移植瘤模型上验证了FXR的抑癌效应。并进一步在HCC患者的肿瘤组织中明确了FXR和SOCS3G表达水平的正向相关性。以上研究结果阐明FXR通过上调SOCS3抑制HCC的新机制,从而为探索以FXR-SOCS3为靶标的HCC治疗提供新的实验依据和理论基础。
[Abstract]:Chapter 1 GATA6 promotes the invasion and metastasis of cholangiocarcinoma (CCA) by up-regulating LOXL2. Background Cholangiocarcinoma (CCA) is a common malignant tumor of the hepatobiliary system. Epithelial-mesenchymal transition (EMT) is an important process for cancer cells derived from epithelial cells to acquire the ability of migration and invasion. Lysyl oxidase-like 2 protein (LOXL2) is a modified enzyme that plays an important role in maintaining the homeostasis of extracellular matrix and cell movement. GATA binding protein 6 (GATA6) is an important transcriptional molecule that regulates cell proliferation and differentiation during embryonic development. Recent studies have shown that LOXL2 and GATA6 are not uniformly expressed in various tumors and play different roles. They are considered as tumor suppressor in some tissues and promote in other tumors. Therefore, it is necessary to study the expression of LOXL2 and GATA6 in CCA and their roles in CCA and to determine whether they promote or inhibit CCA progression. Bioinformatics analysis revealed that there was a possible binding site of GATA6 in the promoter region of LOXL2 gene, suggesting that GATA6 may be an upstream regulator of LOXL2. In addition, literature suggests that LOXL2 may be a tumor-promoting molecule. Therefore, we speculate that GATA 6 in CCA can regulate the expression of LOXL2, promote the occurrence of EMT and enhance the invasion and migration of CCA cells by mediating the down-regulation of the expression of E-cadherin.Material methods and research results 1, CCA tumor tissue and metastasis. The expression of LOXL2 and GATA6 was significantly correlated. Paraffin specimens of 90 CCA patients and 24 corresponding adjacent tissues were collected from the Department of Hepatobiliary Surgery of Southwest Hospital from 2005 to 2012. The expression of LOXL2 and GATA6 in CCA tumor tissues was significantly higher than that in adjacent tissues by immunohistochemistry. The expression of LOXL2 was detected by RT-q PCR and Western blot in CCA cells (QBC939 and RBE). It was found that the expression of LOXL2 in mRNA and protein levels was higher than that in RBE cells. The above results showed that the expression of LOXL2 and GATA6 in CCA was correlated. 2. The high expression of LOXL2 and GATA6 in CCA tumor tissues and cells was related to invasion and metastasis. The data of 90 cases (including follow-up data) were collected. The high expression of LOXL2 and GATA6 in CCA tumor tissues and clinical cases were statistically analyzed. The expression levels of LOXL2 and GATA6 were significantly correlated with lymph node metastasis. Further analysis of Kaplan-Meier model showed that the high expression of LOXL2 and GATA6 resulted in poor prognosis. The invasion of QBC939 cells with high expression of LOXL2 and GATA6 was detected by Transwell invasion test and damage repair test. The above results showed that the functions of LOXL2 and GATA6 were similar in CCA. 3. Bioinformatics analysis showed that GATA6 might have a binding site in the promoter region of LOXL2 gene, suggesting that GATA6 might be an upstream regulator of LOXL2. By RNA interference and overexpression strategy, the effects of GATA6 on LOXL2 expression were detected in both positive and negative ways. After interfering with GATA6 expression in QBC939 cells, the mRNA and protein levels of LOXL2 decreased, and the mRNA and protein levels of LOXL2 increased when GATA6 was overexpressed in RBE cells. GATA6 can affect the invasion and migration of CCA cells by regulating the expression of LOXL2. GATA6 can interfere with the expression of LOXL2 in CCA cells by "recovery test", that is, by interfering with the expression of GATA6 and then interfering with the expression of LOXL2 in reverse direction. The ability of invasion and migration of CCA cells was detected by Transwell invasion test and damage repair test. Overexpression of GATA6 in RBE cells can significantly enhance the ability of invasion and migration, and this inhibition can be partially weakened by reverse interference with LOXL2 expression. GATA6-LOXL2 pathway enhanced the invasion and migration of CCA cells by stimulating EMT. Immunohistochemical staining showed that E-cadherin, an epithelial marker, was low in CCA tumor tissues with high expression of GATA6 and LOXL2, but Vimentin, an interstitial marker, was high in CCA cells. The expression levels of E-cadherin and Vimentin were significantly correlated with the expression levels of GATA6 and LOXL2, respectively. Further, the expression of LOXL2 in CCA cells was detected by "recovery test" after GATA6 expression was interfered, and