Indian J Med Res 151, April 2020, pp 311-318 Quick Response Code:

DOI: 10.4103/ijmr.IJMR_501_18
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Epigenetic downregulation of desmin in gall bladder cancer reveals its

potential role in disease progression

Shushruta Bhunia1, Mustafa Ahmed Barbhuiya3, Sanjiv Gupta2, Braj Raj Shrivastava2 & Pramod Kumar Tiwari1
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Department of Molecular & Human Genetics, Centre for Genomics, Jiwaji University, 2Department of
1

Pathology, Cancer Hospital & Research Institute, Gwalior, Madhya Pradesh, India & 3Department of
Pathology & Laboratory Medicine, Pennsylvania State University College of Medicine, Milton S. Hershey
Medical Center, Hershey, PA, USA

Received March 12, 2018

Background & objectives: Gall bladder cancer (GBC) is a fatal neoplasm, with a globally variable
incidence rates. To improve the survival rate of patients, a newer set of biomarkers needs to be discovered
for its early detection and better prognosis. Our earlier studies on GBC proteomics and whole-genome
methylome data revealed expression of desmin to be significantly downregulated with correlated
promoter hypermethylation during gall bladder carcinogenesis. Thus, to evaluate desmin as a potential
biomarker for GBC, we carried out a detailed follow up study.
Methods: Methylation-specific polymerase chain reaction (MS-PCR) (n=17, GBC and n=23,
non-tumour control), real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
[n=14, GBC and n=14, adjacent non-tumour (ANT)], immunohistochemistry (n=27, GBC and n=14,
non-tumour) and immunoblotting (n=13, GBC and n=13, ANT) were performed in surgically removed
gall bladder tissue samples.
Results: MS-PCR analysis showed methylation of desmin in 88.23 per cent (15/17) gall bladder tumour
samples as compared to non-tumour tissues (39.13%, 9/23). Real-time qRT-PCR analysis revealed a
significant downregulation of desmin expression in GBC as compared to ANT tissue. This was further
confirmed by western blot, showing reduced expression of desmin protein in GBC, as compared to
non-tumour tissue. Immunohistochemical analysis also showed a decreased level of desmin i.e., more
than 95 per cent (26/27) in tumour cells compared to non-tumours (35.71%, 5/14).
Interpretation & conclusions: The increased frequency of desmin promoter methylation which could be
responsible for its significant downregulation, indicates its potential as a candidate biomarker for GBC.
This requires further validation in a large group of patients to evaluate its clinical utility.

Key words Desmin - disease progression - DNA methylation - epigenetics - gall bladder cancer - molecular diagnostic

Gall bladder cancer (GBC) is the most common radical cholecystectomy is the only treatment
neoplasm of the biliary tract. Early detection of strategy for resectable cancer that includes stage I,
GBC is difficult due to the non-availability of II and III cancers not spread outside the gall bladder.
well-defined set of specific biomarkers. At present, However, due to the lack of early detection tools,

© 2020 Indian Journal of Medical Research, published by Wolters Kluwer - Medknow for Director-General, Indian Council of Medical Research
311
 312 INDIAN J MED RES, APRIL 2020

majority of gall bladder cancers are diagnosed at an types and our previous global methylation analysis
advanced stage and unresectable cancers1. Better and proteomic study in gall bladder cancer, we
understanding of the molecular pathogenesis of GBC hypothesized that this gene was downregulated during
is important to identify biomarkers of diagnostic, disease progression in gall bladder cancer through
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therapeutic, and prognostic significance. Our earlier epigenetic mechanisms. To validate the hypothesis, we
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epidemiological studies revealed that gall bladder investigated the pattern of expression of desmin and
cancer-related morbidity was high in north central its epigenetic regulation in gall bladder cancer patients.
region of India2,3. Our objective was to provide a proof of concept on
the epigenetic silencing of desmin through promoter
In our isobaric tags for relative and absolute
methylation in gall bladder cancer.
quantitation (iTRAQ) based proteomic study of
the gall bladder tumours and adjacent non-tumour Material & Methods
(ANT) tissue4, we observed a 3.3-fold downregulation
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The present study was conducted in the Centre

