發(fā)布時間：2018-05-05 01:07

  本文選題：結直腸癌 + YAP　； 參考：《安徽醫(yī)科大學》2016年博士論文

【摘要】：大腸癌是最常見的惡性腫瘤之一,每年以2%的速度遞增。2012年統(tǒng)計全球大腸癌患者約140萬人,其中6.939萬人死亡該疾病,5年存活率從臨床Ⅰ期患者的90%至臨床Ⅳ期患者的10%不等。大腸癌早期癥狀不明顯,多數(shù)大腸癌診斷時已屬于晚期,甚至出現(xiàn)肝臟或其它器官的轉移。大腸癌最基本的治療方法是手術治療,術后輔助化療及放療。隨著診斷技術的發(fā)展和治療方法的改進,大腸癌的預后得到逐步改善,但死亡率依然較高,主要原因是腫瘤的復發(fā)、轉移以及耐藥的產(chǎn)生。此外,化療和放療缺乏特異性和敏感性,副作用較大。因此迫切需要研究闡明大腸癌發(fā)生的分子機制,尋找更好的診斷及預后指標和治療策略。Hippo信號通路與細胞增殖、分化、凋亡及腫瘤發(fā)生有關,是近來的研究熱點。在多種腫瘤中Hippo信號通路成分下調(diào),其轉錄因子YAP1表達水平是大腸癌重要的預后因素,Hippo信號通路負調(diào)節(jié)YAP1,YAP1作為轉錄共激活因子可誘導與細胞增殖及凋亡有關的靶基因的轉錄和表達,如BIRC5,即survivin基因,它屬于凋亡蛋白抑制家族成員(IAP)。Hippo/YAP1信號通路與Wnt/β-catenin信號通路在維持細胞穩(wěn)定方面�？上嗷ソ徊孀饔�,并與大腸癌細胞凋亡及增殖有關。β-catenin是Wnt/β-catenin信號通路中最重要的轉錄共激活因子,可調(diào)節(jié)下游與細胞增殖、凋亡及分化有關的靶基因,survivin是Wnt/β-catenin信號通路的下游靶基因。因此。survivin是Hippo/YAP1及Wnt/β-catenin信號通路的共同靶基因。本研究的目的是觀察Hippo/YAP1及Wnt/β-catenin信號通路在大腸癌發(fā)生中的作用及臨床意義以及si RNA沉默YAP1和survivin基因對大腸癌細胞生物學行為的影響。為此,本研究分為三個部分。第一部分目的:探討大腸癌組織中YAP1,β-catenin及survivin表達、相互關系及臨床意義。方法:采用免疫組織化學S-P法檢測181例大腸癌組織和30例正常粘膜中YAP1,β-catenin和Survivin的表達狀況。結果:1.在181大腸癌組織中,YAP1,β-catenin(細胞核)及survivin陽性率分別為73.5%(133/181),56.9%(103/181)及65.2%(118/181),均明顯高于正常粘膜組(P0.05)。2.大腸癌組織中YAP1表達與分化程度、Duke’s分期有關(P0.05);YAP1細胞核及核/漿表達與患者的臨床Duke’s分期有關(P0.05)。YAP1高表達與分化程度、Duke’s分期及淋巴結轉移有關(P0.05)。β-catenin細胞核表達與分化程度、淋巴結轉移及臨床Duke’s分期有關(P0.05),β-catenin細胞膜不完整表達與組織分化程度、浸潤深度、淋巴結轉移及臨床Duke’s分期等均有關(P0.05)。大腸癌組織中Survivin表達及不同染色定位與患者性別、年齡、分化程度、浸潤深度、淋巴結轉移及臨床Duke’s分期等臨床病理因素均無關(P0.05)。3.大腸癌組織中YAP1高表達與β-catenin細胞核/漿表達及survvin表達有關(P0.05)。β-catenin細胞核表達與survivin表達相關(P0.05)。4.在82例隨訪病例中,YAP1高表達、β-catenin細胞核陽性及survivin陽性表達患者5年生存率均明顯低于對照組(P0.05)。YAP1,β-catenin(細胞核)及survivin任何兩者或三者同時陽性者,其5年生存率均明顯低于相對應的對照組(P0.05)。結論:1.Hippo/YAP及Wnt/β-catenin信號通路在大腸癌發(fā)生、發(fā)展中起重要作用。2.檢測YAP1,β-catenin及survivin表達狀況有助于反映大腸癌的進展和預后。3.Survivin是Hippo/YAP及Wnt/β-catenin信號通路下游的共同靶基因;YAP,β-catenin及survivin均有望成為大腸癌基因診斷及基因治療的新靶點。第二部分目的:探討si RNA沉默YAP及Survivin基因后對人大腸癌細胞YAP,β-catenin及Survivin表達、細胞增殖及凋亡的影響。方法:以脂質(zhì)體Lip-2000為載體,用針對YAP及Survivin特異靶點的si RNA分別轉染入人大腸癌細胞系RKO、HCT116,應用免疫細胞化學S-P法、Western Blot檢測YAP、β-catenin及Survivin蛋白表達變化;RT-PCR檢測m RNA表達;CCK8法檢測細胞增殖;流式細胞術檢測細胞凋亡。結果:免疫細胞化學結果顯示,與對照組相比,YAP在轉染組陰性或僅見可別細胞弱陽性表達。si RNA-YAP轉染RKO細胞后β-catenin及Survivin蛋白表達明顯降低(P0.05)。si RNA-survivin轉染人大腸HCT116細胞后,Survivin蛋白表達明顯降低。Western blot結果顯示si RNA-YAP轉染后,RKO細胞YAP,β-catenin及Survivin蛋白條帶灰度值明顯低于對照組(P0.05)。si RNA-survivin轉染HCT116細胞后,Survivin蛋白條帶灰度值明顯低于對照組(P0.05)。RT-PCR結果顯示,si RNA-YAP轉染后,YAP m RNA表達水平降低約(76.423±9.074%),si RNA-survivin轉染HCT116細胞后,survivin m RNA表達水降低約(69.705±6.151%)(P0.05)。CCK8檢測結果顯示si RNA-YAP及si RNA-survivin分別轉染RKO及HCT116細胞后,細胞生長出現(xiàn)明顯抑制(P0.05)。流式細胞術檢測結果顯示,si RNA-YAP轉染組細胞凋亡比例為23.90±2.07%,si RNA-survivin轉染組細胞凋亡比例為25.73±2.02%,明顯高于對照組(P0.01)。結論:在大腸癌細胞系中,si RNA-YAP可抑制YAP,β-catenin及Survivin表達,si RNA-survivin可抑制Survivin表達,兩者均可抑制大腸癌細胞增殖,促進細胞凋亡;YAP和Survivin有望成為大腸癌基因治療的新靶點。第三部分目的:觀察sh RNA-survivin質(zhì)粒對大腸癌裸鼠移植瘤生長的影響及其分子機制。方法:15只3-5周齡雌性裸鼠隨機分為3組,分別將SW1116細胞、sh RNA對照組細胞及sh RNA-survivin轉染組細胞注入裸鼠右側腋窩皮下組織,建立大腸癌裸鼠移植瘤模型。每兩天測量一次腫瘤大小,計算腫瘤體積,28天后處死裸鼠并稱腫瘤重量,繪制腫瘤生長曲線。Western blot及RT-PCR方法檢測survivin及其下游Ki-67和BCL-2基因的蛋白及m RNA表達。結果:成功建立人大腸癌裸鼠皮下移植瘤模型。sh RNA-survivin組移植瘤的大小、重量及體積明顯減小(P0.05)。與SW1116及sh RNA對照組相比,平均抑留率分別為76.1%及76.3%(P0.05)。Western blot及RT-PCR結果顯示sh RNA-survivin轉染組移植瘤組織中survivin及其下游Ki-67,BCL-2基因蛋白及m RNA表達水平均明顯降低。結論:sh RNA-survivin質(zhì)�？擅黠@抑制survivin及其下游基因Ki-67和BCL-2的表達,抑制大腸癌裸鼠移植瘤生長、促進細胞凋亡,Survivin可作為大腸癌治療的靶基因。總之,本研究應用多種方法對大腸癌組織標本、細胞系體內(nèi)及體外進行研究,所得結論概括如下:1.Hippo/YAP及Wnt/β-catenin信號通路在在大腸癌發(fā)生、發(fā)展中起重要作用,檢測YAP1,β-catenin及survivin表達有助于反映大腸癌的進展和預后。2.Survivin是Hippo/YAP及Wnt/β-catenin信號通路下游的共同靶基因。3.在體外細胞實驗中,si RNA-YAP抑制YAP,β-catenin及Survivin表達,si RNA-survivin抑制Survivin表達,兩者均可抑制大腸癌細胞增殖,促進細胞凋亡;YAP和Survivin有望成為大腸癌基因治療的新靶點。4.在體內(nèi)裸鼠瘤實驗中,sh RNA-survivin質(zhì)�？擅黠@抑制survivin及其下游基因Ki-67和BCL-2的表達,抑制大腸癌裸鼠移植瘤生長、促進細胞凋亡,Survivin可作為是大腸癌分子治療的靶基因。
