C14orf166调节p-Snail亚细胞定位促进IL-8诱导的乳腺癌EMT

发布时间：2018-07-03 10:21

本文选题：C14orf166 + Snail　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的探讨炎症细胞因子IL-8(interleukin-8)通过C14orf166调控Snail并促进乳腺癌上皮间质转化(Epithelial-mesenchymal transition,EMT)分子机制,解析C14orf166在乳腺癌疾病进程中的作用。方法1、将乳腺癌细胞株MCF-7/SK-BR-3分为以下四组:PBS、IL-8、IL-8+LY294002(PI3K/Akt信号通路抑制剂)及IL-8+DMSO,采用免疫印迹试验(Western blotting,WB)和半定量PCR(Semi-quantitative Real-time PCR)检测C14orf166表达情况;并在乳腺癌细胞中加入C14orf166特异性化学合成小干扰RNA(Small interfering RNA,si RNA),观察Snail蛋白水平是否受IL-8的调控。2、在乳腺癌细胞株MCF-7、SK-BR-3中转染C14orf166特异性si RNA或过表达质粒(p CMV-tag2B-C14orf166),采用Western blotting及实时荧光定量PCR(Quantitative Real-time PCR,Q-PCR)观察Snail及EMT相关分子标志物E-cadherin、Vimentin、Fibronectin蛋白水平变化,同时通过划痕试验(Scratch assay)和侵袭试验(Transwell assay)检测细胞侵袭、迁移功能变化。3、在乳腺癌细胞中过表达C14orf166,同时用翻译抑制剂放线菌酮(Cycloheximide,CHX)处理乳腺癌细胞,通过Western blotting及灰度值扫描,检测Snail半衰期变化,确定C14orf166是否调控Snail翻译,IL-8是否能上调乳腺癌细胞磷酸化Snail(p-Snail(S246))。加入C14orf166 sh RNA慢病毒或过表达C14orf166,Western blotting观察p-Snail水平,并进一步分离核浆蛋白观察p-Snail在核浆分布变化情况。4、用C14orf166 sh RNA慢病毒感染乳腺癌细胞MCF-7,嘌呤霉素(Puromycin)筛选C14orf166稳定干扰细胞株,接种至裸鼠脂肪垫,待成瘤后于原位注射PBS或IL-8,观察肿瘤体积变化。4周后处死小鼠,取移植瘤小鼠肿瘤及肝、肺组织,通过免疫组织化学(immunohistochemistry,IHC)方法观察C14orf166、Snail、p-Snail及EMT相关分子标志物的变化。HE染色观察肿瘤转移情况。5、收集乳腺癌及正常乳腺组织临床标本,通过IHC技术检测CXCRB、C14orf166、Snail及EMT分子标志物等表达水平,以及分析与肿瘤恶性程度和迁移转移的相关性。通过Kaplan-Meier Plotter分析,预测C14orf166表达水平与乳腺癌病人生存的相关性。结果1、Western blotting及半定量PCR结果均表明,PI3K/Akt信号通路抑制剂LY294002可抑制IL-8对C14orf166的上调。当C14orf166被特异性干扰,IL-8则失去上调Snail的能力。2、Western blotting结果表明,在MCF-7、SK-BR-3细胞中敲减C14orf166,Snail蛋白水平明显下降,同时上皮标志物随之上升,而间质标志物随之下降,细胞侵袭迁移能力明显受到抑制;在MCF-7、SK-BR-3细胞中过表达C14orf166,Snail蛋白水平明显上升,同时EMT相关标志物也发生了相应变化,细胞侵袭迁移能力明显上升。然而,定量PCR结果表明,Snail的m RNA水平在这两种处理因素下并未受到明显影响。3、Western blotting结果表明,CHX处理细胞后,C14orf166过表达组中Snail半衰期明显延长,且p-Snail在IL-8刺激后随时间上升。当敲减C14orf166时,p-Snail蛋白水平随之下降,并在细胞核中分布减少。同时,当过表达C14orf166时,p-Snail蛋白水平也随之上升,在细胞核中聚集增多。4、IL-8能促进乳腺癌移植瘤小鼠肿瘤生长,当干扰C14orf166时,IL-8对乳腺肿瘤的促进作用明显受到抑制。IHC结果证明,IL-8可在体内促进C14orf166、Snail、p-Snail、Vimentin及Fibronectin的表达,抑制E-cadherin的表达,而C14orf166的缺失明显抑制了IL-8引起的上述变化。IL-8能促进乳腺癌转移,而C14orf166的缺失抑制了乳腺癌发生肝肺转移。5、IHC染色表明,随着乳腺组织恶性程度增加,CXCRB、C14orf166、Snail、Vimentin、Fibronectin表达明显增强,E-cadherin表达明显减弱。生存曲线表明,高水平的C14orf166与乳腺癌病人高死亡率具有明显相关性。结论1、IL-8通过PI3K/Akt通路上调C14orf166而介导Snail的表达。2、C14orf166在乳腺癌细胞中通过调控Snail而影响乳腺癌细胞EMT发生及细胞功能变化。3、C14orf166对乳腺癌细胞侵袭迁移功能的影响是通过调控p-Snail核转位发挥作用的。4、体内试验证明,干扰C14orf166可抑制乳腺癌生长及侵袭转移。5、临床标本中,C14orf166与乳腺癌恶性程度的相关基因表达具有明显相关性,且高水平的C14orf166可促进乳腺癌患者死亡率升高。
[Abstract]:Objective to investigate the role of inflammatory cytokine IL-8 (interleukin-8) to regulate Snail by C14orf166 and to promote the molecular mechanism of Epithelial-mesenchymal transition (EMT), and to analyze the role of C14orf166 in the process of breast cancer. Method 1, the breast cancer cell strains are divided into four groups: PBS, IL-8, IL-8+LY294002 ( PI3K/Akt signaling pathway inhibitors) and IL-8+DMSO were detected by Western blot test (Western blotting, WB) and semi quantitative PCR (Semi-quantitative Real-time PCR), and C14orf166 specific chemical synthesis was added to the breast cancer cells to observe whether the protein level was affected. The regulatory.2 of IL-8 was transferred to the C14orf166 specific Si RNA or the overexpressed plasmid (P CMV-tag2B-C14orf166) in the breast cancer cell line MCF-7 and SK-BR-3. The cell invasion was detected by the scratch test (Scratch assay) and the invasion test (Transwell assay), the migration function was changed.3, the C14orf166 was overexpressed in the breast cancer cells, and the breast cancer cells were treated with the translation inhibitor actinomycone (Cycloheximide, CHX). The Snail half life changes were detected by the Western blotting and the gray value scan, and C14 was determined. Whether or not orf166 regulates Snail translation, whether IL-8 can