贵州医科大学学报

2018, v.43;No.219(12) 1365-1369

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LncRNA-GHET1过表达胃癌细胞株的构建与验证
Construction and Identification of LncRNA-GHET1 Overexpression Cell Line

刘娟娟;周春欢;夏英;赵娟娟;万颖;黄海;
LIU Juanjuan;ZHOU Chunhuan;XIA Ying;ZHAO Juanjuan;WAN Ying;HUANG Hai;Department of Medical Laboratory Sciences,Guizhou Medical University;1st Affiliated Hospital of Guiyang University of Chinese Medicine;The Affiliated Hospital of Guizhou Medical University;

摘要(Abstract):

目的:构建与验证LncRNA-GHET1过表达的胃癌细胞株MGC803,观察LncRNA-GHET1在胃癌细胞株MGC803中表达情况。方法:从人胃癌细胞株MGC803中扩增出LncRNA-GHET1,将其克隆到pc DNA3. 1(-)载体上,构建真核表达载体pc DNA3. 1-GHET1;利用脂质体法将构建好的pc DNA3. 1-GHET1转染胃癌细胞株MGC803,并用G418筛选出LncRNA-GHET1稳定表达的细胞株;通过RT-qPCR验证筛选的细胞株。结果:经双酶切分析以及测序验证,成功构建真核表达载体pc DNA3. 1-GHET1; pc DNA3. 1-GHET1转染胃癌细胞株MGC803,转染效率达95%以上,经RT-qPCR验证,成功构建LncRNA-GHET1过表达稳定转染胃癌细胞株MGC803。结论:成功构建真核表达载体pc DNA3. 1-GHET1以及LncRNA-GHET1过表达稳定转染胃癌细胞株MGC803,为进一步研究LncRNA-GHET1基因在胃癌发生发展中的作用奠定基础。
Objective: To construct and identify overexpressing gastric cancer cell line MGC803 of LncRNA-GHET1 and observe the expression level of LncRNA-GHET1 in gastric cancer cell line MGC803. Methods: LncRNA-GHET1 was amplified from human gastric cancer cell line MGC803 and cloned into pcDNA3. 1(-) vector to construct eukaryotic expression vector pcDNA3. 1-GHET1. Pc DNA3-GHIT1,which was constructed by liposome assay,was transfected into gastric cancer cell line MGC803,and the cell line which stably expressing LncRNA-GHET1 were screened by G418 and verified by RT-qPCR. Results: The eukaryotic expression vector pcDNA3. 1-GHET1 was successfully constructed by double digestion analysis and sequencing validation. pcDNA3. 1-GHET1 was transfected into gastric cancer cell MGC803 with transfection efficiency over 95%,and overexpressing stablytransfected gastric cancer cell line MGC803 of LncRNA-GHET1 was successfully constructed by RTqPCR verification. Conclusions: The successful construction of eukaryotic expression vector pcDNA3. 1-GHET1 and overexpressing stably-transfected gastric cancer cell line MGC803 of LncRNAGHET1 could be utilized for further study in LncRNA-GHET1 gene in the development of gastric cancer.

关键词(KeyWords): 长链非编码RNA;LncRNA-GHET1;过表达;胃肿瘤;癌;真核表达载体
long non-coding RNA;LncRNA-GHET1;overexpression;gastric neoplasms;carcinoma;eukaryotic expression vector

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(81460364,81760429);; 贵阳市科技计划项目(20161001015)

作者(Author): 刘娟娟;周春欢;夏英;赵娟娟;万颖;黄海;
LIU Juanjuan;ZHOU Chunhuan;XIA Ying;ZHAO Juanjuan;WAN Ying;HUANG Hai;Department of Medical Laboratory Sciences,Guizhou Medical University;1st Affiliated Hospital of Guiyang University of Chinese Medicine;The Affiliated Hospital of Guizhou Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2018.12.01

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