MicroRNA (miRNA) refers to small, noncoding RNA molecules that function in regulating the stability or activity of target messenger RNA (mRNA) transcripts, thereby controlling gene expression. Through these actions, miRNAs are key regulators of biological processes, such as cell differentiation, development, and homeostasis. Dysregulated expression of miRNAs are correlated with diseases such as cardiovascular disease, retinal disorders, neurodegenerative diseases, diabetes, and cancer. miRNAs can serve as diagnostic and prognostic biomarkers to detect and monitor disease as well as therapeutic targets.
miRNAs are transcribed as individual genes or as clusters of several miRNA genes that share one promoter. There are several steps in the formation of miRNA. First, miRNA genes are transcribed as primary microRNA (pri-miRNA) transcripts. Then pri-miRNA transcripts are processed in the nucleus by the Microprocessor complex, containing RNAse III-type endonuclease Drosha and DGCR8, to form stem-loop miRNA precursors (pre-miRNAs) that are about 70 nucleotides in length.
Mirtrons are a class of miRNAs located within introns of protein-coding genes. Mirtrons are not processed by the Microprocessor complex, but instead their pre-miRNA is generated directly by splicing.
Pre-miRNA transcripts are processed in the cytoplasm by a second RNAse III-type endonuclease called Dicer, which cleaves the loop structure resulting in a miRNA duplex. The miRNA duplex has two RNA strands bound together by Watson-Crick base paring, with each strand having a length of approximately 21-22 nucleotides. miRNA duplexes are imperfectly paired and therefore have bulges due to mismatches between strands. This distinguishes miRNA from small interfering RNAs (siRNAs) that form perfectly paired duplexes.
Image attribution: Hajarnis S, Lakhia R, Patel V. MicroRNAs and Polycystic Kidney Disease. In: Li X, editor. Polycystic Kidney Disease [Internet]. Brisbane (AU): Codon Publications; 2015 Nov. Chapter 13. doi: 10.15586/codon.pkd.2015.ch13, licensed under Creative Commons Attribution 4.0 International (CC BY 4.0)
One strand of the miRNA duplex is selectively anchored into the Argonaute protein to form the miRNA-induced silencing complex (miRISC), which targets the 3’UTR of target mRNA. Either strand can be loaded into the miRISC; one strand is usually favored over the other. The strand that became part of the miRISC became known as the miR, or “guide” strand. The other strand, which is ejected and gets degraded, was called miR* or “passenger,” and this nomenclature has been suggested to continue for the typically dominant versus ejected/degraded strands.
In addition to Argonaute, the miRISC includes the necessary effector protein GW182 (Glycine-Tryptophan Repeat-Containing Protein of 182 kDa). GW182 proteins bridge Argonaute proteins and downstream effector complexes that mediate miRNA-dependent translational repression. Translation appears to be repressed due to inhibition of translation initiation and destabilization of mRNA.
The mRNA target specificity, which is determined by base pairing at nucleotides 2-7 of the Argonaute-loaded miRNA, is referred to as the miRNA “seed” sequence. There is additional gene targeting through additional pairing between nucleotides in the 3’ half of the miRNA and the target mRNA. miRNA families share a common seed sequence and can target the same group of mRNA substrates with partial redundance. If the initial strand selection is altered, the miRISC will repress a different set of target genes. Tissue-specific differences in strand selection have been found in animal models. Alternative miRNA strand switching is sometimes called “arm switching.”
miRNAs can be intracellular (inside cells) and also extracellular in blood serum/plasma, serum, urine, saliva, seminal fluid, ascites, amniotic pleural effusions, and cerebrospinal fluid. Movement of miRNA from the intracellular environment to the extracellular environment has been reported to occur by passive leakage associated with apoptosis, necrosis or inflammation. miRNAs are also actively secreted by exosomes/microvesicles, lipoproteins, and RNA-protein complexes. Extracellular miRNA in biofluids is also known as circulating miRNA.
Unlike conventional RNA molecules, circulating miRNAs are very stable and resistant to RNase activity and extreme pH temperatures. Bodily fluids contain ribonucleases that break down RNA, but miRNAs are thought to be protected from degradation by lipid vesicles by being in a complexes with RNA binding proteins or both.
miRNAs have a role in cancer-associated biological processes such as proliferation, differentiation, apoptosis, metabolism, invasion, metastasis and drug resistance. In the tumor microenvironment, specific miRNAs are selectively packed into exosomes and become circulating miRNAs. Circulating miRNAs are correlated with the degree of tumor progression and different miRNA profiles are associated with different stages of cancer.
The plasma and serum of cancer patients have shown hundreds of cases of dysregulated miRNAs compared to healthy subjects. Circulating miRNAs are potential biomarkers for diagnosis and prognosis in cancer. Circulating miRNAs include tumor suppressor miRNAs that control of oncogenes and oncomiRs that control tumor suppressor genes.
