Journal of Radiation Research Advance Access published March 16, 2016

Journal of Radiation Research, 2016, pp. 1–6

doi: 10.1093/jrr/rrw009
Supplement – ICRR highlights

The complexity of microRNAs in human cancer

Jennifer Y. Y. Kwan1,2,3, Pamela Psarianos1, Jeff P. Bruce1, Kenneth W. Yip1
and Fei-Fei Liu1,2,3,4*
1
Ontario Cancer Institute, University Health Network, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada
2
Department of Radiation Oncology, Princess Margaret Cancer Centre, University Health Network, 610 University Avenue, Toronto, ON, M5G 2M9, Canada
3
Department of Radiation Oncology, Faculty of Medicine, University of Toronto, 149 College Street, Suite 504, Toronto, ON, M5T 1P5, Canada
4
Department of Medical Biophysics, University of Toronto, 101 College Street, Room 15-701, Toronto, ON, M5G 1L7, Canada
*Corresponding author: Department of Radiation Oncology, Princess Margaret Cancer Center/Ontario Cancer Institute, 610 University Ave, Toronto, Ontario,
M5G 2M9, Canada. Tel: +1-416-946-2123; Fax: +1-416-946-2038; E-mail: [email protected]
Received September 24, 2015; Revised January 4, 2016; Accepted January 15, 2016

A B S T R AC T
MicroRNAs (miRNAs) are small non-coding RNA molecules that have key regulatory roles in cancer, acting as
both oncogenes and tumor suppressors. Due to the potential roles of miRNAs in improving cancer prognostic, pre-
dictive, diagnostic and therapeutic approaches, they have become an area of intense research focus in recent years.
MiRNAs harbor attractive features allowing for translation to the clinical world, such as relatively simple extraction
methods, resistance to molecular degradation, and ability to be quantified. Numerous prognostic, predictive and
diagnostic miRNA signatures have been developed. To date however, miRNA analysis has not been adopted for
routine clinical use. The objectives of this article are to provide an overview of miRNA research and review a selec-
tion of miRNA studies in breast cancer, cervical cancer, sarcoma, and nasopharyngeal carcinoma to highlight
advances and challenges in miRNA cancer research.

KE YWOR DS: microRNA, breast cancer, cervical cancer, sarcoma, nasopharyngeal carcinoma

I N T RO D U C T I O N MiRNAs are dysregulated in almost all human cancers [3] and

MicroRNAs (miRNAs) are non-coding RNAs composed of 18–25 can function as either oncogenes or tumour suppressors, depending
nucleotides [1], which were first described in C. elegans by Lee et al. upon their target transcripts. For example, miRNA-21 (miR-21), one
in 1993 [2]. Since their discovery, miRNAs have been demonstrated of the most overexpressed miRNAs in human epithelial malignancies,
to have a key role in gene regulation in many different systems. There downregulates a myriad of tumour suppressors such as phosphatase
are several important steps involved in the synthesis of miRNAs and tensin homolog (PTEN) [4], ras homolog gene family member
(Fig. 1) [1]. Beginning in the nucleus, RNA polymerase II produces B (RhoB) [5], tropomyosin 1 (TPM1) [6], and programmed cell
pri-miRNA transcripts. These are structurally similar to protein- death 4 (PDCD4) [7], thereby resulting in increased tumour cell pro-
coding gene transcripts with an additional stem-loop structure. This liferation, invasion, and metastasis [4–7]. MiRNA levels are also cor-
stem-loop interacts with the ribonuclease Drosha and double- related with response to ionizing radiation. Downregulating proteins
stranded RNA binding protein, DiGeorge syndrome critical region in its biosynthesis pathway (e.g. Drosha and Dicer) increases radiore-
gene 8 (DGCR8), to generate a precursor miRNA ( pre-miRNA). sistance via a decreased DNA-damage response [8].
Upon transport to the cytoplasm, the ribonuclease Dicer1 and trans- MiRNAs harbor attractive features allowing for translation to clin-
activation-responsive RNA-binding protein 2 (TARBP2) convert the ical practice, such as simple extraction, resistance to molecular degrad-
pre-miRNA into a double-stranded miRNA duplex. Following strand ation, and accurate, reproducible quantification using quantitative
separation, the mature miRNA strand combines with Argonaute and real-time reverse transcription PCR (qRT-PCR) and other analytical
forms the RNA-induced silencing complex (RISC). Gene regulation methods [9]. Combined with their potential to improve cancer diag-
then occurs through either binding to target mRNA transcripts with nosis, classification, prognosis and therapies, miRNAs have garnered
perfect complementarity, resulting in transcript degradation, or more significant research attention in recent years. For example, with the
frequently, binding with imperfect complementarity, leading to inhib- identification of upregulated miR-141 in prostate cancer patients
ition of protein translation. [10], circulating serum miRNAs were speculated as a possible

© The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-
nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use,
please contact [email protected]

• 1
2 • J.Y.Y. Kwan et al.

