Expression of long noncoding RNA GAS5 associated with clinic pathologic factors of gastric cancer

JOURNAL OF MEDICAL RESEARCH

EXPRESSION OF LONG NONCODING RNA GAS5 ASSOCIATED
WITH CLINIC PATHOLOGIC FACTORS OF GASTRIC CANCER
Nguyen Dang Bao1, Nguyen Trong Tue2, Tran Hieu Hoc3,
Nguyen Van Hung3, Tran Van Khanh2, Pham Duc Huan4, Ta Thanh Van2
1

General surgery Department, Gialai General Hospital, 2Center for Gene - Proteine Research,
Hanoi Medical University, 3Bach mai Hospital, 4Hanoi Medical University Hospital

Gastric cancer is the world’s third most common cause of cancer-related death. It remains difficult to cure,
particularly in less developed countries. Several recent studies revealed that long noncoding RNAs
(lncRNAs) could play a critical role in tumor biology. Dysregulation of lncRNA expression has been reported
in different types of cancers, including gastric cancer, but its role in gastric cancer remains unknown. This
study was designed to investigate the expression pattern, biological role and clinical significance of lncRNA
GAS5 in gastric cancer. LncRNA GAS5 was isolated from 48 cancer tissues and 48 non - cancer gastric
tissues of gastric cancer patients in Vietnam. LncRNA GAS5 levels were determined via quantitative reverse
-transcription polymerase chain reaction (Qrt - PCR) and normalized to the expression of GAPDH.
Differences between groups were tested for significance using the 2 -ΔΔCt method. Our results show that
expression of GAS5 was lower in gastric cancer tissues than in normal gastric tissues and was associated
with tumor size, invasion depth and pathologic stage. GAS5 may represent a new marker of prognosis for
gastric cancer patients.

Keywords: Gastric cancer, Long noncoding RNA, GAS5, tumor suppressor, marker cancer

I. INTRODUCTION
Worldwide,

gastric

of cancer mortality is tumor metastasis; thus,
cancer

currently

prevails as the third most common cause of

early diagnosis of gastric cancer is of particular significance to prevent early death.

cancer - related death. Estimations suggest

Eukaryotic genomes encode numerous

that 35% of affected patients present with syn-

long non - coding RNAs (lncRNAs), which

chronous distant metastases [1]. Approxi-

represent a class of RNA molecules longer

mately 9.5 million new gastric cancer cases

than 200 nucleotides without open reading

and 7.2 million deaths are recorded annually

frames of significant length or with limited

[2]. Despite improvements in diagnostic tech-

protein - coding potential [4]. Recently, many

niques and advances in treatments, patients

studies have revealed that lncRNAs could play

with advanced or metastatic gastric cancer

a critical role in regulating gene expression at

continue to face a poor prognosis. Moreover,

various levels, e.g., chromatin modification,

the average survival period after diagnosis is

transcription and post - transcriptional proc-

less than 1 year [3]. One of the major causes

essing [5], Furthermore, they also play an
importnat role in many biological processes,

Corresponding author: Nguyen Trong Tue, Center for
Gene and Protein Research, Hanoi Medical University
E-mail:
Received: 06 November 2016
Accepted: 10 December 2016

JMR 105 E1 (7) - 2016

including cellular differentiation, invasion, and
metastasis [6; 7]. Based on their functions,
lncRNAs can be roughly divided into oncogenic and tumor - suppressor groups [8].

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JOURNAL OF MEDICAL RESEARCH
Indeed, it is now widely acknowledged that

ncRNA in other cancers. For example, GAS5

lncRNAs are likely to be of crucial importance

and/or its snoRNAs have been shown to be

in the pathogenesis of cancer; therefore, a

aberrantly expressed in breast cancer, head

better understanding of lncRNA biology may

and

lead to novel and better approaches for cancer

glioblastoma multiforme and non-small-cell

diagnosis and treatment.

lung cancer [18]. Abnormally low levels of

neck

squamous

cell

carcinoma,

LncRNA expression has been reported in

GAS5 expression may therefore reduce the

different types of cancers, including lung can-

effectiveness of chemotherapeutic agents [19].