Breast cancer: miR-10b and miR-196a are upregulated and miR-4417 is downregulated.
Ovarian cancer: miR-200 cluster is upregulated and dysregulated genes include miR-506, let-7 cluster, miR-183 and miR-22.
Cervical cancer: miR-21a and miR-944 are upregulated and miR-138 is downregulated.
miR-1, miR-133a, miR-208a/b and miR-499 are upregulated shortly after myocardial infarction.
Sepsis is an abnormal host response to pathogenic microorganisms consisting of excessive inflammatory response followed by multiple organ failure. miRNA misexpression is associated with sepsis and non-infective systemic inflammatory response syndrome (SIRS). Decreased expression of miR-25 was found in sepsis patients and lower levels of this miRNA were associated with increased mortality. miR-233 has a role in the exaggerated immune responses through its targets Stathmin, STAT3, Granzyme B, IGFR1 and Artemin. Another inflammatory response regulator is miR-155. Dysregulation of miR-150, expressed mainly in immune cells, is also associated with sepsis. Specific profiles or patterns of miRNA dysregulation are associated with infections with different pathogens.
Nervous system disorders such as Alzhiemer’s disease, epilepsy, Parkinson’s disease, glioblastoma, multiple sclerosis and myasthenia gravis are thought to be partly caused by aberrant expression and/or dysfunction of miRNAs.
In Alzheimer’s disease, downregulated miRNAs are miR-107, miR-298, miR-328. Upregulated miRNAs are miR-9, miR-34a, miR-125b, miR-146a and miR-155. Altered expression of miRNAs can be detected in the brain and cerebrospinal fluid (CSF) as well as plasma and serum.
In Parikinson’s disease, downregulated miRNAs are miR-29a-3p, miR-29c-3p, miR-19a-3p and miR-19b-3p. Upregulated miRNAs are miR-103a-3p, miR-30b-5p, miR-29a-3p, miR-4639-5p.
Glioblastoma is a type of brain cancer. In glioblastoma, downregulated miRNAs include miR-124, miR-137, miR-218 and miR-451. Upregulated miRNAs include miR-21, miR-221, miR-222, miR-335.
In multiple sclerosis, downregulated miRNAs include miR-15a, miR-15b, miR-181c, and miR-328. Upregulated miRNAs include miR-19a, miR-21, miR-22, miR-142-3p, miR-146a, miR-146b, miR-155, miR-210, miR-326.
Myasthenia gravis is an autoimmune condition characterized by weakness and the quick fatigue of voluntarily controlled muscles. The disease is caused by abnormal communication between nerves and muscles. Symptoms include muscle weakness, double vision, drooping eyelids and difficulties with speech, chewing, swallowing and breathing. miRNAs miR-15b, miR-122, miR-140-3p, miR-185, miR-192, miR-20b, miR-885-5p are downregulated in myasthenia gravis. Upregulated miRNAs include miR-21-5p, miR-150-5p, miR-151a-3p, let-7a-5p, let-7f-5p, miR-423-5p.
Many miRNAs are predicted computationally. The next step is to define the role or function of novel miRNAs in the cell and which mRNAs they target. More than 60% of mammalian genes are estimated to be controlled by miRNAs, but most target sites are not known. It is difficult to predict miRNA targets because many miRNAs match imperfectly to the 3’-untranslated region (UTR) or their target mRNA. The following techniques are used to study miRNAs in cells and tissues.
The following methods are based on nucleic acid hybridization, which is the formation of a duplex between two complementary strands of nucleic acid through hydrogen bonding between base pairs. The two main types of nucleic acids are DNA and RNA.
Northern blot is a technique in which RNA separated by gel electrophoresis is transferred to a membrane and exposed to a nucleotide probe that will hybridize at the spot where the nucleotide sequence is complementary RNA on the membrane. Northern blots can be used to assess quantity and size of different miRNAs.
Microarrays are used for high throughput profiling of miRNA expression and can analyze the expression levels of hundreds of miRNA genes in a single assay.Known oligo sequences are spotted on a glass slide and RNA to be analyzed is labeled so it can be detected when it hybridizes or sticks to complementary nucleotide sequence on the slide.
Bead-based profiling is a hybridization technique in which hybridization takes place in solution on glass beads instead of on a glass surface as it does more microarrays. This allows higher specificity for closely related miRNAs. Flow cytometry is used to measure the color of beads and the intensity of signal to give information about identity and abundance of miRNAs.
In situ hybridization (ISH) is the hybridization of a nucleotide probe labelled with radioactivity, fluorescent dyes, or dioxygenin to the RNA of interest in tissue samples, cells, or model organisms.
RT-qPCR combines reverse transcription polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) to measure RNA levels. Reverse transcription is performed, and then the resulting cDNA is used in the qPCR reaction.