of desmin (DES) gene. Our genome-wide for Genomics, Jiwaji University, Gwalior, India, after
methylation analysis using Illumina’s Infinium obtaining approval from the Institutional Human Ethics
HumanMethylation450 BeadChip array5 revealed Committee. Written informed consent was taken from
desmin to be hypermethylated in the promoter region all the patients or their family members included in the
in gall bladder tumours compared to non-tumours. present study.
Methylation-based dysregulation of genes is a
hallmark of tumorigenesis and cancer progression6. Sample collection: Gall bladder cancer tissue and
Dysregulation of cytoskeletal proteins is one of the adjacent non-tumour tissue samples from patients
major events in tumorigenesis and tumour progression. undergoing radical cholecystectomy were collected
Earlier studies proposed that desmin could be helpful at the department of Pathology, Cancer Hospital and
in staging urothelial carcinomas and detecting invasion Research Institute (CHRI), Gwalior, India, during 2007
in muscularis propria7. The process of invasion and to 2016. The patients were selected randomly based
metastasis starts with the remodelling of cellular on their ultrasound findings about gall bladder lesions.
architecture, which also includes the interaction of Based on macroscopic findings post cholecystectomy,
cytoskeletal proteins. Intermediate filaments form a paired gall bladder tissue samples were excised during
flexible but resilient framework that gives structural gross examination from tumour and ANT sites, and
support to epithelium and functions in tissue repair and this was confirmed later with microscopic findings by
regeneration8. Desmin is a 52 kDa protein, composed the surgical pathologist (Table I). The excised tissue
of 470 amino acids and plays an essential structural samples for molecular studies were frozen immediately
role in the maintenance of muscle integrity. It is a main at −80○C. Tissue microarray (TMA) was constructed
intermediate filament protein of type III in skeletal using archival paraffin blocks of gall bladder cancer
and cardiac muscle cells9,10. It resides at chromosome tissue and non-tumour tissue. For methylation-specific
2q35 and has a functional role in muscle contraction11. polymerase chain reaction (MS-PCR), 17 GBC,
Desmin is reported to interact with several other corresponding 17 ANT tissues and 6 cholecystitis tissues
cytoskeletal proteins (e.g., VIM, SYNC, SYNM, DSP, as non-tumour control, for real-time quantitative reverse
TCAP, TMOD1 and DMD), cancer-related proteins transcription-polymerase chain reaction (qRT-PCR), 14
(e.g., NEB, S100B, S100A1 and SHBG) and many GBC and 14 ANT tissues, for immunoblotting 13 GBC
other functional proteins (e.g., CAPN1, AURKB, and 13 ANT tissue samples and for IHC, 27 GBC and
TRIM7, PKD1, ROCK1, SPTAN1 and AURKB)12,13. 14 non-tumour tissue samples were taken.
Immunostaining [immunohistochemistry (IHC)] for
Genomic DNA isolation and bisulphite modification:
desmin showed a weak staining in more than 50 per
Genomic DNA (gDNA) was isolated from gall
cent of cells in leiomyosarcoma and positive staining in
bladder tissue samples using QIAamp DNA mini kit
tubular epithelial cells of normal kidney14. In contrast,
(Qiagen, Germany) and quantitated by NanoDrop
the gastrointestinal and extra-gastrointestinal stromal
(Implen, Germany). The DNA quality was assessed on
tumours cells showed a negative staining pattern for
0.8 per cent agarose gel. One microgram of DNA was
desmin15,16.
taken for bisulphite modification using Zymo EZ DNA
With collective evidence from the previous studies Methylation kit (Zymo Research, USA) following the
demonstrating the role of desmin in different cancer manufacturer’s protocol.
 BHUNIA et al: EPIGENETIC DOWNREGULATION OF DESMIN IN GALL BLADDER CANCER 313

