摘要
目的:构建胃癌细胞核转录因子SP1的3′非翻译区(3′UTR)双荧光素酶报告基因载体,验证microRNA-7(miR-7)调控SP1的分子机制。方法:应用相关生物信息学软件预测miR-7靶向作用于SP1基因的3′UTR区;PCR扩增人胃黏膜上皮细胞SP1的3′UTR,插入至psiCHECK2载体双荧光素酶基因下游,构建野生型(w-3′UTR)和突变型重组表达载体(m-3′UTR),鉴定正确后,分别与miR-7模拟物(mimics)/抑制物(inhibitor)/阴性对照物(NC)共转染胃癌细胞MKN45,检测荧光素酶活性及SP1表达情况。结果:成功构建SP1的3′UTR野生型(psiCHECK2-SP1-w-3′UTR)和突变型(psiCHECK2-SP1-m-3′UTR)表达载体,双荧光素酶报告基因检测显示w-3′UTR/miR-7-mimics组的相对荧光素酶活性表达受抑制,与miR-7 NC组比较降低75%,差异具有统计学意义(P<0.001),而m-3′UTR/miR-7-mimics组的活性表达没有受到影响(P>0.05)。RT-PCR和Western blot检测结果显示miR-7-mimics组SP1表达水平低于miR-7 NC组(P<0.001),而miR-7-inhibitor组SP1表达水平高于miR-7 NC组(P<0.001)。结论:成功构建SP1的3′UTR野生型双荧光素酶报告基因表达载体,miR-7能够显著降低其荧光素酶活性,提示miR-7能靶向负调控SP1的表达。
Objective:To construct the dual luciferase reporter plasmid psiCHECK2-SP1-w-3′UTR and its mutant plasmid psiCHECK2-SP1-m-3′UTR in order to clarify the molecular mechanism of microRNA-7(miR-7)regulating gastric cancer cell SP1 expression.Methods:Relevant bioinformatics software was used to predict miR-7 targeting the 3′UTR region of SP1 gene.The full sequence of SP13′UTR gene was amplified and inserted into the downstream of luciferase gene in psiCHECK2 vector.A wild type of dual luciferase reporter plasmid of psiCHECK2-SP1-w-3′UTR was constructed.Meanwhile,a modified type of dual luciferase reporter plasmid psiCHECK2-SP1-m-3′UTR was constructed by fusion PCR.When both of vectors were verified by PCR,restriction endonuclease and DNA sequencing to be true,they were cotransfected into gastric cancer MKN45 cells with miR-7 mimics,miR-7 inhibitor and miR-7 NC totally.Afterwards,the relative luciferase activity and SP1 was detected.Results:The two kinds of double luciferase reporter plasmid psiCHECK2-SP1-w-3′UTR and its mutant plasmid psiCHECK2-SP1-m-3′UTR were constructed and confirmed by enzyme digestion and sequences.The double luciferase reporter experiment showed that overexpression of miR-7 could significantly inhibit the relative luciferase activity of psiCHECK2-SP1-w-3′UTR,and the difference was statistically significant(P<0.001),with no effect on the expression of psiCHECK2-SP1-m-3′UTR(P>0.05).Rt-PCR and Western blot results showed that SP1 expression level of miR-7-mimics group was lower than that of miR-7 NC group(P<0.001),while SP1 expression level of miR-7-inhibitor group was higher than that of miR-7 NC group(P<0.001).Conclusion:We successfully constructed the dual luciferase reporter plasmid and its mutant plasmid based on psiCHECK2-SP1-3′UTR,which can be used to elucidate the molecular mechanism of miR-7 regulating gastric cancer cell SP1 expression.
作者
刘文莉
国东
刘加霏
王玉珊
张焕虎(指导)
宋文刚(指导)
LIU Wen-Li;GUO Dong;LIU Jia-Fei;WANG Yu-Shan;ZHANG Huan-Hu;SONG Wen-Gang(School of Medical Technology,Beihua University,Jilin 132013,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2021年第1期72-77,共6页
Chinese Journal of Immunology
基金
北华大学研究生创新项目(北华研创合字[2019]074)
威海市医学微生物与免疫学重点实验室项目(2017GGH08)
山东省自然科学基金(ZR2020QC073)。
