The Observed Association Between miRNA and Gene Expression
An examination of the inverse relationship between microRNA and gene expression, from the underlying cellular mechanism to the evidence that validates the interaction.
MicroRNAs (miRNAs) are small molecules of non-coding RNA that function within cells. Gene expression is the process where a gene’s instructions create a functional product, such as a protein. These two components are linked in a regulatory relationship where miRNAs act as modulators of gene expression. This interaction is a widespread form of gene regulation in many organisms, and it allows cells to fine-tune the production of proteins with high specificity.
The Mechanism of miRNA-Mediated Gene Regulation
The relationship between a microRNA and its target gene is primarily an inverse one: high levels of a specific miRNA correspond to low levels of its target gene’s protein product. This regulation occurs after the gene has been transcribed into messenger RNA (mRNA), a process known as post-transcriptional regulation. The process begins when a mature miRNA is loaded into a multi-protein assembly called the RNA-induced silencing complex (RISC), which carries out the gene silencing.
Within the RISC, the miRNA acts as a guide. A specific portion of the miRNA, a sequence of about six to eight nucleotides known as the “seed sequence,” identifies target mRNAs. The RISC is directed to mRNA molecules that contain a sequence complementary to this seed region, most often located in the 3′ untranslated region (3′ UTR).
Once the RISC binds to the target mRNA, it can trigger one of two main outcomes. The first is mRNA degradation, where the complex initiates the destruction of the mRNA molecule. The second outcome is translational repression, where the bound RISC physically obstructs the ribosome from translating the mRNA into a protein. Both mechanisms reduce the amount of protein produced from the target gene.
Observing the Inverse Correlation Experimentally
Scientists confirm the inverse relationship between a miRNA and its target gene through perturbation experiments. These studies involve artificially altering the amount of a specific miRNA inside cultured cells and then measuring the resulting changes in gene expression. This approach provides direct evidence of the miRNA’s regulatory function.
A common technique is to introduce a synthetic “miRNA mimic” into cells, a process called transfection. This mimic is identical to the endogenous miRNA, causing its overexpression. Researchers then quantify the levels of the predicted target mRNA and its corresponding protein. A successful experiment will show a marked decrease in the target mRNA, measured using methods like quantitative real-time PCR (qRT-PCR) or RNA-sequencing.
To confirm specificity, a corresponding decrease in the protein level is also expected, which is observed using a Western blot. The inverse experiment uses a “miRNA inhibitor,” or antagomir, which binds to and sequesters the specific endogenous miRNA, blocking its function. In this scenario, researchers would expect to see an increase in both the target mRNA and protein levels, reinforcing the miRNA’s suppressive role.
Validating the Direct miRNA-Target Interaction
Observing that a miRNA’s level is inversely correlated with a gene’s expression suggests a relationship, but it does not prove a direct physical interaction. To confirm that a miRNA binds directly to a specific mRNA target site, scientists use a reporter assay, with the luciferase reporter assay being the most common. This technique provides definitive evidence of a direct binding event between the miRNA and the mRNA’s 3′ untranslated region (3′ UTR).
The process involves creating a plasmid where the gene for luciferase, an enzyme that produces light, is placed before the 3′ UTR sequence from the suspected target gene. This construct is introduced into cells along with the miRNA being studied. If the miRNA recognizes and binds to the target sequence, it will trigger repression of the luciferase mRNA. This leads to a quantifiable reduction in the amount of luciferase protein produced and a decrease in light emitted by the cells.
A critical component is the control. A second plasmid is constructed that is identical to the first, except the specific nucleotide sequence in the 3′ UTR where the miRNA binds is mutated. When this mutated plasmid is introduced into cells with the miRNA, the miRNA should no longer be able to bind. As a result, the expression of the luciferase gene is not repressed, and light production remains high, confirming the interaction is direct and specific.
Biological Significance of the Association
The regulatory partnership between miRNAs and genes is fundamental to healthy biological systems. During embryonic development, for instance, miRNAs help guide the differentiation of stem cells into specialized cell types by controlling the timing and levels of key proteins. This regulation is also active in adult organisms, helping to maintain cellular balance, or homeostasis, and enabling cells to respond to environmental signals.
The importance of this association is highlighted when it becomes dysregulated, a common feature in numerous human diseases. In cancer, some miRNAs can function as oncogenes; when overexpressed, they can suppress tumor-suppressor genes, promoting uncontrolled cell growth. Conversely, other miRNAs act as tumor suppressors, and if these are lost, their function of reining in cancer-promoting genes is removed, allowing oncogenes to become overactive.
This connection extends to cardiovascular diseases, neurodegenerative disorders, and metabolic conditions. For example, specific miRNAs are involved in processes like inflammation and lipid metabolism, which can contribute to heart disease when their regulation is disturbed. The discovery that miRNA expression is altered in many disease states has opened new avenues for diagnostics and for therapeutics aimed at restoring normal miRNA function.