title: Construction, modification and evaluation of apolipoprotein A-I promoter-driven shRNA expression vectors against hTERT.
abstract: Liver-specific gene knockdown cannot be achieved by short hairpin RNA (shRNA) generated by RNA polymerase III promoter. Here we constructed, modified and evaluated apolipoprotein A-I (ApoA-I) promoter-driven shRNA expression vectors against human telomerase reverse transcriptase (hTERT) in SMMC-7721 cells. The roles of the cis-acting hammerhead ribozyme and the specific pausing site MAZ, the liver-specific promoter ApoA-I, as well as SV40 and CMV enhancers were first individually evaluated, and then they were incorporated to construct a liver-specific shRNA expression plasmid against hTERT, and the inhibitory effects on hTERT were examined in SMMC-7721 cells. The results showed that the introduction of the cis-acting hammerhead ribozyme and the specific pausing site MAZ did not change gene knockdown efficiency significantly, but eliminated the off-target effect. Green fluorescent protein (GFP) expressing plasmid under the control of ApoA-I promoter can produce liver-specific GFP expression, but at a much lower level compared to the CMV promoter. The CMV or SV40 enhancer-modified ApoA-I promoter caused a four or two folds increase in mRNA expression of GFP relative to ApoA-I alone, respectively. The liver-specific shRNA expression plasmid against hTERT under the control of CMV enhancer-modified ApoA-I promoter with the sequences of the cis-acting hammerhead ribozyme and MAZ, induced significant inhibitory effect on hTERT at both mRNA and protein levels in SMMC-7721 cells. Therefore, liver-specific gene therapy is made possible by utilizing shRNA expression vector under the control of CMV enhancer-modified ApoA-I promoter.
authors.affiliation: School of Pharmacy, Zhengzhou University, 100 Science Road, Zhengzhou 450001, China.
authors.forename: Xueling
authors.initials: X
authors.lastname: Ren
authors.forename: Yue
authors.initials: Y
authors.lastname: Ma
authors.forename: Min
authors.initials: M
authors.lastname: Xu
authors.forename: Tingting
authors.initials: T
authors.lastname: Chen
authors.forename: Zhenzhong
authors.initials: Z
authors.lastname: Zhang
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D024506:5' Flanking Region / Q000235:genetics; D016632:Apolipoprotein A-I / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D016632:Apolipoprotein A-I; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D012319:RNA Polymerase II; C509186:TERT protein, human; D019098:Telomerase