MicroRNAs (miRNAs) are a class of small RNAs that have revealed a new level of gene regulation in the cell. After being processed by Drosha and Dicer RNase III endonucleases, mature miRNAs can inhibit the translation of mRNA by directing a RNA-induced silencing complex (RISC) to the target mRNA. miRNAs are making an impact in our understanding of cancer biology. Acting as either tumor suppressors or oncogenes, miRNAs regulate several genes known to play important roles in cancer. With the discovery of miRNAs comes the need for new techniques to study their activity. Bioinformatic tools can be used to predict mRNA targets of miRNA, but validation of miRNA regulation of predicted targets is imperative. miRNAs are differentially expressed in normal and tumor cells as well as between tumor subtypes. These differences may be useful as prognostic and predictive markers in cancer patients. The study of miRNAs holds much promise for improving diagnosis and treatment of cancer. [Mol Cancer Ther 2008;7(12):3655–60]

MicroRNAs (miRNAs) were first discovered by researchers screening for genes controlling development in Caenorhabditis elegans (1). Since their discovery, miRNAs have been found in many organisms, including mammalian systems (2, 3). miRNAs are short, non-protein-coding RNAs ∼22 nucleotides in length that are known to alter gene expression at a post-transciptional level (4–7). miRNAs are first transcribed into long primary miRNAs up to several thousand nucleotides in length (7–9). A Drosha RNase III endonuclease, in complex with the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region gene 8), then cleaves the primary miRNA leaving a hairpin loop structure ∼70 nucleotides in length known as a precursor miRNA (4, 7, 8, 10). The precursor miRNA is exported from the nucleus to the cytoplasm by Exportin-5 (4, 11). Once in the cytoplasm, a complex including Dicer RNase III endonuclease, TAR RNA-binding protein, and protein kinase R-activating protein further cleaves the precursor miRNA generating a short double-stranded miRNA:miRNA* complex intermediate (4, 7, 9, 12, 13). One strand of this miRNA complex (the mature miRNA) is incorporated into a RNA-induced silencing complex (RISC; refs. 4, 6, 7, 14). The miRNA directs the RISC to the 3′-untranslated region (UTR) of target mRNAs by way of a 7- to 8-nucleotide complementary sequence known as the seed sequence (7, 15, 16). The main component of the RISC is an Ago protein from the Argonaute family. Human cells contain four Ago proteins (Ago1-4), and the specific Ago protein found in the RISC determines how the RISC will interact with the target mRNA. Only a miRNA bound to an Ago2-RISC may result in cleavage of its target mRNA (4, 5). The extent of complementarity between the miRNA and its target is also significant in determining whether the end result will be translational inhibition or mRNA cleavage (refs. 6, 7; Fig. 1). One miRNA may be involved in the regulation of many different mRNAs or may bind in several places within the 3′UTR of one mRNA. Work is ongoing to address exactly how miRNAs function to regulate gene expression, including mediating mRNA destabilization, mRNA sequestration, and translational repression during initiation, elongation, and/or termination. Some mRNAs may require the binding of multiple miRNAs for effective regulation.

Figure 1.
Figure 1. miRNA processing. RNA polymerase II transcribes a primary miRNA sequence, which is then cleaved by a complex including Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region gene 8) resulting in a precursor miRNA. Exportin-5 exports the precursor miRNA from the nucleus to the cytoplasm where it is cleaved by Dicer in a complex with TAR RNA-binding protein and protein kinase R-activating protein leaving a miRNA:miRNA* complex intermediate. The miRNA strand of the complex is incorporated into a RISC and guides the complex to the 3′UTR of the target mRNA by way of a 7- to 8-nucleotide seed sequence. This binding will result in either translational repression or mRNA degradation depending on several factors including the extent of complementarity between the miRNA seed sequence and the 3′UTR and the Ago protein in the RISC.
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miRNA processing. RNA polymerase II transcribes a primary miRNA sequence, which is then cleaved by a complex including Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region gene 8) resulting in a precursor miRNA. Exportin-5 exports the precursor miRNA from the nucleus to the cytoplasm where it is cleaved by Dicer in a complex with TAR RNA-binding protein and protein kinase R-activating protein leaving a miRNA:miRNA* complex intermediate. The miRNA strand of the complex is incorporated into a RISC and guides the complex to the 3′UTR of the target mRNA by way of a 7- to 8-nucleotide seed sequence. This binding will result in either translational repression or mRNA degradation depending on several factors including the extent of complementarity between the miRNA seed sequence and the 3′UTR and the Ago protein in the RISC.