Fig. 1. MicroRNA biosynthesis.

Fig. 2. MicroRNA profiling workflow.

non-invasive diagnostic tool. MiRNA profiling may also facilitate carcinoma (NPC) conducted by our group to underscore recent
better classification of tumors into treatment subgroups [11], espe- advances and challenges in miRNA cancer research.
cially for poorly differentiated tumors that are difficult to define by
histology. A number of prognostic miRNA signatures have been gen- B R E A S T CA N C E R
erated and validated in various cancers [12, 13]. Additionally, Breast cancer is the second most commonly diagnosed cancer, and
miRNAs have been investigated for drug therapy (e.g. miR-122 fifth most common cause of cancer mortality worldwide [17].
antagonists for decreasing risk of hepatocellular carcinoma [14]). Pre- MiRNA dysregulation has been identified as a frequent phenomenon
dictive signatures have also been proposed based on select miRNAs in breast cancer. Four main mechanisms of dysregulation have been
(e.g. miR-16, -29b, -150, -1254 and let-7e [15]) associated with radio- proposed, including epigenetic modification, genetic alteration,
sensitivity and radioresistance. changes in miRNA biogenesis, and transcriptional repression [18].
The general approach to miRNA profiling (Fig. 2) involves: (i) Some validated dysregulated miRNAs are highlighted in Table 1 [19–
specimen selection (cells, organisms, fresh tissue, fixed tissue, body 25]. As shown in this Table, global miRNA profiling in breast cancer
fluids); (ii) RNA extraction; (iii) sample quality control; (iv) profiling has yielded a number of potential biomarkers and therapeutic targets.
miRNA with qRT-PCR, microarray, or RNA-sequencing; and (v) We will focus specifically on two studies of miRNA profiling in breast
data analysis [16]. qRT-PCR is the gold standard because it provides cancer from FFPE specimens to highlight its use [19, 26].
the best absolute miRNA quantification with the greatest sensitivity Prior to 2008, the majority of global miRNA profiling in cancer
and dynamic range compared with the other methods [16], although utilized frozen tissues; however, formalin fixation is a common prac-
several other platforms are commonly used in miRNA research. tice for specimen preservation. Thus, Hui et al. [19] examined the
Global miRNA profiling of cancers has yielded important research feasibility of miRNA analysis on FFPE tissues. They performed
findings. In particular, profiling of formalin-fixed, paraffin-embedded miRNA expression profiling on 40 archived FFPE breast lumpectomy
(FFPE) samples from human tumors, which are correlated with clin- specimens using Taqman Low-Density Arrays (TLDAs). The expres-
ical details, has significantly improved our understanding of miRNAs sion of 365 miRNAs in 34 invasive ductal cancers and 6 reduction
in this disease. The purpose of this article is to provide an overview mammoplasty normal epithelial breast tissues was assessed and con-
of miRNA research and highlight a selection of miRNA studies firmed using the gold standard single-well qRT-PCR. The results
in breast cancer, cervical cancer, sarcoma, and nasopharyngeal from both methods were highly correlated and confirmed the
 Micro-RNAs in human cancer • 3

