Introduction of miRNAs is to suppress genes by specifically

Introduction 

Micro-RNAs (abbreviated to miRNAs) are small (21-23 nucleotides), highly conserved non-coding RNA molecules (1). These small molecules count for a large proportion of the human genome, at just under 50%, with over 1000 known human miRNAs to date. Their role of miRNAs is to suppress genes by specifically binding to target mRNAs. By doing so, they are estimated to regulate around one third of the human genome. Due to this crucial role in gene regulation, miRNAs are essential for a whole range of metabolic processes in humans. (2)

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MiRNAs were originally discovered in the 90s in Caenorhabditis elegans. Research was being carried out into Lin-4, a gene involved in controlling larval development, which was found to be responsible for the production of two short RNA molecules. It was later discovered that the longer of these two RNA molecules, 70 nucleotides long, was in fact the precursor for a 22 nucleotide miRNA. Since this discovery many more miRNAs, as well as other non-coding RNAs, such as Ait, H19, Ipw and Tsix have been found to act as gene regulators and silencers. (1)

Since miRNAs have such a pivotal role in human development they can be an extremely prevalent tool for preventing, as well as causing, disease.  The up or down regulation of certain miRAs offsets the balance of gene expression within developmental processes, and in turn, can cause disease. They have been linked to a large range of diseases, from myocardial infarction to autoimmune diseases. This essay outlines role of miRNAs in cancer and how mutations in miRNA or the biogenesis machinery used to produce the miRNA can lead to this disease. Studying the relationship between miRNAs and cancer also provides a useful insight about how we may be able to use miRNAs in the prevention and treatment of cancer in the future. 

Biogenesis of miRNA  

The biogenesis of miRNA is usually characterised by two main cleavage events; although the mechanism of biogenesis changes slightly depending on the origin of each individual miRNA. 
A double-stranded RNA stem loop, around 70base pairs long and found in introns and intervening clusters, encodes each miRNA. RNA polymerase II or III transcribes the gene to generate a primary microRNA (pri-miRNA) precursor molecule. Next, whilst still inside the nucleus the precursor molecule is then cleaved by a microprocessor made up of DGCR8 (an RNA-binding protein) and type III RNase Drosha. This cleavage step produces a roughly 85 nucleotide long precursor microRNA (pre-miRNA). (3) 
From here the pre-miRNA is transported into the cytoplasm via a Ran/GTP/Exportin 5 complex. Exportin 5 (XPO5) is a double-stranded RNA-binding protein and is the part of the complex which attaches to the pri-mRNA. (See Fig 1). The XPO5-RanGTP complex is able to recognise the pre-miRNA as it has an overhang of two nucleotides at its 3′ end. This ensures that XPO5 is specific in transporting only pri-mRNA into the cytoplasm. Once the miRNA is bound a ‘ternary complex’ has been formed; this newly formed complex is then able to diffuse across the nuclear envelope  through a pore into the cytoplasm.(4) Once in the cytoplasm the first cleavage takes place producing a microRNA duplex (miRNA:miRNA*, the asterisk denotes the passenger strand) containing the now mature miRNA. 
The duplex soon unwinds and the mature portion of miRNA folds itself into the RNA-induced silencing complex (RISC). The RISC complex of miRNA is the able bind with target mRNA to direct gene silencing. The gene silencing effect is achieved either simply by translation repression or by cleavage. The method of silencing used is dependant on the degree of complementarity between the miRNA and its target. Figure 2 shows a schematic diagram of miRNA biogenesis. 

miRNAs in Cancer 

Changes in a specific miRNAs expressions can cause issues at opposite ends of biological spectrum. For example, if a certain miRNA that is complementary to a Tumour-suppressor gene is over expressed, then this could results in apoptosis. Whereas, if the same miRNA is down-regulated then it may function as an oncogene, causing cancer. As shown in figure 3, each type of cancer has its own different miRNA expression profile. Hopefully, by compiling data for the miRNA profile of each type of cancer, we will eventually be able to use these profiles for cancer diagnosis and possibly treatment.  

Why do miRNAs become dysregulated?

1) Mutation of the miRNA

The up and down regulation of certain miRNAs can be due to amplification or deletion of miRNA genes. For example, in the case of lung cancer, the 5q33 region, which contains the genes for miR0143 and miR-145, is frequently deleted. This causes both of the miRNAs to br down regulated. On the other hand, amplification of the mi-R-17-92 cluster gene has been linked to many different cancers, including lung cancer, breast cancer, ovarian cancer and T-cell acute lymphoblastic leukaemia. (3)

2) Defects in the biogenesis machinery 

In addition to the up or down regulation of miRNAs causing cancer, it is thought that defects in the biogenesis machinery of miRNA may also be linked to cancers. As described previously, miRNA involves multiple enzymes and proteins, including Drosha, Dicer, DGCR8, Argonaute and Exportin 5. Therefore, if any if this biogenesis machinery experiences a mutation effecting its conformation, or is over or under expressed, then this could lead to abnormal expression profiles of miRNAs.

