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Oncogenic microRNAs as biomarkers of oral tumorigenesis and minimal

residual disease

W.S. Santhi a, R. Prathibha a, Sona Charles a, K.G. Anurup a, G. Reshmi a, Surya Ramachandran a, V.T. Jissa a,Paul Sebastian b, M. Radhakrishna Pillai a,⇑

a Cancer Research Program, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram 695 014, Kerala, Indiab Division of Surgical Oncology, Regional Cancer Centre, Thiruvananthapuram 695 011, Kerala, India

a r t i c l e i n f o

Article history:

Received 31 May 2012

Received in revised form 3 January 2013

Accepted 6 January 2013

Available online xxxx

Keywords:

MicroRNAs

Oral tumor

Minimal residual disease

Biomarker

Leukoplakia

s u m m a r y

Objectives: Classical diagnostic methods are not sensitive enough in detecting oral lesions that may pro-

gress to cancer and in assessing minimal residual disease (MRD) in oral surgical margins. Altered expres-

sion of microRNAs (miRNAs) contributes to human cancer, including oral cancer. Although there are

many studies on microRNAs in oral cancer, there is no reported study comparing the expression of

microRNAs during oral tumor progression and in oral surgical margins.

Materials and methods: This study analyzed the expression of 72 miRNAs that were reported (till June

2011) to be differentially expressed in oral cancer, during phases of oral cancer progression and in oral

surgical margins.

Results: Of the 72 miRNAs analyzed, four (hsa-miR-125a, hsa-miR-184, hsa-miR16 and hsa-miR-96)

showed a common pattern of expression in both sets of tissues. We further analyzed the downstream tar-

get genes of hsa-miR-16 BCL2 and CCND1. The in silico network analysis of these four microRNAs and

their target genes revealed presence of genes involved in tumor progression and transcription factors.

Conclusions: The findings suggest that the combinatorial regulation by these miRNAs and their target

transcription factors might play a substantial role in oral tumorigenesis. Here we report for the first time

that a decreased expression of hsa-miR-125a, hsa-miR-184 and hsa-miR-16 and an increased expression

of hsa-miR-96 could be useful in predicting oral tumorigenesis and importantly in the detection of MRD

and decision-making process for postoperative treatment modalities.

2013 Elsevier Ltd. All rights reserved.

Introduction

MicroRNAs (miRNAs) are a group of endogenous, non-coding,

18-24 nucleotide length, single-stranded RNAs that mediate gene

expression at the post-transcriptional level through mRNA degra-

dation or translational repression.1 It is estimated that the human

genome may have more than 1000 miRNAs. Although they account

for only a minor fraction of the expressed genome, miRNAs areessential regulators of diverse cellular processes, including prolif-

eration, differentiation, apoptosis, survival, motility, invasion and

morphogenesis. Most of the miRNA genes are in cancer-associated

genomic regions or in fragile sites.2 Many miRNAs are implicated

as proto-oncogenes or as tumor suppressors and are aberrantly ex-

pressed in various cancers,3 including Oral Squamous Cell Carci-

noma (OSCC). By modulating oncogenic and tumor suppressor

pathways miRNAs could contribute to tumorigenesis. Recent stud-

ies have identified aberrant miRNA expression profiles in OSCC tis-

sues and/or cell lines compared with matched normal controls.1

Studies by Lu et al.4 have shown that miRNA expression profiles

can be used in cancer diagnosis and also in human cancer

classification.

A high incidence of oral cancer is observed in the Indian subcon-tinent, which accounts for a third of the world burden.5 Oral cancer

is the commonest cancer in India and accounts for the most cancer-

related deaths among men in India. The oral cancer incidence in

Kerala is one among the highest in India. Malignancy of the oral

cavity is often preceded by premalignant lesions, the most com-

mon of which is leukoplakia. The molecular mechanisms leading

to and involved in the malignant transformation of normal oral tis-

sue and leukoplakia are still poorly understood. The annual per-

centage of malignant transformation of leukoplakia varies in

different parts of the world owing to the difference in the use of

tobacco and dietary habits. Despite advances made in the manage-

ment of oral cancer over the last few decades, the survival rate of

1368-8375/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.oraloncology.2013.01.001

⇑ Corresponding author. Tel.: +91 471 2347973; fax: +91 471 2348096.