Stem-loop RT-based assays are used for mature miRNA detection and can discriminate between miRNA species that vary by a single nucleotide. The target miRNA is reverse transcribed using a stem-loop primer annealed to the 3’ end of the miRNA. This template is used to amplify miRNA with a specific forward primer and universal reverse primer or TaqMan probe. The stem-loop primer has a constant region that forms a stem loop and a variable extension of six nucleotides. The stem-loop portion provides an extension so the PCR product length from about 22 nucleotides to around 60, allowing traditional PCR in the next steps. The six-nucleotide extension is the reverse compliment of the 3’ end of the miRNA of interest. Stem-loop RT techniques overcome the challenges of low sensitivity and low specificity in PCR amplifying short RNA targets.
Other amplification techniques:
Loop-mediated isothermal amplification (LAMP), RT-LAMP
Rolling circle amplification (RCA)
Hybridization chain reaction (HCR)
Strand displacement amplification (SDA)
Exponential isothermal amplification assay (EXPAR)
Next-generation sequencing (NGS) can be used to profile and quantify miRNA expression as well as identify unknown miRNA variants. New variants are not found using RT-qPCR and microarrays. miRNA sequencing is similar to DNA sequencing, but there are specific steps required in the RNA library generation, such as enrichment of small RNAs.
The following are web servers and programs for analyzing processed miRNA sequences:
- miRExpress
- miRanda
- miRDeep
- DIANTA microT
- PicTar
The Invader assay is an enzymatic reaction that uses fluorescence resonance energy transfer (FRET) as a measurable signal. In this method, an oligonucleotide probe hybridizes to the target miRNA and an Invader oligonucleotide hybridizes to an overlapping region on the miRNA. This produces an overlap-flap, which is a substrate for the 5’ nuclease called cleavase. After this flap is cleaved, it binds to a secondary reaction template where it binds in an overlapping manner with a FRET oligo with a fluorescent and a quencher molecule. Cleavage of this flap separates the fluorescent molecule from the quencher molecule, and fluorescence is emitted. Single nucleotide variance can be detected with the invader assay as well as parallel analysis of mature and pre-miRNAs.
The quantity of miRNA is only an approximation of active miRNA. miRNAs can be silenced and not able to engage in post-transcriptional suppression of mRNAs, by mono nucleotide addition or binding to proteins. miRNA activity reporters can provide data about active miRNA without cell lysis or in-patient assessment of miRNA activity. miRNA activity reporters may use fluorescence, bioluminescence, magnetic resonance imaging (MRI), and positron emission tomography (PET).
Reporters measure the activity of a gene and can be used to visualize when and where in tissues or cells, miRNA or any transcripts are active. In reporter assays, the region called the promoter that regulates gene activity is fused to a reporter gene, such as luciferase or GFP, which produces bioluminescence or fluorescence respectively when the gene is active. The mRNA targets of miRNA can also be validated using reporter systems. To do this, the 3’UTR of the target gene is fused to the reporter. It is possible to use two reporter constructs to simultaneously analyze miRNAs and their target mRNA.
Luciferase reporter assays require input of luciferin, making them less suitable for analysis of miRNA expression or activity over time. The green fluorescent protein (GFP) does not require input substrate allows for measurement over time and space in living cells.
Reporter assays may not be able to ascertain whether a lost signal is due to reduced gene expression or loss of cells that produced the signal. Reporter systems that use plasmid DNA cannot be used in a living patient. The molecular beacon (MB) in vivo imaging system overcomes these challenges. MB is an oligonucleotide in a hairpin shape fused to a fluorescent dye at the 5’ end and quencher molecule at the 3’ end. In the default state, there is no fluorescence because it is quenched. Binding to the complementary sequence causes the quencher to be displaced and fluorescence is emitted. MB using magnetic nanoparticles can be used to monitor miRNA binding to target by magnetic resonance imaging (MRI) imaging. Radiolabeled tracer oligonucleotides complementary to target miRNAs are used in MB imaging detected using positron emission tomography (PET).
miRbase (http://mirbase.org/) is a public repository and online resource for microRNA sequences and annotation. The repository catalogs, names, and distributes microRNA gene sequences. miRbase also provides sequences, their biogenesis precursors, genome coordinates and context, literature references, deep sequencing expression data and community-driven annotation. miRbase contained 38,000 annotated miRNAs as of 2019.
The following companies are developing therapeutics that target miRNA. miRNA therapeutics in development mainly include miRNA mimics and antagomiR products. miRNA mimics mimic endogenous miRNAs and aim to re-establish the concentration of miRNA that has been suppressed in the progression of disease. AntagomiRs, also known as anti-miRs or blockmirs, aim to suppress the function of miRNAs that are overexpressed and are involved in the disease. Chemical modifications are used to stabilize miRNA mimics, which are double-stranded RNA molecules. Antagomir molecules are engineered DNA or RNA oligonucleotides.
Ionis Pharmaceuticals
Opko Health (Opko CURNA)