Figure 1.
Figure 1. miRNA processing. RNA polymerase II transcribes a primary miRNA sequence, which is then cleaved by a complex including Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region gene 8) resulting in a precursor miRNA. Exportin-5 exports the precursor miRNA from the nucleus to the cytoplasm where it is cleaved by Dicer in a complex with TAR RNA-binding protein and protein kinase R-activating protein leaving a miRNA:miRNA* complex intermediate. The miRNA strand of the complex is incorporated into a RISC and guides the complex to the 3′UTR of the target mRNA by way of a 7- to 8-nucleotide seed sequence. This binding will result in either translational repression or mRNA degradation depending on several factors including the extent of complementarity between the miRNA seed sequence and the 3′UTR and the Ago protein in the RISC.
View largeDownload slide

miRNA processing. RNA polymerase II transcribes a primary miRNA sequence, which is then cleaved by a complex including Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region gene 8) resulting in a precursor miRNA. Exportin-5 exports the precursor miRNA from the nucleus to the cytoplasm where it is cleaved by Dicer in a complex with TAR RNA-binding protein and protein kinase R-activating protein leaving a miRNA:miRNA* complex intermediate. The miRNA strand of the complex is incorporated into a RISC and guides the complex to the 3′UTR of the target mRNA by way of a 7- to 8-nucleotide seed sequence. This binding will result in either translational repression or mRNA degradation depending on several factors including the extent of complementarity between the miRNA seed sequence and the 3′UTR and the Ago protein in the RISC.

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Calin et al. first reported the association between miRNAs and cancer after observing that miR-15-a and miR-16-1 are located on chromosome locus 13q14, a site frequently deleted in B-cell chronic lymphocytic leukemia cases (17). Consequently, miR-15-a and miR-16-1 are down-regulated or deleted in a majority of chronic lymphocytic leukemia cases. Since their discovery, researchers have identified abnormal expression of miRNAs in other malignancies including lymphomas, colorectal carcinoma, breast cancer, lung cancer, thyroid cancer, and hepatocellular carcinomas (18–20). Mapping efforts have revealed that many miRNAs are located in fragile regions of the genome, and several miRNAs located in these regions often have decreased expression in cancer cells (21).

Depending on the target mRNA, miRNAs can act as either tumor suppressors or oncogenes. miRNAs have been shown to regulate several genes known to play key roles in cancer. Two genes significantly correlated with decreased disease-specific survival in breast cancer patients, HER2 and HER3 (22), are suppressed by miR-125a or miR-125b (23). The let-7 family of miRNAs can target several genes associated with cell cycle and cell division including the RAS oncogene (24) and HMGA2 (25). Due to their tumor suppression capability, it is not surprising that this miRNA family is often down-regulated in non-small cell lung cancer (NSCLC). Increased let-7 represses cell proliferation in both NSCLC cells and liver cancer cells (26) and represses tumorigenesis in mouse models (27). Another miRNA family with tumor suppressor properties is the miR-34 family. This family of miRNAs is a direct target of the transcriptional activator p53, and overexpression of the miR-34 family leads to cell growth arrest of both primary and cancer cells (28). Inhibition of epidermal growth factor receptor by miR-128b in NSCLC (29) and miR-7 in glioma (30) provides yet another pertinent example of miRNAs acting as tumor suppressors. A cluster of miRNAs known as miR-17-92, which encodes six miRNAs transcribed together as one polycistronic transcript, act as oncogenes in numerous cancers (31).

miRNAs play highly diverse roles in cancer. They can be involved in metastasis, invasion, proliferation, cell cycle, and apoptosis. Several studies have investigated the roles of specific miRNAs in these cellular events. Tavazoie et al. reported that, as breast cancer cells metastasize, expression of miR-126 and miR-335 is lost. Furthermore, overexpressing these miRNAs in cancer cells decreases lung and bone metastasis in vivo (32). Conversely, miR-373 and miR-520c enhance tumor invasion and metastasis both in breast cancer cells and in vivo (33), which suggests that these miRNAs may target genes required to suppress cell growth and metastasis.