E-mail addresses: [email protected] (W.S. Santhi), [email protected]

(R. Prathibha), [email protected] (S. Charles), [email protected]

(K.G. Anurup), [email protected] (G. Reshmi), [email protected]

(S. Ramachandran), [email protected] (V.T. Jissa), [email protected]

(P. Sebastian), [email protected] (M. Radhakrishna Pillai).

Oral Oncology xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Oral Oncology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / o r a l o n c o l o g y

Please cite this article in press as: Santhi WS et al. Oncogenic microRNAs as biomarkers of oral tumorigenesis and minimal residual disease. Oral Oncol

(2013), http://dx.doi.org/10.1016/j.oraloncology.2013.01.001

http://dx.doi.org/10.1016/j.oraloncology.2013.01.001

mailto:[email protected]










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patients remains unacceptably low. The most important factor in

surgical management of oral cancer is the completeness of surgical

removal of tumor. The success rate of many cancer treatments is

largely dependent on the often microscopically undetectable tu-

mor cells that remain in the body (Minimal Residual Disease or

MRD). Classical diagnostic modalities such as histopathology and

radiology are not always sensitive enough to detect these small

numbers of cells that remain in the body. The increased local–re-

gional recurrences and metastasis of oral cancer, even after treat-

ment points to the importance of molecular markers in the

detection of oral tumor progression and in the assessment of com-

pleteness of surgery. The genetic alterations harbored by tumor

cells can be used as molecular markers for the detection of oral le-

sions that has the capacity for tumor progression and also in the

assessment of MRD in oral surgical margins.

Development of malignancy results from the disruption of fine

tuned signaling homeostasis for proliferation, accompanied by

malfunctional signals for differentiation, cell cycle and apoptosis.6

miRNAs preferentially regulate genes that have a high transcrip-

tional regulation complexity7 and that preferentially target down-

stream genes in cellular signaling flows.8

Complexity of cancer progression mechanisms in higher organ-

isms is thought to also be achieved through controlled and coordi-

nated gene regulatory networks of miRNAs. A miRNA-gene

regulatory network provides a global perspective on the miRNAs

that are dysregulated in cancer as well as the pattern of dysregula-

tion, namely, upregulation or downregulation.9 Several miRNAs are

differentially expressed in various cancers, suggesting common al-

tered regulatory pathways. The effect of miRNAs on cell pathology

and physiology is complex because their activity is exerted in a

one-to-many fashion, and a single messenger can be controlled

by more than one miRNA.10 The cancer miRNA-gene network

throws up several new and interesting biological insights which

will become evident when studied in the global perspective.

Recent work has revealed intriguing changes in the global state

of miRNA expression in cancers including oral cancer. Despite

remarkable recent progress in understanding of functional mecha-nisms of miRNA in various human cancers, the mechanisms, tar-

gets and extent of miRNA-mediated gene expression in oral

cancer is unclear. Studies have compared expression of various

miRNAs in oral cancer and normal oral tissues.11–14 Few have

examined miRNA expression in oral precancerous lesions.15,16

However no previous study has reported expression of miRNAs

during oral tumor progression and in oral surgical margins. This

investigation looked into the expression profile of all reported miR-

NAs (till June 2011) deregulated in oral cancer in two sets of tis-

sues, one involved in oral tumorigenesis and the other in oral

surgical margins. Furthermore, we constructed a bilayered regula-

tory network formed between the miRNAs (that showed the simi-

lar pattern of expression in the two sets of samples) and their

target genes. To the best of our knowledge, this is the first studycomparing the expression profiles of miRNAs in oral tumorigenesis

and in the assessment of MRD in oral surgical margins besides

looking at a computational network analysis. Understanding the

functions of miRNAs provides new insights on the molecular basis

of oral cancer as well as new biomarkers for diagnosis, classifica-

tion, therapy and prognosis evaluation.

