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

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 MGC803of LncRNA-GHET1and observe the expression level of LncRNA-GHET1in gastric cancer cell line MGC803.Methods:LncRNA-GHET1was amplified from human gastric cancer cell line MGC803and cloned into pcDNA3.1(-)vector to construct eukaryotic expression vector pcDNA3.1-GHET1.PcDNA3-GHIT1,which was constructed by liposome assay,was transfected into gastric cancer cell line MGC803,and the cell line which stably expressing LncRNA-GHET1were screened by G418and verified by RT-qPCR.Results:The eukaryotic expression vector pcDNA3.1-GHET1was successfully constructed by double digestion analysis and sequencing validation.pcDNA3.1-GHET1was transfected into gastric cancer cell MGC803with transfection efficiency over95%,and overexpressing stably-transfected gastric cancer cell line MGC803of LncRNA-GHET1was successfully constructed by RT-qPCR verification.Conclusions:The successful construction of eukaryotic expression vector pcDNA3.1-GHET1and overexpressing stably-transfected gastric cancer cell line MGC803of LncRNA-GHET1could be utilized for further study in LncRNA-GHET1gene in the development of gastric cancer.