摘要
目的构建真核细胞延长因子2(eukaryotic elongation factor 2,eEF2)基因的RNA干扰(RNAi)慢病毒质粒及稳定转染的乳腺癌细胞模型,为后续研究eEF2在乳腺癌细胞发生发展中的作用提供实验依据。方法根据eEF2的基因序列和短发夹RNA序列的设计原则设计合成3对shRNA序列,将shRNA序列复性后插入慢病毒载体LV-U6-shRNA-ZSgreen-Puro,构建3种不同eEF2基因敲低靶点的重组质粒sh1、sh2、sh3,以空载体作为阴性对照组(shNC)。采用慢病毒三质粒包装系统共分别转染人肾上皮细胞HEK293T,进行病毒包装和扩增。经酶切及测序验证正确后,将重组慢病毒感染对数生长期的乳腺癌细胞MCF-7,72 h后荧光显微镜观察绿色荧光强度,实时荧光定量PCR(qPCR)法检测eEF2 mRNA转录水平,Western blot法检测eEF2蛋白表达水平。结果eEF2 shRNA载体测序与原设计序列完全一致。重组慢病毒感染的MCF-7中可见绿色荧光蛋白表达。与shNC组相比,eEF2敲低的sh2和sh3组MCF-7细胞中eEF2 mRNA的转录水平明显降低(t分别为9.244和5.938,P分别为0.001和0.004);sh1、sh2和sh3组细胞中eEF2蛋白的表达水平均明显降低(t分别为3.552、9.614和4.432,P分别为0.024、0.001和0.011)。结论成功构建了eEF2基因低表达的慢病毒质粒及稳定转染的乳腺癌细胞模型,有望为临床乳腺癌的治疗提供基于翻译靶向治疗的新靶点。
Objective To construct RNA interference(RNAi)lentivirus plasmid of eukaryotic elongation factor 2(eEF2)gene and stably transfected breast cancer cell model,so as to provide experimental basis for further study on the role of eEF2in the occurrence and development of breast cancer.Methods According to eEF2 gene sequence and the design principles of short hairpin RNA sequence,three pairs of shRNA sequences were designed and synthesized,which were refolded and inserted into lentivirus vector LV-U6-shRNA-ZSgreen-Puro.Three recombinant plasmids sh1,sh2 and sh3 with different knock-down targets of eEF2 gene were constructed,and empty plasmid vectors were used as negative control group(shNC).Human renal epithelial cells HEK293T were co-transfected with three lentivirus plasmid packaging system for virus packaging and amplification respectively.After identification by digestion and sequencing,the recombinant lentivirus was used to infect breast cancer cells MCF-7 in logarithmic growth period.After 72 h,the green fluorescence intensity was observed under a fluorescence microscope,the mRNA transcription level of eEF2 was determined by fluorescence quantitative PCR(qPCR),and the expression level of eEF2 protein was detected by Western blot.Results The sequence of eEF2shRNA vector was identical to the original sequence.The expression of green fluorescent protein was observed in breast cancer cells MCF-7 infected with recombinant lentivirus.Compared with shNC group,the mRNA transcription levels of eEF2 in sh2 and sh3 group MCF-7 cells with eEF2 knockdown decreased significantly(t=9.244 and 5.938,P=0.001and 0.004,respectively).The expression levels of eEF2 protein in sh1,sh2 and sh3 groups decreased significantly(t=3.552,9.614 and 4.432,P=0.024,0.001 and 0.011,respectively).Conclusion The lentivirus vector with low expression of eEF2 gene and the stably transfected breast cancer cell model were successfully constructed,which is expected to provide a new target for clinical treatment of breast cancer based on translation-targeted therapy.
作者
张婷婷
薛媛
朱超
段明廷
陈伟豪
常欣悦
王艳红
贾红燕
ZHANG Tingting;XUE Yuan;ZHU Chao;DUAN Mingting;CHEN Weihao;CHANG Xinyue;WANG Yanhong;JIA Hongyan(School of Basic Medicine,Basic Medical Sciences Center,Shanxi Medical University,Jinzhong 030600,Shanxi Province,China;不详)
出处
《中国生物制品学杂志》
CAS
CSCD
2024年第9期1050-1055,共6页
Chinese Journal of Biologicals
基金
山西省归国留学人员科研基金(2021-157)
山西省回国留学人员科研资助项目(2022-119)。