the expression level of E-cadherin, an EMT marker, was detected. The results showed that GATA6-mediated up-regulation of LOXL2 in QBC939 cells was inhibited. In CCA cells, E-cadherin expression was increased by GATA6-mediated LOXL2 down-regulation, while in RBE cells, LOXL2 down-regulation promoted E-cadherin expression. These results suggest that GATA6 affects EMT by regulating LOXL2 expression, thereby promoting EMT occurrence and invasion and metastasis. It was found that the high expression of GATA6 in CCA was closely related to lymph node metastasis and poor prognosis. Furthermore, in CCA cell model, the positive and negative effects of GATA6 on LOXL2 expression were confirmed by RNA interference and overexpression strategy, and GATA6 up-regulated LOX by "recovery test". L2 expression promotes EMT and thus promotes invasion and migration of CCA cells. These results may not only reveal the mechanism of LOXL2 expression in CCA invasion and metastasis, but also provide theoretical and experimental evidence for future clinical treatment of CCA based on GATA6-LOXL2. Chapter 2 Nuclear receptor FXR inhibits liver fineness by up-regulating SOCS3. Background Hepatocellular carcinoma (HCC) is a worldwide malignant tumor with high mortality. The STAT3 signaling pathway plays an important role in the development of HCC. As an important endogenous negative feedback regulator of STAT3 pathway, low expression in many cancers leads to abnormal activation of STAT3. More and more evidences also show that the restored expression of SOCS3 (i.e. functional recovery) has significant antitumor activity in HCC, inflammatory colon cancer, malignant mesothelioma and other tumors. Farnesoid X receptor (FXR) plays an important role in the prevention and treatment of HCC and other malignant tumors. In addition to its close involvement in substance metabolism and anti-inflammation, FXR plays a significant inhibitory and protective role in HCC and other malignant tumors, but the specific mechanism remains to be further explored. In view of the important role of FXR and SOCS3 in HCC, can FXR play an anticancer role by regulating the expression of SOCS3? Therefore, by analyzing the specific binding sites of FXR in the promoter region of SOCS3 gene in HCC cells, we clarify the new mechanism of FXR inhibiting HCC by up-regulating SOCS3, so as to explore the HCC targeting FXR-SOCS3. Material methods and research results 1. FXR can inhibit the proliferation of hepatocellular carcinoma by up-regulating SOCS-3. FXR agonist GW4064 can inhibit the proliferation of human HCC cells (HepG2 and Huh7) and induce cell cycle by MTS cell proliferation, flow cytometry, RT-qPCR and Western blot. Blocking the expression of p21, the key molecule of cell cycle, and inhibiting the phosphorylation and activation of STAT3 in G1 phase, was demonstrated by RNA interference strategy. Further in vivo experiments showed that GW4064 could inhibit the growth of HCC xenograft tumor in nude mice. 2. To clarify the role of GW4064 in up-regulating the expression of SOCS3. Bioinformatics analysis showed that FXR reaction elements may exist in the promoter region of SOCS3 gene. Further, through reporter gene test (binding point mutation strategy), EMSA test and ChIP test, FXR was confirmed to be up-regulated by activating FXR in the promoter region of human SOCS3 gene. Three, the expression of FXR and SOCS3 in hepatocellular carcinoma tissues was significantly decreased, while STAT3 abnormal activation was detected in 66 HCC clinical specimens from the Department of Hepatobiliary Surgery, Southwest Hospital from 2013 to 2014. Immunohistochemical method was used to detect the expression of FXR and SOCS3 in the corresponding adjacent tissues. The expression levels of FXR and SOCS3 in HCC tumors were significantly lower than those in HCC tumors (statistical analysis showed that there was a significant correlation between the expression levels of FXR and SOCS3), and the activation level of STAT3 (i.e. the expression level of p-STAT3) was significantly higher in HCC tumors. Using hepatocellular carcinoma (HCC) cells as a model, the specific binding sites of FXR in the promoter region of SOCS 3 gene were analyzed in detail, and the molecular mechanism of up-regulation of SOCS 3 by FXR was elucidated. Phosphorylation activation of T3, inhibition of FXR on HCC xenograft in nude mice, and positive correlation between FXR and SOCS3G expression levels were further clarified in HCC patients. The above results clarify the new mechanism of FXR inhibiting HCC by up-regulating SOCS3, thus providing new insights into HCC therapy targeting FXR-SOCS3. The experimental basis and theoretical basis.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735

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