Bioinformatic tools can be used to predict a potential miRNA and the binding sites of its target mRNA. With the rapid pace of newly identified and registered miRNAs, the miRNA registry list is updated several times a year. The most recent, Sanger version 12.0,3

3

http://microRNA.sanger.ac.uk

lists 695 human miRNAs. However, only a fraction of these miRNAs have been experimentally confirmed to regulate their predicted mRNA targets.

Once a miRNA has been identified as a potential regulator of a gene, there are at least two in vitro experiments required to validate the ability of a miRNA to regulate its predicted target mRNA. The first experiment should address miRNA binding to the 3′UTR of the target mRNA. The luciferase reporter assay is one method to show miRNA 3′UTR binding. This assay measures the ability of a miRNA to decrease luciferase activity in cells when its potential binding site is cloned downstream of the luciferase gene (Fig. 2; ref. 34). The second experiment should address miRNA regulation of endogenous mRNA expression or protein translation. It is imperative to show that the miRNA regulates the expression of the target mRNA or protein. This validation can be assessed by (a) transiently transfecting cells with a mimic and inhibitor of the miRNA of interest or (b) stably overexpressing the miRNA of interest in a cell line using lentiviral transfection of a plasmid vector that expresses the miRNA of interest (23, 35). The mimic miRNA is a sequence of miRNA identical to the endogenous miRNA. An antisense strand of RNA acts as the inhibitor miRNA by binding to the miRNA and inhibiting its activity. After the miRNA of interest is added to the cells (or inhibited), the putative target message and protein levels are measured and compared with normal cells (36). When the miRNA levels are increased in the cell, miRNA regulation should result in decreased mRNA and/or protein expression, whereas decreased levels of miRNA should result in increased mRNA and/or protein expression. An advantage to using stably overexpressing cells is that they can be used for many other experiments investigating the role of the miRNA in events such as migration, invasion, cell proliferation, and cell cycle, with less concern over transient transfection efficiencies. Because the miRNA is constitutively overexpressed, phenotypic changes and other oncogene pathway changes in the cells can be observed that may not be apparent with transient transfection (23, 33, 35).

Figure 2.
Figure 2. Luciferase reporter assay used to validate miRNA binding at 3′UTR seed sequence. Condition A: the luciferase reporter plasmid with no 3′UTR insert (control plasmid) is transfected into cells and luciferase activity is measured. These cells should have the highest luciferase activity because the mRNA transcript is lacking the miRNA binding sequence. The relative luciferase activity is depicted in the bar graph. Condition B: the 3′UTR of the putative target mRNA is cloned downstream of the luciferase gene in the reporter plasmid (3′UTR vector). Here, the luciferase activity in transfected cells should be lower than the control cells because the miRNA will bind to the seed sequence in the 3′UTR and inhibit translation of the luciferase gene (see bar graph). Condition C: when mimic miRNAs are transfected into cells with the 3′UTR vector, the luciferase activity should be decreased even further than in condition B (see bar graph). Condition D: a mutation is added in the miRNA binding sequence of the target mRNA 3′UTR and then cloned downstream of the luciferase gene (mutant vector). Cells transfected with the mutant vector should have luciferase activity comparable with the control vector-transfected cells (see bar graph). Condition E: when mimic miRNAs are transfected into cells with the mutant vector, the luciferase activity may decrease slightly, but it will still be greater than the cells transfected with 3′UTR vector (see bar graph).
View largeDownload slide

