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W J C C World Journal of
Clinical Cases
Submit a Manuscript: https://www.f6publishing.com World J Clin Cases 2019 August 6; 7(15): 1954-1963
DOI: 10.12998/wjcc.v7.i15.1954 ISSN 2307-8960 (online)
ORIGINAL ARTICLE
Basic Study
Vestigial like family member 3 is a novel prognostic biomarker for
gastric cancer
Li-Hua Zhang, Zhuo Wang, Long-Hai Li, Yan-Kui Liu, Lin-Fang Jin, Xiao-Wei Qi, Chun Zhang, Teng Wang,
Dong Hua
ORCID number: Li-Hua Zhang
(0000-0002-0931-5039); Zhuo Wang
(0000-0001-8206-6035); Long-Hai Li
(0000-0003-4387-8932); Yan-Kui Liu
(0000-0003-4387-8942); Lin-Fang Jin
(0000-0002-2487-5886); Xiao-Wei Qi
(0000-0001-7761-5095); Chun Zhang
(0000-0001-6422-3367); Teng Wang
(0000-0001-6092-9522); Dong Hua
(0000-0001-5569-1708).
Author contributions: Zhang LH
and Wang Z contributed equally to
this work; Wang T and Hua D
designed the research; Li LH, Liu
YK, Jin LF, Qi XW, and Zhang C
performed the research; Wang T
and Hua D contributed new
reagents and analytic tools; Li LH,
Zhang LH, and Wang Z analyzed
the data; Zhang LH and Wang Z
wrote the paper.
Supported by the Natural Science
Foundation of Jiangsu Province,
No. BK20171150; the National
Natural Science Foundation of
China, No. 81502042; Research
Project of Health and Family
Planning Commission of Wuxi,
No. Q201758; and Nanchang
Hongda Jianghua Educational
Foundation.
Institutional review board
statement: This study was
reviewed and approved by
Affiliated Hospital of Jiangnan
University Institutional Review
Board.
Informed consent statement: Any
personal item or information will
not be published without explicit
consent from the involved persons.
Li-Hua Zhang, Dong Hua, School of Pharmaceutical Sciences, Jiangnan University, Wuxi
214122, Jiangsu Province, China
Li-Hua Zhang, Teng Wang, Dong Hua, Department of Oncology, Affiliated Hospital of Jiangnan
University, Wuxi 214062, Jiangsu Province, China
Zhuo Wang, Department of Geriatrics, Affiliated Wuxi First People's Hospital of Nanjing
Medical University, Wuxi 214122, Jiangsu Province, China
Long-Hai Li, Chun Zhang, Wuxi Medical College, Jiangnan University, Wuxi 214122, Jiangsu
Province, China
Yan-Kui Liu, Lin-Fang Jin, Xiao-Wei Qi, Department of Pathology, Affiliated Hospital of
Jiangnan University, Wuxi 214062, Jiangsu Province, China
Corresponding author: Dong Hua, MD, PhD, Chief Doctor, Professor, Department of
Oncology, Affiliated Hospital of Jiangnan University, No. 200, Huihe Road, Wuxi 214062,
Jiangsu Province, China. wx89211@163.com
Telephone: +86-510-88682109
Fax: +86-510-85808820
Abstract
BACKGROUND
Vestigial like family member 3 (VGLL3) is associated with the prognosis of
epithelial ovarian cancer and soft tissue sarcoma, but its role in gastric cancer
(GC) is unclear.
AIM
To explore the expression pattern and clinical significance of VGLL3 in GC.
METHODS
Integrative analysis was performed on the GC transcriptome profiles and
survival information deposited in the ONCOMINE, GEPIA, and ONCOLNC
databases. The expression levels of VGLL3 mRNA and protein were analyzed in
the freshly resected tumor and normal gastric tissues from GC patients by
quantitative RT-PCR and Western blot, respectively. In addition, the in situ
expression of VGLL3 in the GC tissues was determined by
immunohistochemistry (IHC), and the patients were accordingly classified into
the high and low expression groups. The correlation of VGLL3 expression status
with patient prognosis was then determined by univariate and multivariate Cox
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August 6, 2019 Volume 7 Issue 15
1954
Conflict-of-interest statement: The
authors report no conflicts of
interest in this work.