cer, breast cancer, hepatocellular carcinoma

Previous studies have also shown that lncRNA

and gastric cancer [9 - 13]. In addition, levels

GAS5 was downregulated and served as a

of circulating lncRNAs have been used for

tumor-suppressor lncRNA in prostate cancer

cancer diagnosis and prognosis [14; 15] and

cells, renal cell carcinoma, cells and breast

have also been considered as potential

cancer cells [8; 19; 20]. In this study, we

biomarkers and therapeutic targets for gastric

investigated the potential relationship between

cancer [12]. GAS5 lncRNA is a transcript of

GAS5 lncRNA level in gastric cancer tissues

the growth arrest - specific 5 (GAS5) gene, a

and the clinicopathologic features of the

non-protein coding gene. It was first isolated in

disease, as well as the clinical outcome.

1988 during a search for novel tumour

II. SUBJECTS AND METHODS

suppressors, where subtractive cDNA cloning
was used to identify genes that are preferentially expressed in growth - arrested cells [16].
GAS5 lncRNA negatively regulates the growth

1. Subjects
Tissue collection

of cell line in various cancers [17] and the ot-

Samples were obtained from 48 patients

her systems demonstrate that GAS5 exerts

who had undergone surgery at the Hanoi

complementary effects on cell proliferation

Medical University Hospital, Viet Duc Hospital,

(inhibitory) and apoptosis (stimulatory) and

or Bach Mai Hospital from October 2015 to

together these are likely to form the main ba-

August 2016. The patients were diagnosed

sis of its tumour suppressor activity in vivo

with gastric cancer based on histopathological

[17]. Furthermore, another study showed that

evaluation. Clinicopathologic information was

GAS5 was significantly downregulated in

available for all samples (Table 1). These pa-

gastric cancer tissues of Chinese patients and

tients did not receive any treatment before the

that GAS5 expression was associated with

operation. All specimens were immediately

larger tumor size and advanced pathologic

frozen in liquid nitrogen and stored at −80°C

stage. Patients with low GAS5 expression

until RNA extraction.

levels had poorer disease - free survival and

2. Methods

overall survival than those with high GAS5

expression [12]. Nevertheless, the overall

RNA extraction and qRT-PCR analyses

pathophysiological contributions of lncRNAs to

Total RNA was extracted from tissues

gastric

unknown.

using the Rneasy Minikit QIAgen for qRT-

Additionally, some evidence implicates GAS5

PCR. RNA was reverse transcribed to cDNA

48

cancer

remain

largely

JMR 105 E1 (7) - 2016


JOURNAL OF MEDICAL RESEARCH

by using qScript™ cDNA SuperMix (Quanta

GAS5 RNA levels in 48 pairs of gastric

BioScience, America). Real-time PCR analy-

cancer samples and adjacent, histologically

ses were performed with "PerfeCTa® SYBR®

normal tissues were examined by qRT - PCR

Green

BioScience,

and normalized to GAPDH. Figure 1 showed

America). Results were normalized to the

that the GAS5 level was significantly de-

expression of GAPDH. The PCR primers for

creased in 62.5% (30/48) of gastric cancer

GAS5 or GAPDH were as follows:

tissues compared with corresponding adjacent

FastMix®

(Quanta

GAS5 - F1: 5’ -CCTGTGAGGTATGGTGCTGG

non - tumorous tissues. Moreover, in cancerous tissues, GAS5 expression in the cancer

- 3’
GAS5 - F2: 5’ -CTGTGTGCCAATGGCTTGAG

tissues was at a lower level than that of the
normal comparison specimens. These data

- 3’
GAPDH - F1: 5’- CTTCTTTTGCGTCGCC-

indicate that abnormal GAS5 expression may
be related to gastric cancer pathogenesis.

AGCCG - 3’
GAPDH - F2: 5’- CTTCCCGTTCTCAGCCTTGAC
- 3’.