Materials and methods

Identification of miRNAs involved in oral cancer

OrCa-dB (www.rgcb.res.in/orcadb)17 is a specialized database

that contains all experimentally determined human oral cancer re-lated information and their interactomics along with clinical data

till June 2011. This database revealed 72 miRNAs that are differen-

tially expressed in human oral cancer.

Computational analysis

The targets of these miRNAs were retrieved from Tarbase data-

base (diana.cslab.ece.ntua.gr/tarbase/). We classified identified

genes by function with DAVID (Database for Annotation, Visualiza-

tion and Integrated Discovery; National Institute of Allergy and

Infections, National Institutes of Health) available at (http://davi-

d.abcc.ncifcrf.gov/) and sorted by Gene Ontology (GO) Consortium

terms. The subgroups assigned were enriched with a set of genes

participating in apoptosis and cancer progression. Most of the

genes in this list were found to be targeted by the four miRNAs

hsa-miR-125a, hsa-miR-184, hsa-miR16 and hsa-miR-96. The

molecular interactions of miRNA-gene regulatory network for the

clustered genes were constructed using Cytoscape Network Analy-

sis Tool. These four miRNAs were chosen for our further analysis.

Study subjects and tissue specimens

Tissue samples for this study were obtained at the Regional

Cancer Centre, Thiruvanthapuram. The Institutional Review Board

and Human Ethics Committee of Regional Cancer Centre, Thiru-

vanthapuram and Rajiv Gandhi Centre for Biotechnology, Thiru-

vanthapuram both approved the study. Informed consent was

obtained from all study subjects. Oral cancer patients, who re-

ceived any preoperative chemotherapy or radiotherapy, were ex-

cluded from the study.

Two groups of tissue specimens were analyzed. The first group

included tissues from various phases of oral cancer progression,

namely 31 normal oral mucosa, 49 leukoplakia and 84 oral cancer

tissues. The second group included tissues from 84 patients under-

going surgical resection as the primary treatment modality for oral

cancer. In this set, three types of samples were obtained from the

same patient, which included the primary tumor, surgical margin

(tissue taken from the actual surgical margin) and an extra margintissue (taken 2 cm away from the actual surgical margin). Normal

oral mucosa was obtained from persons undergoing oral and max-

illofacial reconstructive surgery. None of the leukoplakia patients

had received any treatment. Histologically the leukoplakias were

all dysplastic and had moderate to severe dysplasia. The cancer tis-

sues were all Squamous Cell Carcinomas. The normal tissues were

matched for age and smoking status. The surgical margin tissues

were histologically mild to moderate dysplastic. The extra margin

tissues were histologically normal oral tissues and were compara-

ble with the normal oral mucosa of the first group of tissues. The

tissue specimens for qRT-PCR were collected in RNAlater (Ambion,

USA) and stored at 80 C till use while the specimens for immu-

nohistochemical analysis were collected in 10% buffered formalin.

Total RNA isolation

Histopathological assessment was first done on the tissue spec-

imens to conform the pathological status of the tissues. Total RNA

was isolated from tissues using mirVana miRNA Isolation Kit (Ap-

plied Biosystems, USA) following manufactures protocol. In brief,

tissue was disrupted in a denaturing lysis buffer. The samples were

then subjected to Acid-Phenol:Chloroform extraction which pro-

vided a robust front-end purification that also removes most

DNA. For isolation of total RNA, samples were treated with ethanol,

and passed through a Filter Cartridge containing a glass-fiber filter

which immobilizes the RNA. The filter was then washed a few

times, and finally RNA was eluted with a low ionic-strength solu-

tion supplied with the isolation kit. The RNA concentrations weredetermined with a NanoDrop instrument (Thermo Scientific, USA).