Open-Access: This article is an
open-access article which was
selected by an in-house editor and
fully peer-reviewed by external
reviewers. It is distributed in
accordance with the Creative
Commons Attribution Non
Commercial (CC BY-NC 4.0)
license, which permits others to
distribute, remix, adapt, build
upon this work non-commercially,
and license their derivative works
on different terms, provided the
original work is properly cited and
the use is non-commercial. See:
http://creativecommons.org/licen
ses/by-nc/4.0/
Manuscript source: Invited
Manuscript
Received: March 22, 2019
Peer-review started: March 22, 2019
First decision: May 13, 2019
Revised: June 22, 2019
Accepted: July 3, 2019
Article in press: July 3, 2019
Published online: August 6, 2019
P-Reviewer: Bang YJ, Kruszewski
WJ
S-Editor: Cui LJ
L-Editor: Wang TQ
E-Editor: Liu JH
regression analyses.
RESULTS
Analysis of the ONCOMINE and GEPIA databases showed that VGLL3 was
significantly up-regulated in GC tissues (P = 0.003), and associated with the
tumor TNM stage (P = 0.0163). The high VGLL3 expression group had a
significantly worse prognosis compared to the low expression group, as per both
GEPIA (P = 0.0057) and ONCOLNC (P = 0.01). The bioinformatics results were
validated by the significantly higher VGLL3 mRNA and protein levels in the GC
tissues compared to the adjacent normal tissues (P < 0.001) in a cohort of 30 GC
patients. Furthermore, high in situ expression of VGLL3 protein was associated
with more advanced N and TNM stages and HER2 mutation (P < 0.05) in a
cohort of 172 patients. Kaplan-Meier analysis showed that the high VGLL3
expression group had a worse prognosis compared to the low expression group
(P = 0.019). Multivariate analysis showed that VGLL3 expression status was an
independent risk factor for prognosis. In addition, the prognostic risk model
nomogram showed that VGLL3 was the most important indicator, with an area
under the receiver operating characteristic (ROC) curve (AUC) of 0.613 for 3-year
survival and 0.706 for 5-year survival. Finally, the protein interaction network
analysis revealed that VGLL3 is likely involved in the Hippo signaling pathway.
CONCLUSION
VGLL3 is overexpressed in GC tissues and associated with a poor prognosis,
indicating its potential as a novel prognosis biomarker and therapeutic target for
GC.
Key words: Vestigial like family member 3; Stomach adenocarcinoma; HER2 mutation;
Gastric cancer; Bioinformatics analysis
©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
Core tip: The present study for the first time revealed the expression of vestigial like
family member 3 (VGLL3) in gastric cancer (GC) and its correlation with HER2
mutation. Overall, the findings of the present study suggest that VGLL3 is a novel
prognostic biomarker for GC and highlight the significance of VGLL3 as a promising
therapeutic target for GC.
Citation: Zhang LH, Wang Z, Li LH, Liu YK, Jin LF, Qi XW, Zhang C, Wang T, Hua D.
Vestigial like family member 3 is a novel prognostic biomarker for gastric cancer. World J
Clin Cases 2019; 7(15): 1954-1963
URL: https://www.wjgnet.com/2307-8960/full/v7/i15/1954.htm
DOI: https://dx.doi.org/10.12998/wjcc.v7.i15.1954
INTRODUCTION
Gastric cancer (GC) is a frequently occurring malignancy of the digestive tract[1], and it
was the fifth most commonly diagnosed and the third most common cause of cancer-
related mortality worldwide in 2018[2]. Lack of early diagnosis and ineffective
treatment options for the advanced stages are the primary reasons for the dismal 5-
year survival rate of GC[3]. Therefore, it is imperative to identify novel biomarkers for
early diagnosis and accurate prediction of prognosis. Vestigial like family member 3
(VGLL3) is a member of the vestigial like family of proteins[4], and is associated with
epithelial ovarian cancer[5] and soft tissue sarcoma[6]. The aim of this study was to
determine the expression status and prognostic utility of VGL33 in GC. To this end,
we mined the transcriptomic data of GC from the ONCOMINE and GEPIA databases,
and determined its correlation with the survival data from GEPIA and ONCOLINC.
