| Colorectal cancer(CRC) is one of the most common malignant tumor types, and the incidence of CRC is increasing by 2% per year worldwide. It is estimated that about 1.4 million cases and 693,900 deaths occurred in 2012. Five-year survival ranges from more than 90% in patients with stage I disease to about 10% in patients with stage IV disease. The early symptom of CRC is not easy to be detected. When the patients being diagnosed as colorectal cancer, it is often in the advanced stage, even metastatic to live or other organs. The principle therapeutic method is surgery, assisted by radiotherapy and chemotherapy. Although the prognosis for CRC has greatly improved with the continuous development of diagnostic techniques and improvement of treatment method, the mortality rate remains high at the moment. The major causes of CRC-associated mortality are recurrence and metastasis and traditional anticancer drug resistance. Besides, chemotherapy and radiotherapy lack of specificity and sensitivity, and often accompanied by strong toxicity and side effects.Therefore, it is important to study molecular mechanisms of colorectal cancer carcinogenesis, look for better diagnostic and prognostic markers and optimized treatment strategies.The Hippo pathway has recently become subject of intense investigations since it plays a crucial role in cell proliferation, differentiation, apoptosis and tumourigenesis. Components of the Hippo pathway are deregulated in various human malignancies and expression levels of its major signal transducers were proposed as prognostic factors in colorectal cancer. Hippo negatively regulates its major effectors YAP1, which act as transcriptional co-activators inducing expression of genes involved proliferation and apoptosis, such as BIRC5/survivin gene, which belongs to a member of the inhibitor of apoptosis protein family(IAP). The interactions between Hippo/YAP1 and Wnt/β-catenin signalling, crucial roles for the maintenance of cell homeostasis, have been described in relation to the proliferation and apoptosis of colorectal cancer. The effector of the Wnt/β-catenin signal pathway is the transcriptional co-activator β-catenin. It plays an important role by regulating downstream target gene in several developmental processes, such as proliferation, apoptosis or differentiation. Survivin is in the downstream of Wnt/β-catenin signal pathway. Therefore, survivin is the common target gene of Hippo/YAP and Wnt/β-catenin signaling. Our studies aimed to observe role and clinic significance of Hippo/YAP and Wnt/β-catenin signaling pathways in colorectal carcinogenesis and impact of si RNA silencing YAP and survivin genes on biological behavior of colorectal cancer cells. This study is divided into three parts.PartⅠObjective: To investigate the expression and clinical significance of YAP1, β-catenin and survivin in colorectal cancer. Methods: The expressions of YAP1, β-catenin and survivin were detected in 181 cases of CRC and 30 cases of normal colorectal mucosa using immunohistochemistry. Results: 1.The expression rate of YAP1, β-catenin and survivin were 73.5%(133/181),56.9%(103/181) and 65.2%(118/181) in CRC, and higher than these in normal colorectal mucosa, respectively(P<0.05). 2. There was an excellent correlation between YAP expression and histological differentiation, lymph node metastasis and Duke’s stage in CRC(P<0.05). Importantly, Nuclear and nuclear/cytoplasmic YAP expressions were positively correlated with lymph node metastasis and Duke’s stage(P<0.05). The high YAP expression was related with histological differentiation, lymph node metastasis and Duke’s stage in CRC(P<0.05). The overexpression of nuclear β-catenin and incomplete member expression of β-catenin were significantly correlated with histological differentiation, the depth of infiltration, lymph node metastasis and Duke’s stage in CRC(P<0.05). The survivin expression and its different location did not associate with other variables, such as lymph node involvement, histological grade, and Duke’s stage in CRC(P>0.05). 3. There were relationship between high and nuclear/cytoplasmic YAP expression and the expression of nuclear and nuclear/cytoplasmic β-catenin and survivin(P<0.05). The expression of nuclear β-catenin was associated with survivin expression in CRC(P<0.05). 4. In the follow-up data of 82 cases of CRC, the expression of YAP, nuclear β-catenin and survivn were associated with poor prognosis, and the 5-year survival rate were significantly lower than that of self-control groups(P<0.001). Furthermore, we observed the 5-year survival rate in patients with any two or co-expression of YAP, nuclear β-catenin and survivn were significantly lower than that of corresponding control groups(P<0.001). Conclusions:1.Hippo/YAP and Wnt/β-catenin signaling pathway play an important role in colorectal carcinogenesis and progression. 