Genetically Encoded Reporter Genes for MicroRNA Imaging in Living Cells and Animals

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by base paring with the complementary sequences of the target mRNAs, and then exert their function through degrading mRNA or inhibiting protein translation. They play a significant role as a regulatory factor in biological processes of organism development, cell proliferation, differentiation, and cell death. Some of the traditional methods for studying miRNAs, such as northern blot, real-time PCR, or microarray, have been extensively used to investigate the biological properties and expression patterns of miRNAs. However, these methods often require considerable time, cell samples, and the design of effective primers or specific probes. Therefore, in order to gain a deeper understanding of the role of miRNAs in biological processes and accelerate the clinical application of miRNAs in the field of disease treatment, non-invasive, sensitive, and efficient imaging methods are needed to visualize the dynamic expression of miRNAs in living cells and animals. In this study, we reviewed the recent progress in the genetically encoded reporter genes for miRNA imaging. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by base paring with the complementary sequences of the target mRNAs, and then exert their function through degrading mRNA or inhibiting protein translation. They play a significant role as a regulatory factor in biological processes of organism development, cell proliferation, differentiation, and cell death. Some of the traditional methods for studying miRNAs, such as northern blot, real-time PCR, or microarray, have been extensively used to investigate the biological properties and expression patterns of miRNAs. However, these methods often require considerable time, cell samples, and the design of effective primers or specific probes. Therefore, in order to gain a deeper understanding of the role of miRNAs in biological processes and accelerate the clinical application of miRNAs in the field of disease treatment, non-invasive, sensitive, and efficient imaging methods are needed to visualize the dynamic expression of miRNAs in living cells and animals. In this study, we reviewed the recent progress in the genetically encoded reporter genes for miRNA imaging. MicroRNAs (miRNAs) are endogenous non-coding small RNAs whose length is about 20–25 nt, encoded by approximately 3% of the human genomes.1Sekar T.V. Mohanram R.K. Foygel K. Paulmurugan R. Therapeutic evaluation of microRNAs by molecular imaging.Theranostics. 2013; 3: 964-985Crossref PubMed Scopus (21) Google Scholar The first miRNA was found in Caenorhabditis elegans, and thousands of miRNAs have been discovered in plants, animals, and viruses to date.2Di Leva G. Garofalo M. Croce C.M. MicroRNAs in cancer.Annu. Rev. Pathol. 2014; 9: 287-314Crossref PubMed Scopus (1138) Google Scholar miRNAs completely or partially bind to the 3′ untranslated regions (UTRs) of target mRNAs to degrade mRNAs or repress protein translation.3He D. Wong K.-W. Dong Z. Li H.-W. Recent progress in live cell mRNA/microRNA imaging probes based on smart and versatile nanomaterials.J. Mater. Chem. B Mater. Biol. Med. 2018; 6: 7773-7793Crossref PubMed Google Scholar A single miRNA can regulate the expression activity of multiple mRNAs, and its effector molecules play a role in different sites of cell signaling pathways and networks. At the same time, an mRNA is also regulated by multiple miRNAs; over 60% of all mRNA expression is regulated by miRNAs.4Bartel D.P. Chen C.Z. Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs.Nat. Rev. Genet. 2004; 5: 396-400Crossref PubMed Scopus (1090) Google Scholar Therefore, miRNAs can switch quickly between cellular programs to form a dense molecular regulatory network. miRNAs are negative gene regulators at the post-transcriptional level. Mature miRNAs are mainly formed in the nucleus and cytoplasm, and depend on RNA polymerase II, Drosha (double-stranded RNA-specific ribonuclease), and Dicer (RNase III endonuclease) in the formation process (Figure 1). First, under the action of RNA polymerase II, miRNA coding genes are transcribed into primary miRNAs (pri-miRNAs) in the nucleus. Subsequently, the precursor miRNAs (pre-miRNAs) are produced to form a hairpin structure with the specific enzyme action of Drosha. Next, the pre-miRNAs are transported from the nucleus to the cytoplasm under the action of Exportin 5 and then processed into mature double-strand miRNAs by Dicer. Finally, a strand of the duplex mature miRNA is brought into RNA-induced silencing complex (RISC) to base pair with the target mRNA and result in protein translation inhibition or mRNA degradation.5Oh S.W. Hwang D.W. Lee D.S. In vivo monitoring of microRNA biogenesis using reporter gene imaging.Theranostics. 2013; 3: 1004-1011Crossref PubMed Scopus (22) Google Scholar, 6Bhaskaran M. Mohan M. MicroRNAs: history, biogenesis, and their evolving role in animal development and disease.Vet. Pathol. 2014; 51: 759-774Crossref PubMed Scopus (230) Google Scholar, 7Xie M. Steitz J.A. Versatile microRNA biogenesis in animals and their viruses.RNA Biol. 2014; 11: 673-681Crossref PubMed Scopus (40) Google Scholar, 8Treiber T. Treiber N. Plessmann U. Harlander S. Daiβ J.-L. Eichner N. Lehmann G. Schall K. Urlaub H. Meister G. A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis.Mol. Cell. 2017; 66: 270-284.e13Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar In recent years, through analysis of a large amount of animal and human experimental data, it has been found that miRNAs play a key role as a regulatory factor in various biological or pathological processes, such as immune regulation, physiological metabolism, cell proliferation, differentiation, growth, and development by degrading or inhibiting the translation of target RNAs.9Ambros V. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing.Cell. 2003; 113: 673-676Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar Overexpression or underexpression of miRNAs can cause various diseases. For instance, miR-122 plays an important role in the replication of hepatitis C virus (HCV).10Sarnow P. Sagan S.M. Unraveling the Mysterious Interactions Between Hepatitis C Virus RNA and Liver-Specific MicroRNA-122.Annu. Rev. Virol. 2016; 3: 309-332Crossref PubMed Scopus (35) Google Scholar miR-33 can lead to the significant increase of liver ABCA1 expression and plasma high density lipoprotein (HDL) level,11Rayner K.J. Sheedy F.J. Esau C.C. S. of miR-33 in and of PubMed Scopus Google Scholar and the of can result in in G. V. P. MicroRNA and specific 2016; PubMed Google Scholar In miRNAs have been as a such as the Therefore, understanding biological of miRNAs and their of biological processes through efficient and methods is for and clinical the expression and of miRNAs mainly by northern blot, real-time PCR, microarray, methods require a of and are to to a miRNAs with small and specific primers or probes are to Therefore, in order to biological about miRNAs with high and high that are non-invasive, and that are to are needed to the expression of miRNAs. molecular imaging imaging and imaging have the of and the to in and are used to and the biogenesis of miRNAs. In this we first reviewed the traditional methods for and miRNAs, then the reporter gene imaging of miRNA biogenesis and in living cells and animals. is a process of of specific RNA by of with RNA to of microRNAs by northern Biol. 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A complementary imaging to visualize using PubMed Scopus Google Scholar a imaging using as a reporter the of high expression of the expression of the reporter gene and then to high used to design a reporter gene to H. for of by in 2018; 11: Full Text Full Text PDF PubMed Scopus Google Scholar target sequences into the 3′ of the that complementary to and the the reporter gene to in living cells and animals. In the of reporter genes are and is produced to the of the of in cells to is to the inhibition of reporter and In to a can also as the target for imaging that activity by the reporter through the of enzyme and However, of experimental we of in this the of as a reporter gene for and of miRNA expression in the biological process in imaging can to the imaging are used to design miRNA the miRNA and the miRNA target S.W. Hwang D.W. Lee D.S. 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