MicroRNA As a Biomarker in Cancer

MicroRNA (miRNA) represents a diminutive, non-coding ribonucleic acid ranging from 18 to 25 nucleotides in length. This intricate biomolecule plays a vital role in a multitude of biological phenomena, including cellular division, differentiation, morphological transformations, metabolic regulation, and apoptosis. By selectively binding to the 3′-untranslated region of specific target genes, miRNA exerts its influence by either promoting the degradation of messenger RNA molecules or impeding their translation. Intriguingly, aberrations in miRNA genomic and epigenetic structures have been observed, leading to irregularities manifested as mutations characterized by deletions, duplications, and translocations.

You may interested in other types of RNA, please refer to our article Overview of Competing Endogenous RNA (ceRNA).

Circulating microRNAs as biomarkers. (Sundarbose et al., 2013)

What is miRNA?

MicroRNAs (miRNAs), the highly conserved, small-size, endogenous non-coding RNAs, play a crucial role in regulating post-transcriptional gene expression levels. They achieve this by mediating the silencing of specific target genes, leading to the degradation of target messenger RNAs (mRNAs) and the inhibition of protein synthesis.

The regulatory landscape of miRNAs is characterized by its complexity and dynamism. A single miRNA molecule possesses the capacity to interact with one or more mRNA targets, and it is not uncommon for certain mRNAs to harbor multiple binding sites for distinct miRNAs within the same miRNA family. This intricate interplay between miRNAs and mRNAs facilitates the precise fine-tuning of gene expression and the harmonization of diverse cellular processes.

microRNA biogenesis. (Peng et al., 2016)

More than half of the miRNA genes are situated in chromosomal regions prone to fragility, rendering them vulnerable to genomic alterations. These alterations encompass gene amplification, deletion, or translocation, which can disrupt the normative functioning of miRNAs and contribute to the genesis of diseases.

As of now, over 700 human miRNAs have been characterized, and it is estimated that they govern approximately one-third of human genes. MiRNAs partake in a myriad of physiological processes, encompassing individual development, organogenesis, and metabolic homeostasis. Additionally, miRNAs have emerged as pivotal actors in pathological processes such as viral infections and tumorigenesis. Gaining comprehensive insights into the functions and mechanisms of miRNAs in these contexts offers invaluable understanding of disease mechanisms and furnishes potential targets for therapeutic interventions.

The Structure and Function of MicroRNAs

MicroRNAs (miRNAs) are intrinsic RNA molecules that lack coding potential and exist as single-stranded structures. They play crucial roles in the intricate control of gene expression and epigenetic processes. Originating from various cellular sources, including blood cells, miRNAs are renowned for their stability. Their protection from degradation is achieved through encapsulation in extracellular particles or by binding to proteins or high-density lipoproteins (HDL), facilitating their transportation and functional activity within biological fluids.

Recommended reading: Biofluid microRNA Sequencing: Introduction, Platforms, and Challenges.

A fundamental mechanism through which miRNAs govern gene expression involves the complementary base pairing with target messenger RNAs (mRNAs). The miRNA specifically binds to the 3'-untranslated region (UTR) of its target mRNA, leading to the silencing, degradation, or inhibition of protein synthesis. Notably, a single miRNA can regulate multiple target mRNAs, while a single mRNA can be influenced by multiple miRNAs. This intricate interplay between miRNAs and their target mRNAs enables precise and coordinated control of gene expression.

The mechanism of Drosha and Dicer. (Starega-Roslan et al., 2011)

The binding of miRNAs to their target mRNAs relies on sequence complementarity. By recognizing specific sequences or motifs within the mRNA, the miRNA orchestrates the repression or degradation of the mRNA molecule. This regulatory interaction can have a broad impact on gene expression, as one miRNA can target multiple mRNAs, and multiple miRNAs can regulate a single gene. Consequently, miRNAs possess the capacity to exert substantial control over gene expression networks, playing vital roles in various biological processes.

In addition to their involvement in gene regulation, miRNAs have emerged as key players in epigenetic modifications. They possess the ability to influence chromatin remodeling and DNA methylation patterns, thereby modulating the accessibility of genes to the transcriptional machinery and impacting gene expression profiles. Through their modulation of these epigenetic mechanisms, miRNAs contribute to the regulation of cellular processes and the maintenance of cellular homeostasis.

MiRNAs As Biomarkers in Cancer

MiRNAs have emerged as promising biomarkers in cancer diagnosis, prognosis, and treatment. Abnormalities in miRNA genomes, including genomic and epigenetic alterations such as deletions, duplications, and translocations, have been observed in cancer. More than 50% of miRNA genes are located in cancer-associated gene regions and function as proto-oncogenes or tumor suppressors, influencing tumorigenesis.

Each type of cancer exhibits its specific miRNA expression patterns, and these dysregulated miRNAs can serve as biomarkers for different cancer types. For example, in breast cancer, miRNAs have been found to be dysregulated at all stages of the disease. In triple-negative breast cancer (TNBC), a highly aggressive subtype, numerous miRNAs show abnormal expression and regulate multiple signaling pathways that contribute to cancer development and progression. Therefore, miRNAs hold potential as biomarkers for early screening and improved patient survival in TNBC, which is associated with high malignancy, aggressiveness, and poor prognosis.

