Int J Clin Exp Pathol 2016;9(2):1608-1616
www.ijcep.com /ISSN:1936-2625/IJCEP0018084
Original Article
Analysis of mRNA profile in different strategy of bladder
cancer carcinoma by deep sequencing
Xiaoyuan Xu1, Xinping Wang1, Bin Fu2, Lirong Meng3, Bin Lang3
Key Laboratory of System Bio-medicine of Jiangxi Province, Jiujiang University, Jiujiang 332000, China; 2Department of Urology, The First Affiliated Hospital of Nanchang University, Nanchang 330006, China; 3School of Health
Sciences, Macao Polytechnic Institute, Macao, China
1
Received October 18, 2015; Accepted November 28, 2015; Epub February 1, 2016; Published February 15, 2016
Abstract: Harboring gene mutations is an essential step that causes the onset and progression of tumors, and
diverse phenotypes of tumors. Analyzing the bladder cells with different malignance is a significant method seeking possible genes that lead to tumor development, malignant transformation and migrating differentiation. This
study aimed to find the key genes controlling the progression of bladder cancer. We performed a large-scale RNA
sequencing on SV-HUV-1, RT4 (low grade bladder cancer), T24 (malignant bladder cancer), and 5637 (malignant
bladder cancer) cells. We screened the differentially expressed genes (DEGs) in comparison pairwise between SVHUV-1, RT4 and T24; as well as compared DEGs between SV-HUV-1, RT4 and 5637. Further, we performed trend
analysis approach on DEGs with k-mean cluster. Gene ontology and pathway analysis were performed on DEGs to
observe the biological functions. The molecular signature of SV-HUV-1-RT4-5637 was found to be distinct to that
of SV-HUV-1-RT4-T24. In SV-HUV-1-RT4-5637, the majority downregulated DEGs were enriched in Notch pathway
were greatly decreased. In the SV-HUV-1-RT4-T24, profile3 showed insignificant expression in SV-HUV-1 and RT4,
but remarkably decreased in T24. Profile3 gene mainly enriched in the biological processes of fat metabolism, the
gradual increased DEGs in SV-HUV-1-RT4-T24 enriched in the mTOR and HIF-a pathway. In summary, this study
revealed that Notch pathway played pivotal roles in the formation process of bladder cancer and may be a potential
target for the treatment of bladder cancer; reveal molecular mechanisms of different biological characteristics of
various bladder cancers.
Keywords: Bladder cancer, RNA-seq, notch pathway, differentially expressed genes, fatty acid metabolic process
Introduction
Bladder cancer is one of the most common
malignancies, which can threaten to human
health and has raised the fourth position
among malignancies in western countries [1,
2]. When the tumor developed clinically detectable, tumor genome would acquire numerous
mutations [3], the genome mutation would lead
to shift of the whole cell gene expression structure, and these abnormal expressions of genes
often promoted the incidence and development, malignant transformation, invasion and
differentiation of the tumor, as well as determined the different biological traits such as different malignancy, sensitivity to drugs, and
clinical prognosis. Analysis of the changes of
gene expression profiles in different stages of
bladder cancer cells progress plays an important role in tumor biology. The existing researches mainly focus on the culture of tumor lines, to
study the function of individual genes [4]. This
study would analyze expression profiles of bladder cancer cells with diverse malignance
through RNA sequencing, attempted to changes of gene expression as a whole in bladder
cancer progression and find the key pathway of
tumor formation.
Notch signaling pathway, is a novel tumor suppressor which has been firstly found in mouse
skin cancer [4] and human keratinocytes [5].
Previous evidences showed that Notch pathway
was involved in mediating signaling in myeloid
[6], and was associated with the development
of bladder cancer, lung epithelial adenocarci-
mRNA profile in urinary bladder cancer
noma, and head and neck cancer [7-9]. Due to
the diversity of Notch signaling pathway functions, the opinions of its function in bladder
cancer was diverse, suggesting it might be a
cancer-promoting gene while might also have
anticancer activity [10]. Under different circumstance, it plays a distinct role, positive or negative impact on the proliferation and differentiation of cells and apoptosis [10]. Therefore,
exploring its role indifferent states of bladder
cancer is necessary.
