长链非编码RNA-ROR通过Wnt通路诱导高级别卵巢癌EMT特性的机制研究

发布时间：2018-07-01 21:48

本文选题：高级别卵巢浆液性癌 + 长链非编码RNA　；参考：《青岛大学》2017年硕士论文

【摘要】：目的:分析基因间长链非编码RNA-ROR(Linc-ROR)在高级别卵巢浆液性癌组织中的表达,探讨Linc-ROR表达与高级别卵巢浆液性癌细胞生物学功能的关系,并分析Linc-ROR通过Wnt/β-catenin通路对高级别卵巢浆液性癌细胞上皮间质转化过程的影响。方法:(1)实时荧光定量PCR检测34例高级别卵巢浆液性癌组织、20例正常卵巢上皮组织和20例正常输卵管伞端组织中Linc-ROR m RNA的表达,并分析Linc-ROR表达与高级别卵巢浆液性癌FIGO分期、淋巴结转移的关系。(2)常规培养卵巢浆液性乳头状囊腺癌细胞系SKOV3,合成Linc-ROR si RNA和Linc-ROR过表达质粒(p IRES2-EGFP-Linc-ROR重组载体),分别转染SKOV3细胞,采用活细胞计数(CCK-8)法、体外细胞划痕实验和穿膜(transwell)小室侵袭实验分别检测转染后SKOV3细胞的增殖、迁移、侵袭能力,蛋白印迹实验检测上皮间质转化相关标志物E-cadherin、β-catenin、vimentin和Wnt/β-catenin通路靶基因c-myc的表达变化。(3)应用Wnt/β-catenin通路激活剂氯化锂(Li Cl)作用于SKOV3细胞后,CCK-8法观察细胞增殖情况,western blot法检测E-cadherin、β-catenin、vimentin和c-myc的蛋白表达变化;将Li Cl+Linc-ROR si RNA共同作用SKOV3细胞,另设Li Cl+si RNA-NC组作为阴性对照,western blot法检测E-cadherin、β-catenin、vimentin和c-myc的蛋白表达变化。结果:(1)实时荧光定量PCR实验显示,Linc-ROR m RNA在高级别卵巢浆液性癌组织中的表达水平为4.38±2.55,明显高于正常卵巢上皮组织中的1.49±1.69、正常输卵管伞端组织中的1.45±1.57(F=8.62,P0.01);Linc-ROR m RNA的表达水平随着高级别卵巢浆液性癌FIGO分期的递增明显升高(F=95.70,P0.01),且伴有淋巴结转移者明显高于无淋巴结转移者(t=7.40,P0.01)。(2)转染Linc-ROR si RNA组细胞中Linc-ROR m RNA的表达水平明显低于其阴性对照si RNA-NC组(F=26.29,P0.05);与阴性对照si RNA-NC组相比,si RNA组SKOV3细胞的增殖、迁移和侵袭能力均明显降低(P均0.01),E-cadherin蛋白表达量明显升高,β-catenin、vimentin和c-myc蛋白表达量明显减少(P均0.01)。(3)转染过表达质粒ROR组细胞中Linc-ROR m RNA的表达水平明显高于其阴性对照Vector组(t=6.304,P0.01);与阴性对照Vector组相比,ROR组SKOV3细胞的增殖、迁移和侵袭能力均明显增强(P均0.01),E-cadherin蛋白表达量明显降低,β-catenin、vimentin和c-myc蛋白表达量明显增加(P均0.01)。(4)不同浓度Li Cl处理SKOV3细胞不同时间后,对细胞的增殖差异有统计学意义(P0.05),以10mmol/L Li Cl作用24小时对SKOV3细胞增殖的促进最明显。与空白对照组相比,Li Cl能明显下调SKOV3细胞E-cadherin蛋白表达,上调β-catenin、vimentin和c-myc蛋白表达,差异具有统计学意义(P0.01);与Li Cl+si RNA-NC组相比,Li Cl+Linc-ROR si RNA组E-cadherin蛋白表达量明显升高,β-catenin、vimentin和c-myc蛋白表达量明显降低,差异具有统计学意义(P均0.01)。结论:Linc-ROR异常表达与高级别卵巢浆液性癌的侵袭、转移密切相关。高表达Linc-ROR能促进卵巢癌细胞上皮间质转化,可能通过Wnt/β-catenin通路而发挥调控作用。因此,Linc-ROR可能介导高级别卵巢浆液性癌侵袭、转移的重要分子之一。
[Abstract]:Objective: to analyze the expression of INTERGENE long chain non coding RNA-ROR (Linc-ROR) in high grade ovarian serous carcinoma and explore the relationship between the expression of Linc-ROR and the biological function of high grade serous serous carcinoma cells, and to analyze the effect of Linc-ROR on the process of epithelial mesenchymal transition of high grade ovarian serous carcinoma cells through Wnt/ beta -catenin pathway. Methods: (1) the expression of Linc-ROR m RNA in high grade ovarian serous carcinoma tissue, 20 normal ovarian tissue and 20 normal oviduct end tissues was detected by real time fluorescence quantitative PCR, and the relationship between Linc-ROR expression and FIGO staging of high grade ovarian serous carcinoma and lymph node transfer was analyzed. (2) normal ovarian serous papilloma was cultured. Adenocarcinoma cell line SKOV3, synthesized Linc-ROR Si RNA and Linc-ROR overexpressed plasmid (P IRES2-EGFP-Linc-ROR recombinant vector), transfected SKOV3 cells respectively, using live cell count (CCK-8) method, cell scratch test in vitro and membrane (Transwell) chamber invasion test to detect the proliferation, migration, invasion ability and Western blot of SKOV3 cells after transfection respectively. Detection