What is miRNA?

MicroRNA (miRNA) is a non-coding RNA, meaning it is not translated into protein. Instead, miRNAs partake in post-transcriptional gene regulation and silencing to control the translation of messenger RNA (mRNA) into proteins. miRNAs have been found to be critical regulators of essential cellular processes in many organisms. Their functions in health and disease make them attractive candidates for gene therapy targeting cancer and autoimmune diseases.

What Does miRNA Do, and How Was It Discovered?

miRNA consists of 19-24 nucleotides that regulate gene expression by binding complementary mRNA sequences to inhibit translation into proteins.

The discovery of miRNA dates back to 1993 when Lee et al. discovered that the nematode Caenorhabditis elegans transitioned from the first to the second larval stage by downregulating the expression of the LIN-14 gene¹. The downregulation was achieved by a second gene called lin-4, a 21-nucleotide long RNA that was not translated into a separate protein. Initially thought of as a mechanism specific to LIN-14, the same downregulation was observed with another protein, lin-41, regulated by the small RNA let-7². The early 2000s saw a flood of studies discovering non-coding small RNAs in many other species, including humans. Their roles in cellular functions are discussed in the next sections.

miRNA Biogenesis

miRNA synthesis and processing of consists of several steps:

• RNA polymerase (Pol II) transcribes miRNA into primary miRNA (pri-miRNA), a long sequence harboring several miRNA transcripts.
• Pri-miRNA is cleaved in the nucleus by the Drocha-DGCR8 complex, releasing a precursor miRNA (pre-miRNA) of 70 nucleotides long³.
• Pre-miRNA is exported to the cytoplasm, where it is processed further by the RNase III enzyme Dicer. Dicer generates a 19-24 nucleotide-long miRNA duplex⁴, One strand of which incorporates into the RNA-induced silencing complex (RISC) tasked with inhibiting complementary mRNA targets⁵.

miRNA Mechanism of Action

miRNA-mediated regulation involves multiple mechanisms and occurs in a miRNA-induced silencing complex (miRISC). In general, miRNAs bind to target mRNAs at a specific sequence in the 3'untranslated region (3'UTR), although more recent studies uncovered binding at 5'UTR and coding regions.

Depending on the level of complementarity, this might serve to degrade mRNA and repress or activate translations. Generally, if a full complementarity occurs, the mRNA poly(A) tail is cleaved off with the help of Argonaute (AGO2) proteins and degraded by cellular exonucleases⁶ . However, many mRNA target sites do not possess the full complementary sequences, in which case the miRISC suppresses translational activity by interfering with the binding of translation initiation factors. Wu et al. showed that miRNA binding accelerated mRNA deadenylation and destabilization⁷ .

More rarely, mRNA engagement by miRNA can activate the target mRNA, which was observed during cell cycles for the expression of proteins that helped maintain quiescent cell states⁸.

Cellular Functions of miRNAs

miRNAs have been shown to be instrumental in embryonic development and organ formation. Studies showed that knocking down Dicer during biogenesis caused morphological abnormalities and increased the likelihood of embryonic death in mice and fruit flies, which was attributed to impaired stem cell development⁹'¹⁰.

miRNAs are key regulators that play a role in stem cell renewal or differentiation, as determined in Dicer-deficient mice that displayed impaired organ formation¹¹. Researchers discovered a subset of miRNAs termed embryonic stem cell cycle (ESCC) – promoting miRNA, which promotes G1-S transition by inhibiting the expression of cell cycle-inhibiting proteins¹². Thus, ESCC miRNAs may be influential in maintaining stem cell pluripotency. On the other hand, other miRNAs like let-7 were upregulated upon differentiation, suppressing pluripotency in differentiated stem cells¹³.

Immune response regulation can also be attributed to miRNAs. For example, modulation of macrophage activation and differentiation are overseen by miR-125b, which regulates the nuclear factor (NF)–κB pathway¹⁴.

