Being Small and Intronic: miRNAs That Count!

The biological regulation of gene expression in prokaryotes by noncoding antisense RNAs was well studied in the 1980s (1), while trans-acting small RNAs were proven to exert important functions related to virulence; for example, the 514-nucleotide (nt) long transcript RNAIII, which is involved in infection aggressiveness by the Gram-positive opportunistic human pathogen Staphylococcus aureus. Through this wealth of knowledge, at the beginning of the 2000s, it was discovered that small noncoding RNAs (ncRNA), named miRNAs, were not an eccentricity of the Caenorhabditis elegans genome where they were initially discovered (2), but a commonality for most known species through well-conserved evolutionary miR families (3), and this discovery took the scientific community by storm. The further identification of their roles in human diseases (first in the most common form of adult leukemia; ref. 4) magnified the interest in the study of ncRNAs in both flavors, as small RNAs (by definition under 200-nt long) and as long ncRNA (lncRNA; ref. 5). Consequently, in the last two decades, more than 110,000 articles on the topic of miRNAs and more than 30,000 on lncRNAs were published, with half of each focusing on cancer. Most of these focus on the roles of one noncoding transcript on one coding gene downstream target with subsequent in vitro and in vivo characterization in a few cell systems.

miRNAs, as well as lncRNAs, are located everywhere in the human genome: out of all the human mature miRNAs identified by experiments, more than half are located in the introns of “host” genes (6). Functional studies of ncRNAs located in intergenic spaces are straightforward, however, for intronic miRNAs, coregulation of the miRNA and the host gene, which can be either protein-coding or a long ncRNA, adds a new layer of complexity. This colocalization also brings potential technical hassles during experimental studies due to the difficulties in separating the phenotypic effects of the intronically expressed noncoding transcript from the actual “host” gene.

In this issue, Kwok and colleagues have taken significant steps toward deciphering the complex interactor network pinpointed at a genomic locus implicated in the occurrence of colon cancer (7). Using integrative multi-transcriptomics analysis, the authors identified the FTX locus, comprising the lncRNA, FTX, and multiple intronic miRNAs, as a highly upregulated miRNA:host locus in colon cancer. These data show the power of the wise use of publicly available genomics databases, such as Gencode, miRbase, The Cancer Genome Atlas, or Gene Expression Omnibus colon cancer datasets. Nowadays, the preparation of a well-designed genomics project through the integrated multi-platform analyses of such databases offers a solid ground for the start of the project and helps in the identification of the best genetic elements to be further analyzed.

The authors continued by proving that FTX possesses oncogenic properties by significantly increasing anchorage-dependent and -independent proliferation in colon cancer models and by boosting xenograft tumor growth. Mechanistically, through the use of RNA pull-downs coupled with stable isotope labeling with amino acids in cell culture–based quantitative mass spectrometry, they found that FTX interacts with the RNA helicase, DHX9, and with the ribonuclease, DICER, potentially through a ternary complex formation between FTX, DHX9, and DICER. By transcriptome and small RNA sequencing of HCT116 colon cancer cells upon the knockdown of FTX, it was proven that this lncRNA can regulate A-to-I RNA editing. In addition, also upon FTX knockdown, a significant reduction in global miRNA expression, but not of the expression of protein-coding genes, was observed, in agreement with the role of DICER in miRNA processing (8). Furthermore, intronic miR-374b and miR-545 promoted PI3K–AKT signaling and increased anchorage-dependent and -independent growth of colon cancer cells, which were in turn inhibited by tumor suppressors PTEN and RIG-I (retinoic acid-inducible gene I, also known as DDX58; not to be confounded with retinoid-inducible gene 1, or RIG-1, which is also known as PLAAT4). Finally, intronic miR-421 may exert an autoregulatory effect on miR-374b and -545 through a mechanism not fully deciphered, but at least partially Dicer dependent.

What makes this study different from many ncRNAs studies in cancers? First, it deciphers the function of each of the noncoding genes from a multigenic locus. This adds a more holistic perspective and is closer to the reality of how complex genomic loci contribute to cancer development. Second, it proves the downstream targets of each of the short and long ncRNAs and links these into a functional network. Taken together, such data reveal the elaborate interplay between intronic miRNA and their host transcripts in the modulation of important cancer signaling pathways. Third, it shows that classical RNA editing is under the influence of ncRNAs, adding a new layer of complexity to the already complex and yet to be understood mechanism of various types of RNA editing. Fourth, it identifies an instance of miRNA to miRNA regulation, a type of interaction scantly investigated to date, which is potentially more widespread than considered when taking into account that the number of known human miRNAs has just surpassed 2,600 (9).

This report, as most well-designed studies, answers important scientific questions and also initiates a series of questions to be solved in the future. What is the clinical significance of these data? For a large number of ncRNAs, correlations with overall survival were identified. Therefore, it would be of interest to know whether these genetic elements have additive or antagonistic effects on the time from diagnosis to time of death (either having same effects or having contrary effects, respectively) for patients with tumors harboring alterations at the FTX locus. Is such a new functional landscape of multigenic loci linked to tumor initiation or to response to therapy, such as with the use of PI3K-AKT inhibitors? Biomarkers for early cancer diagnosis or for response to therapy are in great need in medical practice. One of the most successful lncRNA-based biomarkers is prostate cancer associated 3 transcript (PCA3) lncRNA (named Progensa). Progensa is the first urine-based molecular test to help determine the need for repeated prostate biopsies in men with a suspected diagnosis of prostate cancer. PCA3 is a spliced lncRNA that is highly overexpressed in most types of prostate cancer. The transcript regulates the levels of the host gene PRUNE2 through formation of a double-stranded RNA that undergoes RNA-dependent adenosine-to-inosine RNA editing (10). Does it make sense to block cancer progression through the development of the anti-FTX therapy? Targeting overexpressed ncRNAs with antisense RNAs or with small molecules directly binding to the RNA sequences or structures, or restoring the expression of lost miRNAs through mimics, is in full development in recent years. Targeting master regulators of complex downstream networks of coding and noncoding transcripts (such as FTX) could be a successful strategy for cancer therapy in the near future. Certainly, more good news will come from the research performed by the groups reporting these exciting data such as Kwok and colleagues, and more discoveries with translational potential will come from the study of the FTX locus.

No disclosures were reported.