兰州大学机构库 >第二临床医学院
RIPK4对骨肉瘤细胞上皮-间质转化的影响及机制研究
Alternative TitleEffect of RIPK4 on epithelial-mesenchymal transition in osteosarcoma cells and its mechanism
移志刚
Subtype硕士
Thesis Advisor王栓科
2017-03-01
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword骨肉瘤 RIPK4 上皮-间质转化 Wnt/β-catenin信号通路
Abstract

目的:探讨受体相互作用蛋白激酶4(receptor interacting protein kinase 4,RIPK4)在骨肉瘤组织和细胞中的表达及其临床意义;进一步研究RIPK4对骨肉瘤MG-63、U-2OS细胞上皮间质转化(epithelial-mesenchymal transition,EMT)的影响及分子机制。方法:(1)应用免疫组织化学法检测36例骨肉瘤和15例骨软骨瘤组织中RIPK4和β-catenin蛋白的表达情况,并分析二者与骨肉瘤患者的临床病理特征及预后的相关性;(2)分别提取4例骨肉瘤和正常骨组织、骨肉瘤MG-63和U-2OS细胞、人正常成骨细胞hFOB1.19中蛋白,采用蛋白质免疫印迹法进一步验证RIPK4骨肉瘤组织和细胞中表达水平。(3)采用脂质体法将特异性针对RIPK4基因的小分子干扰RNA(small interfering RNA,siRNA)分别转入骨肉瘤MG-63、U-2OS细胞,成功构建RIPK4基因沉默表达的骨肉瘤MG-63、U-2OS细胞系,采用蛋白质印迹法检测沉默RIPK4基因表达后骨肉瘤MG-63、U-2OS细胞系中RIPK4表达的变化;采用CCK-8法检测沉默RIPK4基因后对骨肉瘤MG-63、U-2OS细胞增殖活力的影响;Transwell小室法检测骨肉瘤MG-63、U-2OS细胞侵袭和迁移能力的变化;倒置光学显微镜下观察骨肉瘤MG-63、U-2OS细胞形态的变化。最后,釆用蛋白质印迹法和免疫荧光染色法检测骨肉瘤MG-63、U-2OS细胞中EMT相关分子E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)以及β-catenin总蛋白和核蛋白的表达变化。结果:(1)骨肉瘤组织中RIPK4、β-catenin阳性表达率分别为63.9%(23/36)、75.0%(27/36),显著高于其在骨软骨瘤组织中的阳性表达率6.7%(1/15)、20.0%(3/15)(P值均<0.05)。RIPK4和β-catenin在骨肉瘤组织的表达均与Enneking分期密切相关(P值均<0.05);RIPK4的表达还与肿瘤大小相关(P<0.05)。骨肉瘤组织中RIPK4的表达与β-catenin表达之间呈正相关(r=0.634,P<0.01)。Kaplan-Meier生存分析发现,RIPK4、β-catenin阳性表达组中患者的总生存时间均短于阴性表达组(P值均<0.05)。单因素COX回归分析发现,年龄、Enneking分期、转移、RIPK4蛋白的阳性表达与患者总生存时间相关(P值均<0.05)。(2)蛋白质印迹法结果显示,RIPK4无论是在骨肉组织还是骨肉细胞中的表达水平均高于正常骨组织和正常成骨细胞。(3)RIPK4-siRNA转染骨肉瘤MG-63、U-2OS细胞后,RIPK4的表达水平显著下调(P<0.05);RIPK4-siRNA转染组MG-63、U-2OS细胞的增殖、迁移以及侵袭能力明显降低(P值均<0.05);倒置光学显微镜下观察发现RIPK4-siRNA转染后,骨肉瘤MG-63、U-2OS细胞由间质形态转变成上皮细胞形态。此外,蛋白质印迹法结果显示:RIPK4-siRNA转染组MG-63、U-2OS细胞中E-cadherin的表达水平显著上调(P<0.05),而N-cadherin、vimentin以及β-catenin总蛋白和核蛋白的表达水平明显降低(P<0.05)。同时,免疫荧光染色也得到了相同结论。结论:(1)骨肉瘤组织中RIPK4、β-catenin表达明显上调,且与骨肉瘤患者的病理特征及预后密切相关;同时,二者表达呈正相关,提示RIPK4和β-catenin可能共同协调骨肉瘤发生发展。(2)沉默RIPK4可能通过抑制Wnt信号通路逆转骨肉瘤MG-63、U-2OS细胞EMT的发生。