VGLL3 levels were upregulated in GC samples and associated with a poor prognosis.
The bioinformatics data were successfully validated on the tumor and normal gastric
tissues resected from GC patients. Based on the VGLL3 levels, the patients were
stratified into the high and low expression groups, and a prognosis prediction model
was established on the basis of VGLL3 status and clinical data. Our findings show a
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Zhang LH et al. VGLL3 is a novel prognostic biomarker for gastric cancer
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strong prognostic role of VGLL3 in GC, which can potentially translate to clinical
applications.
MATERIALS AND METHODS
Patients and tissue samples
A total of 172 tissue samples were obtained from gastric adenocarcinoma patients
who underwent major surgery at the Affiliated Hospital of Jiangnan University
between 2009 and 2012. The tissues were immediately fixed in formalin and
embedded in paraffin for further analysis. In addition, fresh samples were dissected
from 30 patients at the Affiliated Hospital of Jiangnan University in November 2018,
and immediately stored in liquid nitrogen for molecular analysis. The mean follow-up
duration was 43.1 mo and ranged from 0.2 to 86 mo. None of the patients received
chemotherapy or radiotherapy before surgery[7]. The tumor stage classification was
determined by three pathologists who were blinded to the patient data according to
the guidelines of the American Joint Committee on Cancer (AJCC). Freshly resected
tissues were obtained from 202 patients, and were fixed in formalin and embedded in
paraffin. In addition, tissue specimens from 30 patients were flash frozen in liquid
nitrogen. All participating clinicians and patients provided written informed consent.
Bioinformatics analysis
The VGLL3 mRNA levels in the GC and normal gastric tissues were determined by
integrated analysis of the Oncomine database (www.oncomine.org)[8] using
Coexpedia (www.coexpedia.org)[9]. ONCOLINC (http://www.oncolnc.org/cancer/)
and Coexpedia were used to analyze the prognostic value of WISP1 in STAD. The
STRING database (http://www.string-db.org) was utilized to construct the protein-
protein interaction (PPI) network[10].
RNA isolation and RT-qPCR
Total RNA was extracted from frozen tissue samples using Trizol reagent (Invitrogen,
Carlsbad, CA) according to the manufacturer’s instructions, and reverse transcribed
using the PrimeScript RT-PCR kit (Takara, Japan)[11]. RT-qPCR was performed on the
ABI 7500 RealTime PCR System (Applied Biosystems, Inc. USA) using SYBR Green
Master Mix (Takara, Japan), and the VGLL3 levels were normalized to β-actin. The
following primers were used: Forward primer, 5’-CCAACTACAGTCACC-
TCTGCTAC-3’ and reverse primer, 5’-ACCACGGTGATTCCTTACTCTTG-3’ for
VGLL3; forward primer, 5’-CCTGTGGCATCCACGAAACT-3’ and reverse primer, 5’-
GAAGCATTTGCG GTGGACGAT-3’ for β-actin. All reactions were performed in
triplicates, and the 2-∆∆Ct method[12] was used to quantify the relative expression levels
of VGLL3.
Western blot analysis
Total protein was extracted from GC and para-cancerous tissues using the RIPA lysis
buffer (Pierce, Thermo Scientifc, Cramlington, United Kingdom)[13], and quantified
with the enhanced BCA protein assay kit (KeyGEN BioTECH, Jiangsu, China). Equal
amount (40 mg) of proteins per sample were separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SD-PAGE), and then transferred to
polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, United States). After
blocking with 5% non-fat milk for 1 h at room temperature (RT), the membranes were
incubated overnight with anti-VGLL3 (ab83555, Abcam) and β-actin (ab8226, Abcam)
primary antibodies at 4 °C. After washing thrice with TBST, the membranes were
incubated with HRP-conjugated secondary antibody (1:1000) for 1 h at RT. The
protein bands were visualized with an ECL chemiluminescence system after short
exposure to X-ray films (Kodak, Japan). Densitometric analysis was performed with
Image Pro-Plus software, and the relative expression levels of VGLL3 were
normalized to tubulin.