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http://www.rgcb.res.in/orcadb

http://david.abcc.ncifcrf.gov/






http://www.rgcb.res.in/orcadb

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Reverse transcription and quantitative RT-PCR of miRNAs and mRNAs

Quantitative RT-PCR analyses for miRNAs and mRNAs were per-

formed using TaqMan miRNA and mRNA assays. All reagents and

primers were obtained from Applied Biosystems, USA. Reverse

transcriptase reactions and real-time PCR were performed accord-

ing to the manufacturer’s protocols. This technique, unlike the con-

ventional assay of random-primed RT, consists of using a target-

specific stem-loop structure and a reverse-transcriptase primer fol-

lowed by a specific real-time assay. cDNA was synthesized on a

15ll RT reaction mix containing 100 mM dNTP, MultiScribe Re-

verse Transcriptase (50 U/ll), RT buffer, RNase Inhibitor, RNA sam-

ple (10 ng/15 ll) and specific 5X TaqMan MicroRNA RT Primers for

miRNA analysis, while random primers were used for mRNA anal-

ysis. The RT product was diluted 15 times. The qRT-PCR reactions

were carried out in a 20 ll reaction mixture containing TaqMan

2X Universal Master Mix (No AmpErase UNG), 20X TaqMan Micr-

oRNA Assay or 20XTaqMan mRNA Assay and diluted cDNA. RNU

44 was used as endogenous control for miRNAs and GAPDH for

mRNAs. Real-Time PCR were performed in a 7500 Real Time PCR

System (Applied Biosystems, USA). Relative expression was calcu-

lated using the comparative threshold cycle (Ct) method.

Immunohistochemistry

Immunohistochemical reactions were carried out as described

by us earlier.18 Briefly, sections were deparaffinized in xylene

and rehydrated through graded alcohol to water. Endogenous per-

oxidase activity was blocked with 1.5% H2O2 in methanol for

15 min. Sections were then incubated with 3% bovine serum albu-

min (BSA) for 30 min followed by incubation at 4 C overnight with

primary antibodies specific for bcl-2 and cyclin d1 (Santa Cruz Bio-

technology, USA) at dilutions 1:50. The reactions were visualized

using Super Sensitive Polymer-HRP Detection System (BioGenex,

USA) following manufacturer’s protocol. The sections were coun-

terstained with Mayer’s hematoxylin, dehydrated in graded alco-hol, cleared in xylene and mounted.

Statistical analysis

Data analysis was done using Intercooled Stata 8.02 software

package. mRNA and miRNA expression along tissues involved in

oral surgical margins and along oral tumor progression were ana-

lyzed by spearman rank correlation coefficient test. Analysis show-

ing a confidence interval above 95% ( p < 0.05) was considered

significant.

Results

Our main objective for this study was to identify deregulated

microRNA expression associated with both oral cancer progression

and in oral surgical margins and further network analysis to eluci-

date the underlying miRNA-Target gene regulatory network in can-

cer progression and apoptosis. Two sets of tissues were used in this

study; the first set included tissues from various phases of tumor

progression (including normal oral mucosa, premalignant leuko-

plakia and oral cancer tissues) and the second set that included tu-

mor, surgical margin and an extra margin tissue taken from the

same patient for assessing MRD in oral surgical margins. For

qRT-PCR, in case of tissues involved in oral cancer progression, nor-

mal oral mucosa was taken as the calibrator for calculating the fold

change and in the evaluation of oral surgical margins, the extramargin tissues from that set were used as calibrator.

In silico network analysis

Computational functional clustering and target prediction tools

revealed the presence of 17 validated genes which were the targets

of hsa-miR-184, hsa-miR-96, hsa-miR-125a and hsa-miR-16 in-

volved in cancer progression and apoptosis. The regulatory net-

work formed between these four miRNAs and the target genes

was then constructed (Fig. 1). Out of these 17 target genes 3 were

transcription factors.

