A Time For Non-Coding RNA To Shine

Once considered the dark matter of the human genome, non-coding RNAs now shed light on early signs of cancer and, soon – a wide range of human diseases.

There are about 3 billion nucleotides in the human genome, but only 1% of all genes are protein-coding. The remaining 99% are non-coding genes that may give rise to transcripts known as non-coding RNA (ncRNA).

Because ncRNAs were thought not to have any role in metabolic processes, they were long considered merely “junk” or “noise.” It wasn’t until 45 years ago—when Mizuno et al. established micF, a small ncRNA in E. coli that regulates response to environmental stress—that this view began to change. The discovery is now considered one of the earliest pieces of evidence that demonstrate the cellular function of ncRNAs.

Ironically, a plethora of RNAs were discovered much earlier. But in all these instances, scientists used up a lot of time, energy and resources between discovering the RNA and understanding its regulatory function. For instance, in 1967, British pathologist George Brownlee successfully discovered 6SRNA, a ubiquitous regulatory RNA in bacteria—and the very first non-coding RNA to be sequenced. In 1988, Ikemura et al. found Spot 42, a regulatory RNA in carbohydrate metabolism. From there, it took a few more decades for the regulatory role of both RNAs to be fully outlined.

Today, the pace of research has changed. Technological advancements in sequencing workflows, as well as data handling tools, have led to an explosion of ncRNA-centred papers in recent years. Many of these studies focus on exploring the clinical relevance of ncRNA, such as in early disease detection, and hold a lot of promise in the lab and in the field.

In Singapore, Assistant Professor Cheong Jit Kong, Director of the Yong Loo Lin School of Medicine ncRNA Core Facility (NUSMed ncRNA Core) at the National University Singapore, is at the forefront of this movement. He investigates how ncRNAs can be used as clinically- relevant biomarkers for human disease detection, including the identification of early stages of cancer.

“RNA research and development has very much been challenging, because of various aspects. Compared to known biological analytes like DNA, messenger RNA and proteins, ncRNA research has a pretty short history,” he shares. “While it is an emerging field, at its very nascent stage, we want to get to know ncRNAs betterwhat they do and how they control normal cellular processes in our body.”


Peering through a black hole

Today, scientists understand that the ncRNA is a collective term for a group of genetic moieties that do not get translated into a protein. “They come in different forms, including microRNA (miRNA), long non-coding (IncRNA), circular (circRNA), and other small RNAs,” Dr. Cheong explains.

Of these, miRNA remains the tool of choice for potential use at the bedside. The miRNome, an atlas of all miRNAs, is a smaller subset than other ncRNA groups. Out of approximately 2,500 mature miRNAs, 800 of these have already been cross-validated using different workflowsInterestingly, it was a previous study Dr. Cheong conducted in the miRNA space that inspired him to pursue ncRNA research in cancer.

“I came across a research article that described how miRNAs control the expression of my favourite gene, Casein kinase 1α (CK1α) via its noncoding 3’-untranslated region in its transcript,” Dr. Cheong says. CK1α has been cited for its role in “diverse cellular, physiological, and pathological processes”—all properties that make it a suitable target for newly developed therapeutics.

“More recently, we have shown that the non-coding 5’-untranslated region in the CK1α transcript recruits RNA-binding proteins to regulate protein translation of CK1α in mutant RAS-driven cancers,” he adds. RAS genes are a family of genes involved in modulating cell growth and apoptosis, with their mutations accounting for a third of all human cancers.


The dawn of ncRNA biomarkers

In the paper Noncoding RNome as Enabling Biomarkers for Precision Health, Dr. Cheong and his collaborators proposed ncRNA-based liquid biopsy as a non-invasive alternative to standard surgical biopsies for informing clinicians of the best cancer management strategy for each patient.

“The promise of ncRNAs in altering the clinical trajectory of human diseases is best demonstrated in the context of human cancer,” says Dr. Cheong. “ncRNA research is making significant contributions to advance our understanding of the complexity of cancer, as well as the many challenges in its management and therapy.”

The success of the concept of liquid biopsies as a non-invasive test for cancer screening is hinged on the dynamics of miRNAs. They display unique patterns at the onset and progression of disease while remaining in a stable state in healthy cells. These patterns are referred to as biomarkers, or biological indicators of the state of a disease. And since there is conclusive evidence of miRNAs being secreted in circulation, obtaining a liquid biopsy could provide the same molecular granularity as information otherwise obtained through more invasive means.

Dr. Cheong worked with a local biotechnology company, MiRXES, to introduce its industry-leading miRNA profiling platform. “MiRXES’ research collaboration with the Singapore Gastric Cancer Consortium culminated in the discovery and development of a world-first miRNA-based clinical assay for early detection of gastric cancer.

The said assay is now CE-marked and an HSA-approved Class IV in vitro diagnostics (IVD) medical device that is currently undergoing registration trials in China and Japan. Aside from promising better healthcare outcomes, the project has also cemented Singapore as a leader in the ncRNA sphere.


A bright future in precision medicine

In 2020, as a result of the growing prominence of ncRNA research, Singapore hosted a global ncRNA Symposium. The event not only became a venue for clinical researchers to share their groundbreaking work, but more importantly, Dr. Cheong says that it also set the stage for “the development of a national standard to guide the design, development and performance evaluation of miRNA-based molecular diagnostic assays.”

Of course, the path to standardising ncRNA-based tools in the clinical landscape does not come without roadblocks. “We still have to enhance data reproducibility and translation of miRNA clinical assays, as well as validation studies,” Dr. Cheong shares. The publication of Singapore Standard SS656 helped lay the foundation of best practices for the isolation, handling, and analysis of ncRNA biomarkers. And while it has become Singapore’s valued contribution to the International Nucleic Acid IVD standard, much and more needs to be done.

The underrepresentation of the Asian population in genomic studies is a recurring theme. “The more we unravel truths in the biomedical sciences, the more we know that our genetic makeup is slightly different. This can affect the decision-making process in clinical management,” Dr. Cheong points out. Discoveries based on our own clinical cohort, then, are imperative—and fortunately, within reach. He adds, “We have excellent public healthcare institutions and institutes of higher learning with strong research capabilities in Singapore, as well as a trove of private biotechnology companies that work together to accelerate bench-to-bedside translation.”

Dr. Cheong’s vision? To champion the golden era of RNA research and complement existing genome data with nuanced insights. “If anything, the COVID-19 pandemic catapulted RNA-powered in vitro diagnostics and medicine into prominence,” he says. “For many years, we’ve been dreaming about applying RNA medicine to clinical applications and we’ve done that exactly in a span of two years, as you can see in the creation of COVID-19 mRNA vaccines and implementation of COVID vaccination programs.”

This, Dr. Cheong hopes, is only the beginning: Moving forward, ncRNAs will be a key modality for developing therapeutics for a range of human diseases.