Table 1. Dysregulated miRNAs in breast cancer Table 2. Sources of expression heterogeneity in cervical tissues
Upregulated miRNAs [19–21] Downregulated miRNAs Challenges in miRNA profiling of cervical cancer tissues
[19, 21–25]
1. Interpatient variability [29]
miR-9, miR10b, miR-21, miR- miR-30a, miR-31, miR-34a, miR- 2. Intratumor heterogeneity [30]
27a, miR-29a, miR-96, miR- 125, miR-126, miR-146a, miR- 3. Specimen preservation differences [30]
146a, miR-155, miR-181, 146b, miR-195, miR-200, miR- 4. Profiling platform differences [30]
miR-191, miR-196a, miR- 205, miR-206, miR-221, and
221/222, miR-373, miR-375, let-7
miR-520c, and miR589 intratumor heterogeneity, poor correlation between miRNAs in
frozen and FFPE samples, and poor reliability of profiling platforms
(Table 2).
dysregulation of miR-21, miR-155, miR-191, miR-196a, miR-125b Despite difficulties with profiling in cervical tissues, positive find-
and miR-221, which were previously reported. Thus, this study pro- ings have been reported in the literature as well. For example, based
vided support for performing miRNA profiling on FFPE tissues. on the global miRNA profiling of 79 cervical cancer fresh frozen
Clinical trial materials are a rich source of uniformly collected and punch biopsies and 11 normal samples, Kogo et al. [31] identified a
processed archival FFPE tumor tissues for evaluation and are ideal for difference in miR-218 expression between malignant cervical and
addressing primary or secondary research aims involving miRNA pro- normal cervical epithelial tissues. Downregulation of miR-218 was
filing [26, 27]. For example, 71 FFPE blocks from participants of a prognostic for reduced DFS, overall survival and lymph node recur-
Phase III clinical trial comparing Tamoxifen alone vs Tamoxifen plus rence. Further investigations of miR-218 biology determined that it
breast radiotherapy in women with node-negative breast cancer [28] was targeting the survivin axis to modulate cancer proliferation,
served as the cohort for a subsequent miRNA study by Shi et al. [26]. migration and invasion. In this process, YM155 was identified as a
Through global miRNA profiling with qRT-PCR, they identified a promising small molecule inhibitor of survivin, which could reduce
6.7-fold higher expression of miR-301 in tumours as compared with nodal metastasis in cervical cancer xenograft models [31].
normal breast tissue [26]. Overexpression of this miRNA was also
associated with increased incidence of nodal and distant relapse [26].
SAR COM A
However, the prognostic value of miR-301 required further validation
Sarcomas are a heterogeneous group of rare tumors that provide
in a larger patient cohort.
diagnostic and prognostic challenges. Its 5-year overall survival is
∼60–80%, with distant metastasis (DM) as the main contributor
C E RVI CA L CA N C E R to mortality [32]. Tumor size and grade have demonstrated prognos-
Cervical cancer is the fourth most common cancer in females and tic significance, but variability in clinical outcome suggests the need
accounts for 7.5% of all female cancer deaths [17]. Identifying prog- for more robust biomarkers. Few prognostic signatures for soft tissue
nostic and predictive biomarkers are important aims of miRNA sarcomas have been validated. Wong et al. [33] were the first to valid-
research in cervical cancers; however, cervical tissues have presented ate a miRNA signature for distant metastasis–free survival (DMFS)
several challenges in miRNA profiling. in undifferentiated pleomorphic sarcomas (UPS), the most common
In 2010, Pereira et al. [29] studied miRNA expression in human and aggressive subtype of adult soft-tissue sarcomas. Using TaqMan
cervical tissues using a 381-probe microarray platform on a mixed set Human Micro-RNA Array-A, 110 fresh frozen UPS samples were
of 19 normal, 14 pre-cancerous, and four cancerous cervical tissues analyzed to identify and validate a six-miRNA signature for DMFS.
derived from 25 patients. During their investigation, they noted This signature, comprised of miR-132, miR-138, miR-143, miR-221,
highly variable miRNA expression among the normal samples. This miR-224 and miR-491–5p, was independently prognostic of known
variability was confirmed to be non-technical and related to the inter- clinical factors. Functional studies demonstrated that these miRNAs
patient biological differences, such as human papillomavirus (HPV) were targeting the Rho adhesion pathway in UPS metastasis.
status and age. This study emphasized the importance of considering
interpatient variations that may influence miRNA expression profiles, N A S O P H A RY N G E A L CA R C I N O M A
including natural genetic variation, age, viral infections, and non-neo- Nasopharyngeal carcinoma (NPC) is an epithelial malignancy of the
plastic diseases, when interpreting miRNA profiling results in cervical head and neck that has a preponderance in Southeast Asia. Patients
tissues [29]. with early stage NPC have a favorable outcome when treated with
A subsequent study in cervical cancers further underscored add- radiotherapy, with 5-year local control rates exceeding 90% [34].
itional considerations in miRNA analyses. In 2015, How et al. [30] However, late stage disease (Stage III–IVB) has a worse 5-year overall
described their investigation of developing a nine-miRNA signature survival rate of only ∼68% or less [34]. Furthermore, DM continues
for cervical cancers. They formulated a candidate prognostic signature to occur and is the major factor contributing to mortality in this
for disease-free survival (DFS), which included well-characterized population. Improved prognostic markers associated with DM are
miRNAs involved in neoplasia, based on a training cohort of frozen therefore clearly required. Bruce et al. [35] profiled 734 miRNAs on a
tissues. However, there was difficulty validating this signature in an training set comprised of 125 NPC FFPE biopsy samples, and a four-
independent cohort comprised of FFPE tissue samples. They identi- miRNA expression signature for DM was validated on an independ-
fied several reasons contributing to this challenge including ent cohort of 121 FFPE biopsies. This signature demonstrated
4 • J.Y.Y. Kwan et al.

