REVIEW

Non-coding RNAs for Medical Practice in Oncology
Tetsuro Setoyama,1,2 Hui Ling,1 Shoji Natsugoe2 and George A. Calin1

1Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center,

Texas, USA

2Department of Surgical Oncology and Digestive Surgery, Graduate School of Medical and Dental Sciences,

Kagoshima University, Kagoshima, Japan(Received for publication on April 1, 2011)(Revised for publication on May 7, 2011)(Accepted for publication on May 19, 2011)

Alterations in microRNA (miRNA) and other short or long non-coding RNA (ncRNA) are involved in
the initiation, progression, and metastasis of human cancer. The main molecular alterations result fromvariations in gene expression, which are usually minor but have consequences for a vast number of target protein-coding genes. The causes of the widespread differential expression of ncRNAs in malignant
cells compared with normal cells can be explained by the location of these genes in genomic regions
associated with cancer, by epigenetic mechanisms, and by alterations in the processing machinery. Expression profiling of human tumors based on the expression of miRNAs and other short or long ncRNAs
has identified signatures associated with diagnosis, staging, progression, prognosis, and response totreatment. In addition, profiling has been exploited to identify ncRNAs that may represent downstream
targets of activated oncogenic pathways or that target protein-coding genes involved in cancer. Recent
studies found that miRNAs and non-coding ultraconserved genes are the main candidates for the elusive class of cancer-predisposing genes and that other types of ncRNAs participate in the genetic puzzle
that gives rise to the malignant phenotype. These discoveries could be exploited for the development of
useful markers for diagnosis and prognosis in cancer, as well as for the development of new RNA-based
cancer therapies. (Keio J Med 60 (4) : 106–113, December 2011)

Keywords: non-coding RNAs, MicroRNAs, cancer, oncogenes, tumor suppressors

Introduction
Non-coding RNAs (ncRNAs) are RNAs that do not encode proteins. Until recently the central dogma of genetics was that RNA played the role of messenger between
the gene and the final proteins codified by the gene, andncRNAs were ignored in the field of genome sequencing.1,2 In the past decade, genome-wide transcriptional
analyses have estimated that most transcribed mammalian genomic sequences are ncRNAs.3–5 In addition, newncRNAs have been recognized as important participantsin several mechanisms of gene regulation.5
These new ncRNAs can be categorized as either shortor long. Long ncRNAs (lncRNAs) are processed in fullfrom their templates and are typically >200 nt long; small
ncRNAs, conversely, are processed from longer precursors and include microRNAs (miRNAs), small interfering RNAs, piwi-interacting RNAs, tiny RNAs, and cryptic unstable transcripts.5
Many classes of ncRNAs have been described in the literature, and their roles in cellular biology are recognized
as being sophisticated. miRNAs are well-known ncRNAs
molecules that regulate various gene expressions at the
RNA level. In addition, lncRNAs such as ultraconserved
genes (UCGs) and HOX antisense intergenic RNA (HOTAIR) have been recently characterized.6,7
In various human diseases, the alteration of several
ncRNA types has been reported. A role for ncRNA in hu-

Presented at the 1790th Meeting of The Keio Medical Society in Tokyo, November 25, 2010.
Reprint requests to: George A. Calin, MD, PhD, Department of Experimental Therapeutics, Unit 0036, The University of Texas MD Anderson
Cancer Center, Houston, TX 77030, USA, E-mail: gcalin@mdanderson.org
Copyright © 2011 by The Keio Journal of Medicine