Table I. Demographic details of the patients included in the study

Characteristics MS‑PCR Real‑time PCR IHC Western blotting
n (%) (n=23) n (%) (n=14) n (%) (n=27) n (%) (n=13)
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Gender
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Male 5 (21.74) 4 (28.57) 6 (34.78) 6 (46.16)

Female 18 (78.26) 10 (71.43) 21 (65.22) 7 (53.84)
Age (yr)
≤45 12 (52.17) 2 (14.29) 9 (33.33) 4 (30.77)
>45 11 (47.83) 12 (85.71) 18 (66.67) 9 (69.23)
Mean±SD (range) 47.9±12.36 (22‑80) 52.71±9.13 (30‑65) 47.47±12.83 (26‑72) 51.92±10.69 (32‑65)
Grades of cancer
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Grade I 1 (5.88)* 5 (35.71) 3 (11.11) 3 (23.08)

Grade II 8 (47.06)* 2 (14.29) 9 (33.33) 4 (30.77)
Grade III 8 (47.06) *
7 (50) 15 (55.56) 6 (46.15)
Location of ANT/non‑tumour
Neck 19 (82.60) 6 (42.86) 13 (92.86)# 6 (46.16)
Body 2 (8.70) 3 (21.43) 0 (0)# 2 (15.38)
Fundus 2 (8.70) 5 (35.71) 1 (7.14) #
5 (38.46)
Tumour (with/without stone) *

With stone 4 (23.53)* 9 (64.29) 6 (22.22) 4 (30.77)

Without stone 13 (76.47) *
5 (35.71) 21 (77.78) 9 (69.23)
*
Information on pathological tumour grades, location of the tumour in gall bladder and the presence and/or absence of stones were available
for 17 patients; #location available for 14 patients only. ANT, adjacent non‑tumour; SD, standard deviation; IHC, immunohistochemistry;
MS‑PCR, methylation‑specific polymerase chain reaction

Detection of promoter methylation by mini kit (Qiagen, Germany). Qiagen’s cDNA

methylation-specific (MS)-PCR: The following synthesis kit was used to prepare cDNA following the
primer sequences were used for DES promoter manufacturer’s protocol.
for MS-PCR: for methylated sequence, forward
primer: 5ˈ-TTTTTACGGTTACGGGTC-3ˈ; reverse Real-time PCR: Real-time qRT-PCR11 (n=14, GBC and
primer: 5ˈ-CGTCGACACTATTTATATCCC-3ˈ and n=14, ANT) was performed using 0.75 µg of total RNA
for unmethylated sequence, forward primer: 5ˈ-GT in technical duplicates for DES (Qiagen, Germany)
TTTTATGGTTATGGGTT-3ˈ; reverse primer: 5ˈ-CAT and normalized to RRN18S (Qiagen, Germany) using
CAACACTATTTATATCCCT-3ˈ. The primers were QuantiTect SYBR Green RT-PCR kit (Qiagen,
designed using Methyl Primer Express® Software v1.0, Germany) on the Applied Biosystems 7500, USA,
(Thermo Fischer Scientific, USA). The PCR thermal platform. The data were graphically presented as a per
conditions were as follows: initial denaturation at 95°C cent fold change in tumour tissue normalized to that of
for 5 min, 35 cycles of denaturation 35 sec, annealing at ANT tissue.
53°C for 30 sec, extension at 72°C for 45 sec and final
extension for 10 min. The PCR products were resolved Western blotting: Tissue samples corresponding to those
on 8% native polyacrylamide gel electrophoresis used for real-time qRT-PCR were used for western
(PAGE). The MS-PCR was performed as described by blotting (n=14 GBC and n=14 ANT). Protein lysates were
Herman et al17 with some modifications. prepared from tissues in lysis buffer containing protease
inhibitor cocktails (Roche Diagnostics, Germany) using
RNA isolation and cDNA synthesis: Total RNA a manual homogenizer and quantified using Bradford
from gall bladder tissue samples (14 - tumour method18. Equal quantity of protein (30 µg) was loaded
and 14 - non-tumour) was extracted using TRIzol on a 110 per cent sodium dodecyl sulphate (SDS)-PAGE
solution (Qiagen, Hilden, Germany) and the RNeasy gel and transferred (blotted) onto a polyvinylidene
 314 INDIAN J MED RES, APRIL 2020