In addition to breast cancer, miRNAs have been successfully employed as biomarkers in the diagnosis and management of other cancers, including colon cancer, gastric cancer, and ovarian cancer. The use of miRNAs as biomarkers offers several advantages, such as non-invasiveness, low cost, and ease of detection through circulating tests. These approaches reduce patient discomfort during the diagnostic process and enable early detection and intervention. Furthermore, miRNAs are abundant, stable, and resistant to degradation by RNA enzymes, making them well-suited for clinical diagnostic applications.

Please refer to our article The Role of Exosome RNA in Cancer for more details.

Sequencing Techniques for miRNA Biomarker Discovery

The advent of next-generation sequencing (NGS) technologies has revolutionized the field of miRNA research, enabling comprehensive profiling of miRNAs and facilitating biomarker discovery.

Sequencing Platforms For miRNA Sequencing

Next generation sequencing: Illumina sequencing is the most widely used NGS platform for miRNA sequencing. It utilizes a reversible terminator-based sequencing-by-synthesis approach, allowing high-throughput parallel sequencing of millions of DNA fragments.

Steps Involved in miRNA Sequencing Workflow

• Sample preparation: The first step in miRNA sequencing involves extracting total RNA from the biological sample, followed by enrichment of small RNA molecules. This is typically achieved using size selection or miRNA-specific capture techniques.
• Library preparation: The enriched small RNA molecules are converted into sequencing libraries through enzymatic processes. Adapters containing unique barcodes are ligated to the ends of the miRNA molecules, enabling multiplexed sequencing.

Library preparation for small RNA sequencing. (Baran-Gale et al., 2015)

• Sequencing: The prepared libraries are loaded onto the NGS platform of choice, and the sequencing run is performed according to the platform's specifications. This step generates millions of short sequencing reads that represent the miRNAs present in the sample.
• Data preprocessing: The raw sequencing data undergoes quality control, adapter trimming, and removal of low-quality reads to ensure the reliability of downstream analyses.
• Mapping and quantification: The processed reads are aligned to a reference genome or miRNA sequence database to identify and quantify miRNAs. Specialized bioinformatics tools are used for this purpose.
• Differential expression analysis: Comparative analysis is performed to identify differentially expressed miRNAs between different sample groups or conditions. Statistical methods are employed to determine significant miRNA expression changes.
• Functional analysis: Further analysis, such as target prediction and pathway enrichment analysis, is carried out to understand the biological implications of the differentially expressed miRNAs.

Considerations For Data Analysis and Interpretation in miRNA Sequencing Studies

• Normalization methods: Various normalization strategies are available to account for technical variations and normalize miRNA expression levels across samples. Popular methods include global mean normalization, quantile normalization, and spike-in normalization.
• Statistical analysis: Rigorous statistical approaches, such as the use of appropriate tests (e.g., t-tests, ANOVA) and multiple testing corrections, are crucial to identify statistically significant miRNA biomarkers.
• Validation techniques: Experimental validation of miRNA biomarkers using independent sample sets or alternative techniques (e.g., qPCR, in situ hybridization) is essential to confirm their reliability and reproducibility.
• Integration with other omics data: Integrating miRNA sequencing data with other omics data, such as mRNA expression or DNA methylation profiles, can provide a more comprehensive understanding of regulatory networks and potential miRNA-mRNA interactions.
• Functional interpretation: Functional interpretation of miRNA sequencing data involves the identification of target genes regulated by differentially expressed miRNAs. Bioinformatics tools and databases can be utilized to predict miRNA-target interactions and investigate the biological pathways and processes influenced by the miRNAs of interest. Gene ontology analysis and pathway enrichment analysis provide insights into the functional roles and potential disease associations of the identified miRNA biomarkers.
• Cross-validation and replication: It is crucial to validate miRNA biomarkers in independent cohorts or replication studies to ensure the robustness and generalizability of the findings. Replication in different populations or experimental conditions strengthens the reliability of identified miRNA biomarkers as potential diagnostic, prognostic, or therapeutic targets.
• Integration into clinical practice: The ultimate goal of miRNA biomarker discovery is their translation into clinical practice. Before incorporating miRNA biomarkers into clinical settings, further validation in larger patient cohorts and assessment of their diagnostic or prognostic utility, sensitivity, specificity, and predictive value are necessary. Development of standardized protocols, guidelines, and assays is vital for their successful integration into routine clinical workflows.

References:

1. Sundarbose, K.; Kartha, R.V.; Subramanian, S. MicroRNAs as Biomarkers in Cancer. Diagnostics 2013, 3, 84-104.
2. Peng, Yong, and Carlo M. Croce. "The role of MicroRNAs in human cancer." Signal transduction and targeted therapy 1.1 (2016): 1-9.
3. Baran-Gale, Jeanette, et al. "Addressing bias in small RNA library preparation for sequencing: a new protocol recovers microRNAs that evade capture by current methods." Frontiers in genetics 6 (2015): 352.
4. Starega-Roslan, Julia, et al. "The role of the precursor structure in the biogenesis of microRNA." Cellular and molecular life sciences 68 (2011): 2859-2871.