How to Study the Function of miRNA

What is miRNA?

miRNA is a kind of non-coding single-stranded RNA widely stored in the RNA molecule with a length of about 18-22 nucleotides, which can regulate gene expression through a variety of pathways, and plays an important role in the regulation of gene expression, cell cycle, developmental chronology of organisms, and disease process. The three regulatory pathways of miRNAs that are relatively clear in current research are:

1, affecting RNA transcriptional activity;

2, inhibiting the translation of mature mRNAs; and 3, affecting DNA methylation.

Generally speaking, functional miRNAs are genes that are transcribed to form Pri-miRNAs (hundreds of nucleotides) with the participation of RNA polymerase II (RNA pol II), which undergo a two-step cleavage. The first step of the cleavage reaction takes place in the nucleus and is catalyzed by the Drosha/DGCR8 complex, in which Drosha is a type III RNA cleaving enzyme, which is the core catalytic component, and DGCR8 is a double-stranded RNA-binding protein responsible for recruiting pri-miRNA substrates. Intranuclear cleavage produces pre-miRNA around 60-70 bases in length, and then the pre-miRNA exits the nucleus, where the second step of cleavage is accomplished in the cytoplasm by Dicer RNAase. The first step of the cleavage reaction in the nucleus is particularly important, on the one hand, to remove the long irrelevant sequences, from the thousands of bases length of pri-miRNA to produce only 60-70 bases length of Pre-miRNA, on the other hand, cleavage produces the 3' end of the final mature miRNA, which is critical for the function of miRNA, so it requires the cleavage site is very precise.

miRNA is a small single-stranded non-coding RNA molecule.

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Mechanisms of miRNA Action

The mechanism of action of miRNA mainly involves two aspects. Firstly, miRNA binds to the 3'UTR region of mRNA through its own seed sequence, leading to translational repression and thus affecting gene expression. Secondly, it can halt ribosome progression during translation elongation, leading to translation termination. miRNA can affect mRNA expression and translation, thereby involving corresponding phenotypic changes. Therefore, predicting and validating miRNA target genes play a crucial role in miRNA functional studies.

What Are 3'UTRs Doing?

3'-UTR, short for three prime untranslated region, is a sequence on mRNA that immediately follows the coding region. Besides sequences that can be translated into proteins, there are also untranslated sequences, such as the 5' cap and 5'-UTR at the 5' end, as well as the 3'-UTR and polyadenylate tail at the 3' end. The 3'-UTR contains the sequence AAUAAA, which can be recognized by specific proteins to initiate polyadenylation, resulting in mRNA cleavage and addition of approximately 250 adenosine monophosphates at the 3' end, forming the polyadenylate tail. The sequence, length, and secondary structure of the 3'-UTR may be involved in post-transcriptional regulation, affecting mRNA polyadenylation, translation efficiency, mRNA localization within cells, mRNA stability, and other functions through various mechanisms. Many mutations in the 3'-UTR are associated with specific diseases.

Methods for miRNA Functional Studies

For gene-related functional studies, the methods typically involve either overexpression or knockdown, and miRNA research is no exception. If this step follows target gene prediction, it can serve as validation for the predicted target genes. For example, after overexpression or knockdown, one can use techniques such as PCR, Western blotting, or sequencing to observe the expression of target genes, thereby validating the binding relationship between miRNA and its targets. Conversely, if this step precedes target gene prediction, sequencing can be used to identify differentially expressed genes, thereby identifying potential target genes. Subsequently, fluorescence enzyme experiments can be employed to validate the screening results. Therefore, although the techniques are the same, the sequence in which they are employed can have different implications.

Three commonly used methods for miRNA overexpression are as follows:

• The first is constructing an overexpression vector for miRNA. Typically, the precursor of miRNA is cloned into an overexpression vector. After overexpression, the precursor is cleaved to produce mature miRNA.
• The second method involves chemically synthesized miRNA mimics. These mimic molecules, when transiently transfected into cells, increase the level of miRNA, thus achieving overexpression.
• The third method is miRNA agomir, which is a chemically modified derivative of mimics. It exhibits better efficacy in in vivo experiments compared to mimics alone.

Through overexpression techniques, the phenotypes resulting from excessive miRNA levels can be observed, providing insights into the potential functions of miRNA.

Regarding miRNA interference, there are generally four methods:

• The first method is constructing miRNA sponge, which is a commonly used technique. It involves designing plasmid vectors with tandem arrays of miRNA binding sites based on the interaction between miRNA and mRNA. After transfection into the host cells, the miRNA sponge is transcribed into mRNA in the cytoplasm, inducing binding of miRNA with similar binding domains, thereby relieving its suppression of target genes.
• The second method is chemically synthesized miRNA inhibitors, which are single-stranded molecules completely complementary to the corresponding miRNA. When transiently transfected into cells, they directly bind to the miRNA complementary sequence, thus relieving the suppression of target genes.
• The third method is miRNA antagomir, a chemically modified derivative of Inhibitors, which shows better efficacy in in vivo experiments.
• The fourth method is miRNA knockout, which disrupts the coding genes of miRNA. While the previous methods prevent miRNA from binding to target genes, this approach disrupts miRNA from the perspective of its coding genes. These techniques can reduce the inhibitory effect of miRNA on target genes, thereby increasing the expression levels of their proteins.

Research Hot Spots in miRNA

With the rapid development of high-throughput sequencing technology, miRNA sequencing based on high-throughput sequencing has emerged as a new and powerful tool for identifying and quantitatively analyzing miRNAs, overcoming the limitations of other research methods. In recent years, the biological roles of miRNAs have gradually become well-known. As a mature non-coding RNA for research, miRNAs have gradually become more comprehensive and deep in the following application areas:

Molecular Markers

Research has shown that the levels of microRNAs in cells and tissues are closely related to their states. Timely monitoring of microRNA levels within cells may reveal the cell's status, and even predict the next steps in cell development, which is one of the important principles for applying microRNAs in cancer monitoring.

Disease Treatment Research

In clinical medicine, by introducing microRNAs or microRNA antagonists (often sequences paired with microRNAs) into patients, the levels of microRNAs in the body can be increased or decreased, thereby regulating downstream gene expression and ultimately achieving therapeutic effects.

Artificial miRNAs

Artificial microRNA (amiRNA) technology is designed based on the generation and action principles of natural miRNAs. It involves designing small RNA molecules targeting one or more specific genes. These molecules efficiently and specifically inhibit gene expression. AmiRNAs can silence different proteins within the same family, making them highly popular.