"New Star" Technology in Scientific Research - MicroRNA Real-time PCR

Introduction to Micro RNA
MicroRNAs (miRNAs) are non-coding single-stranded small-molecule RNAs of approximately 20-23 nucleotides in length, which are negatively expressed at the post-transcriptional level by complementary pairing with target mRNAs. Regulation leads to degradation of mRNA or translational inhibition. It is produced by Dicer enzyme processing of a single-stranded RNA precursor of about 70-90 bases in size with a hairpin structure, which is different from siRNA (double strand) but closely related to siRNA. The first confirmed miRNA was lin-4 and let-7, first discovered in nematodes, and several research groups subsequently identified hundreds of miRNAs in a variety of biological species including humans, fruit flies, and plants. So far, thousands of miRNAs have been reported to exist in multicellular eukaryotes such as animals, plants, and fungi, and are highly conservative in evolution.

Micro RNA production mechanism
The miRNA is part of a long post-transcriptional RNA molecule (pri-miRNA) that is first treated in the nucleus by a double-stranded RNA-specific nuclease Drosha into a 70-100 nucleotide hairpin RNA (pre-miRNA). . This hairpin structure RNA is transported to the cytoplasm and cleaved by another double-stranded RNA-specific nuclease Dicer, resulting in a 19-23 nucleotide mature miRNA that binds to an RNA-mediated silencing complex (RISC) In a similar or identical complex, this complex is involved in RNA interference. Base pairing of miRNA and target gene mRNA directs RISC to degrade the target fragment or hinder its translation process. The base pairing of the miRNA and its target mRNA complements the specificity of this process. The effect of inhibition by translation or degradation is determined by the degree of mismatch between the miRNA and its mRNA of interest, and the target mRNA with a high degree of matching will be degraded. Since miRNAs can pair incompletely complementary mRNAs to inhibit protein translation, each miRNA can have multiple target genes, and several miRNAs can regulate the same gene. This complex regulatory network can economically regulate the expression of multiple genes through a single miRNA, or it can fine-regulate the expression of a gene through a combination of several miRNAs. The gradual deepening of research based on miRNA-regulated gene expression will help us understand the complexity of the genome of higher eukaryotes and the complex network of gene expression regulation. As shown below


Micro RNA characteristics
The miRNAs that have been identified are presumably mostly produced by a single-stranded RNA precursor having a hairpin structure and a hairpin structure of about 70 bases, which are processed by Dicer, having a 5' terminal phosphate group and a 3' hydroxyl group. A small RNA fragment of approximately 21-25 nt in size, localized to the 3' or 5' end of the RNA precursor.
Fifteen percent of the 100 new miRNAs identified by the recent three research groups from nematodes, Drosophila, and Hela cells are highly conserved across the nematode, Drosophila, and mammalian genomes (only 1-2 bases) The difference), colleagues at Lau and Bartel Labs believe that all miRNAs may have orthologs in other species (Ortholog, which refers to those genes that originate from the same ancestor and perform the same function in different organisms). As a category, these similar genes are called "orthologs").
Bantam was first thought to be a genetic locus involved in cell proliferation in Drosophila. It is known that the insertion of several transposons containing an enhancer into a 12.3 kb region spanning this site results in repeated growth of the eye and wing of Drosophila, whereas a transposon-mediated deletion of a 23 kb fragment spanning that site is The mutant flies are smaller than the wild type flies. Cohen and colleagues used a 3.85 kb fragment to introduce a 21 kb fragment-deficient flies into their original size. But the strange thing is that the EST in this 3.85kb fragment has no similar effect. Cohen compared this fragment with the homologous sequence of Anopheles gambiae and found a highly conserved region of 90 bp. The RNA folding program (mfold) found that this conserved sequence can form a hairpin structure, making this segment much like a miRNA. Precursor. This result confirmed that the larvae of the mutant Drosophila lacked a 21 bp bantam miRNA by Northern blot, and the role of bantam miRNA in cell proliferation was confirmed by a series of "functional deletion"-"function recovery" experiments using this 90 bp mRNA precursor. The researchers used a computer program to search for three potential binding sites for bantam in the 3' non-coding region of hid mRNA (hid is an apoptosis-inducing gene in Drosophila) and confirmed that bantam miRNA inhibits the translation of hid instead of Transcription.
The expression patterns of miRNAs vary. Some worms and Drosophila miRNAs are expressed in all cells at all developmental stages, while other miRNAs are based on a more restrictive phase and temporal expression pattern. There are significant differences in the levels of miRNAs in different tissues and different developmental stages.