RNA extraction and sequencing
Abnormal metabolic pathway is an important
symbol of a tumor [11]. Oncogenes and tumor
suppressor genes can normally regulate metabolic pathways, and different gene mutations
of a tumor have different metabolic states [12,
13]. We analyzed the abnormal gene in SV-HUV1-RT4-T24 progression and found several
abnormal expressions involved in multiple metabolic pathways, especially a large number of
mTOR and HIF-a pathway expression in advanced bladder cancer.
The DEGs in different groups were selected
using bioinformatics methods. The genes gradually reducing with the increase of malignancy
were assorted as group 0; those RT4 reduced,
5637 or T24 had little difference to RT4 as
group 1; those RT4 was lower than SV-HUV-1,
5637 or T24 had little difference to SV-HUV-1
as group 2; those RT4 had little difference to
SV-HUV-1, 5637 or T24 decreased significantly
as group 3; those RT4 had little difference to
SV-HUV-1, 5637 or T24 significantly increase
das group 4; those RT4 was higher than
SV-HUV-1, 5637 or T24 had little difference to
SV-HUV-1 as group 5; those RT4 increased,
5637 or T24 had little difference to RT4 as
group 6; those RT4 expression was higher than
SV-HUV-1, 5637 or T24 was higher than RT4 as
group 7.
Our research addressed to various stages of
differentially expressed genes (DEGs) in progress and development of bladder cancer cells,
combining trend analysis with functional
assays, to explain the role of genes differentially expressed in the development of tumorigenesis. Through large-scale sequencing, we
first analyzed the trend of differential expression gene SV-HUV-1, RT4 and 5637. GO function and pathway analysis showed that there
was little difference in expression of SV-HUV-1,
RT4, but a steep increase of DEGs in 5637
which mainly concentrated in the Notch signaling pathway. The escalating SV-HUV-1-RT4-T24
gene mainly concentrated in mTOR and HIF-1
pathway. Finally, we demonstrated the variation
of gene expression in related pathways by
RT-PCR.
Materials and methods
Cell lines
Bladder cell lines SV-HUV-1, RT4, 5637, and
T24 were all purchased from ATCC and cultured
in RPMI-1640 medium containing 10% fetal
serum bovine (FBS) at 37°C in an incubator
with 5% CO2. When the mixture confluent reaches to 80%, 107 tumor cells were collected for
the application of RNA sequencing.
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Different bladder cancer cells were counted in
3 × 106 and cellular RNA was extracted by
TRziol (Invitrogen Life Technologies, Carlsbad,
CA, USA). The amount and integrity of RNA were
detected using ND-1000 spectrophotometer
(Nanodrop) and Agilent Bioanalyzer 2100.
Illumina RNA-Seq chips were the platform for
hybridization and sequencing.
Differentially expressed genes screening
Gene ontology (GO) database and DAVID web
tool v6.7 were employed to analyze the biological functions of selected DEGs [14, 15] in each
group. False discovery rate (FDR) < 0.2 was
considered as significantly different.
RT-PCR analysis
RT-PCR analysis was used to analyze the
expressions of DEGs to confirm the changes of
mRNA in different tumor cells. RNA in tumor
cells was extracted, reverse transcribed, then
quantified with specific primers PCR. RT-PCR
was performed under Real-Time PCR System
with cycling conditions of 94°C for 10 min, followed by 94°C for 15 s and 58°C for 30 s, 72°C
for 20 s, 45 cycles in total. The relative expression levels of mRNA were calculated by 2-ΔΔCT.
Specific primer sequences are as follow.
Results
DEGs in bladder epithelial cells
We would first perform RNA sequencing on four
different urothelial cells SV-HUV-1, RT4, 5637,
Int J Clin Exp Pathol 2016;9(2):1608-1616
mRNA profile in urinary bladder cancer
Figure 1. Expression changes of the genes in different kinds of cells. A: Expression changes of genes in SV-HUV1-RT4-T24 group; B: Expression changes of genes in SV-HUV-1-RT4-5637.