Immunohistochemistry (IHC)
The formalin-fixed tissues were dehydrated, embedded in paraffin, and cut into 5 μm
thick sections. IHC was performed according to the manufacturer’s protocol[14].
Briefly, the sections were heated in citrate buffer in a microwave, cooled, and
incubated overnight with anti-VGLL3 antibody (1:50, clone PA5-68441, Invitrogen) at
4 °C. After labeling with the secondary antibody for 1 hour at RT, color was
developed using liquid DAB substrate. Three pathologists blinded to the patient
identity observed and graded VGLL3 positivity as 0, 1 (1%–29% positively-stained
cells), 2 (30%–69%), or 3 (70%-100%), and the staining intensity as 0 (negative), 1
(weak staining), 2 (moderate staining), or 3 (strong staining). The immunoreactive
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Zhang LH et al. VGLL3 is a novel prognostic biomarker for gastric cancer
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score (IRS) was then calculated for each sample by multiplying the staining intensity
and positivity scores, and graded as: 0-1, “-”; 2-3, “+”; 4-6, “++”; and 6-9, “+++”[15].
Based on the IRS, the samples were stratified into the high (4-9) and low (0-3) VGLL3
expression groups.
Fluorescence in situ hybridization (FISH)
The tissue sections were treated with denaturing solution to denature the DNA into
single strands, and hybridized with the PathVysion probes (No.36-161060, PathVysion
HER-2DNA Probe Kit). After washing the unbound probe with DNA, the sections
were counterstained with the nuclear dye DAPI (4’, 6 diamidino-2-phenylindole).
Positive LSI HER-2/neu and CEP 17 signals were counted under a fluorescence
microscope, and the ratio of the copy number of HER-2/neu gene to that of
chromosome 17 was calculated.
Statistical analysis
Statistical analyses were performed with R*64 version 3.5.2 software and Graphpad
6.01. HER2 status (positive or negative) was designated on the basis of the combined
FISH and IHC results as per the American Society of Clinical Oncology/College of
American Pathologists Clinical Practice Guidelines. VGLL3 expression levels in the
tumor and normal samples were compared by the Student's t-test, and the association
between VGLL3 and clinico-pathological features was assessed by the chi-square test.
The overall survival (OS) curve was plotted by the Kaplan-Meier method and
analyzed by the log-rank test. Univariate and multivariate analyses of the prognostic
factors were performed using the Cox proportional hazard regression model. A
nomogram was formulated based on the results of the multivariate analysis with the
package of rms in R version 3.5.2 (http://www.r-project.org/)[16]. The receiver
operating characteristic (ROC) curve was plotted to determine the sensitivity and
specificity of the VGLL3-based prognostic score[17], and the area under the curve
(AUC) was calculated. All P-values were two-tailed and considered statistically
significant when less than 0.05.
RESULTS
VGLL3 mRNA and protein levels are upregulated in GC samples
GEPIA database profiling showed that although VGLL3 was significantly
overexpressed in GC samples of advanced TNM stages relative to those at the lower
stages (P = 0.0163), there was no significant difference between the GC and normal
tissues (P > 0.05). Integrated analysis of multiple VGLL3 transcriptome datasets from
the ONCOMINE database, however, showed that VGLL3 was significantly
overexpressed in GC (P = 0.003), and notably associated with a poor prognosis
according to the ONCOLINC database analysis (P = 0.01, n = 378) (Figure 1). In
addition, GEPIA online survival analysis further confirmed that VGLL3 was a marker
of poor prognosis in GC (P = 0.0062, n = 384). To validate the in silico data, we
analyzed the expression levels of VGLL3 in the tumor and normal gastric tissues
resected from 30 GC patients, and observed significantly higher levels of VGLL3
mRNA (P < 0.001) and protein (P < 0.001) in the GC tissues compared to normal
tissues (Figure 2). Taken together, VGLL3 is upregulated in GC and possibly
associated with a worse prognosis.
