Ras-GTP酶蛋白RASAL2对卵巢癌上皮间质转化与转移的调控作用以及相关机制的研究

发布时间：2018-04-09 12:04

本文选题：卵巢癌　切入点：RASAL2　出处：《天津医科大学》2015年博士论文

【摘要】：上皮性卵巢癌(EOC)是最常见的妇科恶性肿瘤之一,也是女性第五大癌症死亡原因。2015年,在全美预计的新发病人数与死亡人数分别为21,290与14,180。其中50%以上的卵巢癌患者年龄超过65周岁。因此卵巢癌是目前威胁女性健康的首要疾病之一。上皮间质转化(Epithelial-mesenchymal transition,EMT)是上皮细胞逐渐失去其代表性的细胞极性与细胞间连接,而获得间质细胞所具有的侵袭与迁移能力的过程。目前普遍认为上皮间质转化参与癌症转移的起始过程,促进癌症细胞离开原始位置向远端散播。卵巢癌是转移高发癌症,EMT对卵巢癌转移的调控作用已有较多报道,然而卵巢癌EMT的调控机制尚不清晰。细胞外信号利用多种跨膜信号传递通路调控细胞内的生物过程。Ras-GTP酶正是介导这些信号传递通路的关键分子。Ras蛋白(H-Ras,N-Ras,and K-Ras),是Ras-GTP酶家族最重要的蛋白。其生物学功能被两种相反功能的蛋白调控,鸟嘌呤核酸交换因子(guanine nucleotide exchange factors,GEFs)和GTP酶活化蛋白(GTPase-activating proteins,GAPs)。RASAL2是一种近期新发现RasGAP。有报道称其在乳腺癌中具有抑癌作用。但其具体生物学功能尚不明确。本研究卵巢癌细胞系为模型,并辅以卵巢癌患者样本,旨在探索RASAL2在卵巢癌的发生与转移中的作用与机制,并期望以此提出新的卵巢癌治疗靶点。通过对57例卵巢癌患者肿瘤样本cDNA以及8例正常卵巢组织样本cDNA分析发现,虽然RASAL2的表达与卵巢癌患者的年龄和组织学分型无关,但其与卵巢癌的病理分级以及临床分期显著相关。提示RASAL2在卵巢癌中可能作为一种抑癌因子发挥作用。我们在三种卵巢癌细胞系SKOV3,OVCAR3和A2780中分别敲低了RASAL2的表达。利用软琼脂集落形成实验,侵袭与迁移实验,裸鼠体内成瘤实验等对RASAL2在卵巢癌生物学中作用进行了初步探索。实验结果显示,RASAL2敲低促进卵巢癌细胞软琼脂集落形成以及体内成瘤能力。而且细胞体外侵袭与迁移能力都有显著提升。这说明RASAL2在卵巢癌中具有抑癌作用,特别是具有抑制侵袭迁移能力。EMT常被癌症利用从而促进其自身转移。我们利用qPCR,免疫印记与免疫荧光实验检测了RASAL2敲低对卵巢癌细胞EMT标志分子的分布与表达的影响。并且对卵巢癌细胞裸鼠成瘤模型样本进行免疫组织化学染色或免疫印记实验检测其EMT标志分子表达变化。实验结果表明,RASAL2敲低可以显著降低上皮表型蛋白的表达,与之相反的,间质表型标志蛋白却发生显著上调。免疫荧光实验显示E-cadherin在细胞表面聚集减少,而vimentin却发生上调。体内EMT标志蛋白检测实验结果与体外一致。上皮表型蛋白的表达在RASAL2敲低后下调,而间质表型标志蛋白发生上调。此外,我们检测了卵巢癌患者cDNA中RASAL2与EMT的表达,并进行了相关性分析。结果显示,RASAL2的表达与E-cadherin呈现正相关。这些结果说明在卵巢癌中RASAL2是EMT的负向调控因子。由于RASAL2属于Ras-GAP蛋白家族。其发挥调节抑癌与抑制EMT作用很可能是通过抑制Ras-ERK信号通路完成的。因此我们检测了RASAL2敲低对Ras-ERK通路活化状态的影响。结果显示在体外RASAL2下调后,Ras-GTP,pRaf-1,p-MEK1/2和p-ERK1/2等Ras-ERK核心调控蛋白都发生上调。在体内,免疫组化以及免疫印记实验显示,RASAL2敲低卵巢癌组织表现出与体外实验一致的结果。这提示RASAL2可能具有抑制Ras-ERK通路活性的功能。另外,利用MEK抑制剂PD98059体外抑制ERK活化后,RASAL2敲低所介导的软琼脂集落形成,侵袭迁移能力增强以及EMT都发生了逆转。这些是实验结果共同表明RASAL2对卵巢癌的抑癌作用以及EMT的抑制作用可能是依赖于其RasERK活化作用。综上,我们证实RASAL2是卵巢癌的抑癌因子,特别是抑制卵巢癌侵袭转移能力。其抑癌功能可能主要是通过抑制Ras-ERK通路活化,进而抑制卵巢癌细胞EMT来达到的。以上研究增进了我们对卵巢癌转移调控过程的了解。通过对RASAL2的深入研究我们第一次证明了其在卵巢癌中的作用,并发现了其调控肿瘤的具体分子机制。增强了我们对Ras-ERK通路的理解,为以后Ras通路的癌症治疗提供了新的靶点与新的思路。
[Abstract]:Epithelial ovarian cancer (EOC) is one of the most common gynecologic malignant tumor, but also women's fifth major causes of cancer death in.2015 years, the incidence of the new expected and the death toll was 21290 and 14180. in ovarian cancer patients aged 50% or more than 65 years of age. So ovarian cancer is one of the most important disease threatening women's health at present. Epithelial mesenchymal transition (Epithelial-mesenchymal transition EMT) is a cell polarity and epithelial cells gradually lose their representative cell junction, and has obtained between Leydig cell invasion and migration ability in the process. It is generally believed that the epithelial mesenchymal transition in the initiation of cancer metastasis, promote cancer cells left the original location to the distal spread. Ovarian cancer is the high incidence of cancer metastasis, EMT on ovarian cancer metastasis regulation has been widely reported, but the regulation mechanism of EMT in ovarian cancer is still not clear The use of a variety of extracellular signals. Transmembrane signaling pathway regulates the biological processes of.Ras-GTP enzyme in cells is mediated by these signaling pathway molecule.Ras protein (H-Ras, N-Ras, and, K-Ras, Ras-GTP) is the most important enzyme family protein. Its biological function is regulated to two opposite functions, guanine nucleic acid exchange factor (guanine nucleotide exchange factors, GEFs) and GTP (GTPase-activating proteins, enzyme activated protein GAPs.RASAL2) is a recent discovery of RasGAP. said it has tumor suppressor role in breast cancer. But its biological function is not clear. The study of ovarian cancer cell line as a model, and samples of ovarian cancer with the patients, and to explore the mechanism of RASAL2 in the occurrence and metastasis of ovarian cancer in the role, and expect to put forward a new target in the treatment of ovarian cancer. 