FZD2调控肝细胞癌拟态血管形成和肿瘤干细胞特性的初步研究

发布时间：2018-06-26 12:11

本文选题：肝细胞癌 + FZD2　；参考：《南方医科大学》2017年硕士论文

【摘要】：肝细胞癌是我国最常见的恶性肿瘤之一,新型治疗方法逐步增多,但根治性手术和肝移植仍是主要治疗手段,其高复发率和高死亡率仍然为得到明显改善。卷曲蛋白2(frizzled2,FZD2)是一种新近发现的肿瘤相关分子,它高表达于多种恶性肿瘤,参与调节肿瘤的生物学行为,影响肿瘤患者的预后,有望成为肝细胞癌的治疗靶点。本文旨在探究FZD2与上皮-间质转化(epithelial-mesenchymal transition,EMT)、拟态血管形成(vasculogenic mimicry,VM)以及肿瘤干细胞(cancer stem cells,CSCs)特性三方面的关系,将有助于阐明肝细胞癌的转移复发机制,为患者的早期诊断、靶向治疗以及预后改善研究提供理论基础。1、FZD2在肝细胞癌组织中的表达及其临床意义我们采用荧光定量PCR和免疫组化染色分别从基因转录水平和蛋白水平检测100例肝细胞癌患者癌组织和癌旁组织FZD2的表达水平,发现其在癌组织的表达量显著高于癌旁组织(P0.05);统计学分析表明,FZD2表达量与肿瘤大小、病理分化程度、微癌栓、TNM分期、BCLC分期、转移以及早期复发等多种病理参数有关(P0.05);生存分析显示,FZD2高表达的患者术后无瘤生存时间和总生存时间明显缩短(P0.05),提示FZD2可能是肝细胞癌潜在的预后指标。2、FZD2调控肝癌细胞的增殖、迁移和侵袭能力为了探究FZD2在肝细胞癌中的促癌作用,我们分别构建siRNA瞬转FZD2低表达肝癌细胞模型和慢病毒稳转FZD2过表达细胞模型,采用CCK-8法、平板克隆形成法、流式细胞仪、Transwell法以及Westernblot检测发现,敲低FZD2表达抑制肝癌细胞增殖、迁移侵袭能力以及EMT进程,诱导肝癌细胞凋亡,而过表达FZD2则促进细胞增殖、迁移侵袭以及EMT特性;采用FZD2低表达稳转细胞株构建裸鼠皮下移植瘤模型证实,FZD2也可以调控肝癌细胞体内增殖能力和EMT进程。3、FZD2调控肝细胞癌的VM形成能力VM是肝细胞癌转移复发的危险因素,其与FZD2的关系尚未明确。我们采用CD34/PAS双染法观察肝细胞癌和裸鼠移植瘤组织VM的表达情况,发现FZD2与VM表达呈正相关(P0.05);细胞三维培养条件下,敲低FZD2抑制肝癌细胞的成管能力,而过表达FZD2则增强这种能力,提示FZD2介导肝细胞癌VM形成。4、FZD2调控肝癌细胞的CSCs特性除VM外,CSCs特性也与肝细胞癌转移复发密切相关。流式细胞仪检测到,敲低FZD2表达后肝癌细胞干细胞亚群CD44(+)细胞减少(P0.05),Westernblot和免疫组化染色进一步证明,肝癌细胞和移植瘤组织中CSCs转录因子Nanog和SOX2表达下调,而过表达FZD2上调肝癌细胞上述干细胞表型。因此,FZD2参与调节肝癌细胞CSCs特性。结论:FZD2可能通过调控EMT、VM以及CSCs特性促进肝细胞癌的转移复发,有望成为肝细胞癌新的预测指标和治疗靶点。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China, but radical surgery and liver transplantation are still the main treatment methods. The high recurrence rate and high mortality rate are still significantly improved. Frizzled2FZD2 (frizzled2FZD2) is a newly discovered tumor-related molecule, which is highly expressed in many kinds of malignant tumors. It is involved in regulating the biological behavior of tumors and affecting the prognosis of tumor patients. It is expected to be a therapeutic target for hepatocellular carcinoma. The purpose of this study was to explore the relationship between FZD2 and the characteristics of epithelial-mesenchymal transition, vasculogenic mimicrysma VM and (cancer stem cells of tumor stem cells, which would be helpful to elucidate the mechanism of metastasis and recurrence of hepatocellular carcinoma. The expression and Clinical significance of FZD2 in Hepatocellular carcinoma We detected 100 cases of Hepatocellular carcinoma by fluorescence quantitative PCR and Immunohistochemical staining from Gene transcription level and protein level Expression of FZD2 in hepatocellular carcinoma tissues and adjacent tissues, It was found that the expression of FZD2 in cancer tissues was significantly higher than that in adjacent tissues (P0.05). Statistical analysis showed that FZD2 expression was correlated with tumor size, pathological differentiation, TNM stage and BCLC stage. Survival analysis showed that tumor free survival time and total survival time of patients with high expression of FZD2 were significantly shortened (P0.05), suggesting that FZD2 might be a potential prognostic index of hepatocellular carcinoma. Proliferation of hepatoma cells, In order to investigate the role of FZD2 in the promotion of hepatocellular carcinoma, we constructed a cell model of low expression of FZD2 by siRNA and a model of overexpression of lentivirus into FZD2 by using CCK-8 method and plate clone formation method. Flow cytometry and Western blot analysis showed that low expression of FZD2 inhibited proliferation, migration, invasion and EMT process of HCC cells, and induced apoptosis of HCC cells, while overexpression of FZD2 promoted cell proliferation, migration and invasion, and EMT characteristics. It was proved that FZD2 could also regulate the proliferation of hepatoma cells in vivo and the VM formation ability of hepatocellular carcinoma regulated by EMT process by using FZD2 low expression stably transformed cell line in nude mice, which was a risk factor for metastasis and recurrence of hepatocellular carcinoma (HCC). Its relationship with FZD2 is not clear. We used CD34 / pas double staining method to observe the expression of VM in hepatocellular carcinoma and xenografts in nude mice, and found that FZD2 was positively correlated with VM expression (P0.05). Overexpression of FZD2 enhances this ability, suggesting that FZD2 mediates the formation of VM in hepatocellular carcinoma. 4FZD2 regulates the characteristics of CSCs in hepatocellular carcinoma cells. Besides VM, the characteristics of CSCs are also closely related to the metastasis and recurrence of HCC. Flow cytometry showed that the expression of CSCs transcription factors Nanog and SOX2 was down-regulated in hepatoma cells and transplanted tumor tissues, and the CD44 (P05) cell subsets were decreased by Western blot and immunohistochemical staining after knockout FZD2 expression was lowered, and the expression of CSCs transcription factors Nanog and SOX2 were down-regulated in HCC cells and transplanted tumor tissues. Overexpression of FZD2 upregulated the above stem cell phenotype. Therefore, FZD2 is involved in regulating the characteristics of CSCs in hepatoma cells. Conclusion: FZD2 may promote the metastasis and recurrence of hepatocellular carcinoma by regulating the characteristics of EMTV and CSCs, and may be a new predictor and therapeutic target of HCC.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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