fluoride (PVDF) membrane (GE Healthcare, USA). in blocking solution for one hour, and sections
The membrane was blocked for 1 h at room temperature were incubated overnight at 4°C in a humidified
with blocking buffer [(5% non-fat skimmed milk in chamber with a mouse monoclonal anti-desmin
1x Tris-buffered saline with 0.1% tween-20 (TBST)] antibody (Santa Cruz Biotechnology Inc., USA),
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and incubated overnight at 4°C with anti-desmin diluted to 1:500. The Vectastain Elite ABC kit
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monoclonal antibody at a dilution of 1:1000 (Vector Laboratories, USA) was used according to
(Santa Cruz Biotechnology Inc., USA) and monoclonal the manufacturers’ instructions to visualize the signal.
anti-β-actin (Santa Cruz Biotechnology Inc., USA) After incubation with primary antibody, sections were
at a dilution of 1:1000 in 1x TBST. Three washes of incubated with secondary antibody (Goat anti-mouse
1x TBST were given, followed by incubation with an immunoglobulin G) conjugated with biotin-labelled
anti-mouse, horseradish peroxidase (HRP)-conjugated polymer (Santa Cruz Biotechnology Inc., USA) for
secondary antibody dilution (1:10,000) at room 60 min at room temperature, followed by incubation
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temperature for two hours. The blot was then washed with avidin-biotin conjugates. The slides were washed
three times with 1x TBST and twice with 1x TBS. The in 1x phosphate-buffered saline, pH 7.4 (3 times for
blot was developed using a supersensitive enhanced 5 min each) between each incubation step. Antibody
chemiluminescence reagent (GE Healthcare, USA) detection was performed with 3,3’-diaminobenzidine
according to the manufacturer’s protocol on the BioRad (DAB) tetrahydrochloride peroxidase substrate, and
Gel Doc System. Densitometry analysis was performed tissues were counterstained with haematoxylin for one
using the AlphaImager 2200 software (USA). minute. The samples were dehydrated in 100 per cent
ethanol and xylene and mounted with glass coverslip
Tissue microarray and immunohistochemistry: TMA using DPX mountant for histology. The scoring
was constructed using paraffin sections (5 µm) of gall (staining) was considered as follows: negative or no
bladder tissue samples (GBC=27 and non-tumour staining; 1+, weak positive, when less than 10 per cent
tissues, n=14). The sections were deparaffinized in of the cells were positive; 2+, positive, with 11-50 per
xylene and rehydrated in graded concentrations of cent positive cells, and 3+, strongly positive, with more
alcohol (90, 70 and 30%). The slides were treated in a than 50 per cent positive cells. The cellular localization
microwave at 80 watts in 10 mM citric acid (pH 6.0) of the immunostaining was also recorded.
for antigen retrieval, and endogenous peroxidase was
quenched with 0.03 per cent H2O2 for 20 min. Figure 1 shows the investigations done in the present
Non-specific binding was blocked by pre-incubation study.

Fig. 1. Workflow demonstrating systematic investigations leading to identification of desmin (DES) as an epigenetically regulated gene in
gall bladder cancer. MS-PCR, methylation-specific polymerase chain reaction; qRT-PCR, quantitative reverse transcription-polymerase chain
reaction; IHC, immunohistochemistry; iTRAQ, isobaric tags for relative and absolute quantitation; LC-MS/MS, liquid chromatography-tandem
mass spectrometry; DMRs, differentially methylated regions.
 BHUNIA et al: EPIGENETIC DOWNREGULATION OF DESMIN IN GALL BLADDER CANCER 315

Statistical analysis: Student’s t test and Fisher’s exact Protein expression [Western blot and
test or Chi-square test were performed to test the immunohistochemistry (IHC)]: The western blot
significance of this study at 95 per cent confidence analysis revealed significant loss of expression of DES
interval and 5 per cent level of significance using
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(P=0.0081) in GBC, compared to the corresponding