Expression of miRNAs during oral tumor progression and in oral

surgical margins

In this study we considered those miRNAs that were found to

have a differential expression both in oral tumor progression and

in oral surgical margins. Only four miRNAs (miR-125a, miR-184,

miR-16, and miR-96) fell into this criterion from both the groups

of tissues. In the first set we analyzed 16 normal oral mucosa, 28

leukoplakia and 56 oral cancer tissues. The expression of miR-

125a, miR-184 and miR-16 decreased where as miR-96 increased

with oral tumorigenesis (Fig. 2 and Table. 1). The second group in-

cluded 56 sets of tissues (each set including oral tumor, surgicalmargin and an extra margin tissue). The expression of these

microRNAs were found to be reversed in the second set of tissues.

miR-125a, miR-184 and miR-16 expression increased in oral surgi-

cal margins while miR-96 decreased (Fig. 2 and Table 2)

Expression of mRNAs BCL2 and CCND1 during oral tumor progression

and in oral surgical margins

We further evaluated the target genes of miR-16, BCL2 and

CCND1. These two genes are regulators of apoptotic pathway. Five

each of normal, leukoplakia and tumor samples, in the first group

and five sets, each including tumor, surgical margin and an extra

margin, in the second group were evaluated to study mRNA

expression of BCL2 and CCND1. These two mRNAs had a reverse

pattern of expression compared to miR-16 (Fig. 3 and Tables 3

and 4).

Protein level expression of BCL2 and CCND1 were further eval-

uated by immunohistochemistry in paraffin embedded tissues.

Here also two groups of tissues were studied, first group included

10 normal oral tissues, 16 leukoplakias and 23 oral tumor samples

and the second group was composed of 23 sets of tissues, each in

including tumor, surgical margin and an extra margin. The cyto-

plasmic expression of bcl2 and the cytoplasmic as well as nuclear

immunoreactivity of cyclin d1 increased with histological progres-

sion of oral cancer, from normal oral mucosa to leukoplakias to oral

cancer (Fig. 4). There was a significant decrease in the expression of

bcl2 along with cyclin d1 from tumor to surgical margin to extra

margin (Fig. 4). The results were statistically significant and hada p-value of less than 0.001.

Discussion

By regulating expression of target genes, miRNAs play a crucial

role in the initiation and progression of human cancer. Different

sets of miRNAs are up-regulated or down-regulated in cancers of

different cellular origins. In this study we looked into the expres-

sion profiles of 72 miRNAs that were reported (till June 2011) to

be deregulated in human oral cancer tissues. We studied the

expression of these miRNAs in two sets of tissues: during oral tu-

mor progression and in oral surgical margins. It was observed that

of the 72 miRNAs studied, only miR-125a, miR-184, miR16 andmiR-96 gave a constant pattern of expression in each type of

W.S. Santhi et al. / Oral Oncology xxx (2013) xxx–xxx 3





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tissues involved in oral tumor progression and in oral surgical

margins.

In this study the first set of samples evaluated were pathologi-cally squamous cell carcinomas, dysplasia and normal oral mucosa,

and the second set of samples was clinically tumor, surgical margin

and extra margin tissues. The oral cancer tissues of both sets were

pathologically squamous cell carcinomas, while the surgical mar-

gin tissues had varying degree of dysplasia similar to the leukopla-

kia tissues of the first set. The extra margin tissues of the second

set of samples were histologically normal oral tissues resembling

the normal oral mucosa in the first set. Like the normal oral muco-

sa tissues, the apparently normal looking extra margin tissues had

the highest expression of the miRNAs, miR-125a, miR-184 and

miR-16, while least expression was observed in OSCC tissues. On

the other hand miR-96 showed a reverse pattern of expression,

the highest expression was observed in oral cancer tissues mean

while the normal oral tissues and the extra margin tissues showedmaximum expression.