The relationship between GAS5 expression

qRT - PCR and data collection were performed on Effpendor Realplex mastercycler.
The relative expression of GAS5 was analysed using the 2−ΔΔCt method for relative quantitation and normalized to GAPDH.

and

clinicopathologic

factors

in

patients with gastric cancer
The clinical pathology of the 48 gastric carcinoma patients included in this study are
shown in table 1. The patients were divided
into two groups according to the relative GAS5
expression in their tumor tissues: a relative

Statistical analysis
Group comparisons, correlations and associations were performed using SPSS statistical
software (version 16.0, SPSS Inc) and the Chi
-Square test. A p-value less than 0.05 was
considered statistically significant when comparing the differences between patient groups.

high - GAS5 expression group (n = 30, GAS5
expression ratio ≥ 1) and a relative low - GAS5
expression group (n = 18, GAS5 expression
ratio ≤ median ratio) (Figure 1). Clinicopathologic features were compared between the
two groups (table 2). The low - GAS5 group
was found to have greater tumor size (p =

3. Research ethics: This research was
approved by the ethics committee of the Hanoi
Medical

University,

Approval

No.187-

HMUIRB, signed February 20, 2016.

III. RESULTS
Expression of GAS5 is down - regulated
in human gastric cancer tissues

JMR 105 E1 (7) - 2016

0.032), higher TNM stage (p = 0.001), greater
invasion depth (p = 0.037), more regional
lymph nodes (p = 0.001) and more lymphatic
metastases (p = 0.001). However, GAS5
expression level was not associated with other
parameters such as gender (p = 0.431), age
(p = 0.662), tumor location (p = 0.179) and
histologic differentiation (p = 0.574) (table 2).

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JOURNAL OF MEDICAL RESEARCH
Table 1. Clinicopathological characteristics and GAS5 expression in 48 tissue samples of
patients with gastric cancer
Clinical parameter

n (%)

Age (years)
< 50

8 (16.7)

> 50

40 (83.3)

Gender
Male

34 (70.8)

Female

14 (29.2)

Location
Distal

35 (72.9)

Middle

13 (27.1)

Size
< 2 cm

5 (10.4)

2 - 5 cm

24 (50)

> 5 cm

19 (39.6)

Histologic diffentiation
Moderately differentiated

11 (22.9)

Poorly differentiated

25 (52.1)

Undifferentiated

12 (25.0)

Invasion depth
T1

3 (6.2)

T2

14 (29.2)

T3

25 (52.1)

T4

6 (12.5)

TNM Stages
IA

3 (6.2)

IB

4(8.3)

II

11 (22.9)

IIIA

20 (41.7)

IIIB

2 (4.2)

IV

8 (16.7)

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Clinical parameter

n (%)

Lymphatic metastasis
Yes

35 (72.9)

No

13 (27.1)

Regional lymph nodes
N0

13 (27.1)

N1

20 (41.7)

N2

11 (22.9)

N3

4 (8.3)

Distant metastasis
Yes

6 (12.5)

No

42 (87.5)
Low GAS5 expression

High GAS5 expression

Figure 1. Relative expression of GAS5 in gastric cancer tissues (n = 48) compared with
corresponding non - tumor normal tissues (n = 48)
GAS5 expression was examined by qRT - PCR and normalized to GAPDH expression. Data
was presented as fold - change in tumor tissues relative to normal tissues. Patients’ GAS5

expression was classified into two groups based on relative GAS5 expression in tumor tissues: a
relative high - GAS5 expression group (n = 18, red columns) and a relative low-GAS5 expression
group (n = 30, blue columns).

JMR 105 E1 (7) - 2016

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JOURNAL OF MEDICAL RESEARCH
Table 2. Correlation between GAS5 expression and clinicopathological characteristics in
patients with gastric cancer
GAS5 (number of case)
Clinical parameter
High - GAS5 group

Low - GAS5 group

< 50

3

5

> 50

15

25

Male

12

22

Female

6

8

Distal

15

20

Middle

3

10

< 2 cm

4

1

2 - 5 cm

9

11

> 5 cm

5

18

Moderately

4

7

Poorly

8

17

Undifferentiated

6

6

T1

2

1

T2

9

5

T3

6

19

T4

1

5

Chi - Square test
p-value

Age (years)
0.662
Gender

0.431
Location
0.179
Size

0.032

Histologic differentiation

0.574

Invasion depth

0.037

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GAS5 (number of case)
Clinical parameter