Luciferase reporter assay used to validate miRNA binding at 3′UTR seed sequence. Condition A: the luciferase reporter plasmid with no 3′UTR insert (control plasmid) is transfected into cells and luciferase activity is measured. These cells should have the highest luciferase activity because the mRNA transcript is lacking the miRNA binding sequence. The relative luciferase activity is depicted in the bar graph. Condition B: the 3′UTR of the putative target mRNA is cloned downstream of the luciferase gene in the reporter plasmid (3′UTR vector). Here, the luciferase activity in transfected cells should be lower than the control cells because the miRNA will bind to the seed sequence in the 3′UTR and inhibit translation of the luciferase gene (see bar graph). Condition C: when mimic miRNAs are transfected into cells with the 3′UTR vector, the luciferase activity should be decreased even further than in condition B (see bar graph). Condition D: a mutation is added in the miRNA binding sequence of the target mRNA 3′UTR and then cloned downstream of the luciferase gene (mutant vector). Cells transfected with the mutant vector should have luciferase activity comparable with the control vector-transfected cells (see bar graph). Condition E: when mimic miRNAs are transfected into cells with the mutant vector, the luciferase activity may decrease slightly, but it will still be greater than the cells transfected with 3′UTR vector (see bar graph).

Figure 2.
Figure 2. Luciferase reporter assay used to validate miRNA binding at 3′UTR seed sequence. Condition A: the luciferase reporter plasmid with no 3′UTR insert (control plasmid) is transfected into cells and luciferase activity is measured. These cells should have the highest luciferase activity because the mRNA transcript is lacking the miRNA binding sequence. The relative luciferase activity is depicted in the bar graph. Condition B: the 3′UTR of the putative target mRNA is cloned downstream of the luciferase gene in the reporter plasmid (3′UTR vector). Here, the luciferase activity in transfected cells should be lower than the control cells because the miRNA will bind to the seed sequence in the 3′UTR and inhibit translation of the luciferase gene (see bar graph). Condition C: when mimic miRNAs are transfected into cells with the 3′UTR vector, the luciferase activity should be decreased even further than in condition B (see bar graph). Condition D: a mutation is added in the miRNA binding sequence of the target mRNA 3′UTR and then cloned downstream of the luciferase gene (mutant vector). Cells transfected with the mutant vector should have luciferase activity comparable with the control vector-transfected cells (see bar graph). Condition E: when mimic miRNAs are transfected into cells with the mutant vector, the luciferase activity may decrease slightly, but it will still be greater than the cells transfected with 3′UTR vector (see bar graph).
View largeDownload slide

Luciferase reporter assay used to validate miRNA binding at 3′UTR seed sequence. Condition A: the luciferase reporter plasmid with no 3′UTR insert (control plasmid) is transfected into cells and luciferase activity is measured. These cells should have the highest luciferase activity because the mRNA transcript is lacking the miRNA binding sequence. The relative luciferase activity is depicted in the bar graph. Condition B: the 3′UTR of the putative target mRNA is cloned downstream of the luciferase gene in the reporter plasmid (3′UTR vector). Here, the luciferase activity in transfected cells should be lower than the control cells because the miRNA will bind to the seed sequence in the 3′UTR and inhibit translation of the luciferase gene (see bar graph). Condition C: when mimic miRNAs are transfected into cells with the 3′UTR vector, the luciferase activity should be decreased even further than in condition B (see bar graph). Condition D: a mutation is added in the miRNA binding sequence of the target mRNA 3′UTR and then cloned downstream of the luciferase gene (mutant vector). Cells transfected with the mutant vector should have luciferase activity comparable with the control vector-transfected cells (see bar graph). Condition E: when mimic miRNAs are transfected into cells with the mutant vector, the luciferase activity may decrease slightly, but it will still be greater than the cells transfected with 3′UTR vector (see bar graph).

Close modal

As new roles for miRNAs in cancer continue to be discovered, their future effect on diagnosis, prognosis, and treatment of patients remains to be seen. miRNA profiling holds potential for differentiating between normal and tumor cells and between different tumor subtypes. miRNA expression profiling can classify several different human cancers, including poorly differentiated tumor, as well as distinguish from benign tissue (37). The ability to classify over 22 different tumor types using only 48 different miRNAs holds promise for patients with carcinoma of unknown primary (38). With an incidence of up to 4% of all newly diagnosed cancers, carcinoma of unknown primary is a diagnostic and treatment challenge. Providing an oncologist with the tissue of origin allows for improved treatment decision planning when traditional diagnostic modalities are not helpful.