GraphPad Prism (www.graphpad.com). Pearson’s Chi- ANT tissues (Fig. 3C and D). The result of western
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square test was performed for categorical values. To blot was reconfirmed by IHC of tumour and
analyze the difference between two data sets, two-tailed non-tumour tissues which showed adenocarcinoma
Wilcoxon rank-sum test or Student’s t test was performed cells to have significantly decreased level of desmin
for real-time qRT-PCR and immunoblotting. Cohen’s expression (26/27, 96.30%), compared to non-tumours
unweighted kappa and linearly weighted kappa tests (5/14, 35.71%) (P<0.001). It also revealed that the
were carried out to measure concordance of MS-PCR-
subcellular localization of desmin is cytoplasmic.
based methylation and IHC-based expression of desmin.
Intense positive staining was observed in muscle
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Results cells of the gall bladder tissue (data not shown). The
expression and staining patterns are shown in Figure
Methylation-specific PCR: Our previous whole-genome 3A, B and Table II.
methylation analysis showed hypermethylation in
5’ region (5’UTR) of desmin gene. The MS-PCR Concordance of promoter methylation and protein
reconfirmed the methylation status of DES promoter, expression: Cohen’s kappa statistics weighs the
about 88 per cent (15/17) of GBC patients showed frequencies of two methods and analyzes the
promoter hypermethylation (P=0.004) as compared to proportions of agreement. The observed kappa of more
non-tumours, where only 39 per cent (9/23) showed than 80 per cent is considered as a strong agreement
promoter methylation (Fig. 2A). The studied cases
between the two methods19. The methylation of desmin
comprised all grades of GBC (grade I, II and III)
by MS-PCR and downregulation of protein expression
(Table I).
using IHC in tumours showed a 88.74 per cent
Real-time polymerase chain reaction (PCR): The (kappa value=0.7244) agreement [95% confidence
real-time qRT-PCR results revealed that at transcript interval (CI): 0.523-0.925] observed as a proportion
level, the expression of DES gene was significantly of maximum possible agreement of methylation and
downregulated in gall bladder cancer tissues downregulation of desmin in gall bladder cancer. The
(P=0.001) as compared to its corresponding ANT reverse was observed in non-tumour samples with
tissues. All grades of GBC (grade I, II and III) in 12 no agreement of methylation and protein expression
of 12 GBC tissues showed downregulation of desmin with 10 per cent (kappa value=0.0727) agreement
(Fig. 2B and Table I). (95% CI: 0-0.361).

A B

Fig. 2. (A) Histogram showing statistical difference in methylation in tumours and non-tumours. (B) Relative fold change of desmin RNA
expression by real-time polymerase chain reaction in gall bladder tumours and non-tumours. Fold change in tumour tissue normalized to
non-tumour after subtracting the control 18S rRNA Ct values.
 316 INDIAN J MED RES, APRIL 2020

A B
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C D

Fig. 3. (A) Representative images of immunohistochemistry staining of desmin in gall bladder tumours (no diaminobenzidine, DAB stain) and
adjacent non-tumour showing positive DAB stain, counterstain used was haematoxylin. (B) Histogram showing percentage of immunohistochemical
staining patterns of desmin in gall bladder tumours and adjacent non-tumours. (C) Western blotting showing expression of desmin in five
representative samples of gall bladder tumour (T1-T5) and their adjacent non-tumour (N1-N5). (D) Histogram showing the densitometric
quantitation of desmin expression in gall bladder tumours and non-tumours from the western blotting (n=5 in both groups).