2. The analysis of YAP, β-catenin and survivn may be useful for evaluating the progress and prognosis of CRC. 3. Survivin may be the common downstream target gene of Hippo/YAP and Wnt/β-catenin signaling pathway, and YAP, β-catenin and survivin are expected to become new target for diagnosis and treatment of CRC in future.PartⅡObjective: To explore expression of YAP, β-catenin and Survivin protein and the effect of si RNA-YAP and si RNA-survivin on the proliferation and apoptosis of human colorectal cancer RKO and HCT116 cells. Methods: si RNA targeted YAP and survivin were transfected into RKO and HCT116 cells with lipofectamine 2000, respectively. YAP,β-catenin and Survivin protein expressions were detected by Immunocytochemistry and Western blot. m RNA level of YAP and Survivin were analyzed by RT-PCR, the proliferation of cells was measured by CCK8 assay. The apoptosis of cells was analyzed by Flow Cytometry(FCM). Results: Compared with YAP positive expression in control group, YAP was negative expression in si RNA-YAP transfected group. Occasionally, a small number of RKO cells showed weakly positive expression. The expression of β-catenin and survivin protein distinctly decreased in RKO cells after si RNA-YAP transfected. Survivin showed weakly expression in si RNA-survivin transfected group in HCT116 cells. The protein grey value of YAP, β-catenin and survivin were 0.384 ± 0.033, 0.219 ± 0.021 and 0.402 ± 0.017 in si RNA-YAP transfected group,respectively; and significantly lower than these in the control group by Western blot(P<0.05). The protein level of survivin in HCT116 in si RNA-survivin transfected group was lower than that in the control group by Western blot(P<0.05).The m RNA level of YAP and survivin significantly reduced in transfected group compared with control group by RT-PCR(P<0.05), the m RNA of YAP and survivin were inhibited by 76.423±9.074% in si RNA-YAP transfected group and 69.705±6.151% in si RNA-survivin transfected group(P<0.05).Compared with control group, the cells growth was significantly inhibited in si RNA-YAP interfered group in RKO cells and in si RNA-survivin interfered group in HCT116 cells by CCK8 assay(P<0.05). The apoptosis rates of RKO and HCT116 cells were 23.90±2.07% in si RNA-YAP transfected group and 2.73±2.02% in si RNA-survivin transfected group,respectively; and significantly higher than these in control group by FCM(P<0.05). Conclusions: si RNA-YAP could inhibit expression of YAP, β-catenin and Survivin protein in RKO cells. si RNA targeting survivin gene can inhibit expression of survivin in HCT116 cells. They could inhibit the proliferation of colorectal cancer cells and induce apoptosis. YAP and Survivin may provide a novel approach for gene therapy target of colorectal cancer.Part ⅢObjective: To observe the effects of the sh RNA-survivin vector on inhibiting human colorectal cancer SW1116 cell growth in xenografts in nude mice and its molecular mechanisms. Methods: Fifteen three-to-five-week-old female nude mice [BALB/c A-nu(nu/nu)] were randomly divided into three group, with five mice per group. SW1116 cells, SW1116 cells with sh RNA control and SW1116 cells expressing sh RNA survivin were subcutaneously injected into the right armpit tissue of nude mice to establish colorectal xenografts models in nude mice. Bidimensional tumor measurements were measured with calipers once every two days. Tumor volumes were calculated according to the formula:(width2×length)/2. The mice were euthanized after 28 days, and tumors were weighed. The tumor growth curve was plotted. The expressions of protein and m RNA level of survivin and its downstream Ki-67 and BCL-2 genes were detected by Western blot and RT-PCR,respectively. Results: Implanted human colorectal cancer xenografts model in nude mice was successively established.Compared with SW1116 and sh RNA control groups, the size, weights and tumor volume of xenografts in nude mice markedly decreased in sh RNA-survivin group(P<0.05). The average inhibitory rates in sh RNA-survivin group were 76.1% and 76.3% compared with SW1116 and sh RNA control group(P<0.05). The expressions of protein and m RNA level of survivin and its downstream Ki-67 and BCL-2 genes significantly decreased by Western blot and RT-PCR in sh RNA-survivin group(P<0.05). Conclusion: The sh RNA-survivin vector inhibits the growth of colorectal cancer xenografts and induces apoptosis by inhibiting the expressions of survivin and its downstream Ki-67 and BCL-2 genes. Survivin is an attractive target gene for colorectal cancer therapy.In conclusion, in this studies, YAP, β-catenin and Survivin were analyzed in the colorectal cancer tissues, colorectal cancer cell lines in vivo and in vitro by using a variety of methods. Conclusions are summarized as follows:1. Hippo/YAP and Wnt/β-catenin signaling pathway play an important role in colorectal carcinogenesis and progression. The analysis of YAP, β-catenin and survivn may be useful for evaluating the progress and prognosis of CRC.2.Survivin may be the common downstream target gene of Hippo/YAP and Wnt/β-catenin signaling pathway.3. si RNA-YAP could inhibit expression of YAP, β-catenin and Survivin protein in vitro. si RNA targeting survivin gene can inhibit expression of survivin in vitro. They could inhibit the proliferation of colorectal cancer cells and induce apoptosis in vitro. YAP and Survivin may provide a novel approach for gene therapy target of colorectal cancer.4. The sh RNA-survivin vector inhibits the growth of colorectal cancer xenografts and induces apoptosis by inhibiting the expressions of survivin and its downstream Ki-67 and BCL-2 genes in vivo. Survivin is an attractive target gene for colorectal cancer therapy. |
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