T24 to analyze the changes of their expression
profiles. SV-HUV-1 was considered as normal
bladder epithelial cell line, RT4 was low-grade
urothelial or benign cell, 5637 and T24 were
two different high-grade bladder cancer cell
lines. Sequenced genes were comprehensive,
including expression of non-coding RNA (Table
S1).
Cluster analysis of DEGs
We compared SV-HUV-1-RT4-5637 DEGs pairwise (Tables S2-S4). DEGs were divided into 8
groups; grouping method is seen in ‘approach’.
We obtained 336 DEGs in group 0, 1165 DEGs
in group 1, 640 DEGs in Group 2, 288 DEGs in
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group 3, 702 DEGs in group 4, 768 DEGs in
group 5, 366 DEGs in group 6, 350 DEGs in
group 7 (Figure 1A and Table S5).
DEGs in SV-HUV-1-RT4-T24 were clustered
(Tables S6-S8), and DEGs were divided into 8
groups. We obtained 188 DEGs in group 0, 533
in group 1, 143 in Group 2,124 in group 3, 345
in group 4, 369 in group 5, 111 in group 6, 131
in group 7 (Figure 1B and Table S9).
Functional analysis of DEGs
In order to further explore the function of each
differential gene, we would carry out the GO
data analysis on DEGs in each group, as shown
in the results, DEGs in group 0 in SV-HUV-1Int J Clin Exp Pathol 2016;9(2):1608-1616
mRNA profile in urinary bladder cancer
RT4-5637 were mainly concentrated in gene
expression, DNA damage repair pathways, RNA
metabolic pathways; group 1 mainly concentrated in the cell cycle and cell division; group 2
mainly concentrated in protein translocation
and chromatin regulation; group 3 concentrated in the gene expression; Notch receptors in
group 4 processed the highest score; group 5
concentrated in the apoptotic pathway; group 6
concentrated in JNKK activation and cell adhesion; group 7 was IL-1 receptor signal and ribosome biogenesis (Figure 2A).
that Jagged 1 (JAG1), Histone Deacetylase 5
(HDAC5), JAG2, Recombination Signal Binding
Protein for Immunoglobulin Kappa J Region
(RBPJ) was significantly increased in Notch signaling pathway in SV-HUV-1-RT4-5637. In
SV-HUV-1, RT4, 5637, we detected genes EIF4
and EBP1 in HIF-a pathway and VEGFB in mTOR
pathway. With the increasing degree of malignancy of bladder cancer, the expression of
these genes gradually increased.
Meanwhile, we conducted GO gene differential
expression data analysis on each group of
SV-HUV-1-RT4-T24, as shown in the results,
DEGs in group 0 inSV-HUV-1-RT4-T24 mainly
concentrated in mitochondrial ATP synthesis
and respiratory oxidation of anion chain; group
1 mainly concentrated in mitosis; group 2 mainly concentrated in mitosis and cell differentiation; group 3 concentrated in fatty acid metabolism; group 4 concentrated in cell proliferation
regulation; group 5 concentrated in lipid metabolism and interferon inducing cell signaling
pathways; group 6 concentrated in polyamine
catabolism; group 7 concentrated in platelet
activation and insulin receptor signaling pathway (Figure 2B).
It has been widely known that genomic mutations leads to the expression structure mutations in bladder cancer, and heterogeneity is an
important feature of bladder cancer [15]. mRNA
and DNA chip technology [16] have been used
to detect abnormal expression of genes in bladder cancer cells, related prognosis marks [1719], and parsed the biological nature of bladder
cancer [20, 21]. However, due to the self-limitation of chip technology such as narrow coverage [22], more and more studies tended to use
second-generation sequencing technology for
the changes of expression profiles in bladder
cancer [23]. The next generation RNA sequencing technology not on lycan detects mRNA but
also discover non-coding RNA. In this study, we
first used the next generation sequence to analyze the gene expression profiles in bladder
cancer cell lines of different degree of malignancy, attempted to discover the key cancer
progression pathway and molecules through
trend analysis and GO, pathway analysis and
other functions.