MiRNAs are differentially expressed in various cancer cells com-

pared with normal cells. In the first set of tissues (during oral tu-

mor progression), it was observed that miR-125a, miR-184 andmiR-16 decreased with the progression of oral tumor. In this sec-

ond set of samples (those involved in oral surgical margins),

miR-125a, miR-184, miR-16 had a statistically significant increase

in its expression form tumor to surgical margin to extra margin tis-

sues. Down regulation of miRNAs is more common than up-regu-

lation in cancers.19,20,4,21 The global down-regulation of miRNAs

in cancer cells has been suggested to reflect the lower differentia-

tion stage of the tumor cells compared with normal cells.4 In both

sets of tissues least expression of miRNAs miR-125a, miR-184,

miR-16 were observed in oral cancer tissues which suggested a tu-

mor suppressor role for these miRNAs.

Park et al.12 have found that two salivary miRNAs, miR-200a

and miR-125a, which were present at lower levels in oral squa-

mous cell carcinoma patient saliva than in healthy controls. Iden-tical to our results of miR-125a in oral squamous cell carcinoma,

Figure1 Gene regulatory network formedbetween thedifferentially expressedmicroRNAs andtheir targetgenes involved in tumorigenesis. Thetargets of miRNAswhich are

involved in the process of tumorigenesis were identified and a network was constructed. This network consists of three potential transcription factors that are involved in

cancer progression. The network was constructed using Cytoscape 2.8.2.






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down regulation of miR-125a was also observed in prostate can-

cer.22 Iorio et al.23 have shown that along with miR-125b (a homo-

logue of 125a), miR-10b and miR-145, was down-regulated in

breast cancer and this group has also suggested that this down-

regulated miRNAs may potentially act as tumor suppressor gene.Through transient transfection studies, miR-125a, have shown to

reduce ERBB2 and ERBB3 oncogenic protein levels in a human

breast cancer cell line.24

Similar to the expression of miR-125a in oral cancer, miR-184

also had a down-regulated expression during oral cancer progres-

sion (form tumor to leukoplakia to cancer tissues) and in oral sur-gical margins (from tumor to surgical margin to extra margin

Figure 2 MicroRNA expressions were detected by qRT–PCR. Four microRNAs hsa-miR-16, hsa-miR-125a, has-miR-184 and hsa-miR-96 showed a common pattern of

expression during oral tumorigenesis and in oral surgical margins. (A) Shows the expression of hsa-miR-16, hsa-miR-125a, has-miR-184 and hsa-miR-96 during oral

tumorigenesis (form normal oral mucosa to leukoplakia to oral tumor) and (B) shows the expression of the same microRNAs in oral surgical margins (form oral tumor to

surgical margin to extra margin).

Table 1

Mean qRT expression of hsa-miR-16, hsa-miR-125a, has-miR-184 and hsa-miR-96

during oral tumor progression. The p-values were all less than 0.0001. The range

values are given in brackets.

microRNAs Tissues involved in oral tumorigenesis

Normal Leukoplakia Tumor

miR-125a 1 0.39 (0.24–0.42) 0.23 (0.15–0.40)

miR-184 1 0.14 (0.06–0.22) 0.1 (0.07–0.19)

miR-16 1 0.6 (0.47–1.10) 0.38 (0.27–0.45)

miR-96 1 5.42 (3.89–6.67) 78.64 (56.04–81.66)

Table 2

Mean qRT expression of hsa-miR-16, hsa-miR-125a, has-miR-184 and hsa-miR-96 in

oral surgical margins. The p-values were all less than 0.0001. The range values are

given in brackets.

microRNAs Surgical margin tissues

Tumor Surgical margin Extra margin

miR-125a 0.23 (0.15–0.40) 0.39 (0.18–0.51) 1

miR-184 0.21 (0.11–0.34) 0.28 (0.12–0.41) 1

miR-16 0.38 (0.27–0.45) 0.58 (0.51–0.65) 1

miR-96 78.64 (56.04–81.66) 6.19 (5.11–6.89) 1






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tissues). Normal oral mucosa of the first set of samples and extramargin tissues of the second set of samples had higher expression

for mir-184, while the cancer tissues showed the lowest expression

for miR-184. Our result of miR-184 in oral cancer is in sharp con-

trast to the results of the same miRNA in Squamous Cell Carcinoma

of Tongue.14 Wong et al.14 have shown that miR-184 levels were

significantly higher in tongue cancer patients in comparison with

normal individuals, and the levels were significantly reduced after

surgical removal of the primary tumors.

