MicroRNA in Cancer: An Overview
Ken Doyle, PhD., Principal Consultant, Loquent LLC
Introduction to miRNA
The wealth of information uncovered by genome sequencing projects over the past few decades has revealed that the vast majority of eukaryotic genomes—over 98% in humans—is transcribed into noncoding RNA species. Of these RNAs, microRNA (miRNA) has attracted considerable attention ever since the discovery of transcriptional repression of the lin-4 gene in C. elegans (1). miRNAs are small noncoding RNAs, typically 20–24 nucleotides, and are highly conserved in plants and animals. They exert negative regulatory effects on gene expression largely by binding to short motifs in messenger RNA (mRNA) that are complementary to the miRNA. These effects result from: i) interference with translation of mRNAs; ii) cleavage of the mRNA; or iii) destabilization of the mRNA by shortening of the poly(A) tail.
Most miRNAs are transcribed from intergenic regions by RNA polymerase II. The primary transcripts are precursor molecules (pri-miRNA) that are processed by two ribonucleases—Drosha in the nucleus and Dicer in the cytoplasm—into mature miRNAs (2,3) .
Through feedback mechanisms, miRNAs are involved in regulating a wide variety of cellular processes in animals, including differentiation, proliferation, and apoptosis. Thus, it is not surprising that dysregulation of even a single miRNA-mediated pathway can have profound physiological effects. In addition, many miRNA genes are located at genomic breakpoints or other unstable regions. These observations, coupled with widespread evidence from gene expression studies demonstrating aberrant miRNA expression in a variety of tumor cells and tissues, have established the involvement of miRNAs in cancer (4) . Different classes of miRNAs can play oncogenic as well as tumor-suppressing roles; the same group of miRNAs can exhibit oncogenic activity in one tissue type but act as a tumor suppressor in another (5). Thus, it is important to consider the cellular context when drawing any conclusions about the activity of a specific miRNA. Nonetheless, miRNAs have gained significant importance as diagnostic markers in cancer, and they constitute potentially attractive therapeutic targets.
Dysregulation of miRNA Gene Expression
Gene expression studies of tumor:normal tissue pairs often reveal lower levels of miRNA in the tumor samples (6) . Various mechanisms for downregulation of miRNA expression have been proposed, including mutations in miRNA genes, epigenetic regulation, and alterations in miRNA pathways mediated by transcription factors.
Initial evidence suggested that many miRNA genes are located at cancer-specific translocation sites, CpG islands, and fragile sites within the genome (7); however, the relationship between deactivation of miRNA genes and oncogenesis is complex and varies depending on the type of cancer (8). Single-nucleotide polymorphisms (SNPs) have been identified in both miRNA genes and their targets, playing opposing roles: some enhance the tumor-suppressor function of the miRNA, while others result in increased miRNA expression and consequent oncogenic activity (9). Further, mutations in the genes encoding miRNA-processing transcription factors Drosha and Dicer1 also resulted in increased tumorigenic activity (10, 11) .
Epigenetic alterations have long been a hallmark of many types of cancer (12). Silencing of miRNA genes by hypermethylation has been observed in breast cancer and colorectal cancer (13) (14) , while the expression of oncogenic miRNAs was increased by DNA hypomethylation in ovarian cancer (15) . Mapping studies have shown that miRNA silencing by methylation of miRNA promoter regions is associated with breast cancer development and metastasis (16). In addition to DNA methylation, altered histone acetylation has been identified to play a role in reducing the expression of antioncogenic miRNAs in breast cancer cells (17) .
A host of transcription factors are involved in expression, processing, and transcription of miRNAs. The oncogenic transcription factor Myc binds to the promoter region of many miRNAs and, typically, acts as a negative regulator of miRNA gene expression (18). Myc also has indirect effects on miRNA activity through its activation of secondary factors that, in turn, downregulate the antiproliferative, tumor-suppressive, and proapoptotic activities of let-7, miR15a/16-1, miRNA-26a, and miR-34 family members (19). Other miRNAs have been implicated in oncogenic transcription factor pathways, including those mediated by Ras (20) , ZEB1/ZEB2 (21), and p53 and cyclin-dependent kinases (22). Some miRNA regulation also occurs at the post-transcriptional stage (23); for example, the serine/threonine protein kinase IRE1a, which also possesses endoribonuclease activity, cleaves specific pre-miRNAs, thereby reducing the translation of the proapoptotic factor caspase 2 (24) .