miRNAs may also be useful as prognostic or predictive tools in cancer. A five-miRNA signature has been reported to predict disease-free and overall survival in resected NSCLC patients (38). These miRNA signatures may become potential tools for therapeutic decision making. It remains to be seen how the miRNA signature performs against the various prognostic mRNA expression signatures in resected NSCLC, which can include from 3 to >30 mRNAs, with little overlap (39). Although miRNA expression profiling appears to holds more promise than mRNA profiling for discriminating between tumor types, it remains uncertain if this is due to technical or biological reasons. From a technical perspective, the current number of human miRNAs (695) is several-fold less than mRNA transcripts (∼47,000), and probe sizes on miRNA platforms tend to be shorter and more uniform in length. A true technical advantage for miRNA profiling is that their small size lessens their susceptibility to degradation in paraffin-embedded, formalin-fixed tissue, allowing for wider application of miRNA profiling on banked tissue samples for future discovery and validation of hypotheses (40).

More important than predictors of prognosis, miRNA expression may also help oncologists determine the best course of treatment. In a retrospective study of advanced NSCLC patients treated with an epidermal growth factor receptor tyrosine kinase inhibitor, both response and survival significantly correlated with tumor miR-128b loss of heterozygosity (29). Another study determined that let-7i, miR-16, and miR-21 expression levels affect the response of tumor cell lines to several different anticancer agents (36). In another example, decreased expression of let-7g and miR-181b in colon cancer correlated with patient response to S-1-based chemotherapy treatment (41).

The potential to use miRNAs as biomarkers for disease is strengthened by their stability in human serum and plasma (42, 43). Instead of using invasive procedures to extract tissue from patients’ tumors, miRNAs can be measured directly from the patients’ blood products. Using Solexa sequencing, the miRNA expression profile from serum of healthy individuals was shown to be significantly different from that of patients with lung cancer, colorectal cancer, and diabetes (43). Another study investigating the expression of miRNAs in plasma found that miR-141 can serve as a biomarker for prostate cancer (42).

miRNAs may one day even be administered in cancer therapeutics either as single agents or in combination therapies. Studies have already shown that miRNAs can play a direct role in drug effectiveness. By overexpressing let-7 in lung cancer cell lines and in C. elegans, let-7 can repress resistance to radiation therapy both in vitro and in vivo (44). In another study, inhibition of miR-21 and miR-200b enhanced sensitivity to gemcitabine in cholangiocytes (45). Delivery systems such as locked nucleic acid increase the stability of miRNAs for in vivo application (46) and may be promising as cancer therapy. In humans, what remains to be seen is the potential of miRNA for off-target effects on unintended mRNA targets possessing identical 3′UTR miRNA binding sequences. These potential untoward effects of miRNA-targeted therapy may differ from small interfering RNA-targeted agents currently in clinical trials.

As the miRNA field advances, new methods of study are being put to use. Researchers have used coimmunoprecipitation of Ago proteins followed by microarray analysis of bound mRNAs as a means to identify direct targets of miRNAs (47). Large-scale proteomic approaches have recently confirmed that overexpression or knockdown of a miRNA can affect the expression of hundreds of proteins and that the 3′UTR contains the primary motif for miRNA controlled protein repression (48, 49).

The study of miRNAs is an exciting and rapidly growing field. Because miRNAs were first discovered in C. elegans, we have greatly increased our understanding of their formation and function in the cell and have developed several experiments to validate predicted targets of miRNA regulation. With this knowledge, several tools to improve patient care may be on the horizon covering diagnostic, prognostic, and predictive measures for treatment decision making. miRNA-targeted therapy in clinical trials may one day be feasible. Although we have come a long way in a short period with regards to our understanding of miRNAs and cancer, we still have much to learn. As our understanding of these small RNAs increases, we hope they can be used to improve cancer diagnostics and therapeutics.

G.J. Weiss: coinventor on patent application for the measurement of miRNA in tumors expressing EGFR. No other potential conflicts of interest were disclosed.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Bob Duggan, Mary Beth Cunney, Michael Beveridge, and Candice Nulsen for assistance with figures for this article.

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