106Keio J Med 2011; 60 (4): 106–113

man carcinogenesis is most certain, providing evidence
for targeting these molecules as anticancer agents. However, at present, a clear relationship with cancer has beenreported for only a few ncRNA classes (miRNA, UCG,
and HOTAIR). Therefore, the purpose of this review is todescribe recent findings on UCGs, HOTAIR, and especially miRNAs and their effect on cancer.
HOTAIR and Ultraconserved Regions (UCRs)
lncRNAs range in size from approximately 200 nt toover 100 kb. Like messenger RNAs (mRNAs), lncRNAs
seem to be transcribed mostly by RNA polymerase II, butmany lncRNAs do not undergo the subsequent standard
mRNA processing steps.8 Because of this characteristic,
lncRNAs often have regulatory functions involving nuclear retention close to transcription sites.9 Although themechanisms and functions of lncRNAs are mostly unclear, they seem to mirror those of protein-coding genes.6
The mechanism of lncRNA function is generally unknown, but evidence suggests that it is similar to that of
HOTAIR, which is itself an lncRNA. HOTAIR regulates
the chromatin methylation state of the HOXD locus intrans through polycomb repressive complex 2. HOTAIR
is related to polycomb repressive complex 2 and lysinespecific demethylase 1/RE1-silencing transcription factor
co-repressor 1/RE1-silencing transcription factor protein
complexes; HOTAIR coordinates the chromatin targetingof these proteins and it couples histone H3K27 methylation with H3K4 demethylation to create epigenetic genesilencing.10,11 HOTAIR was recently reported to play arole in cancer metastasis and may be an indicator of poor
prognosis in patients with primary breast cancer.7
UCGs, one of the new ncRNAs, are transcribed from
UCRs12 and have a tissue-specific expression pattern.12,13
Researchers became aware of UCRs when the alignments of human, rat, and mouse genomes demonstrated
that despite 300 million years of negative selection, somegenomic regions remained highly conserved (100% identity).5,14 In fact, UCRs are even more conserved than coding genes are, and UCRs are now believed to have fundamental functions in vertebrate evolution, including thatof mammals.2,14–16 UCRs are frequently found in fragile
genomic regions that are usually associated with cancer,
and UCG expression is aberrant in several human carcinomas and leukemias compared to that of their normal
tissue counterparts.12 Deregulated UCG signatures arecancer specific and have prognostic implications. As isalso the case for miRNAs, UCGs can act as oncogenes
or tumor-suppressor genes, and UCG expression is controlled by miRNAs.12
miRNAsmiRNAs are ncRNAs of 19–25 nt in length and areclassic examples of ncRNAs. By degrading or blocking

107translation of mRNA targets, these miRNAs regulate
more than 30% of the mammalian genome.17 miRNAs
are also involved in the initiation, progression, and metastasis of human cancer18,19 and can behave as either oncogenes or tumor suppressors.17
miRNA profiling, which has been achieved by various
methods, has identified signatures associated with diagnosis, staging, progression, prognosis, and response totreatment of human tumors. Therefore, miRNA profiling
represents a new addition to the diagnostic and prognostic
tools available for use by medical oncologists. The main
paradigms for miRNA involvement in human cancers are
summarized in the sections that follow.
Alteration of miRNAs in All Human Malignancies
Alterations of miRNAs, first identified in the mostcommon form of adult leukemia, i.e., B cell chronic lymphocytic leukemia (CLL),20 have since been detected inmany types of human tumors (Table 1)18,20–68. The main
mechanism of alterations of microRNoma (defined as thefull complement of miRNAs present in a genome) in cancer cells seems to be aberrant gene expression, which ischaracterized by abnormal levels of expression of mature
and/or precursor miRNA sequences. The causes of thewidespread differential expression of miRNA genes between malignant and normal cells can be explained by
the genomic location of these genes in cancer-associatedregions and genomic alterations,18 by epigenetic mechanisms, and by alterations in the processing machinery
Several developments in high-throughput miRNAprofiling include oligonucleotide miRNA microarrays,
as described previously69; the use of a bead-based flow
cytometric technique36–38,70; the quantitative reverse
transcription-polymerase chain reaction for precursor
miRNA71 or active miRNA72,73; the miRNA serial analysis of gene expression (miRAGE), which combines direct
miRNA cloning with serial analysis of gene expression(SAGE)35–38,73,74; and the high-throughput array-based
Klenow enzyme (RAKE) assay.75 miRNAs differentiallyexpressed between tumors and their normal tissue counterparts have been identified for several tumor types,
including lymphoma,57 breast cancer,25 lung cancer,24
thyroid papillary carcinoma,76 glioblastoma,63 hepatocellular carcinoma,77 colorectal carcinoma,74 pancreatic tumors,78 and pituitary adenomas.79
miRNA Alterations May Result in Cancer
Predisposition
Despite extensive research, the molecular basis formost familial cancers is unknown. Because the extent
of miRNA involvement in human cancers is much greater than initially believed, it is tempting to propose thatgermline mutations or polymorphisms in miRNA genes
or interacting sequences from target mRNA might repre-

Setoyama T, et al: Non-coding RNAs for Diagnosis and Therapy of Cancer

108

Table 1 miRNA expression profiles in common types of cancer
miRNA
miR-15a miR-16–1
cluster
let-7 family
miR-29 family

Target genes
Bcl-2, MCL1, Wt-1

PropertyTS

KRAS, NRAS, c-myc,
HMGA2
TCL-1, MCL1, CDK6,
DNMT3s
CDK4, CDK6, Bcl-2, cyclinE2, E2F3, c-Met, c-Myc
ZEB1, ZEB2TS
TS

Expression in cancer
Downregulated in CLL
Downregulated in lung and breast cancers

