本文選題：卵巢癌　切入點(diǎn)：RASAL2　出處：《天津醫(yī)科大學(xué)》2015年博士論文

【摘要】：上皮性卵巢癌(EOC)是最常見的婦科惡性腫瘤之一,也是女性第五大癌癥死亡原因。2015年,在全美預(yù)計(jì)的新發(fā)病人數(shù)與死亡人數(shù)分別為21,290與14,180。其中50%以上的卵巢癌患者年齡超過65周歲。因此卵巢癌是目前威脅女性健康的首要疾病之一。上皮間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)是上皮細(xì)胞逐漸失去其代表性的細(xì)胞極性與細(xì)胞間連接,而獲得間質(zhì)細(xì)胞所具有的侵襲與遷移能力的過程。目前普遍認(rèn)為上皮間質(zhì)轉(zhuǎn)化參與癌癥轉(zhuǎn)移的起始過程,促進(jìn)癌癥細(xì)胞離開原始位置向遠(yuǎn)端散播。卵巢癌是轉(zhuǎn)移高發(fā)癌癥,EMT對(duì)卵巢癌轉(zhuǎn)移的調(diào)控作用已有較多報(bào)道,然而卵巢癌EMT的調(diào)控機(jī)制尚不清晰。細(xì)胞外信號(hào)利用多種跨膜信號(hào)傳遞通路調(diào)控細(xì)胞內(nèi)的生物過程。Ras-GTP酶正是介導(dǎo)這些信號(hào)傳遞通路的關(guān)鍵分子。Ras蛋白(H-Ras,N-Ras,and K-Ras),是Ras-GTP酶家族最重要的蛋白。其生物學(xué)功能被兩種相反功能的蛋白調(diào)控,鳥嘌呤核酸交換因子(guanine nucleotide exchange factors,GEFs)和GTP酶活化蛋白(GTPase-activating proteins,GAPs)。RASAL2是一種近期新發(fā)現(xiàn)RasGAP。有報(bào)道稱其在乳腺癌中具有抑癌作用。但其具體生物學(xué)功能尚不明確。本研究卵巢癌細(xì)胞系為模型,并輔以卵巢癌患者樣本,旨在探索RASAL2在卵巢癌的發(fā)生與轉(zhuǎn)移中的作用與機(jī)制,并期望以此提出新的卵巢癌治療靶點(diǎn)。通過對(duì)57例卵巢癌患者腫瘤樣本cDNA以及8例正常卵巢組織樣本cDNA分析發(fā)現(xiàn),雖然RASAL2的表達(dá)與卵巢癌患者的年齡和組織學(xué)分型無關(guān),但其與卵巢癌的病理分級(jí)以及臨床分期顯著相關(guān)。提示RASAL2在卵巢癌中可能作為一種抑癌因子發(fā)揮作用。我們?cè)谌N卵巢癌細(xì)胞系SKOV3,OVCAR3和A2780中分別敲低了RASAL2的表達(dá)。利用軟瓊脂集落形成實(shí)驗(yàn),侵襲與遷移實(shí)驗(yàn),裸鼠體內(nèi)成瘤實(shí)驗(yàn)等對(duì)RASAL2在卵巢癌生物學(xué)中作用進(jìn)行了初步探索。實(shí)驗(yàn)結(jié)果顯示,RASAL2敲低促進(jìn)卵巢癌細(xì)胞軟瓊脂集落形成以及體內(nèi)成瘤能力。而且細(xì)胞體外侵襲與遷移能力都有顯著提升。這說明RASAL2在卵巢癌中具有抑癌作用,特別是具有抑制侵襲遷移能力。EMT常被癌癥利用從而促進(jìn)其自身轉(zhuǎn)移。我們利用qPCR,免疫印記與免疫熒光實(shí)驗(yàn)檢測(cè)了RASAL2敲低對(duì)卵巢癌細(xì)胞EMT標(biāo)志分子的分布與表達(dá)的影響。并且對(duì)卵巢癌細(xì)胞裸鼠成瘤模型樣本進(jìn)行免疫組織化學(xué)染色或免疫印記實(shí)驗(yàn)檢測(cè)其EMT標(biāo)志分子表達(dá)變化。實(shí)驗(yàn)結(jié)果表明,RASAL2敲低可以顯著降低上皮表型蛋白的表達(dá),與之相反的,間質(zhì)表型標(biāo)志蛋白卻發(fā)生顯著上調(diào)。免疫熒光實(shí)驗(yàn)顯示E-cadherin在細(xì)胞表面聚集減少,而vimentin卻發(fā)生上調(diào)。體內(nèi)EMT標(biāo)志蛋白檢測(cè)實(shí)驗(yàn)結(jié)果與體外一致。上皮表型蛋白的表達(dá)在RASAL2敲低后下調(diào),而間質(zhì)表型標(biāo)志蛋白發(fā)生上調(diào)。此外,我們檢測(cè)了卵巢癌患者cDNA中RASAL2與EMT的表達(dá),并進(jìn)行了相關(guān)性分析。結(jié)果顯示,RASAL2的表達(dá)與E-cadherin呈現(xiàn)正相關(guān)。這些結(jié)果說明在卵巢癌中RASAL2是EMT的負(fù)向調(diào)控因子。由于RASAL2屬于Ras-GAP蛋白家族。其發(fā)揮調(diào)節(jié)抑癌與抑制EMT作用很可能是通過抑制Ras-ERK信號(hào)通路完成的。因此我們檢測(cè)了RASAL2敲低對(duì)Ras-ERK通路活化狀態(tài)的影響。結(jié)果顯示在體外RASAL2下調(diào)后,Ras-GTP,pRaf-1,p-MEK1/2和p-ERK1/2等Ras-ERK核心調(diào)控蛋白都發(fā)生上調(diào)。在體內(nèi),免疫組化以及免疫印記實(shí)驗(yàn)顯示,RASAL2敲低卵巢癌組織表現(xiàn)出與體外實(shí)驗(yàn)一致的結(jié)果。這提示RASAL2可能具有抑制Ras-ERK通路活性的功能。另外,利用MEK抑制劑PD98059體外抑制ERK活化后,RASAL2敲低所介導(dǎo)的軟瓊脂集落形成,侵襲遷移能力增強(qiáng)以及EMT都發(fā)生了逆轉(zhuǎn)。這些是實(shí)驗(yàn)結(jié)果共同表明RASAL2對(duì)卵巢癌的抑癌作用以及EMT的抑制作用可能是依賴于其RasERK活化作用。綜上,我們證實(shí)RASAL2是卵巢癌的抑癌因子,特別是抑制卵巢癌侵襲轉(zhuǎn)移能力。其抑癌功能可能主要是通過抑制Ras-ERK通路活化,進(jìn)而抑制卵巢癌細(xì)胞EMT來達(dá)到的。以上研究增進(jìn)了我們對(duì)卵巢癌轉(zhuǎn)移調(diào)控過程的了解。通過對(duì)RASAL2的深入研究我們第一次證明了其在卵巢癌中的作用,并發(fā)現(xiàn)了其調(diào)控腫瘤的具體分子機(jī)制。增強(qiáng)了我們對(duì)Ras-ERK通路的理解,為以后Ras通路的癌癥治療提供了新的靶點(diǎn)與新的思路。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.31

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