57 cases of tumor samples of patients with ovarian cancer CDNA and 8 cases of normal ovarian tissue samples cDNA analysis found that, although patients with RASAL2 expression of ovarian cancer and age and histological type, but the ovarian cancer and pathological grading and clinical stage were significantly correlated. Suggesting that RASAL2 may act as a tumor suppressor factor play a role in ovarian cancer. In three ovarian carcinoma SKOV3 cells express low RASAL2 and A2780 respectively on OVCAR3. Using the soft agar colony formation assay, invasion and migration experiments in nude mice of RASAL2 in ovarian cancer biology effect were studied. The experimental results showed that knockdown of RASAL2 promotes ovarian cancer cells in soft agar colony the formation and tumorigenicity in vivo and in vitro. Cell invasion and migration ability are significantly improved. It indicated that RASAL2 has a tumor suppressor role in ovarian cancer, especially inhibit the invasion and migration of.EMT is often In order to promote their own cancer metastasis. We use qPCR, Western blot and immunofluorescence assay of RASAL2 knockdown on the expression and distribution of molecular markers of ovarian cancer cell EMT and on ovarian cancer cells in nude mice model samples by immunohistochemistry or Western blotting experiments to detect the EMT expression of molecular markers of change. The experimental results show that RASAL2 knockdown can significantly reduce the expression of epithelial phenotype protein, on the contrary, mesenchymal marker protein was significantly up-regulated. Immunofluorescence assay showed that E-cadherin reduced aggregation on the cell surface, while vimentin was up-regulated. EMT in vivo experimental results consistent with the detection of protein markers in vitro. The expression of epithelial phenotype protein in the RASAL2 knockdown down, while mesenchymal marker protein increased. In addition, we examined the expression of RASAL2 and EMT cDNA in patients with ovarian cancer, And correlation analysis. The results showed that the positive correlation with the expression of E-cadherin and RASAL2. These results indicate that RASAL2 EMT is a negative regulatory factor in ovarian cancer. The RASAL2 belongs to the Ras-GAP protein family. Its function of regulating tumor suppressor and suppression of EMT function may be through inhibition of Ras-ERK signal pathway. So we detected the effect of RASAL2 knockdown on Ras-ERK pathway activation in vitro. The results showed that the down-regulation of RASAL2, Ras-GTP, pRaf-1, p-MEK1/2 and p-ERK1/2 Ras-ERK were up-regulated. Core regulatory proteins in vivo, display immunohistochemistry and immune imprinting experiments, RASAL2 knockdown of ovarian cancer showed consistent results and in vitro experiment. The results suggest that RASAL2 may inhibit Ras-ERK pathway activity. In addition, the inhibition of ERK activation by MEK inhibitor PD98059 in vitro after knockdown of RASAL2 mediated by low soft agar colony shape As the invasion and migration enhanced and EMT were reversed. The experimental results show that RASAL2 is common for ovarian cancer tumor suppressor function and EMT inhibition may be dependent on the activation of RasERK. In conclusion, we demonstrated that RASAL2 is a cancer factor inhibiting ovarian cancer, especially the inhibition of ovarian cancer invasion and metastasis. The tumor suppressor function may be mainly through inhibiting the activation of Ras-ERK pathway, and then inhibit ovarian cancer cell EMT to achieve. The above research improves our understanding of the regulation of metastasis of ovarian cancer. Further studies of RASAL2 we first prove its roles in ovarian cancer, and found that the molecular mechanism of the regulation of tumor. Enhance our understanding of the Ras-ERK pathway, provide new targets and new ideas for the treatment of cancer of the Ras pathway.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.31

【共引文献】