Do small RNAs have potential in disease diagnosis and treatment?

Ribonucleic acids (RNAs) are very complex and their complete functions are yet to be fully elucidated. Noncoding RNAs having no protein-coding capacity are represented by housekeeping noncoding RNAs: transfer (t)RNA, ribosomal (r)RNA, small nuclear (sn)RNA, and small nucleolar (sno)RNA that are engaged in basic cellular and molecular processes and by regulatory noncoding RNA: short noncoding RNA (<200 nt, sncRNA) and long noncoding RNA (>200 nt, lncRNA) that are important for gene expression/transcript stability. snoRNAs are extensively studied ncRNAs, which are mostly responsible for the post-transcriptional modification and maturation of rRNAs, snRNAs, and other cellular RNAs.

Several classes of small RNAs have emerged recently. Numerous aspects of their origins, structures, related effector proteins, and biological roles have led to the identification of three main categories: short interfering (si)RNAs, micro (mi)RNAs, and piwi-interacting (pi)RNAs. These small RNAs are regulated by two recently discovered miRNA-regulatory RNAs, namely competing endogenous (ce)RNA and circular (circ)RNA. Recently, another class of small RNAs (17–18 nt in length) was discovered in animals using deep sequencing approaches and these are found to be associated with transcription initiation (‘tiRNAs’) and splice sites (‘spliRNAs’). Initial studies suggest that they may play a role in nucleosome positioning and/or be involved in chromatin organization. There are also other reports of less distinct classes of promoter-associated RNAs called PASRs, TSSa-RNAs, and PROMPTS, some of which may play a role in RNA-directed transcriptional gene silencing.

Discoveries of these sncRNAs have induced a paradigm shift in our overall understanding of gene expression regulation and different signaling pathways. We now understand that coding and noncoding RNA machinery work in concert to maintain overall homeostasis. These RNAs perform diverse functions such as regulating gene expression, splicing, translation, and post-transcriptional modifications. Alterations and personal variations of RNA interactions have been automatically coupled with disease etiology and phenotypical variations. They have also demonstrated clinical significance as potential RNA biomarkers, RNA mimics, and RNA antagonists for early diagnosis, prognosis, and therapeutic targets for several diseases, including cancer [1,2].

Small RNAs are also involved in specifying and stabilizing distinct chromatin states. But lately, it has become obvious that small RNAs can trigger epigenetic gene silencing. Once initiated by primary small RNAs, the repressed states can be propagated across multiple generations. Hence, there is an assumption that small RNAs might be involved in sensing environmental conditions and triggering epigenetic gene expression changes, which is responsible for increased population fitness of an organism. In this situation, epigenetic changes induced by environmental stressors, including reprotoxicants, can explain some trans-generationally transmitted phenotypes in non-Mendelian ways [3].

A little over two decades ago, two main classes of double-stranded (ds)RNA in gene regulation, short-interfering (si)RNA and micro (mi)RNA, differing in biogenesis and modes of target regulation were discovered. siRNAs and miRNAs share many similarities and both are short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger (m)RNA, yet their mechanisms of action and clinical applications are distinct. They have recently been investigated as novel classes of therapeutic agents for the treatment of a wide range of disorders, including cancers and infections. The major difference between siRNAs and miRNAs is that the former is highly specific with only one mRNA target whereas the latter have multiple targets. The therapeutic approaches of siRNAs and miRNAs are therefore very different.

dsRNA triggers RNA interference (RNAi), which is an efficient mechanism for the sequence-specific inhibition of post-transcriptional gene expression. Hence, this homology-dependent gene silencing has a great latent role in transcriptional regulation and medicine, as a potential diagnostic and therapeutic agent for a wide range of conditions, including cancer, infectious diseases, and metabolic disorders. Moreover, the siRNAs can be used for therapy as a ‘switch off’ for many causing genes. The role of siRNA in the diagnosis of cancerous cells is still not revealed due to the immediate degradation of siRNAs before they can exert their function. Although the utilization of miRNA and siRNA as biomarkers for diagnosing early stages of diseases is still in the pipeline, it is a fast growing field in the area of diagnostics. Furthermore, some snoRNAs have been used as biomarkers for studying the mechanisms of cell transformation and tumorigenesis. They have been found to be highly expressed in blood, plasma, and serum of cancer patients. The expression profile of snoRNA has been used in classification of B-cell chronic lymphocytic leukemia [4]. On the other hand, ceRNA and circRNA have been reported to antagonize the effects of miRNA. These ceRNAs contain binding sites for different miRNAs and play an important role by competing with the miRNA targets. These circRNAs may have a role in promoting the process of carcinogenesis by altering the oncogenic signaling pathways, resulting in increased proliferation, invasion, and metastasis of tumor cells. Their alteration in expression level can serve as a promising tool for the detection of different types of cancers [5].