increase the phosphorylation of Snail (p-Snail (S246)) in breast cancer cells. C14orf166 sh RNA lentivirus or overexpression C14orf166. Cells MCF-7, purinamycin (Puromycin) screening C14orf166 stable interference cell lines, inoculated into the nude mouse fat pad, after the tumor to be injected in situ PBS or IL-8, observe the tumor volume.4 weeks after the death of mice, take the tumor and liver, lung tissue of the transplanted tumor mice, observe C14orf166, Snail, through the immunohistochemical (immunohistochemistry, IHC) method. P-Snail and EMT related molecular markers change.HE staining to observe tumor metastasis.5, collect breast cancer and normal breast tissue clinical specimens, detect the expression level of CXCRB, C14orf166, Snail and EMT molecular markers by IHC technique, and analyze the correlation with tumor malignancy and migration and metastasis. Kaplan-Meier Plotter score. The correlation between C14orf166 expression level and the survival of breast cancer patients was predicted. Results 1, Western blotting and semi quantitative PCR results showed that the PI3K/Akt signaling pathway inhibitor LY294002 inhibited the up-regulated IL-8 on C14orf166. When C14orf166 was specifically disturbed, IL-8 lost the Snail capacity.2. The level of C14orf166, Snail protein in SK-BR-3 cells decreased and the epithelial markers increased, while the interstitial markers decreased, and the cell invasion and migration ability was obviously inhibited. In MCF-7, SK-BR-3 cells overexpressed C14orf166, the level of Snail protein increased obviously, and the EMT related markers changed correspondingly. The ability of cell invasion and migration increased significantly. However, quantitative PCR results showed that the m RNA level of Snail was not significantly affected by.3, and Western blotting results showed that the Snail half life of C14orf166 overexpression group was obviously prolonged after CHX processing cells, and p-Snail was increased after IL-8 stimuli. The level of p-Snail protein decreased and decreased in the nucleus. At the same time, when the C14orf166 was expressed, the level of p-Snail protein increased, and the accumulation of.4 in the nucleus was increased. IL-8 could promote the growth of the tumor of breast cancer transplanted mice. When the C14orf166 was disturbed, the effect of IL-8 on breast cancer was obviously inhibited by.IHC. IL-8 can promote the expression of C14orf166, Snail, p-Snail, Vimentin and Fibronectin in the body, and inhibit the expression of E-cadherin, and the absence of C14orf166 obviously inhibits the above changes caused by IL-8.IL-8 can promote the metastasis of breast cancer, while C14orf166 deletion inhibits the liver and lung metastasis.5 of breast cancer. The expression of CXCRB, C14orf166, Snail, Vimentin, Fibronectin was obviously enhanced and the expression of E-cadherin decreased obviously. The survival curve showed that the high level of C14orf166 had a significant correlation with the high mortality rate of breast cancer patients. Conclusion 1, IL-8 is promoted by PI3K/Akt pathway up C14orf166 and mediates Snail expression.2. The effect of EMT on breast cancer cells and cell function changes by regulating Snail is.3. The effect of C14orf166 on the invasion and migration of breast cancer cells is.4 that regulates the role of p-Snail nuclear transposition. In vivo experiments have shown that interference with C14orf166 can inhibit the growth and invasion of breast cancer, and in clinical specimens, C14orf166 and malignant progression of breast cancer There was a significant correlation between the degree of gene expression and the high level of C14orf166 in patients with breast cancer.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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