High-GAS5 group

Low-GAS5 group

IA

2

1

IB

3

1

II

9

2

IIIA

4

16

IIIB

0

2

IV

0

8

Chi-Square test
p-value

TNM Stages

0.001

Lymphatic metastasis
Yes

8

27
0.001

No

10

3

Regional lymph nodes
N0

10

3

N1

7

13

N2

1

10

N3

0

4

Yes

0

6

No

18

24

0.001

Distant metastasis
0.048

IV. DISCUSSION

metastasis by directly targeting chromatin

Long non - coding RNAs are a class of

modification complexes, indicating that the

recently discovered non-protein coding RNAs

abnormal expression of lncRNAs increases

> 200 nucleotides in length, with a role in

the chance of tumorigenesis and cancer

mammalian

cell

development. However, at present, only a few

differentiation. Their dysregulation has been

lncRNAs have been functionally studied in

detected in various diseases, most notably

detail and many important questions remain

cancer. Recent studies have shown that many

unanswered [21]. Therefore, identification of

lncRNAs are dysregulated in various solid

cancer - associated lncRNAs and investigation

tumors. Several lncRNAs can regulate cancer

of their clinical significance and functions may

development

JMR 105 E1 (7) - 2016

and

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JOURNAL OF MEDICAL RESEARCH
provide a missing piece of the well - known

ciated with increased tumor size, higher TNM

oncogenic and tumor suppressor network

stage, greater invasion depth and increased

puzzle.

regional lymph nodes (table 2). Our findings

Human GAS5 is encoded at 1q25, a locus

were similar to a previous study by Sun, M et

that has been associated with abnormalities in

al (2014) in Chinese gastric cancer patients,

several types

including

but unlike Sun, et. al.’s study, our findings re-

prostate cancer cells, renal cell carcinoma

vealed that low GAS5 was correlated with lym-

cells and breast cancer cells [8; 19; 20]. Low

phatic metastasis (table 2). Furthermore, ecto-

GAS5 expression is an adverse prognostic

pic expression of GAS5 was demonstrated to

factor for survival in breast cancer, cervical

decrease gastric cancer cell proliferation and

cancer, and gastric cancer [22; 12]. Converse-

induce apoptosis, while downregulation of en-

ly, over-expression of GAS5 has been attribu-

dogenous GAS5 could promote cell prolifera-

ted to the prevention of several cancer cell

tion in vitro and in vivo [12]. These findings

lines through regulation of apoptosis and the

indicate that GAS5 may play a key tumor-

cell cycle, under basal conditions as well as in

suppressor

some chemotherapeutic agents. These results

prognostic markers for gastric cancer.

suggest that GAS5 may have clinical signifi-

V. CONCLUSION

of

cancer

cells,

cance in the development of cancer therapies

role

and

may

be

a

novel

Our study reveals that GAS5 expression

[8; 12; 19; 20].
This is the first study to investigate the

level in gastric cancer tissue is associated with

expression pattern and prognostic significance

tumor size, TNM stage, invasion depth, num-

of GAS5 in Vietnamese patients with gastric

ber of regional lymph nodes and extent of lym-

cancer. GAS5 expression was retrospectively

phatic metastasis. Our results indicate that

analyzed in 48 patients with gastric carcinoma.

GAS5 might represent a potential biomarker

Its expression levels in gastric cancer tissues

for the diagnosis and management of gastric

were compared with those found in distal

cancer.

normal tissue from the same patients. A clear
reduction of more than 62% in GAS5 expres-

Acknowledgements

sion level in the gastric cancer tissues

We would like to express our sincerest

compared with normal tissues was observed

gratitude to the Center for Gene-Protein

(Figure 1). This reduction was statistically sig-

research at the Hanoi Medical University for

nificant,

reduction

their help with our experiments. We would also

in GAS5 expression level is related to the

like to thank the Hanoi Medical University

development of gastric cancer.

Hospital, Viet Duc Hospital and Bach Mai

suggesting

that

the

Our results showed that GAS5 expression
was significantly decreased in gastric cancer
tissues. Patients’ GAS5 expression levels

hospital for giving us patient samples.

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