In addition to the mechanisms described, studies have demonstrated that miRNA processing can be regulated by other miRNAs. as an example, the miR-103/107 family is known to target expression of Dicer, reducing the levels of a broad spectrum of miRNAs in breast cancer (25) .
miRNA Purification and Analysis
Multiple methods for purifying miRNA have been developed to address challenges that are specific to the source material. For example, an extraction method for cells or tissue samples, where miRNAs are relatively abundant, may not be optimal for circulating miRNAs in extracellular fluid, such as plasma or serum. Blood samples may pose additional problems, due to high levels of endogenous RNases. In general, miRNA purification methods are similar to those for total RNA, followed by an additional step during which the miRNA fraction is isolated and enriched by size-selection or selective precipitation. Most commercial methods are based on organic extraction of total RNA and purification of miRNA on silica columns. Promega offers miRNA purification methods that overcome the limitations of organic extraction, are easy to automate, and yield purified miRNA suitable for downstream analysis by a variety of techniques.
miRNA profiling experiments, regardless of the method used, require accurate quantitation to ensure reproducible results. The short length of miRNAs can pose particular problems for methods based on qRT-PCR, while microarray and NGS-based analysis can be challenging where absolute quantitation is required. Popular methods for miRNA profiling and quantitation have been reviewed extensively by Pritchard et al. (2012) MicroRNA profiling: approaches and considerations. Nat. Rev. Genet. 13, 358–369.
miRNAs in Cancer
Early assays for miRNA in cancer cells and tissues used quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure miRNA levels, e.g., in tissue and plasma samples derived from tongue squamous cell carcinoma (26). Although qRT-PCR is still used in some miRNA-based diagnostic tests, the wide availability and high throughput of microarray-based technologies has made them the method of choice. More recently, next-generation sequencing has enabled whole-genome analysis of various cancer types including cervical cancer (27), oesophageal squamous cell cancer (28), and urothelial bladder cancer (29). These studies have revealed significantly different miRNA expression profiles in normal vs. tumor samples from the same tissue. Analysis of miRNA expression in breast cancer tissues compared to normal adjacent tissue identified several dysregulated miRNAs; further, the analysis could be used as a predictive tool to classify samples as normal or tumorigenic (30) .
Regardless of the technique, the relative stability of miRNAs in formalin-fixed, paraffin-embedded (FFPE) tissue makes them a more attractive diagnostic tool, compared to mRNA. The discovery of circulating miRNAs in body fluids (31)—such as plasma and later serum, saliva, and urine—has paved the way for the development of noninvasive testing methods. Although the mechanisms by which circulating miRNAs might exert their regulatory effects are not widely understood, these noninvasive miRNA assays have been used in a variety of cancers. However, further research is needed to overcome issues with inconsistent results across studies, potentially due to inaccurate sample collection, processing, and miRNA quantitation as well as lack of consensus for data normalization (31, 32) .
miRNAs As Therapeutic Agents
The inhibition of oncogenic miRNAs (oncomiRs) that are overexpressed has been studied in preclinical models, most commonly in mouse xenografts and in primates (33, 34). This approach relies on the use of antisense miRNAs (antimiRs) that have been chemically modified for increased stability.
However, as with other approaches that rely on delivering nucleic acids into cells, considerable challenges lie in overcoming cellular barriers and ensuring targeted delivery of the therapeutic agent.
A pioneering study in mice demonstrated the feasibility of intravenous delivery of 2´O-methyl-derivatized antimiRs conjugated to cholesterol. These antimiRs targeted miR-10b, and resulted in inhibiting beast cancer metastasis to the lung but did not affect tumor growth at the site of infusion (35). Other model studies have demonstrated success using the antimiR approach in hepatocellular carcinoma (36) , Hodgkin lymphoma (37), neuroblastoma (38), and glioma (39).
A recent, novel approach used nanoparticle-encapsulated peptide nucleic acids in a mouse lymphoma model (40). This method allowed the antimiRs to target the acidic tumor microenvironment, while evading hepatic clearance and facilitating entry into the tumor cells by a nonendocytic pathway. The approach should be widely applicable to ensure targeted delivery of antimiRs against any oncomiR under investigation.
In contrast to the use of antimiRs, an alternative therapeutic approach makes use of reintroducing tumor-suppressive miRNAs that have been downregulated in the target cells. This approach has shown promise in model organisms using let-7 and miR-34 in lung cancer (41) , miR-34 and miR-143/145 in pancreatic cancer (41, 42), and mir-26a in hepatocellular carcinoma (43).
Thus, miRNA shows considerable promise as a therapeutic agent for a variety of cancers. However, it remains to be seen whether these results from model systems can be translated into clinical trials, while addressing concerns about efficacy and safety. At the time of writing, a Phase I trial of miR-RX34 (a mimic of miR-34, which is downregulated in a variety of cancers through a p53-mediated pathway) was underway in patients with liver cancer (44).
Although our knowledge of the precise mechanisms by which miRNAs contribute to transcriptional regulation is incomplete, strong evidence supports a causal link between miRNA dysregulation and the development of many types of cancer. Gene expression studies, particularly those powered by next-generation sequencing, offer predictive power to enable the development of miRNA-based diagnostic assays that can assist with early detection of cancer and inform physicians about personalized treatment options. Therapeutic approaches, designed to silence oncogenic miRNAs using antisense miRNA or to introduce high levels of tumor-suppressive miRNAs, have shown encouraging results in animal models. The extension of these approaches to comprehensive, randomized controlled trials that evaluate the safety, specificity, and effectiveness of miRNA across multiple cancer types can provide a potential new weapon in the arsenal of therapeutic tools being deployed in the clinic.
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