microRNA Primer: The Basics

Since its discovery in 1993, microRNA (miRNA) and research into its function have brought some exciting applications to the field of molecular biology. Many novel miRNAs have been identified and their role in gene expression has become well understood (ref 1).

What is microRNA?

Approximately 18-22 nucleotides in length, these small, single-stranded non-coding RNAs are capable of serving multiple roles related to gene expression. Unlike messenger RNA (mRNA) which is responsible for carrying genetic information from the nucleus into the cytoplasm to facilitate protein synthesis, miRNA interact with mRNA to suppress translation, downregulating protein synthesis. In this way miRNA are responsible for managing which types and what quantities of proteins cells make.

The extracellular presence of miRNA and its motility between cells demonstrate their use in cell-to-cell communication, being transported by exosomes and Argonaute protein complexes (ref 2). Their role as gene regulators and their overall stability and presence in all bodily fluids, tissues, and most cell types make them an ideal candidate for use as biomarkers in diagnostic testing and as prognostic tools (ref 3).

Over 2,600 miRNAs have been cataloged thus far in the human genome, and interactions with many different messenger RNAs have been observed. The biosynthesis of most miRNA occurs in the nucleus resulting from the transcription and processing of precursor RNAs. MicroRNAs are named using the prefix miR- and a number, and may end with 5p or 3p referring to the parent RNA strand from which it was cleaved.

A myriad of miRNA functions has been suggested including interactions with DNA in the nucleus to promote active or inactive chromatin states. In the cytosol miRNA may inhibit or promote translation, and they may trigger mRNA degradation, though more evidence for this is required (ref 3-4).

What can miRNA tell us?

The body of research has concluded that miRNAs are involved with regulating approximately 60% of human RNA from a given cell (ref 5). As such, the role of miRNA has been studied extensively across numerous applications in medicine and microbiology. Exciting potential exists for miRNAs to be utilized as biomarkers for diagnosing infectious and non-infectious diseases, and for assessing conditions pertaining to gut and reproductive health, among others (ref 6-9).

Circulating miRNAs are typically transported as part of a protein complex or in vesicles, which help protect the miRNA and keep it stable. These circulating miRNAs can resist decay for up to four days at room temperature, and have survived extreme temperature fluctuations and a wide range of pH values (ref 8). Quantitative PCR (qPCR), when used as a diagnostic, successfully identifies miRNA using less material and with a lower failure rate than mRNA-based assays (ref17). The small size, stability, abundance and specificity of miRNA will make them one of most utilized biomarkers for developing new, faster, and more sensitive methods of disease detection and therapeutic intervention going forward.

As novel miRNA and the genes they regulate are identified, we can learn much about protein output by measuring miRNA. Indeed, miRNA exhibits a higher correlation with protein output than the related mRNA. In other words, measuring the presence of miRNA gives a better indication of the status of translated proteins than the mRNA itself (ref1). Dysfunction of miRNA regulation by diseases such as cancer and other conditions will likely be more easily detectable by studying miRNA expression.

Modern applications of miRNA

Research is ongoing to identify and catalog miRNA in the context of early disease detection, increasing the efficacy of treatment and dramatically improving prognosis.  Prostate cancer patients show a reliable difference in the expression of miR-143 compared to healthy individuals (ref10). Early miRNA researched identified miR-43 as a potential Lymphoma biomarker (ref11). Novel miRNA in blood and tissue have been identified to indicate the presence of many types of cancer including breast, colorectal, lung and liver cancer (ref 12,13,14).

As a predictor of disease or prognostic indicator miRNA also shows promise. In addition to its reliability as a test for lung cancer, a study of smokers showed detectable changes in miRNA expression years before the onset of disease compared with disease-free smokers (ref 12).

Other diseases studied with respect to miRNA diagnosis involve diabetes and even Alzheimer’s. Not only have disrupted miRNA conditions been observed with non-infectious diseases, but changes in the expression of circulating miRNAs in bodily fluids have been observed in response to infectious disease as well, including HIV, malaria and Ebola virus. Further, miRNA has the benefit of being utilized as a biomarker during early infection stages when typical diagnostics utilizing antibodies or viral RNA are unreliable and the disease undetectable (ref 6).

Using miRNA to develop therapeutic applications is the likely next step. With the specificity of miRNAs, a huge potential exists to utilize them as a therapeutic target and research into the use of miRNA for cancer therapies is being undertaken, with several miRNA-based drugs entering clinical trials (ref 3-5).

How Inti Labs and MIRA™ use miRNA

The increasing global trend towards infertility for hopeful parents is due to several factors, including genetic preconditions, disease, infection, and the age of the mother. As a result, many people are turning to assisted reproductive technology (ART) including in vitro fertilization (IVF). One of the costly and time-consuming challenges presented to parents attempting IVF is implantation failure.

A contributing factor to implantation failure is a displaced Window of Implantation (WOI), referring to the period of time during the menstrual cycle when the endometrium is most receptive to embryo implantation. As many as 30% of infertility patients experience a displaced WOI (ref 16). Utilizing miRNA as an indicator of endometrial receptivity can elucidate this discrepancy, informing IVF providers exactly when is the optimal time to implant.

As miRNAs have been shown in multiple studies to regulate the status of the endometrial lining, approximately 100 novel miRNAs have been identified which interact with over 600 genes related to endometrial receptivity (ref 16, 17). This target group of miRNAs makes up the panel analyzed by MIRA™ to provide insight into whether or not the endometrial biopsy was collected pre- or post-WOI. For example, high expression of miR-145 has been shown to correlate with implantation failure. Upregulation of some miRNAs, such as miR-429 and miR-125b, and downregulation of others can indicate implantation failure, endometrial receptivity decline, or poor pregnancy rates. A subsection of this panel of miRNAs analyzed by MIRA™ has a 90% accuracy rate for attributing implantation failure to a displaced WOI (ref 16-19).

