RNA Biomarkers: Frontier of Precision Medicine for Cancer

Abstract

As an essential part of central dogma, RNA delivers genetic and regulatory information and reflects cellular states. Based on high-throughput sequencing technologies, cumulating data show that various RNA molecules are able to serve as biomarkers for the diagnosis and prognosis of various diseases, for instance, cancer. In particular, detectable in various bio-fluids, such as serum, saliva and urine, extracellular RNAs (exRNAs) are emerging as non-invasive biomarkers for earlier cancer diagnosis, tumor progression monitor, and prediction of therapy response. In this review, we summarize the latest studies on various types of RNA biomarkers, especially extracellular RNAs, in cancer diagnosis and prognosis, and illustrate several well-known RNA biomarkers of clinical utility. In addition, we describe and discuss general procedures and issues in investigating exRNA biomarkers, and perspectives on utility of exRNAs in precision medicine.

Keywords: biomarker; cancer; exRNA; precision medicine.

Figure 1

Studies and examples of RNA biomarkers in cancer. (A) Numbers of publications on RNA biomarker in cancer (The data were collected using keywords of “RNA biomarker” and “cancer” browsed in NCBI PubMed); (B) Typical types of RNAs used as biomarkers in cancer; (C) Gene expression pattern of PAM50 genes, calculated from published TCGA breast cancer data (BRCA). The patients were classified into five subtypes (Basal, HER2, LumB, LumA and Normal-like) based on PAM50 genes’ expression profile; (D) Kaplan-Meier analysis for different subtypes in the TCGA BRCA cohort. Subtypes were classified using the PAM50 panel. p-value was determined based on log-rank test.

Figure 2

Biogenesis and categories of extracellular RNAs (exRNAs). Biogenesis of Extracellular vesicles (EVs) released by tumor cells. EVs include exosomes, micro-vesicles, oncosomes, and apoptotic bodies. Apoptotic tumor cells release apoptotic bodies, while normal or active tumor cells release exosomes, micro-vesicles, and oncosomes. Exosomes are endocytic membrane-derived vesicles released by the fusion of the multivesicular bodies (MVBs) with the cell membrane. However, the cell membrane directly outwards buds to the extracellular milieu and forms the micro-vesicle. Besides, oncosomes are large EVs formed through the budding of the tumor cell membrane. EVs deliver a variety of DNA, protein, and RNA species including miRNA, piwiRNA, lncRNA, mRNA, tRNA, snoRNA, circRNA, etc.

Figure 3

Experimental and analytical procedures for the identification of novel RNA biomarkers. First, tissues and/or bio-fluids are collected from both patients and health individuals. Then RT-qPCR, small RNA-seq or other RNA sequencing methods are performed on isolated and purified RNA samples. RT-qPCR procedure includes cDNA synthesis by reverse transcription from total RNAs and qPCR reactions with the synthesized cDNA templates. RNA-seq procedure includes library preparation, in which RNA transcripts are fragmentized and transcribed into cDNAs, and high-throughput sequencing. Then, RNA-seq data are processed with a pipeline that includes mapping of reads to the reference genome, assembly of transcriptome from mapped reads, calculating each transcript’s expression abundance (i.e., FPKM, fragments per kilobase of transcript, per million fragments sequenced), and differential expression analysis. Differential expression of selected RNA biomarkers can then be validated by RT-qPCR results in different sample groups. Finally, advanced bioinformatics and statistical analyses will integrate clinical data with expression profiles to obtain the biomarkers’ correlations with diagnostic or prognostic properties.