Downregulated in CLL, AML, lung cancer,
breast cancer, and cholangiocarcinoma
miR-34a/b/c
TS
Downregulated in pancreatic, colon, and breast
cancersmiR-200/141 family
TS
Downregulated in breast, renal clear cell,
gastric, and bladder cancers
miR-155
c-maf, SHIP1
OG
Upregulated in high-risk CLL, AML, lymphomas, colon cancer, lung cancer, and breast
cancermiR-17–92 family
E2F1, Bim, PTEN
OG
Upregulated in lymphomas and breast, lung,colon, stomach, and pancreatic cancers
miR-21
PTEN, PDCD4, TPM1
OG
Upregulated in breast, colon, pancreatic, lung,
prostate, liver, and stomach cancers; AML;
CLL; and glioblastomas
miR-372/ miR-373
LATS2
OG
Upregulated in testicular cancer
CLL, chronic lymphocytic leukemia; AML, acute myeloid leukemia; TS, tumor suppressor; OG, oncogene.

sent a mechanism of cancer predisposition (for a review,
see Calin et al. 200618). In fact, a germline mutation in
the pri-miR-16–1/15a precursor in a patient with familial
CLL and breast cancer in first-degree family memberssuggests a possible predisposing effect,30 but the precise
roles of mutations in miRNAs are still unknown. Tumorspecific pri-miRNA sequence abnormalities seem to be amore widespread phenomenon in tumorigenesis, as mutations near the clusters miR-17–92 on chromosome 13
and miR-106–92 on chromosome X were described in amouse model.
These clusters are amplified in human lymphomas80
and accelerate c-MYC-induced tumorigenesis in a mousemodel of B-cell lymphoma,57 suggesting a possible pathogenetic role for such mutations. Furthermore, germline
single nucleotide polymorphisms were identified in two
recognition sites in the c-KIT oncogene for miR-221, miR222, and miR-146, which are all strongly overexpressedin thyroid cancers.76 Therefore, because the thermodynamics of RNA–RNA binding plays an essential role ina miRNA interaction with its target mRNA, sequencevariations influencing this interaction may result in cancer predisposition.
miRNAs as Oncogenes and Tumor Suppressors
The classical models of tumorigenesis postulate alterations in protein coding oncogenes and tumor suppressor
genes. miRNAs are also contributors to oncogenesis,
functioning as tumor suppressors (as in the case of miR15a and miR-16–123 and the let-7 family26) or as oncogenes(as in the case of miR-155,51 the miR17-92 cluster,57,76 andmiR-2151,65). Relatively minor variations in the levels of
expression of a miRNA or mutations that moderately

References
20–23
24–29
24,25,30–34
35–38
39–43
24,25,31,44–51

51–62
18,30,31,47,51,63–67

68

affect the conformation of the miRNA::mRNA pairing
could have important consequences for the cell becauseof the large number of targets of each miRNA. For example, downregulation of the suppressor miR-15a/miR-16–1
induces overexpression of the B cell lymphoma 2 (BCL2)
gene and possibly other genes that may be important
for tumorigenesis,23 while overexpression of oncogenic
miR-17–92 cooperates with c-MYC in stimulating lymphomagenesis.57,76 miRNA alterations in somatic cells
can initiate or contribute to tumorigenesis, and miRNAalterations in germline cells may predispose a person tocancer. A paradigm for this model is human B-cell CLL,
in which miR-15a and miR-16–1 are located in the mostfrequently deleted genomic region, are downregulated in
the majority of cases, harbor mutations in familial cases,
and induce apoptosis in a leukemia mouse model by targeting the antiapoptotic BCL2 gene.
miRNA Profiling Can Improve Diagnosis ofCancer in Patients
To date, numerous profiling studies using tumors ofvarious histotypes have been published.51,70,81 Lu et al70.,
using a bead-based flow-cytometric profiling technology
performed on a set of 334 samples that included multiple
human cancers (mainly leukemias), found that miRNA
expression profiles classify human cancers and reflect
the developmental lineage and differentiation state of
the tumors. Of important diagnostic consequence is thatthe miRNA-based classifier is better at establishing thecorrect diagnosis of poorly differentiated samples with
non-diagnostic histological appearance than is the protein-coding gene’s mRNA classifier. This type of classification represents an important advance in the diag-Keio J Med 2011; 60 (4): 106–113