[Abstract]:Colorectal cancer is one of the most common malignant tumors. About 1 million 400 thousand people in the global colorectal cancer are estimated at a rate of 2% per year at a rate of 2%, of which 69 thousand and 390 people die of the disease. The 5 year survival rate varies from 90% to 10% in clinical stage IV patients. The early symptoms of colorectal cancer are not obvious, and most of the colorectal cancer are diagnosed at the advanced stage. The most basic treatment for colorectal cancer is surgical treatment, adjuvant chemotherapy and radiotherapy. With the development of diagnostic techniques and the improvement of treatment methods, the prognosis of colorectal cancer is gradually improved, but the mortality is still high, mainly due to the recurrence, metastasis and drug resistance. Chemotherapy and radiotherapy are lack of specificity and sensitivity and side effects are large. Therefore, it is urgent to study the molecular mechanism of the occurrence of colorectal cancer and to find better diagnostic and prognostic indicators and therapeutic strategies for.Hippo signaling pathway, which are related to cell proliferation, differentiation, apoptosis and tumor occurrence. The Hippo signal in a variety of tumors The expression level of the transcription factor YAP1 is an important prognostic factor for colorectal cancer, and the Hippo signaling pathway negatively regulates the YAP1. As a transcriptional co activating factor, YAP1 can induce the transcription and expression of target genes related to cell proliferation and apoptosis, such as BIRC5, or survivin gene, which belongs to the.Hippo/YAP1 letter of the apoptotic protein inhibition family (IAP). The signal pathway and Wnt/ beta -catenin signaling pathway are often intersecting in maintaining cell stability and are related to apoptosis and proliferation of colorectal cancer cells. Beta -catenin is the most important transcription co activating factor in Wnt/ beta -catenin signaling pathway, which can regulate the target genes associated with cell proliferation, apoptosis and differentiation, and survivin is Wnt/ beta -catenin. The target genes downstream of the signal pathway. Therefore,.Survivin is a common target gene for Hippo/YAP1 and Wnt/ beta -catenin signaling pathways. The purpose of this study is to observe the role and clinical significance of Hippo/YAP1 and Wnt/ beta -catenin signaling pathway in the development of colorectal cancer and the effect of Si RNA silencing YAP1 and survivin on the biological behavior of colorectal cancer cells To this end, this study is divided into three parts. The first part is to explore the expression of YAP1, beta -catenin and Survivin in colorectal carcinoma tissue, their relationship and clinical significance. Methods: the expression of YAP1, beta -catenin and Survivin in 181 cases of colorectal carcinoma and 30 cases of normal mucosa were detected by immunohistochemistry. Results: 1. in 181 large intestine cancer tissues. The positive rates