Hippo及Wnt信号通路在大肠癌发生中的作用及沉默YAP,Survivin基因对大肠癌细胞生物学行为的影响

发布时间：2018-05-05 01:07

本文选题：结直肠癌 + YAP　；参考：《安徽医科大学》2016年博士论文

【摘要】：大肠癌是最常见的恶性肿瘤之一,每年以2%的速度递增。2012年统计全球大肠癌患者约140万人,其中6.939万人死亡该疾病,5年存活率从临床Ⅰ期患者的90%至临床Ⅳ期患者的10%不等。大肠癌早期症状不明显,多数大肠癌诊断时已属于晚期,甚至出现肝脏或其它器官的转移。大肠癌最基本的治疗方法是手术治疗,术后辅助化疗及放疗。随着诊断技术的发展和治疗方法的改进,大肠癌的预后得到逐步改善,但死亡率依然较高,主要原因是肿瘤的复发、转移以及耐药的产生。此外,化疗和放疗缺乏特异性和敏感性,副作用较大。因此迫切需要研究阐明大肠癌发生的分子机制,寻找更好的诊断及预后指标和治疗策略。Hippo信号通路与细胞增殖、分化、凋亡及肿瘤发生有关,是近来的研究热点。在多种肿瘤中Hippo信号通路成分下调,其转录因子YAP1表达水平是大肠癌重要的预后因素,Hippo信号通路负调节YAP1,YAP1作为转录共激活因子可诱导与细胞增殖及凋亡有关的靶基因的转录和表达,如BIRC5,即survivin基因,它属于凋亡蛋白抑制家族成员(IAP)。Hippo/YAP1信号通路与Wnt/β-catenin信号通路在维持细胞稳定方面常可相互交叉作用,并与大肠癌细胞凋亡及增殖有关。β-catenin是Wnt/β-catenin信号通路中最重要的转录共激活因子,可调节下游与细胞增殖、凋亡及分化有关的靶基因,survivin是Wnt/β-catenin信号通路的下游靶基因。因此。survivin是Hippo/YAP1及Wnt/β-catenin信号通路的共同靶基因。本研究的目的是观察Hippo/YAP1及Wnt/β-catenin信号通路在大肠癌发生中的作用及临床意义以及si RNA沉默YAP1和survivin基因对大肠癌细胞生物学行为的影响。为此,本研究分为三个部分。第一部分目的:探讨大肠癌组织中YAP1,β-catenin及survivin表达、相互关系及临床意义。方法:采用免疫组织化学S-P法检测181例大肠癌组织和30例正常粘膜中YAP1,β-catenin和Survivin的表达状况。结果:1.在181大肠癌组织中,YAP1,β-catenin(细胞核)及survivin阳性率分别为73.5%(133/181),56.9%(103/181)及65.2%(118/181),均明显高于正常粘膜组(P0.05)。2.大肠癌组织中YAP1表达与分化程度、Duke’s分期有关(P0.05);YAP1细胞核及核/浆表达与患者的临床Duke’s分期有关(P0.05)。YAP1高表达与分化程度、Duke’s分期及淋巴结转移有关(P0.05)。β-catenin细胞核表达与分化程度、淋巴结转移及临床Duke’s分期有关(P0.05),β-catenin细胞膜不完整表达与组织分化程度、浸润深度、淋巴结转移及临床Duke’s分期等均有关(P0.05)。大肠癌组织中Survivin表达及不同染色定位与患者性别、年龄、分化程度、浸润深度、淋巴结转移及临床Duke’s分期等临床病理因素均无关(P0.05)。3.大肠癌组织中YAP1高表达与β-catenin细胞核/浆表达及survvin表达有关(P0.05)。β-catenin细胞核表达与survivin表达相关(P0.05)。4.在82例随访病例中,YAP1高表达、β-catenin细胞核阳性及survivin阳性表达患者5年生存率均明显低于对照组(P0.05)。YAP1,β-catenin(细胞核)及survivin任何两者或三者同时阳性者,其5年生存率均明显低于相对应的对照组(P0.05)。结论:1.Hippo/YAP及Wnt/β-catenin信号通路在大肠癌发生、发展中起重要作用。2.检测YAP1,β-catenin及survivin表达状况有助于反映大肠癌的进展和预后。3.Survivin是Hippo/YAP及Wnt/β-catenin信号通路下游的共同靶基因;YAP,β-catenin及survivin均有望成为大肠癌基因诊断及基因治疗的新靶点。第二部分目的:探讨si RNA沉默YAP及Survivin基因后对人大肠癌细胞YAP,β-catenin及Survivin表达、细胞增殖及凋亡的影响。