Novel approaches for gene-specific interference via manipulating actions of microRNAs: examination on the pacemaker channel genes HCN2 and HCN4.
Recent evidence has suggested microRNAs as viable therapeutic targets for a wide range of human disease. However, lack of gene-specificity of microRNA actions may hinder this application. Here we developed two new approaches, the gene-specific microRNA mimic and microRNA-masking antisense approaches, to explore the possibility of using microRNA's principle of actions in a gene-specific manner. We examined the value of these strategies as rational approaches to develop heart rate-reducing agents and "biological pacemakers" by manipulating the expression of the cardiac pacemaker channel genes HCN2 and HCN4. We showed that the gene-specific microRNA mimics, 22-nt RNAs designed to target the 3'untranslated regions (3'UTRs) of HCN2 and HCN4, respectively, were efficient in abrogating expression and function of HCN2 and HCN4. The gene-specific microRNA mimics repressed protein levels, accompanied by depressed f-channel conductance and the associated rhythmic activity, without affecting mRNA levels of HCN2 and HCN4. Meanwhile, we also designed the microRNA-masking antisense based on the miR-1 and miR-133 target sites in the 3'UTRs of HCN2 and HCN4 and found that these antisense oligodeoxynucleotides markedly enhanced HCN2/HCN4 expression and function, as reflected by increased protein levels of HCN2/HCN4 and If conductance, by removing the repression of HCN2/HCN4 expression induced by endogenous miR-1/miR-133. The experimental examination of these techniques and the resultant findings not only indicate feasibility of interfering miRNA action in a gene-specific fashion but also may provide a new research tool for studying function of miRNAs. The new approaches also have the potential of becoming alternative gene therapy strategies.
Xiao
Jiening
J
Research Center, Montreal Heart Institute, Montreal, Canada.
Yang
Baofeng
B
Lin
Huixian
H
Lu
Yanjie
Y
Luo
Xiaobin
X
Wang
Zhiguo
Z
D020413:3' Untranslated Regions; D000818:Animals; D000831:Animals, Newborn; D001145:Arrhythmias, Cardiac / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D002460:Cell Line; D015536:Down-Regulation; D005240:Feasibility Studies; D015316:Genetic Therapy / Q000379:methods*; D006339:Heart Rate / Q000235:genetics; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014162:Transfection
D020413:3' Untranslated Regions; C578710:HCN2 protein, human; C513836:HCN4 protein, rat; C578712:Hcn2 protein, rat; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D015221:Potassium Channels; D012333:RNA, Messenger; D011188:Potassium