Pathway analysis of DEGs
In order to further understand the pathways of
DEGs in each group, we performed gene pathway analysis for SV-HUV-1-RT4-5637 and
SV-HUV-1-RT4-T24 (Figure 3A and 3B). Through
analysis, we found that each group of genes
were consistent with GO analysis pathway in
profile 4 in SV-HUV-1-RT4-5637, and genes
causing 5637 increase were mostly concentrated in the Notch signaling pathway. We found
that those genes gradually increased in profile
7 of the SV-HUV-1-RT4-T24 were mainly concentrated in HIF-1 and mTOR, which all prompted
that T24 and metabolism genes had some
relevance.
RT-PCR analysis
RT-PCR was performed to verify the DEGs in
RNA-seq. In order to further validate the results
of our RNA-seq, we cultured the SV-HUV-1, RT4,
5637 and T24 cells, and extracted their RNA,
and expression levels of these genes were validated in messager RNA level. Results showed
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Discussion
In this study, DEGs in SV-HUV-1-RT4-5637 were
initial elevated, and several DEGs were involved
in the Notch signaling pathway. Notch played
an important role in the development of bladder cancer and suppressed tumor progression
[24, 25]. We found that the expression of JAG1,
HDAC5, JAG2 and RBPJ were significantly
increased, which were coincidence with former
evidences of these genes of JAG1 [26], HDAC5
[27], JAG2 [28], RBPJ [29] in other tumors such
as breast cancer, liver cancer, lung cancer.
However, roles of these DEGs on effecting bladder cancer still remain unclear. HIF-a and mTOR
played a crucial role in the process of occurring
and development of bladder cancer [30], promoted energy metabolism and tumor angiogenesis [31]. We found EIF4EBP1 and VEGFB had
Int J Clin Exp Pathol 2016;9(2):1608-1616
mRNA profile in urinary bladder cancer
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Int J Clin Exp Pathol 2016;9(2):1608-1616
mRNA profile in urinary bladder cancer
Figure 2. Gene ontology analysis of the screened DEGs in different group. A: The significant GO terms of DEGs in SV-HUV-1-RT4-T24 group; B: The significant GO
terms of DEGs in SV-HUV-1-RT4-5637 group.
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Int J Clin Exp Pathol 2016;9(2):1608-1616
mRNA profile in urinary bladder cancer
Figure 3. Pathway analysis of the screened DEGs in each group. A: The enriched significant pathways of DEGs in SV-HUV-1-RT4-T24 group; B: The enriched significant
pathways of DEGs in SV-HUV-1-RT4-5637 group.
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Int J Clin Exp Pathol 2016;9(2):1608-1616
mRNA profile in urinary bladder cancer
significantly increased expression in the
SV-HUV-1-RT4-T24, suggesting that these
developments might involve in the development of bladder cancer.
In future experiments, we will detect the changes of differential expression genes on genome
and protein level, combining with genome
sequencing and protein detection experiments.
Then combined with tumor function experiments, such as apoptosis, proliferation, drug
reactions, we will explain the functions of
detected differentially expressed genes, provide appropriate therapeutic targets for personalized treatment of bladder cancer [32-34].
In conclusion, the data presented in this study
revealed that Notch pathway plays pivotal roles
in the formation process of bladder cancer and
may be a potential target for the treatment of
bladder cancer, reveal molecular mechanisms
of different biological characteristics of various
bladder cancers. Our study may provide theoretical basis for the future exploration of Notch
pathway in bladder cancer.
[4]
[5]
[6]
[7]
[8]
Acknowledgements
This research was supported by the Science
and Technology Development Fund of Macao
(Grant No. 064/2012/A).
Disclosure of conflict of interest
[9]
None.
Address correspondence to: Dr. Bin Lang, School of
Health Sciences, Macao Polytechnic Institute, R. de
Luís Gonzaga Gomes, Macao, China. Tel: +8600853-85993440; E-mail: [email protected]
[10]
[11]
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