方法:以脂质体Lip-2000为载体,用针对YAP及Survivin特异靶点的si RNA分别转染入人大肠癌细胞系RKO、HCT116,应用免疫细胞化学S-P法、Western Blot检测YAP、β-catenin及Survivin蛋白表达变化;RT-PCR检测m RNA表达;CCK8法检测细胞增殖;流式细胞术检测细胞凋亡。结果:免疫细胞化学结果显示,与对照组相比,YAP在转染组阴性或仅见可别细胞弱阳性表达。si RNA-YAP转染RKO细胞后β-catenin及Survivin蛋白表达明显降低(P0.05)。si RNA-survivin转染人大肠HCT116细胞后,Survivin蛋白表达明显降低。Western blot结果显示si RNA-YAP转染后,RKO细胞YAP,β-catenin及Survivin蛋白条带灰度值明显低于对照组(P0.05)。si RNA-survivin转染HCT116细胞后,Survivin蛋白条带灰度值明显低于对照组(P0.05)。RT-PCR结果显示,si RNA-YAP转染后,YAP m RNA表达水平降低约(76.423±9.074%),si RNA-survivin转染HCT116细胞后,survivin m RNA表达水降低约(69.705±6.151%)(P0.05)。CCK8检测结果显示si RNA-YAP及si RNA-survivin分别转染RKO及HCT116细胞后,细胞生长出现明显抑制(P0.05)。流式细胞术检测结果显示,si RNA-YAP转染组细胞凋亡比例为23.90±2.07%,si RNA-survivin转染组细胞凋亡比例为25.73±2.02%,明显高于对照组(P0.01)。结论:在大肠癌细胞系中,si RNA-YAP可抑制YAP,β-catenin及Survivin表达,si RNA-survivin可抑制Survivin表达,两者均可抑制大肠癌细胞增殖,促进细胞凋亡;YAP和Survivin有望成为大肠癌基因治疗的新靶点。第三部分目的:观察sh RNA-survivin质粒对大肠癌裸鼠移植瘤生长的影响及其分子机制。方法:15只3-5周龄雌性裸鼠随机分为3组,分别将SW1116细胞、sh RNA对照组细胞及sh RNA-survivin转染组细胞注入裸鼠右侧腋窝皮下组织,建立大肠癌裸鼠移植瘤模型。每两天测量一次肿瘤大小,计算肿瘤体积,28天后处死裸鼠并称肿瘤重量,绘制肿瘤生长曲线。Western blot及RT-PCR方法检测survivin及其下游Ki-67和BCL-2基因的蛋白及m RNA表达。结果:成功建立人大肠癌裸鼠皮下移植瘤模型。sh RNA-survivin组移植瘤的大小、重量及体积明显减小(P0.05)。与SW1116及sh RNA对照组相比,平均抑留率分别为76.1%及76.3%(P0.05)。Western blot及RT-PCR结果显示sh RNA-survivin转染组移植瘤组织中survivin及其下游Ki-67,BCL-2基因蛋白及m RNA表达水平均明显降低。结论:sh RNA-survivin质粒可明显抑制survivin及其下游基因Ki-67和BCL-2的表达,抑制大肠癌裸鼠移植瘤生长、促进细胞凋亡,Survivin可作为大肠癌治疗的靶基因。总之,本研究应用多种方法对大肠癌组织标本、细胞系体内及体外进行研究,所得结论概括如下:1.Hippo/YAP及Wnt/β-catenin信号通路在在大肠癌发生、发展中起重要作用,检测YAP1,β-catenin及survivin表达有助于反映大肠癌的进展和预后。2.Survivin是Hippo/YAP及Wnt/β-catenin信号通路下游的共同靶基因。3.在体外细胞实验中,si RNA-YAP抑制YAP,β-catenin及Survivin表达,si RNA-survivin抑制Survivin表达,两者均可抑制大肠癌细胞增殖,促进细胞凋亡;YAP和Survivin有望成为大肠癌基因治疗的新靶点。4.在体内裸鼠瘤实验中,sh RNA-survivin质粒可明显抑制survivin及其下游基因Ki-67和BCL-2的表达,抑制大肠癌裸鼠移植瘤生长、促进细胞凋亡,Survivin可作为是大肠癌分子治疗的靶基因。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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