of YAP1, beta -catenin (nucleus) and Survivin were 73.5% (133/181), 56.9% (103/181) and 65.2% (118/181), which were significantly higher than that in the normal mucosa (P0.05).2. large intestine cancer tissues. The expression of Duke 's stage was related to the Duke' s stage (P0.05). The degree of high expression and differentiation, Duke 's staging and lymph node metastasis (P0.05). The expression of beta -catenin is related to the degree of differentiation, lymph node metastasis and clinical Duke' s staging (P0.05). The incomplete expression of the cell membrane of beta -catenin is related to the degree of tissue differentiation, infiltration depth, lymph node metastasis and clinical Duke 's staging. The expression of Survivin in cancer tissues and different staining localization and the sex, age, differentiation, depth of invasion, lymph node metastasis and clinical Duke 's staging are all independent of the clinicopathological factors (P0.05) the high expression of YAP1 in.3. colorectal carcinoma is related to the expression of beta -catenin nucleus / pulp and survvin expression (P0.05). The expression and surv of the cell nucleus of beta -catenin. Ivin expression related (P0.05).4. in 82 follow-up cases, YAP1 high expression, beta -catenin nuclear positive and Survivin positive expression of 5 year survival rates were significantly lower than the control group (P0.05).YAP1, beta -catenin (nucleus) and Survivin any or three both positive, the 5 year survival rate was significantly lower than the corresponding control group (P0.05). Conclusion: 1.Hippo/YAP and Wnt/ beta -catenin signaling pathways occur in colorectal cancer..2. plays an important role in the detection of YAP1. The expression of beta -catenin and survivin is helpful to reflect the progression and prognosis of colorectal cancer, which is the common target gene in the downstream of Hippo/YAP and Wnt/ beta -catenin signaling pathway. New target for gene diagnosis and gene therapy of colorectal cancer. Second objective: To explore the effect of Si RNA silencing YAP and Survivin gene on YAP, beta -catenin and Survivin expression, cell proliferation and apoptosis in human colorectal cancer cells. Methods: liposome Lip-2000 as the carrier and transfected into human colorectal cancer with Si RNA for YAP and Survivin specific targets, respectively. Cell line RKO, HCT116, using immunocytochemical S-P method, Western Blot to detect the expression of YAP, -catenin and Survivin protein, RT-PCR detected m RNA expression, CCK8 method to detect cell proliferation and flow cytometry to detect cell apoptosis. Results: immunocytochemical results showed that compared with control group, the transfection group was negative or only weak cells were weak. After transfection of.Si RNA-YAP to RKO cells, the expression of beta -catenin and Survivin protein decreased significantly (P0.05) after transfection of.Si RNA-survivin into human colorectal HCT116 cells, the expression of Survivin protein decreased significantly. ) after transfection of.Si RNA-survivin to HCT116 cells, the gray value of the Survivin protein band was significantly lower than that of the control group (P0.05).RT-PCR, and the YAP m RNA expression level was reduced by about (76.423 + 9.074%) after Si RNA-YAP transfection. I RNA-YAP and Si RNA-survivin were transfected to RKO and HCT116 cells respectively. The cell growth was