What this means for providers is that patients who have had repeated implantation failure, or who have a limited number of IVF attempts may use MIRA™ to increase the chances of successful implantation. Understanding a patient’s endometrial health is key to giving them the best opportunity for a viable IVF treatment and can additionally serve to rule out a displaced WOI as a cause of implantation failure.

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References

1. Halushka, M. K., Fromm, B., Peterson, K. J., and McCall, M. N. (2018). Big strides in cellular microRNA expression. Trends Genet. 34, 165–167. doi: 10.1016/j.tig.2017.12.015
2. Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, et al. Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci USA (2011) 108:5003–8. doi: 10.1073/pnas.1019055108
3. Barbarotto, E., Schmittgen, T. D., & Calin, G. A. (2008). MicroRNAs and cancer: Profile, profile, profile. International Journal of Cancer, 122(5), 969– 977.
4. Volinia S, Calin GA, Liu CG, et al.: A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006;103(7):2257–61 10.1073/pnas.0510565103
5. Bartel, D. P. (2018). Metazoan MicroRNAs. Cell, 173(1), 20–51. https://doi.org/10.1016/j.cell.2018.03.006
6. Biswas, S., Haleyurgirisetty, M., Lee, S., Hewlett, I., and Devadas, K. (2019). Development and validation of plasma miRNA biomarker signature panel for the detection of early HIV-1 infection. EBioMedicine 42, 307–316. doi: 10.1016/j.ebiom.2019.04.023
7. Hayder H, O’Brien J, Nadeem U, Peng C. MicroRNAs: crucial regulators of placental development. Reproduction (2018) 155:R259–71. doi: 10.1530/REP-17-0603
8. Wu, C. W., Cao, X., Berger, C. K., Foote, P. H., Mahoney, D. W., Simonson, J. A., Anderson, B. W., Yab, T. C., Taylor, W. R., Boardman, L. A., Kisiel, J. B., & Ahlquist, D. A. (2017). Novel approach to fecal occult blood testing by assay of erythrocyte-specific microRNA markers. Digestive Diseases and Sciences, 62(8), 1985–1994. https://doi.org/10.1007/s10620-017-4627-6
9. Hayes J, Peruzzi PP, Lawler S. MicroRNAs in cancer: biomarkers, functions and therapy. Trends Mol Med. (2014) 20:460–9. doi: 10.1016/j.molmed.2014.06.005
10. Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res (2008) 18:997–1006. doi: 10.1038/cr.2008.282
11. Lawrie CH, Gal S, Dunlop HM, et al.: Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma. Br J Haematol. 2008;141(5):672–5 10.1111/j.1365-2141.2008.07077.x
12. Boeri M, Verri C, Conte D, et al.: MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A. 2011;108(9):3713–8 10.1073/pnas.1100048108
13. Ali Ahmed, E., Abd El-Basit, S. A., Mohamed, M. A., and Swellam, M. (2020). Clinical role of MiRNA 29a and MiRNA 335 on breast cancer management: their relevance to MMP2 protein level. Arch. Physiol. Biochem. doi: 10.1080/13813455.2020.1749085 [Epub ahead of print].
14. Ji, J., Yu, L., Yu, Z., Forgues, M., Uenishi, T., Kubo, S., Wakasa, K., Zhou, J., Fan, J., Tang, Z. Y., Fu, S., Zhu, H., Jin, J. G., Sun, H. C., & Wang, X. W. (2013). Development of a miR-26 companion diagnostic test for adjuvant interferon-alpha therapy in hepatocellular carcinoma. International Journal of Biological Sciences, 9(3), 303–312. https://doi.org/10.7150/ijbs.6214
15. Rupaimoole, R., Slack, F. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov 16, 203–222 (2017). https://doi.org/10.1038/nrd.2016.246
16. Chen CH, Lu F, Yang WJ, Yang PE, Chen WM, Kang ST, Huang YS, Kao YC, Feng CT, Chang PC, Wang T, Hsieh CA, Lin YC, Jen Huang JY, Wang LH. A novel platform for discovery of differentially expressed microRNAs in patients with repeated implantation failure. Fertil Steril. 2021 Apr 3:S0015-0282(21)00088-1. doi:10.1016/j.fertnstert.2021.01.055. Epub ahead of print. PMID: 33823989.
17. Reza, A. M., Choi, Y., Han, S. G., Song, H., Park, C., Hong, K., & Kim, J. (2018). Roles of microRNAs in mammalian reproduction: From the commitment of germ cells to peri-implantation embryos. Biological Reviews, 94(2), 415-438. doi:10.1111/brv.12459
18. Liang, J., Wang, S., & Wang, Z. (2017). Role of microRNAs in embryo implantation. Reproductive Biology and Endocrinology, 15(1). doi:10.1186/s12958-017-0309-7
19. Altmäe, S., Martinez-Conejero, J. A., Esteban, F. J., Ruiz-Alonso, M., Stavreus-Evers A., Horcajadas, J. A., & Salumets, A. (2012). MicroRNAs miR-30b, miR-30d, and miR-494 Regulate Human Endometrial Receptivity. Reproductive Sciences, 20(3), 308-317. doi:10.1177/1933719112453507