nosis of metastatic cancer of unknown primary site, as
these patients present with metastases (late-stage disease)without an established primary tumor (i.e., a site where atherapeutically curative or palliative intervention can be
performed).
Volinia et al.51 described a large-scale, detailed microarray analysis using 540 samples from six types of the mostfrequent solid human cancers and found a common signature composed of 61 miRNAs, mainly overexpressedwith respect to corresponding normal tissues. Evidence
has been found that the predicted targets for the differentially expressed miRNAs are significantly enriched forprotein-coding tumor suppressors and oncogenes, supporting the roles of these genes in tumorigenesis. Of the
228 miRNA genes analyzed, 36 were overexpressed and
21 were downregulated in cancer cells compared withnormal cells. Hierarchical clustering analysis showed that
this miRNA signature enabled the tumor samples to begrouped on the basis of their tissue of origin, which cangreatly help in diagnosis.
miRNA Profiling Can Predict Outcome forCancer Patients
In most CLL patients, the prognosis is relatively good
and, after diagnosis, treatment is started only if poor
prognostic markers are evident. Among these markers,
those most commonly used are high expression levelsof the zeta-chain-associated protein of 70 kDa (ZAP-70)
gene and the absence of mutations in the immunoglobulin
variable-region heavy-chain (IgVH) gene. By performing a miRNA profiling screening of 144 CLL patients,
researchers found that a unique signature of 13 miRNAs(of the 190 analyzed) differentiated cases on the basis of agood or poor prognosis and on the presence or absence ofdisease progression.30 Among the 13 miRNAs, miR-16–1
and miR-15a were expressed at lower levels in patientswith a good prognosis, which was in agreement withearly reports that 13q14.3 genomic deletions at the locus
harboring these genes are favorable prognostic features.
Identification of new prognostic markers could represent a significant advance for the identification of patients
who would benefit from more aggressive therapy for lungcancer, which is the leading cause of cancer death in men
worldwide. In univariate analyses, expressions of bothmiR-155 and let-7a-2 were shown to correlate with poor
survival in 104 American patients with lung cancer; in
multivariate analyses, only the expression of miR-155 wascorrelated with poor prognosis when all clinical variables
were considered together.24 In an independent study of
143 Japanese patients with lung cancer, underexpressionof let-7 was found to significantly correlate with a shorter
survival time after potentially curative surgery; multivariate analysis showed that underexpression of let-7 was anindependent prognostic factor for the stage of the diseaseand correlated with significantly shorter survival time.82

109

miRNAs as Therapeutic Targets
Because miRNAs regulate the expression of multiple
genes in a disease pathway, miRNAs and the genes they
influence can be therapeutic targets. The use of miRNAs
as drug therapies is still at the preclinical stage, and theresults of clinical and toxicological studies have not yet
been published. miRNA mimics were shown to induce
cell death and reduce tumorigenic potential of the miR15a/16 cluster in a leukemia model21 and of the membersof the miR-29 family in a lung cancer model.33
Several animal model studies have been reported.83–86
Systemic administration of anti-miRNA (miR-122) oligonucleotide therapeutics in mice and African green monkeys was shown to reduce total plasma cholesterol andhepatitis C viral load. As a result, systemic administration of miR-122 could reduce liver damage.83–85 In addition, when the mimic for miR-26a was administered systemically in a murine model of liver cancer, tumor size
was clearly reduced.86 Thus, in addition to confirming thesafety and efficacy of anti-miRNA oligonucleotides in a
preclinical trial, these studies have established a baseline
for their testing in future clinical trials.
Conclusions
There is no question that miRNAs and other ncRNAs
are involved in the regulation of tumorigenic pathways
involved in tumor development and progression. Whether
miRNAs represent the “dark side” of cancer predisposition will be soon be clearly answered by our future studies in a large series of familial cancer patients. miRNAs
and other ncRNAs have only recently been identified as
new diagnostic and prognostic tools for cancer patients,
and the use of miRNAs in cancer therapy represents a
potential treatment option for medical oncologists in thenear future. The rationale for using miRNAs and otherncRNAs in cancer treatment is that they are natural antisense interactors that regulate many genes involved ineukaryotic survival and proliferation. These discoveries
could be exploited for the development of useful markers
for diagnosis and prognosis in cancer, as well as for thedevelopment of new RNA-based cancer therapies.
Acknowledgments
G.A.C. is supported as a Fellow at The University ofTexas MD Anderson Research Trust, as a University ofTexas System Regents Research Scholar, and by the CLL
Global Research Foundation. Research in Dr. Calin’s laboratory is supported in part by the National Institutes of
Health through MD Anderson’s Cancer Center Support
Grant CA016672, a Department of Defense Breast Cancer
Idea Award, Developmental Research Awards in Breast
Cancer, MD Anderson’s Ovarian Cancer, Brain Cancer,

Setoyama T, et al: Non-coding RNAs for Diagnosis and Therapy of Cancer

110

and Leukemia SPOREs, a CTT/3I-TD grant, a 2009 Seena Magowitz–Pancreatic Cancer Action Network AACR
Pilot Grant, and the Arnold Foundation. We would liketo thank Kristi Speights (Department of Scientific Publications, MD Anderson Cancer Center) for her help withediting this manuscript.
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