of epithelial mesenchymal transition related markers E-cadherin, beta -catenin, vimentin and Wnt/ beta -catenin pathway target gene c-myc expression changes. (3) the application of Wnt/ beta -catenin pathway activator lithium chloride (Li Cl) on SKOV3 cells Protein expression changes; Li Cl+Linc-ROR Si RNA co acted on SKOV3 cells, and Li Cl+si RNA-NC group was set as negative control. Western blot method was used to detect E-cadherin, beta -catenin, and protein expression changes. Results: (1) real time fluorescence quantitative assay showed the expression in the high grade ovarian serous carcinoma tissue The level was 4.38 + 2.55, obviously higher than 1.49 + 1.69 in normal ovarian epithelial tissue, 1.45 + 1.57 (F=8.62, P0.01) in normal oviduct end tissue, and the expression level of Linc-ROR m RNA increased obviously with the increase of FIGO staging of high grade ovarian serous carcinoma (F=95.70, P0.01), and the lymph node metastasis was significantly higher than that of no lymph node metastasis. (t=7.40, P0.01). (2) the expression level of Linc-ROR m RNA in the cells transfected with Linc-ROR Si RNA was significantly lower than that of the negative control Si RNA-NC group (F=26.29, P0.05), and the proliferation, migration and invasion ability of the cells were significantly lower than those in the negative control group (all 0.01). The expression of imentin and c-myc protein decreased significantly (P 0.01). (3) the expression level of Linc-ROR m RNA in the transfected plasmids ROR group was significantly higher than that of the negative control Vector group (t=6.304, P0.01), and the proliferation, migration and invasion ability of the ROR group were significantly enhanced (0.01), and the protein table was significantly higher than that of the negative control Vector group. The expression of beta -catenin, vimentin and c-myc protein increased significantly (P 0.01). (4) after different concentrations of Li Cl at different time, the proliferation of cell proliferation was statistically significant (P0.05). 10mmol/L Li Cl effect was most obvious to the proliferation of SKOV3 cells, compared with the blank control group. The expression of E-cadherin protein in SKOV3 cells, up regulation of the expression of beta -catenin, vimentin and c-myc protein, was statistically significant (P0.01). Compared with the Li Cl+si RNA-NC group, the expression of Li Cl+Linc-ROR Si was significantly higher than that of the Li Cl+si RNA-NC group, and the expression of beta and protein was significantly decreased, and the difference was statistically significant (0.01 Conclusion: the abnormal expression of Linc-ROR is closely related to the invasion and metastasis of high grade ovarian serous carcinoma. High expression of Linc-ROR can promote the epithelial mesenchymal transition of ovarian cancer cells and may play a regulatory role through the Wnt/ beta -catenin pathway. Therefore, Linc-ROR may mediate the invasion and metastasis of high grade ovarian serous carcinoma.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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