Non-coding RNAs (ncRNAs) refer to the RNAs that do not translate into proteins. There are many kinds of ncRNAs, and some of them play key roles in the physiological and pathological processes, such as transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), multiple small RNAs (such as microRNAs, siRNAs, snRNAs, etc.), and some long non-coding RNAs[1, 2]. Many ncRNAs have not yet been discovered, and there are also many ncRNAs whose functions have not been determined or are useless[3, 4]. The first ncRNA to be characterized was alanine tRNA in 1965s [5], and the structure of tRNA was resolved in the 1970s (Figure.1a) [6, 7]. And then as more ncRNAs were discovered, the importance of ncRNAs was determined (Figure.1b). NcRNAs are involved in the pathological process of various diseases, including cancer[8], autism[9], Alzheimer’s disease[10], etc. Therefore, it is considered to be a potential target for the treatment of many diseases and thus has become one of the current research hotspots.
Figure.1 Non-coding RNA. (a) Molecular structure of alanine tRNA derived from yeast. (b) Complementation of the central dogma by noncoding RNAs.
What is MicroRNA (miRNA)?
MicroRNAs (miRNAs) are a kind of small single-stranded non-coding RNA containing about 22 nucleotides[11]. According to genomics analysis, the human genome encodes up to more than 1900 miRNAs, and in the latest bioinformatics-based analysis this number can be as high as 2300[12, 13]. However, after manual sorting, it is finally confirmed that the number of human miRNAs is only about 500, and miRNAs whose physiological functions have been identified are still few[14]. The first miRNA was identified in the 1990s[15]. And after the 2000s, it was identified as an important regulatory factor and gradually became a research hotspot[16]. MiRNAs are involved in many pathological processes of various diseases, including cancer, heart disease, nerve system diseases, kidney diseases, obesity, etc. And some of the miRNA play key roles in the basic life activities of human growth, development, and survival, especially some genetically conserved miRNAs[17].
Molecular mechanism and Research Methods of miRNA
Synthesis and Functions of miRNA
Precursor miRNA (pre-miRNA) is obtained by transcription, and it can form a self-complementary folded hairpin structure (Figure.2). This hairpin-like primary transcript is cleaved through the action of Dicer to form two miRNAs, a miRNA 5p (miRNA) near the 5′ end and a miRNA 3p (miRNA*) near the 3′ end. MiRNA 3p is usually rapidly degraded, and the remaining miRNA 5p is usually considered to be the mature form of miRNAs. Most miRNAs 3p are short-lived and useless, but it was found that some of the miRNAs 3p also have important physiological and pathological functions, identical to miRNAs generally considered mature [18]. MiRNA mainly exerts its function by blocking the expression of target genes by mediating the transcription and translation of target genes. MiRNAs can bind to mRNA by complementary base pairing. This leads to three possibilities, including the degradation of the mRNA, the shortening of the polyA tail of the mRNA, and the blocking of the translation process involving ribosomes, which ultimately lead to blocked translational processes of mRNAs. Besides, the matching sequence of animal miRNAs and the target genes is too short (at least 6-8bp), which makes animal miRNA have many targets and difficult for a single miRNA to block the expression of the target gene[19]. Therefore, combinatorial regulation of multiple miRNAs is a common way for animal miRNAs to function[20].
Figure.2 Structure of Pre-miRNA
Research Methods of miRNA
Quantification of miRNAs can usually be performed by RT-PCR-based methods, such as stem-loop RT-PCR and polyA RT-PCR[21, 22]. But due to the small size of miRNA molecules, the high-throughput analysis process has a high error rate. Therefore, it is a common method to determine the role of miRNA by the level of target gene mRNA[23]. In project design and experimental operation, miRNA mimics, inhibitors, agomir, and antagomir are widely used in animal and cell experiments for miRNA-related research. These additives can mimic miRNA function or as miRNA inhibitors are used in functional experiments. Mimics are a kind of chemically synthesized, double-stranded miRNA-like RNA, typically used for cell transfection[24]. And agomirs are a kind of chemically modified mimics of miRNA, it has stronger membrane binding capacity and is less prone to degradation, typically used in the animal models and enables non-transfected cell treatment[25].
Examples of miRNA-related publications
Combining in vivo experiments with animal models and in vitro experiments with cellular models is a routine strategy for miRNA research. In a publication on liver cancer, Feng, et al. revealed the role of miR-149 3p in hepatocellular carcinoma (HCC) through miRNA mimics-based cellular models and agomir-based mouse models[18]. In the in vivo experimental part, they establish HCC mouse models (LPS or DEN exposed) based on C57 WT and miR-149 3p KO mice and then treat the mice with miR-149 3p agomir. In the in vitro experimental part, they transfected miR-149 3p mimics, LPS, TNF-α, and p65 separately or jointly for liver cancer cell lines. Besides, a tumorigenic model was established by miR-149 3p mimics-treated HCC cell lines and BLAB-C nude mice. Combining the results of the above in vivo and in vitro experiments, they find that miR-149 3p can inhibit the progression of HCC by down-regulate the expression of TNF-α-TRADD-NF-κB signal-related death domain protein.
Conclusion
MiRNA is a very hot research field in the past 10 years, and the number of publications has been at a high level in recent years (Figure.3). For researchers who want to enter this field, it is necessary to master the strategies and methods related to miRNA research.
Figure.3 Number of miRNA-related publications in recent decades (form Pubmed).
Where to get MicroRNA products and services?
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References
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