摘要
目的探讨STGC3基因在鼻咽癌发生发展中的作用机制。方法设计基于miRNA30框架的STGC3特异性shRNA,构建STGC3特异性miR30-shRNA真核表达载体pRNAT-U6/shRNA-STGC3,转染永生化人鼻咽上皮细胞系NP69;采用RT-PCR检测未转染、转pRNAT-U6空质粒和转pRNAT-U6/shRNA-STGC3组NP69细胞中STGC3基因mRNA的表达;使用流式细胞仪检测3组细胞周期分布与细胞凋亡情况。结果与未转染组和转pRNAT-U6空质粒组相比,转染pRNAT-U6/shRNA-STGC3的NP69细胞中STGC3基因mRNA表达下降(P(0.05),G0/G1期细胞比例下降,G2期和S期的细胞比例之和增加(P(0.05)。结论 miR30-based shRNA成功地下调了STGC3在NP69细胞系中的表达;STGC3基因在NP69细胞系的表达下调,使NP69细胞生长增殖速度加快,凋亡减少,提示STGC3对NP69细胞增殖具有抑制作用。
Objective To analyze the suppress tumor mechanism of STGC3 in the development of nasopharyngeal carcinoma.Methods We designed shRNA targeted STGC3 based on the construction features of mcrioRNA30 and constructed the recombinant expression vector.The recombinant expression vector pRNAT-U6/shRNA-STGC3 and the blank vector pRNAT-U6 were transiently transfected into human immortalized nasopharyngeal epithelial cell line NP69 using Lipofectamine 2000,and the expression of fluorescin protein was observed by fluorescence microscope.The mRNA expression level of STGC3 was detected by RT-PCR.Cycle distribution and apoptosis of NP69 were detected after STGC3 gene expression down-regulated by FCM.Results Bacteria Liquid PCR,restriction enzyme digestion and DNA sequencing demonstrated that the recombinant expression vector pRNAT-U6/shRNA-STGC3 was constructed successfully.The expression of fluorescin protein demonstrated that the vectors were transfected into NP69 cell successfully.The result of RT-PCR showed that STGC3 gene expression of pRNAT-U6/shRNA-STGC3/NP69 was repressed compared with pRNAT-U6/NP69 and NP69（p﹤0.05）.FCM analysis indicated that the cell percentage in G0/G1 phase was lowered,and G2＋S phase increased（P﹤0.05）.Conclusion 1.The eukaryotic expression vector pRNAT-U6/shRNA-STGC3 was constructed successfully.2.The miRNA30-based shRNA targeted STGC3 down-regulated the expression level of STGC3 in the cell line NP69.3.The low-expression of STGC3 induced that the growth and proliferation of NP69 Cell Line were increased,and the apoptosis rate was lowered.
出处
《中南医学科学杂志》
CAS
2011年第2期144-148,共5页
Medical Science Journal of Central South China
基金
国家自然科学基金项目(30470967)
湖南省自然科学基金重点项目(09JJ3071)