Other Abstract

Objective: To explore the expression of receptor interacting protein kinase 4 in osteosarcoma tissues and cells, and its clinical significance. To further investigate the effect of RIPK4 on the epithelial-mesenchmal transition in osteosarcoma MG-63 and U-2OS cells and molecular mechanism.Methods: (1) Immunohistochemistry was used to detected the expressions of RIPK4 and β-catenin protein in 36 specimens of osteosarcoma tissues and 15 specimens of osteochondroma tissues, and to analyze the correlation between the clinicopathological characteristics and prognosis of osteosarcoma patients. (2) The proteins of four cases in osteosarcoma and normal bone tissues, osteosarcoma MG-63 and U-2OS cells, normal human osteoblast hFOB1.19 were extracted, and the expression levels of RIPK4 osteosarcoma tissue and cells were further confirmed by Western blotting. (3) The specific small interfering RNA targeting for RIPK4 gene was transferred into osteosarcoma MG-63 and U-2OS cells by liposome, respectively, to successfully construct osteosarcoma MG-63, U-2OS cell lines of which RIPK4 gene was silenced. After silencing RIPK4, the expression of RIPK4 in osteosarcoma MG-63 and U-2OS cell lines was detected by Western blotting. Cell proliferation of osteosarcoma MG-63 and U2OS after silencing RIPK4 were measured by CCK-8 assay. Cell migration and invasion of osteosarcoma MG-63 and U2OS after silencing RIPK4 were measured by transwell chamber assay. The morphological changes of osteosarcoma MG-63 and U-2OS cells were observed under an inverted optical microscope. Finally, the expression changes of E-cadherin, N-cadherin, vimentin and total and necleus β-catenin were detected by Western blotting and immunofluorescence staining.Results: (1) In osteosarcoma, the positive expression rates of RIPK4 and β-catenin were 63.9%(23/36) and 75.0%(27/36), respectively, which were significantly higher than those in osteochondroma tissues 6.7%(1/15 ), 20.0%(3/15) (all P<0.05). The expressions of RIPK4 and β-catenin in osteosarcoma were closely associated to Enneking stage (all P<0.05). Meanwhile, the expression of RIPK4 was also correlated with tumor size (P<0.05). The expressions of RIPK4 and β-catenin in osteosarcoma presented a positive correlation (r = 0.634, P<0.01). Kaplan-Meier survival analysis showed that the overall survival time of patients with RIPK4 and β-catenin positive expression groups were shorter than that of negative expression group (P<0.05). Univariate COX regression analysis revealed that age, Enneking stage, metastasis and RIPK4 positive expression were correlated with overall survival time of patients with osteosarcoma (all P<0.05). (2) Western blotting showed, the expression levels of RIPK4 either in osteosarcoma tissues or cells were higher than normal bone tissues and normal osteoblast cells (P<0.05). (3) The expression level of RIPK4 was significantly down-regulated by RIPK4-siRNA transfected into MG-63 and U-2OS cells (P<0.05). The proliferation, migration and invasion abilities of MG-63 and U-2OS cells in RIPK4-siRNA transfected group were significantly decreased (all P<0.05). In an inverted optical microscope observed that after transfected with RIPK4-siRNA, the morphpological of osteosarcoma MG-63, U-2OS cells are changed from mesenchymal phenotype to epithelial cell morphology. In addition, Western blotting showed that the expression levels of E-cadherin in MG-63 and U-2OS cells were significantly up-regulated (P<0.05), while N-cadherin, vimentin and the protein expression of total and nuclear β-catenin was significantly decreased (P<0.05). At the same time, immunofluorescence staining also received the same conclusion.Conclusion: (1) The expression of RIPK4 and β-catenin in osteosarcoma tissues were significantly up-regulated and correlated with the pathological characteristics and prognosis of osteosarcoma patients. Meanwhile, the expression of RIPK4 and β-catenin was positively correlated, suggesting that RIPK4 and β-catenin may co-coordinate the development of osteosarcoma.(2) Silencing RIPK4 may reverse the occurrence of EMT in osteosarcoma MG-63 and U-2OS cells by inhibiting Wnt signaling pathway.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/200860
Collection第二临床医学院
Recommended Citation
GB/T 7714
移志刚. RIPK4对骨肉瘤细胞上皮-间质转化的影响及机制研究[D]. 兰州. 兰州大学,2017.
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