prognostic value in addition to tumor node metastasis staging, the unreliable in some cases. For example, methods that require a reliable
mainstay of clinical classification in NPC. In addition, this four- estimate of the distribution of miRNA expression values have been
miRNA signature was observed to possess potential predictive value noted to lack applicability in miRNA datasets compared with mRNA
in identifying a low-risk group of locally advanced NPC patients who data, since the majority of global miRNA profiling array platforms test
could be treated with radiotherapy alone, thereby sparing the add- much fewer transcripts than mRNA platforms [37, 39]. This yields a
itional toxicity of chemotherapy. Other signatures comprised of dif- smaller set of miRNAs with stable expression. Thus, the use and
ferent sets of miRNAs have also been reported for NPC, including a selection of controls become paramount in miRNA studies. It has
five-miRNA signature by Liu et al. [36]. Bruce et al. [35] attempted been suggested that a greater number of normalization controls
to compare the four-miRNA with the five-miRNA signature to deter- should be used and controls should be validated for stability before
mine if superiority could be established; however, due to differences use [39]. In general, adapting mRNA methodologies to miRNA
in technical platforms, patient population, and availability of clinical studies has its limitations, and novel approaches for miRNA-specific
data, no definitive conclusions could be drawn. With an accumulating research have been explored. In this era with little consensus on
number of miRNA signatures reported in the literature, prioritization miRNA data normalization methods, it is imperative for researchers
of miRNA signatures to translate for clinical use could become prob- to ensure that signatures generated are not normalization method–
lematic without the ability to compare signatures derived from differ- specific to ensure robustness and generalizability.
ent methodologies. Lastly, the biological validity of some miRNA signatures has been
called into question [35, 40]. MiRNA signatures are currently devel-
oped from algorithms based on finding associations between expres-
L ES S O N S L EA R NE D sion and disease outcome. Hence, causality is often not taken into
In recent years, research in miRNA profiling has garnered significant account, and algorithms may generate signatures that are not biologic-
interest. Dysregulation of miRNAs in a variety of different cancers ally significant, despite their statistical significance [40]. For example,
have been reported, and associated miRNA signatures have been based on our experience, we were able to validate 8.4% of 90 000 ran-
developed. Along the way, a number of challenges in miRNA research domly generated miRNA signatures, comprised of 2–10 miRNAs
have also emerged. Based on the research reviewed in this article, each, using the Bruce et al. training and validation sample set [35]
there are some lessons that can be learned. (Fig. 3). Thus, it is important to consider the limitations of algo-
Three main problems have been identified in the miRNA profiling rithms for miRNA signature generation and how selection of algo-
workflow; they involve tissue preparation, profiling platform selection, rithms may impact results. Utilization of further methods to evaluate
as well as data normalization and analysis. The first issue speaks to signatures of likely biological significance, such as the Bruce et al.’s
the need for more careful consideration of differences between speci- approach of prioritizing highly significant signatures for further
men types. Tissue preservation technique appears to affect miRNA molecular target identification and pathway analysis, is needed.
expression results, as described in How et al.’s study [30], which Currently, a variety of methods exist for generation of miRNA
demonstrated the difficulty in comparing miRNA profiling of signatures. As a result, numerous signatures have been developed.
frozen and FFPE tissues. Thus, when defining training and validation
cohorts for future miRNA signature testing, it would be prudent to
extract RNA from tissues prepared in a similar manner to maximize
comparability.
Technical platform variability has also proven to be an important
consideration in investigating miRNAs. Several platforms have been
used to quantify miRNAs; however, these techniques have significant
methodological differences. For example, TLDAs use multiplex
reverse transcription followed by TaqMan PCR, while NanoString
uses optical quantification of fluorescently tagged miRNAs. Consist-
ency between profiling platforms has been called into question by
studies such as that of How et al. [30], which demonstrated that pro-
filing of FFPE RNA samples by TLDA and NanoString had poor cor-
relation in regards to miRNA abundances and tumor-to-normal
tissue expression differences [37]. This, therefore, increases the diffi-
culty of interpreting results across different platforms. In addition, for
PCR-based techniques, the quantification may not distinguish be-
tween abundances of miRNA at different levels of maturation. Specif-
ically, pri- and pre-miRNA levels cannot be discriminated if the
amplicon is shared by both [38]. Thus, it is important to understand
the limitations of each methodology.
As well, the study of miRNAs has created some new challenges Fig. 3. Validation of randomly generated miRNA
that have arisen since past research with mRNA transcripts. Normal- signatures. Density plot is shown with different color plots
ization of data is required for decreasing technical bias; however, data denoting different signature sizes (2–10 miRNAs). Vertical
normalization methods for miRNA analysis remain varied and line denotes log-rank P < 0.05 in our validation set.
 Micro-RNAs in human cancer • 5

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