摘要
目的:构建microR-185-5p(miR-185-5p)的过表达载体并研究miR-185-5p对人胰腺癌Panc-1细胞体外增殖的影响。方法:将人工合成的miR-185-5p寡聚核苷酸与经EcoR I和HindⅢ双酶切连接上纯化后的真核表达载体pcDNA TM 6.2-GW/EmGFP-miR,接着转化进E.Coli Top10,再经过DNA测序、比对检测进行验证;将构建成功的载体转染进Panc-1细胞中,用倒置荧光显微镜和qRT-PCR方法验证转染效率,用CCK8方法检测miR-185-5p对Panc-1细胞增殖的影响。结果:成功构建了miR-185-5p真核表达载体;构建好的载体瞬时转染Panc-1细胞后发现miR-185-5p的表达显著增加,并且能够抑制Panc-1细胞的体外增殖能力。结论:miR-185-5p的真核表达载体构建成功,同时证明了miR-185-5p过表达可抑制人胰腺癌细胞Panc-1的增殖。
Objective:To construct microRNA-185-5p(miR-185-5p)overexpression vector,and to explore its effect on the proliferation of human pancreatic cancer cell line Panc-1 in vitro.Methods:The synthetic oligonucleotide of miR-185-5p was combined with the purified eukaryotic expression vector pcDNA TM 6.2-GW/EmGFP-miR that digested by EcoRⅠand HindⅢ,and then transformed into E.Coli Top10,then verified by DNA sequencing,comparison and detection.The successfully constructed vector was transfected into Panc-1 cells,and the transfection efficiency was determined by inverted fluorescence microscope and qRT-PCR,and the effect of miR-185-5p on the proliferation of Panc-1 cells was detected by CCK8 method.Results:The eukaryotic expression vector of miR-185-5p was successfully constructed.After transient transfection of Panc-1 cells with the constructed vector,the expression of miR-185-5p was significantly increased,and it could inhibit the expression of Panc-1 cells.Conclusion:The eukaryotic expression vector of miR-185-5p is successfully constructed.It is proved that the overexpression of miR-185-5p can inhibit the proliferation of human pancreatic cancer cell Panc-1.
作者
燕婧
冯芸
李丹
高艺
张静
YAN Jing;FENG Yun;LI Dan;GAO Yi;ZHANG Jing(Medical School of Yan’an University,Yan’an 716000,China)
出处
《陕西医学杂志》
CAS
2021年第11期1323-1326,1332,共5页
Shaanxi Medical Journal
基金
国家自然科学基金资助项目(81660492)。