Several miRNAs were implicated in organ development processes to slow down proliferation and induce differentiation. These include but are not limited to miR-273 for neuronal development¹⁵, miR-1 for cardiac morphology¹⁶, miR-27 for myocyte differentiation during skeletal muscle development¹⁷, and miR-127 for lung branching¹⁸.

miRNA and Disease

Functional miRNA biogenesis and regulation are significant in cell development and differentiation. Because of this, miRNA dysregulation is strongly associated with several human malignancies. The known miRNA dysregulation mechanisms include:

• Upregulated or downregulated miRNA expression,
• Dysregulation of miRNA transcription factors,
• Epigenetic modifications on miRNA, such as hypermethylation and histone acetylation, and
• Impaired miRNA biogenesis caused by aberrant activity of RNase III endonucleases¹⁹

The first evidence linking miRNA dysregulation to cancer emerged in 2002. Calin et al. discovered two miRNA genes, miR-15a and miR16-1, which acted as tumor suppressors and apoptotic regulators, were depleted in B-cell chronic lymphocytic leukemia²⁰. Since then, research has pointed to the deleterious impact of altered miRNA on several cancer-associated phenotypes. Some examples include:

• miR-21 overexpression downregulating the tumor suppressor Leucine zipper transcription factor-like 1 (LZTFL1) and promoting proliferation and metastasis in breast cancer²¹.
• Upregulation of miR-185 and downregulation of miR-133b were detected in highly metastatic colorectal cancer²².
• Downregulation of miR-29b drives epithelial to mesenchymal transition (EMT) in prostate cancer²³
• miR-21 overexpression promoting angiogenesis by targeting PTEN, which activates downstream signaling pathways and upregulates vascular endothelial growth factor (VEGF)²⁴

Overall, studies showed the influence of aberrant miRNA expression on several biological processes linked to cancer.

miRNA dysregulation has been correlated to many other infectious and non-infectious diseases. It was reported that human immunodeficiency virus-1 (HIV-1) suppressed miRNA expression to promote viral replication in the host²⁵. Mycobacterium tuberculosis facilitated host invasion by upregulation of miR-99b to suppress immune response from dendritic cells and macrophages²⁶. Finally, miRNA dysregulation has been detected in autoimmune diseases, from multiple sclerosis (MS)²⁷ to systemic lupus erythematosus (SLE)²⁸.

miRNA in Therapeutic Applications

Given the broad scope of microRNA function and involvement in disease, they have become attractive targets for therapeutic applications. Because a single miRNA can target multiple pathways, miRNA-targeting can ensure a holistic regulation of altered cellular mechanisms in disease.

A number of miRNA-based therapeutics are on trial to treat Huntington's Disease, type 2 diabetes, non-small cell lung cancer, hepatocellular carcinoma, and heart failure, although the mechanism of action varies depending on disease. Some miRNA therapeutics are oligonucleotide-based miRNA inhibitors (anti-miR), targeting dysregulated miRNAs to regulate the cellular mechanisms they disrupt. Other synthetic miRNA therapeutics serve as miRNA mimics to compensate for downregulated counterparts, targeting aberrantly expressed mRNA to regulate protein synthesis.

Although the potential of miRNA therapeutics is promising, they are still under development. A key challenge in their systemic administration are off-target effects due to the wide range of cellular pathways miRNAs can control. One proposed solution is the targeted delivery of miRNA mimics. The anticancer miRNA-mimic therapeutic TargomiR is an ideal example that enhances the targeting capacity of miRNA by packaging it in bacterial-based nano cells coated with tumor-specific antibodies²⁹.

Another avenue of interest is combining miRNA therapeutics with conventional drugs or small-interfering RNAs (siRNA). For instance, the combination of miR-159 miRNA mimic and doxorubicin was shown to counteract drug resistance in triple-negative breast cancer³⁰. Additionally, Petrek et al. demonstrated the efficacy of recombinant plasmid co-expressing miRNA and siRNAs on lung cancer cell lines³¹.

Finally, the development of miRNA sponges with multiple miRNA binding sites shows potential by regulating multiple dysregulated miRNAs. Preliminary studies demonstrated efficacy in mouse models for cardiovascular diseases³² and human breast cancer cell lines³³.

References

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