Micro RNA function
Research on microRNAs is increasing because scientists are beginning to recognize that these ubiquitous small molecules have a broad role in the regulation of eukaryotic gene expression. Most of the hundreds of miRNAs found in species such as nematodes, fruit flies, mice, and humans have the same characteristics as other molecules involved in the regulation of gene expression—the levels of miRNAs are significant in different tissues and stages of development. Difference, this miRNA expression pattern has differential spatial and temporal expression patterns, suggesting that miRNAs may be important as molecules involved in the regulation of gene expression.
The first identified miRNA, lin-4 and let-7, first discovered in nematodes, can be induced in an unknown way by partial complementation to the 3' non-coding regions (3'UTRs) of the target mRNA target. Protein translation inhibition, which in turn inhibits protein synthesis, regulates the development of nematodes by regulating the translation of a set of key mRNAs (reviewed in Pasquinelli 2002).
The bantam miRNA is the first miRNA to be found to have a proto-oncogene function. In addition to lin-4 and let-7, some miRNAs are known to be involved in post-transcriptional regulation of genes that play important roles in cell differentiation and tissue development, such as mir-14, mir-23, and the like.
There are two clues in the study of plant miRNAs suggesting that miRNAs may be involved in plant development. First, the expression levels of three miRNAs in the carpel factory (car) mutant were significantly decreased. CARPEL FACTORY is a Dicer-like enzyme involved in the development of plants, and its deletion mutants are characterized by defects in embryonic and leaf development. The experimental results suggest that this defect is caused by the lack of miRNA processing. The high level of expression of most plant miRNAs in certain tissues also suggests that they may be involved in the development of plant tissues.
Analysis of a subset of miRNAs suggests that miRNAs are involved in a number of important processes in life, including early development (Reinhart 2000), cell proliferation, apoptosis, cell death (Brennecke 2003), fat metabolism (Xu 2003), and cell differentiation. (Kawasaki 2003). In addition, one study showed a significant association between a decrease in two miRNA levels and chronic lymphocytic leukemia, suggesting a possible relationship between miRNA and cancer (Calin 2002).
Due to the breadth and diversity of miRNAs, miRNAs may have a wide variety of biological functions. Although the study of miRNAs is still in its infancy, it is speculated that the regulation of gene expression by miRNAs in higher eukaryotes may be as important as transcription factors. One view is that miRNAs may represent a way to regulate gene expression at a newly discovered level.
However, the function of most miRNAs remains a mystery. The mechanism and function are shown in the figure below.



Micro RNA recognition method
Several research groups have used bioinformatics as a bioinformatics method to conduct research on miRNAs. Because of the presumed degradation of RNA by Dicer, 21-23 bases, 5' hydroxyl and 3' hydroxyl RNA fragments, some laboratories use improved directional cloning methods to screen for the same characteristics. Small molecules - Screening for RNA molecules of a certain size, ligating to 3' and 5' adapters, reverse transcription and amplification by PCR, subcloning and sequencing. The localization and clustering of miRNA precursors on the genome is performed by querying the genome database. This method helps to determine whether miRNAs are degradation products of molecules such as mRNAs, tRNAs, and rRNAs.
Some laboratories use an RNA folding program 'mfold' to determine whether highly conserved regions between C. elegans and C. briggsae contain potential miRNA precursors, and then use Northern Blots to determine whether these miRNAs are actually expressed. It is.
Although hundreds of miRNAs have been identified by biochemical or bioinformatic methods, the identified miRNAs are only a drop in the ocean. Since many of the identified miRNAs have been identified from individual clones, it can be assumed that there are still Many miRNAs are “leaky” during the separation and identification process, and sequencing is far from enough.

Micro RNA and stem cells
Recent studies have shown that microRNAs also play an important role in stem cell self-replication, directed differentiation and tissue regeneration. It is a key regulator of stem cell properties, maintenance, and transformation, and is a focus of current stem cell regulation research. Recently, Gangara Ju. VK and Lin H made a review in Nature Stem Cell, systematically summarizing the regulation of microRNAs on stem cells.

However, the regulation of microRNAs on stem cells goes far beyond this. It is also involved in cell cycle, cell reprogramming, pluripotent stem cell production, signaling, specific differentiation, Riches, repair, regeneration, mutation, metabolism and more.

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