RIPK4对骨肉瘤细胞上皮—间质转化的影响及机制研究

发布时间：2018-04-19 03:40

本文选题：骨肉瘤 + RIPK4　；参考：《兰州大学》2017年硕士论文

【摘要】：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,懫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(P0.05)。骨肉瘤组织中RIPK4的表达与β-catenin表达之间呈正相关(r=0.634,P0.01)。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(P0.05);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(P0.05),而N-cadherin、vimentin以及β-catenin总蛋白和核蛋白的表达水平明显降低(P0.05)。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。
[Abstract]:Objective: To investigate the effects of receptor interacting protein kinase 4 (receptor interacting protein kinase 4, RIPK4) expression in osteosarcoma tissues and cells and its clinical significance; further study of RIPK4 on osteosarcoma MG-63 and U-2OS cells of epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT) and the effects of molecular mechanisms. Methods: (1) expression RIPK4 and -catenin protein was detected by immunohistochemistry in 36 cases of osteosarcoma and 15 cases of osteochondroma tissues, and to analyze the correlation between clinicopathologic features and prognosis of two patients with osteosarcoma; (2) were extracted from 4 cases of osteosarcoma and normal bone tissues, osteosarcoma MG-63 and U-2OS cells, human normal bone cells hFOB1.19 protein, using Western blotting further confirmed the expression level of RIPK4 in osteosarcoma tissues and cells. (3) using Lipofectamine specific RIPK4 gene targeted small molecule RNA (siRNA small interfering interference RNA) were transferred into osteosarcoma MG-63, U-2OS cells, the successful construction of osteosarcoma MG-63 expression of RIPK4 gene silencing, U-2OS cell line was detected by using Western blotting, silencing of RIPK4 gene expression in human osteosarcoma MG-63, expression of RIPK4 in U-2OS cell line; MG-63 silencing RIPK4 gene on osteosarcoma detection using CCK-8 method, U-2OS cell proliferation activity; osteosarcoma MG-63 were determined by Transwell. The changes of U-2OS cell invasion and migration of osteosarcoma MG-63; observed under an inverted microscope, U-2OS changes in cell morphology. Finally, Zhi by Western blotting and immunofluorescence staining of osteosarcoma U-2OS cells in MG-63 assay. EMT related molecules of E- cadherin (E-cadherin), N- cadherin (N-cadherin), vimentin (vimentin) and the expression of beta -catenin total protein and nuclear protein changes. Results: (1) osteosarcoma RIPK4 in the tumor tissues, beta -catenin positive expression rate was 63.9% (23/36), 75% (27/36), significantly higher than the positive expression in osteochondroma tissues was 6.7% (1/15), 20% (3/15) (P 0.05) expression of.RIPK4 and beta -catenin in osteosarcoma tissues were associated with Enneking stage closely related (P 0.05); the expression of RIPK4 was correlated with tumor size (P0.05). The expression of RIPK4 in osteosarcoma and beta -catenin expression was positively correlated (r=0.634, P0.01).Kaplan-Meier survival analysis showed that RIPK4, the total survival time of patients with positive expression in group B -catenin were shorter than the negative group (P 0.05). The single factor COX regression analysis showed that age, Enneking staging, metastasis, RIPK4 protein expression was correlated with survival time (P < 0.05). (2) the results of Western blotting showed that RIPK4 in osteosarcoma tissue or blood cells in the expression level Were higher than that of normal bone tissue and normal osteoblast cells. (3) RIPK4-siRNA was transfected into osteosarcoma MG-63 and U-2OS cells, the expression level of RIPK4 was significantly reduced (P0.05); RIPK4-siRNA MG-63 transfection group, U-2OS cell proliferation, migration and invasion ability decreased significantly (P < 0.05); RIPK4-siRNA transfection was observed by inverted optical microscope after MG-63 of osteosarcoma, U-2OS cells by mesenchymal morphology into epithelial cells. In addition, the results of Western blotting showed that RIPK4-siRNA MG-63 transfection group, the expression level of E-cadherin in U-2OS cells was significantly up-regulated (P0.05), N-cadherin, vimentin and the expression level of beta -catenin total protein and nuclear protein decreased significantly (P0.05). At the same time, immunofluorescence staining also got the same conclusion. Conclusion: (1) RIPK4 in osteosarcoma, beta -catenin expression, which is closely related with the pathological characteristics and prognosis of patients with osteosarcoma; At the same time, the two was a positive correlation, suggesting that RIPK4 and -catenin may coordinate beta osteosarcoma development. (2) silencing RIPK4 could inhibit the Wnt signaling pathway and reverses osteosarcoma U-2OS cells MG-63, EMT.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738

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