miR-15a and miR-16-1 are transcribed as a cluster (miR-15a–

miR-16-1) that resides in the 13q14 chromosomal region. Dele-

tions or point mutations in region 13q14 occur at high frequency

in chronic lymphocytic leukemia, lymphoma and several solid tu-

mors.25–27 miR-16 acts as tumor suppressor gene in various human

tumors.27–30

These results are in agreement with our findings of miR-16 in oral cancer. The squamous cell carcinoma samples had

the least expression of this miRNA, while the normal tissues had

the highest expression. Although there is no direct evidence, our

results also suggest a possible tumor suppressor role of mir-16 in

oral cancer.

miR-96 and miR-183 have been found to be up regulated in var-

ious human cancers such as breast, lung, colon, liver, ovary, pros-tate, testis cancer and lymphoma.30–35 The same miRNA, miR-96

was found to have a similar expression in oral cancer in our studies

also. Oral cancer tissues had the highest expression of this miRNA.

Further Yamada et al.36 have found significant increases in miR-96

and miR-183 expression with advancing tumor grade in urothelial

carcinoma and they have also suggested miR-96 to be a good diag-

nostic marker for urothelial carcinoma in combination with uri-

nary cytology. In contrast to these results tumor suppressor role

of miR-96 has also been reported; this down regulation of miR-

96 was observed in pancreatic cancer by Yu et al.37

The analysis of downstream targets of miRNAs revealed the

presence of genes that are altered during tumor progression. The

regulatory network constructed using 182 targets genes of the four

miRNAs revealed their involvement in diverse processes as metab-olism, ubiquitylation, neuronal patterning, embryogenesis, cyto-

skeleton remodeling, oncogenesis and signaling. These miRNAs

share common targets as well as pathways. Further clustering of

target genes revealed presence of target genes of these miRNAs

which participate in tumor progression and apoptosis. The map

of gene interaction network describes the potential pathways that

may be used by a cell to accomplish a tumorigenic nature.

Further we evaluated the expression of two of the important

targets of miR-16, BCL2 and CCND1. BCL2 protein family plays an

important role in regulating the cellular program of apoptosis.

BCl2 has been known to be over expressed in a wide range of can-

cers in humans including leukemias, lymphomas, and carcinomas.

BCl2 expression is inversely correlated with miR-16 in CLL cells29

both inducing apoptosis. miR-16 targets BCL2 in gastric cancercells at least in part by modulation of apoptosis.38 Decreased

Figure 3 The mRNA level expression of BCL2 and CCND1, downstream target genes of miR-16 were evaluated by qRT-PCR during oral tumorigenesis and in oral surgical

margins. (A)Shows theexpression of BCL2 andCCND1 during oral tumorigenesis, formnormal oral mucosa to leukoplakia to oral tumor and(B) shows theexpression of BCL2

and CCND1 in oral surgical margins, form oral tumor to surgical margin to extra margin.

Table 3

Mean qRT expression of BCL2 and CCND1 during oral tumor progression. The p-values

were less than 0.0001. The range values are given in brackets.

mRNAs Tissues involved in oral tumorigenesis

Normal Leukoplakia Tumor

BCL2 1 4.536 (4.01–4.78) 8.178 (7.87–9.67)CCND1 1 3.49 (2.81–4.92) 5.01 (4.24–5.33)

Table 4

Mean qRT expression of BCL2 and CCND1 during oral tumor progression. The p-values

were less than 0.0001. The range values are given in brackets.

mRNAs Surgical margin tissues

Tumor Surgical margin Extra margin

BCL2 8.178 (7.87–9.67) 4.726 (4.07–5.11) 1

CCND1 5.01 (4.24–5.33) 2.98 (1.45–3.88) 1






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expression of miR-15a and miR-16-1 is associated with increased

