本文選題：高級別卵巢漿液性癌 + 長鏈非編碼RNA��； 參考：《青島大學(xué)》2017年碩士論文

【摘要】：目的:分析基因間長鏈非編碼RNA-ROR(Linc-ROR)在高級別卵巢漿液性癌組織中的表達(dá),探討Linc-ROR表達(dá)與高級別卵巢漿液性癌細(xì)胞生物學(xué)功能的關(guān)系,并分析Linc-ROR通過Wnt/β-catenin通路對高級別卵巢漿液性癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化過程的影響。方法:(1)實(shí)時熒光定量PCR檢測34例高級別卵巢漿液性癌組織、20例正常卵巢上皮組織和20例正常輸卵管傘端組織中Linc-ROR m RNA的表達(dá),并分析Linc-ROR表達(dá)與高級別卵巢漿液性癌FIGO分期、淋巴結(jié)轉(zhuǎn)移的關(guān)系。(2)常規(guī)培養(yǎng)卵巢漿液性乳頭狀囊腺癌細(xì)胞系SKOV3,合成Linc-ROR si RNA和Linc-ROR過表達(dá)質(zhì)粒(p IRES2-EGFP-Linc-ROR重組載體),分別轉(zhuǎn)染SKOV3細(xì)胞,采用活細(xì)胞計(jì)數(shù)(CCK-8)法、體外細(xì)胞劃痕實(shí)驗(yàn)和穿膜(transwell)小室侵襲實(shí)驗(yàn)分別檢測轉(zhuǎn)染后SKOV3細(xì)胞的增殖、遷移、侵襲能力,蛋白印跡實(shí)驗(yàn)檢測上皮間質(zhì)轉(zhuǎn)化相關(guān)標(biāo)志物E-cadherin、β-catenin、vimentin和Wnt/β-catenin通路靶基因c-myc的表達(dá)變化。(3)應(yīng)用Wnt/β-catenin通路激活劑氯化鋰(Li Cl)作用于SKOV3細(xì)胞后,CCK-8法觀察細(xì)胞增殖情況,western blot法檢測E-cadherin、β-catenin、vimentin和c-myc的蛋白表達(dá)變化;將Li Cl+Linc-ROR si RNA共同作用SKOV3細(xì)胞,另設(shè)Li Cl+si RNA-NC組作為陰性對照,western blot法檢測E-cadherin、β-catenin、vimentin和c-myc的蛋白表達(dá)變化。結(jié)果:(1)實(shí)時熒光定量PCR實(shí)驗(yàn)顯示,Linc-ROR m RNA在高級別卵巢漿液性癌組織中的表達(dá)水平為4.38±2.55,明顯高于正常卵巢上皮組織中的1.49±1.69、正常輸卵管傘端組織中的1.45±1.57(F=8.62,P0.01);Linc-ROR m RNA的表達(dá)水平隨著高級別卵巢漿液性癌FIGO分期的遞增明顯升高(F=95.70,P0.01),且伴有淋巴結(jié)轉(zhuǎn)移者明顯高于無淋巴結(jié)轉(zhuǎn)移者(t=7.40,P0.01)。(2)轉(zhuǎn)染Linc-ROR si RNA組細(xì)胞中Linc-ROR m RNA的表達(dá)水平明顯低于其陰性對照si RNA-NC組(F=26.29,P0.05);與陰性對照si RNA-NC組相比,si RNA組SKOV3細(xì)胞的增殖、遷移和侵襲能力均明顯降低(P均0.01),E-cadherin蛋白表達(dá)量明顯升高,β-catenin、vimentin和c-myc蛋白表達(dá)量明顯減少(P均0.01)。(3)轉(zhuǎn)染過表達(dá)質(zhì)粒ROR組細(xì)胞中Linc-ROR m RNA的表達(dá)水平明顯高于其陰性對照Vector組(t=6.304,P0.01);與陰性對照Vector組相比,ROR組SKOV3細(xì)胞的增殖、遷移和侵襲能力均明顯增強(qiáng)(P均0.01),E-cadherin蛋白表達(dá)量明顯降低,β-catenin、vimentin和c-myc蛋白表達(dá)量明顯增加(P均0.01)。(4)不同濃度Li Cl處理SKOV3細(xì)胞不同時間后,對細(xì)胞的增殖差異有統(tǒng)計(jì)學(xué)意義(P0.05),以10mmol/L Li Cl作用24小時對SKOV3細(xì)胞增殖的促進(jìn)最明顯。與空白對照組相比,Li Cl能明顯下調(diào)SKOV3細(xì)胞E-cadherin蛋白表達(dá),上調(diào)β-catenin、vimentin和c-myc蛋白表達(dá),差異具有統(tǒng)計(jì)學(xué)意義(P0.01);與Li Cl+si RNA-NC組相比,Li Cl+Linc-ROR si RNA組E-cadherin蛋白表達(dá)量明顯升高,β-catenin、vimentin和c-myc蛋白表達(dá)量明顯降低,差異具有統(tǒng)計(jì)學(xué)意義(P均0.01)。結(jié)論:Linc-ROR異常表達(dá)與高級別卵巢漿液性癌的侵襲、轉(zhuǎn)移密切相關(guān)。高表達(dá)Linc-ROR能促進(jìn)卵巢癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化,可能通過Wnt/β-catenin通路而發(fā)揮調(diào)控作用。因此,Linc-ROR可能介導(dǎo)高級別卵巢漿液性癌侵襲、轉(zhuǎn)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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Shi-Ping Liu;Jia-Xin Yang;Dong-Yan Cao;Keng Shen;;Identification of differentially expressed long non-coding RNAs in human ovarian cancer cells with different metastatic potentials[J];Cancer Biology & Medicine;2013年03期

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本文編號：2089257