Drosha and Dicer both belong to the RNase III endonuclease family and both play a significant role in miRNA maturation,  forming pre-miRNA and miRNA duplex respectively. When these two enzymes are not regulated correctly they will up or down regulate certain miRNAs, perhaps leading to tumours. For example, in 2015 when studying a sample of 534 patients with Wilms’ tumours, Walz found that single nucleotide mutations in DGCR8 and Drosha were found in 15% of cases.  In terms of miRNAs, this mutation lead to decreased expression of mature Let-7a and miR-200 family; perhaps having a knock on effect large enough to induce the tumours.(5)
Furthermore,  in ovarian cancer high levels of these two enzymes are associated with increased survival, suggesting that the up regulation of certain miRNAs increases chance of recovery. Conversely, decreased Dicer expression significantly correlates with reduced patient survival. This suggests that down regulation of specific miRNAs may be a likely cause, or at least factor, of ovarian cancer. (6)

Similarly, dysregulation of Argonaute has also been linked with cancer.  In another study on Wilms’ tumours, it was found that the Argonaute gene (EIF2C1/Ago) was quite frequently lost.  In other words, a decrease in the Argonaute protein, and therefore a likely down regulation of miRNAs, is linked to Wilms’ tumours.  Similarly, Argonaute expressions is lower in melanoma cells than healthy skin cells, suggesting a link between the down regulation of miRNAs and malignant melanoma. (7) Alternatively, in some cancers Argonaute proteins seem to be over expressed. Patients with gastric cancer, for example, have  a sigifantly highest expression of human argonaute proteins than healthy individuals. (8)

Finally, in terms of biogenesis machinery, Exportin 5 (XPO5) mediates export of pre-miRNA from the nucleus and into the cytoplasm as part of a Ran/GTP/Exportin 5 Complex. As with the other biogensis machinery, dysregulation of Exportin 5 has been linked to multiple cancers,  In Dukes’ type C colorectal adenocarcinoma cells a single point insertion of an alanine into exon 32 produces an early termination signal. This results in frameshift mutation and hugely changes the conformation of Exportin 5, causing it to lose its function to export pre-miRNAs. This leads to the pre-miRNAs building up in the nucleus, without moving onto the next stage of processing, ultimately resulting in reduced expression of miRNA. This potentially means that if we are able to reverse the mutation and restore the protein to its regular conformation  that it may suppress the tumour from spreading further. (9)

The possibility for cancer treatment using miRNAs

By studying miRNA profiles are a few therapeutic methods have been found that could possibly help to suppress tumour growth: miRNA Inhibition theory, miRNA mimetic agents and Small Molecules Inhibitors of miRNAs. (10)
The miRNA Inhibition Therapy involves designing oligonucleotides that will force the miRNA into an unusual conformation, meaning that it cannot by processes by the RISC. These single stranded oligonucleotides include anti-MiR oligonucleotides, antagomirs, miRNA masks and multiple others. This sort of therapy would be possible for when miRNAs are up regulated. Antagomir-122 was injected into mice and was successful, causing a reduction in miR-122 in multiple organs including the heart and lungs for up to 23 days. (10) 
Secondly, miRNA mimics can be used to correct mutations in tumour suppressing miRNAs in order to restore their tumour suppressing function. This has been shown to work in in vitro experiments with miR-15a. When miR-15a mimics was introduced to prostate cancer cell, apoptosis occurred and cell proliferation stopped. This is very positive data which presents a exciting future for cancer therapies, however effective in vivo experiments must be carried out first.
Lastly, small molecule inhibitors of miRNAs (SMIRs) act to inhibit miRNA biogenesis, therefore preventing it from binding to its target; this is useful for diseases I need which miRNAs are up regulated. Although research involving SMIRs is still in it’s very early stages, a few molecules have been found to work. For example, enoxacin binds to TAR RNA-binding protein 2, a protein involved in miRNA biosynthesis. (10)

Conclusion 

In summary, miRNAs have been linked to many cancers, with each individual type of cancer exhibiting specific miRNA profiles. It is thought that increased and decreased expression of specific miRNAs has the ability to both encourage and suppress malignant tumour growth. This change in expression may be due to many reasons, including a mutation in the miRNA gene itself, or conversely by dysregulation of the proteins used in miRNA biogenesis. Lastly, due to fast emerging data on miRNA profiles in disease, therapies such as SMIRS are beginning to be designed and tested. Although they are still in the early stages, most of which are still in in vitro testing, this research marks an exciting future for the use of miRNAs in disease treatment. 

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