Studies on avian endogenous retrovirus ALVE1 env transcripts specifically targeted by miR-155 in vitro
In this study,the DNA gga-miR-155 target site AGCATTA was demonstrated to exist in env transcripts of endogenous retrovirus ALVE1 by bioinformatics analysis.ALVE1 env sequence was amplified from chicken embryo fibroblast (CEF) cell genome and subcloned into pmirGLO vector which contains luciferase gene,thus creating the recombinant plasmid "pmirGLO-ALVE1-ENV-WT".Meanwhile,overlap PCR was performed to obtain the mutate sequences of miR-155 target site and the recombinant plasmid "pmirGLO-ALVE1-ENVMU" was constructed.In addition,gga-miR-155 precursor sequence was cloned into pcDNA 3.1 vector,and the recombinant plasmid "pcDNA-gga-miR-155" was created.Then,these vectors were co-transfected into DF-1 cells and the cells were collected to detect their luciferase activity.The result indicated that gga-miR-155 significantly reduced luciferase activity of pmirGLO-ALVE1-ENV-WT,while in all of control groups and the mutant group no significant change was found in luciferase activity.The results of fluorescence quantitative PCR further revealed that gga-miR-155 agomir in MSB1 cells significantly down-regulated the expression of ALVE1 env transcripts.This study suggests that gga-miR-155 may directly target ALVE1 env transcript.This is the first report that miR-155 can directly target endogenous retroviral sequences.
国家科技期刊平台
NSTL
万方数据
维普