Bcl2 expression and resistance to apoptosis in chronic lymphocytic

leukemia.39 Cyclin D1 (CCND1) is another well-known regulator of

cell-cycle progression and is a member of bcl2 family. It is over ex-

pressed in several types of cancer including breast, lung, squa-

mous, neuroblastoma, and lymphomas.40 Chen et al. have shown

that miR-16-1 in the regulation of CCND1 in Mantle cell lym-

phoma. In agreement with our present study, Bonci et al.27 have

also reported that the miR-15a and miR-16-1 cluster targets

BCL2 as well as CCND1 in prostate cancer and that, they promote

several tumorigenic features, including survival, proliferation,and invasion.

Alterations in miRNA function and regulation have rapidly

emerged as important players in cancer pathogenesis. Individual

miRNAs and miRNA signatures have already been shown to have

prognostic value in many cancers. OncomiRs are involved in cancer

as oncogenes and/or tumor suppressors. Our study provides an in-

sight into the fact that same set of molecular events may be in-

volved in the progression of normal oral mucosa to premalignant

lesion to OSCC and also in oral surgical margin and in extra mar-

gins. We have earlier reported a similar pattern of expression for

two molecules, NF-jB and COX-2 during oral tumorigenesis and

in the assessment of MRD in oral surgical margins.18 Here we re-

port for the first time that of the 72 miRNAs reported (till June

2011) to be deregulated in oral cancer four miRNAs show the samepattern of expression during oral tumor progression and in oral

surgical margins (Fig. 5). The significance of this result lies in the

fact that pathologically the tissues involved in both the set of sam-

ples (that is oral tumorigenesis and in oral surgical margins) were

similar. The in silico network analysis of these four miRNAs and

their target genes revealed the presence of genes involved in tumor

progression and transcription factors. These results suggest that

the combinatorial regulation by these miRNAs and their target

transcription factors plays a substantial role both during oral

tumorigenesis and in oral surgical margins.

A better understanding of the molecular pathways that give rise

to oral cancer is essential in the identification of novel molecularbiomarkers that will have clinical utility in predicting prognosis

and therapeutic efficacy, as well as in designing targeted therapy

for this disease. The study of oral cancer is particularly challenging,

since oral cancer is an important cause of morbidity and mortality,

especially in developing countries. Our clinical as well as in silico

results together suggest that analysis of these molecules (down

regulation of miR-125a, miR-184 and miR-16 and an upregulation

of miR-96) could be useful as biomarkers for predicting prospec-

tive oral cancer lesions and furthermore be used in identifying

residual tumor cells in oral surgical margins. More functional stud-

ies are needed in elucidating the mechanism of action of the puta-

tive oncogenic and tumor suppressor functions of these miRNAs.

To conclude, this is a preliminary study comparing the expres-

sion of all 72 miRNAs that were reported (till June 2011) to bederegulated in oral cancer; in two important aspects of oral cancer,

Figure 4 Immunohistochemical expression of bcl2 and cyclin D1 (A) during oral cancer progression (B) in oral surgical margins (40 magnification).






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during oral tumorigenesis and in the assessment of oral surgical

margins. The two sets of data on differential miRNA expression (tu-

mor progression data and oral surgical margin data) together show

that decreased expression of miR-125a, miR-184 and miR-16 and

an increased expression of miR-96 is associated with oral tumori-

genesis and of value in the detection of MRD in surgical margins.

This would have impact on decision-making process for both pre-

treatment and postoperative treatment modalities. However, more

functional studies are needed in elucidating the mechanism of ac-

tion of the putative oncogenic and tumor suppressor functions of

these miRNAs.

Conflicts of interest statement

No potential conflicts of interest were disclosed.

Acknowledgement

We acknowledge Council for Scientific and Industrial Research,

Government of India, for Senior Research Fellowship (CSIR F. NO.

09/716/(0100)/2008/EMR-I DATED 19.03.2008) to Santhi W.S.

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