obviously inhibited (P0.05). Flow cytometry showed that the percentage of apoptosis in Si RNA-YAP transfection group was 23.90 + 2.07%, and the percentage of apoptotic cells in Si RNA-survivin transfection group was 25.73 + 2.02%, obviously higher than that of the control group (P0.01). In the system, Si RNA-YAP can inhibit the expression of YAP, beta -catenin and Survivin, and Si RNA-survivin can inhibit the expression of Survivin. Both can inhibit the proliferation of colorectal cancer cells and promote cell apoptosis. YAP and Survivin are expected to be a new target for colorectal cancer gene therapy. Third objective: To observe the growth of colorectal carcinoma in nude mice by SH RNA-survivin. Methods: 15 3-5 weeks old female nude mice were randomly divided into 3 groups. The SW1116 cells, sh RNA control group cells and sh RNA-survivin transfected cells were injected into the right axillary subcutaneous tissue of the nude mice to establish the tumor model of nude mice. The size of the tumor was measured every two days and the tumor volume was calculated. The nude mice were killed after 28 days. The tumor growth curve.Western blot and RT-PCR methods were used to detect the protein and m RNA expression of Survivin and its downstream Ki-67 and BCL-2 genes. Results: the size, weight and body volume of the transplanted tumor model of the subcutaneous xenografts in nude mice of human colorectal cancer was successfully established, and the weight and volume of the tumor decreased significantly (P0.05). Compared with the average inhibition rate of 76.1% and 76.3% (P0.05).Western blot and RT-PCR, the results showed that survivin and its downstream Ki-67 in the transplanted tumor tissues of SH RNA-survivin transfected group, BCL-2 gene protein and m RNA expression level were significantly reduced. To inhibit the growth of transplanted tumor in nude mice and promote apoptosis, Survivin can be used as the target gene for the treatment of colorectal cancer. In a word, many methods have been used to study the tissue specimens of colorectal cancer in vivo and in vitro. The conclusions are summarized as follows: 1.Hippo/YAP and Wnt/ beta -catenin signaling pathway in the development of large intestine cancer, the development of heavy cancer The expression of YAP1, beta -catenin and Survivin can help to reflect the progression and prognosis of colorectal cancer..2.Survivin is the common target gene of the downstream of Hippo/YAP and Wnt/ beta -catenin in vitro, and Si RNA-YAP inhibits YAP, beta -catenin and expression, both of which can inhibit the large amount of expression. YAP and Survivin are expected to become the new target of colorectal cancer gene therapy,.4. in the tumor experiment in nude mice in vivo. Sh RNA-survivin plasmid can obviously inhibit the expression of Ki-67 and BCL-2 of Survivin and its downstream genes, inhibit the growth of transplanted tumor in nude mice and promote cell apoptosis. Survivin can be used as large intestine cancer. The target gene for molecular therapy.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.34

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相關期刊論文 前1條

1 ;Significance and relationship between Yes-associated protein and survivin expression in gastric carcinoma and precancerous lesions[J];World Journal of Gastroenterology;2009年32期

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本文編號：1845490