Table II. Immunohistochemistry staining pattern (score) gall bladder tumorigenesis. The progression of cancer
and subcellular localization of desmin protein in tumour and involves a complex cascade of molecular processes,
non‑tumour cells and methylation-based gene silencing is a known
Staining pattern Non‑tumour Tumour mechanism among those. The cytoskeletal proteins are
tissues tissues the major contributors towards the disorder strength,
Total positive (1+, 2+ and 3+) 9/14 1/27 mainly due to their presence everywhere in the cell20.
Total negative (0) 5/14 26/27 These structural proteins are the essential cellular
Subcellular localization Cytoplasmic components carrying out normal cellular functions. The
P <0.001* disintegrity of these can lead to abnormal functioning
*
Comparison between positive vs. negative in respective of the cell. Desmin is one of the main components of
groups the cytoskeletal system. It has been demonstrated that
the phosphorylation of desmin in fasting and muscle
atrophy interferes with its normal assembly process21.
Discussion
It has also been reported that the cytoplasmic presence
Our study revealed a significant downregulation of desmin protein in gall bladder tumour tissues may be
of desmin in tumour tissue (GBC) as compared to due to the fact that mutant desmin causes collapsing of
ANT tissue associated with promoter methylation, pre-existing desmin and aggregation in the cytoplasm9.
suggesting epigenetic regulation of the gene during Desmin has a major role in mitochondrial positioning
 BHUNIA et al: EPIGENETIC DOWNREGULATION OF DESMIN IN GALL BLADDER CANCER 317

and respiratory function22. Moreover, desmin has been types of experiments qRT-PCT, western blotting and
identified as one of the hub genes for colon cancer MS-PCR.
by a network-based study23. Loss of expression of
The present observations further reaffirmed our
desmin has been reported in gastrointestinal stromal
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earlier proteomic and methylome studies, where

tumours24. The expression of desmin, a smooth muscle
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desmin was found 3.3-fold downregulated at protein

cell marker, is found to be reduced in dysplastic
level and found to be methylated in gall bladder cancer.
cells25. The intermediate filament may be considered
The available data from multiple studies32 provide
as a useful diagnostic marker to identify neoplasm convincing evidence to consider desmin as a potential
immunohistochemically, as these show co-expression biomarker for GBC that needs to be probed further
of desmin, vimentin and keratin14. Intermediate with large sample size. Methylated desmin DNA may
filament has a suggestive role in signal transduction, also be investigated as part of the circulatory tumour
which involves mechanosignaling in cells26. Our study DNA in blood serum of a large group of calculus
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finds further support from the report of Raparia et al27, cholecystitis and various stages of gall bladder cancer to
who demonstrated a positive expression of desmin in investigate the potential of methylated desmin DNA as
gall bladder tissue samples, collected from chronic a liquid biopsy tumour marker for gall bladder cancer.
cholecystitis cases. Lower expression of desmin gene To further understand the functional implications and
in myoblasts and myotube is also suggested to occur mechanisms involved in the dysregulation of desmin
due to DNA methylation28. Like desmin, promoter in gall bladder cancer, in vitro and in vivo experiments
methylation in some other genes, such as CDKN2A, using cell lines and animal models should be employed.
ESR1, PGP9.5 and SSBP2, has also been observed
in GBC29. DAPK1, DLC1, TIMP3 and RARβ2 were Financial support & sponsorship: The last author (PKT)
observed to be hypermethylated in tissues from acknowledges the Madhya Pradesh Council of Science and
Technology (MPCST) Bhopal, India, for providing funds to carry
cholecystitis to advance GBC stages30. It is presumed
out this work. The first author (SB) was a recipient of fellowships
that desmin might be acting as a tumour suppressor provided by MPCST and Jiwaji University, Gwalior, India. Authors
gene, since hypermethylation in the promoter of also acknowledge the University Grants Commission-Special
any relevant gene may likely evolve novel tumour Assistance Programme (UGC-SAP) and the Department of Science
suppressor genes31. and Technology (DST), Government of India, for providing partial
financial support in conducting this work.
Our earlier study on quantitative proteomics of
GBC tissue identified desmin as a downregulated Conflicts of Interest: None.
(3.3-fold) protein in gall bladder tumours compared to
ANTs4. Poor expression of desmin is also observed in References
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For correspondence: Dr Pramod Kumar Tiwari, Department of Molecular & Human Genetics, Centre for Genomics, Jiwaji University,
Gwalior 474 011, Madhya Pradesh, India
e-mail: [email protected]