ARA55基因在CNE2鼻咽癌细胞中的功能研究
发布时间：2019-05-29 19:52
【摘要】：目的:1.构建ARA55基因的真核表达载体并完成相关鉴定,研究ARA55基因过表达对CNE2鼻咽癌细胞生物学特性的影响;2.明确ARA55在TGFβ1介导的CNE2细胞上皮间质转化(EMT)及侵袭、迁移中的作用。方法:1.通过PCR扩增获得ARA55基因全长cDNA序列,纯化后用HindIII和XhoI双酶切消化,经T4DNA连接酶作用,连接至pCMV-C-EGFP真核表达载体,连接产物经转化、挑取克隆、扩增培养后,提取小量质粒,进行DNA测序鉴定;用HindIII和XhoI内切酶消化酶消化连接产物,琼脂糖凝胶电泳鉴定。重组质粒经ZLip2000转染至CNE2鼻咽癌细胞,荧光显微镜观察EGFP的表达情况,免疫印迹检测ARA55-EGFP融合蛋白的表达水平;通过CCK-8比色、划痕修复实验、Transwell小室、AnnexinV-PE/7-AAD双荧光染色、DNA梯状电泳及蛋白印迹检测凋亡蛋白变化等实验,探讨ARA55过表达对CNE2鼻咽癌细胞生物学特性的影响。2.利用一定浓度的TGFβ1诱导CNE2细胞株中ARA55的表达,免疫印迹检测ARA55蛋白及EMT相关标志物N-cadherin、Claudin-1等的表达变化;采用ARA55的siRNA质粒,经X-tremeGENEsiRNA转染至CNE2细胞株;通过CCK-8比色、划痕修复、Transwell侵袭迁移等实验,研究TGFβ1介导的ARA55表达上调及沉默以ARA55表达对CNE2鼻咽癌细胞EMT及侵袭迁移的影响。结果:1.DNA测序及双酶切分析显示pCMV-ARA55-EGFP重组载体构建成功。2.CCK-8比色、划痕修复实验、Transwell小室等结果显示pCMV-ARA55-EGFP组细胞生长增殖、侵袭迁移能力明显低于pCMV-C-EGFP空载体组和/或空白对照组(P0.05或0.01);AnnexinV-PE/7-AAD双荧光染色及DNA梯状电泳结果可见,pCMV-ARA55-EGFP组细胞产生凋亡,且凋亡率明显高于pCMV-C-EGFP空载体组(P0.05);免疫印迹显示pCMV-ARA55-EGFP组细胞Bcl-2表达下调,CytochromeC表达上调,同时伴有Caspase-9和Caspase-3的激活。3.TGFβ1诱导后,免疫印迹显示ARA55在CNE2细胞中的表达上调;ARA55诱导组细胞发生间质样改变,N-cadherin的表达上升,Claudin-1表达下降,同时细胞的生长增殖及侵袭迁移能力明显高于对照组(P0.05或0.01);诱导表达的ARA55通过siRNA下调后,siRNA-ARA55组细胞生长增殖及侵袭迁移能力下降(P0.05或0.01)。结论:1.成功构建pCMV-ARA55-EGFP重组载体;2.ARA55过表达可抑制CNE2鼻咽癌细胞生长增殖,诱导凋亡;3.ARA55参与了TGFβ1介导的CNE2细胞EMT及侵袭迁移过程。
[Abstract]:Objective: 1. The eukaryotic expression vector of ARA55 gene was constructed and identified to study the effect of overexpression of ARA55 gene on the biological characteristics of CNE2 nasopharyngeal carcinoma cells. 2. To investigate the role of ARA55 in TGF 尾 1 mediated epithelial stroma transformation, invasion and migration of CNE2 cells. Method: 1. The full-length cDNA sequence of ARA55 gene was obtained by PCR amplification, purified and digested by HindIII and XhoI, and ligated to pCMV-C-EGFP eukaryotic expression vector by T4DNA ligase. The ligated product was transformed, cloned and cultured. A small amount of plasmid was extracted and identified by DNA sequencing. The ligated products were digested by HindIII and XhoI endonuclease and identified by agarose gel electrophoresis. The recombinant plasmid was transformed into CNE2 nasopharyngeal carcinoma cells by ZLip2000. The expression of EGFP was observed by fluorescence microscope and the expression level of ARA55-EGFP fusion protein was detected by immunoblotting. The changes of apoptotic proteins were detected by CCK-8 colorimetric assay, scratch repair test, Transwell chamber, AnnexinV-PE/7-AAD double fluorescence staining, DNA ladder electrophoresis and Western imprinting. To investigate the effect of overexpression of ARA55 on the biological characteristics of CNE2 nasopharyngeal carcinoma cells. 2. The expression of ARA55 in CNE2 cell line was induced by a certain concentration of TGF 尾 1, the expression of ARA55 protein and EMT related marker N 鈮，
本文编号：2488181
[Abstract]:Objective: 1. The eukaryotic expression vector of ARA55 gene was constructed and identified to study the effect of overexpression of ARA55 gene on the biological characteristics of CNE2 nasopharyngeal carcinoma cells. 2. To investigate the role of ARA55 in TGF 尾 1 mediated epithelial stroma transformation, invasion and migration of CNE2 cells. Method: 1. The full-length cDNA sequence of ARA55 gene was obtained by PCR amplification, purified and digested by HindIII and XhoI, and ligated to pCMV-C-EGFP eukaryotic expression vector by T4DNA ligase. The ligated product was transformed, cloned and cultured. A small amount of plasmid was extracted and identified by DNA sequencing. The ligated products were digested by HindIII and XhoI endonuclease and identified by agarose gel electrophoresis. The recombinant plasmid was transformed into CNE2 nasopharyngeal carcinoma cells by ZLip2000. The expression of EGFP was observed by fluorescence microscope and the expression level of ARA55-EGFP fusion protein was detected by immunoblotting. The changes of apoptotic proteins were detected by CCK-8 colorimetric assay, scratch repair test, Transwell chamber, AnnexinV-PE/7-AAD double fluorescence staining, DNA ladder electrophoresis and Western imprinting. To investigate the effect of overexpression of ARA55 on the biological characteristics of CNE2 nasopharyngeal carcinoma cells. 2. The expression of ARA55 in CNE2 cell line was induced by a certain concentration of TGF 尾 1, the expression of ARA55 protein and EMT related marker N 鈮，
本文编号：2488181
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