Currently, the major emerging and reemerging economically important infectious diseases like MERS, Ebola, West Nile, Zika, corona, and chikungunya globally are caused by RNA viruses. miRNAs are seen to be released into host cells in response to these viral infections. There are several studies in which a number of miRNAs are found to be differentially expressed during various stages of viral replication, such as 10 miRNAs in rabies, 11 miRNAs in bovine herpesvirus 5, and some miRNAs in Actinobacillus pleuropneumoniae and pseudorabies infection in pigs [6]. These miRNAs target the viral transcripts, resulting in inhibition of viral replication, suggesting their antiviral roles and possible uses for diagnosis of viral infection in addition to their genome-wide regulatory roles.

Over the last two decades, numerous (>225) virus-encoded miRNAs have been identified, most of which are from DNA viruses. Although the number of RNA-virus-derived miRNAs is currently increasing, current knowledge of their roles in physiological and pathological processes is still unclear. It is assumed that although the molecular mechanisms and machinery of host derived and viral miRNA are similar, the majority of viral miRNAs may utilize a target strategy that differs from host miRNAs. Many viral miRNAs may have evolved to regulate viral-encoded transcripts or networks of host genes that are unique to viral miRNAs. Other important roles of viral miRNA are i) during persistent/latent infection, ii) evading host defenses, and iii) meditation of evolutionarily conserved functions (e.g. immune evasion, cell cycle control, defense against apoptosis, and promotion of latency). However, there is a need for additional functional studies involving synthetic viral miRNA mutants and appropriate models of infection to understand their diagnostic and therapeutic applicability [7,8].

Additionally, the sequencing of dsRNA using dsRNA seq from total RNA present in infected tissues can act as a promising tool for viral identification. dsRNA can be used in the diagnosis of all three types of viral genomes infecting humans or animals. This is due to the property of viral replication that ssRNA+ viruses must go through an intermediate double-stranded stage, which can act as the detection stage of replicating viruses. By performing dsRNA-seq, the full-length RNA viruses having different genomes can be identified in diverse hosts [9]. This strategy involving metagenomics study is a preferable tool for identification of unknown viruses whose genome has not been sequenced. The sequence obtained is de novo assembled for identification of novel replicating viruses and can act as a promising tool for the identification of viral diseases that share little or no similarity with known viruses, including the synthetic viruses.

The profiling of miRNAs is proved to be a better indicator for many diseases, which relies on early prognosis and treatment, especially cancers. These miRNAs are released as tumor-suppressing agents during the formation of cancerous tissue and can be used for early diagnosis of growing cancer cells. Exosomes isolated from the serum have revealed different signatures of miRNAs and non-coding RNAs, which can be utilized for early diagnosis of glioblastoma. In similar diseases, like diabetes and cardiac hypertrophy, many miRNAs are released like miR375 and miR133, which can be used as biomarkers for early onset of diseases [10,11]. The expression changes in levels of miRNA are so rapid that even traumatic venepuncture can result in their upregulation. Also, the tissue specificity index (TSI) needs to be calculated for each miRNA when targeting them for diagnosis.

While dsRNAs act as novel biomarkers for the diagnosis of different diseases, they have unique challenges to face during profiling of miRNA. One of the greatest challenges is the presence of miRNA isomers (isomiRs) having slight differences from the mature sequence. These can be created by the addition of any nucleotide or imprecise cleavage by the enzymes Dicer and Drosha. These are also shown to be biologically and functionally meaningful, but while profiling miRNAs, they are considered as artifacts or noise [12]. These isomiRs may have an effect on miRNAs’ stability and also reduce their values as novel biomarkers. Another challenge is the low yield of circulating miRNA (1–10 ng/μl) in body fluids, such as plasma, serum, and urine [13]. The use of serum for biomarker studies may be biased as blood cells during coagulation are also responsible for the release of miRNAs.

In conclusion, despite the identification of many miRNA and siRNA as diagnostic or predictive biomarkers, their utility is in infancy stage. They require intensive characterization and validation before jumping to a promising diagnostic tool.

Declaration of Interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Funding

This paper was not funded.