VGLL3 is a potential prognostic factor for GC
The relationships between VGLL3 levels and the clinico-pathological characteristics of
the patients are summarized in Table 1. High levels of VGLL3 were positively
correlated with tumor lymph node metastasis (P = 0.028) and TNM stage (P = 0.041).
In addition, the median survival time of the GC patients was significantly lower in the
high VGLL3 expression group than in the low expression group (35 mo vs 49 mo, P =
0.019; Figure 3C). Univariate analysis showed that the VGLL3 expression level (P =
0.019) was the only significant prognostic factor of GC, whereas age, gender,
differentiation, T stage, lymph node status, TNM stage, and HER2 status did not show
any significant results (P > 0.05). After including the significant factors in the Cox
proportional hazards regression model for multivariate prognostic analysis, we found
that VGLL3 expression (P = 0.019) was an independent risk factor for GC. ROC curves
were plotted to determine the power of the VGLL3 prognostic score in predicting the
3- and 5-year survival, and the AUCs were 0.613 and 0.706, respectively (Figure 3I).
We constructed a PPI network of VGLL3 using the STRING database, and identified
YAP1, TEAD1, WWTR1, and VGLL4 as upstream of VGLL3, and POU1F1, TEAD3,
AKAP11, SYT17, CHMP28, TEAD4, and ERBBE2 (HER2) as downstream proteins
(Table 2).
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Figure 1
Figure 1 Vestigial like family member 3 mRNA expression levels in gastric cancer tissues determined by integrated analysis of different databases. A and
B: Vestigial like family member 3 (VGLL3) expression in gastric cancer and normal gastric tissues (A) and in different TNM stages (B) in the GEPIA database; C:
VGLL3 expression reported in the five published studies in the ONCOMINE database; D and E: Kaplan-Meier survival analysis of patients in the GEPIA (D) and
ONCOLNC (E) databases stratified by VGLL3 expression status.
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Figure 2
Figure 2 Vestigial like family member 3 mRNA and protein expression levels in gastric cancer. The expression of vestigial like family member 3 mRNA (A) and
protein (B and C) in 30 pairs of gastric cancer and normal gastric tissues is shown.
DISCUSSION
GC is the third most common cause of cancer-related deaths worldwide, mainly due
to ineffective diagnostic and therapeutic options. It is often detected at the advanced
stage, and is highly heterogeneous. Therefore, novel molecular biomarkers for the
early diagnosis and treatment of GC are urgently needed. VGLL3 is a member of the
vestigial-like family proteins that are related to sex and maturation[18-20], and is
reportedly associated with epithelial ovarian cancer and soft tissue sarcoma. Through
bioinformatics data mining and integrated analysis, we found that VGLL3 mRNA was
not only upregulated in the GC tissues relative to normal gastric tissues, but also
significantly associated with advanced TNM stage GC and poor survival outcomes.
To validate the in silico data, we analyzed the gastric tissues of GC patients, and found
abnormally high levels of VGLL3 in the tumor relative to normal tissues. In addition,
high in situ expression of VGLL3 was correlated with tumor lymph node metastasis,
TNM stage, and HER2 mutation, but not with age, gender, differentiation, T stage, or
M stage. The patients were stratified into the high and low VGLL3 expression groups,
and the former showed a significantly poor survival. Univariate and multivariate
analyses further indicated that VGLL3 expression and TNM stage were independent
risk factors for the prognosis of GC. Finally, the respective AUCs of the 3- and 5-year
survival of the VGLL3-based prognostic model were 0.613 and 0.706, indicating a
somewhat imperfect predictive ability. This could be due to the insufficient number of
samples and other confounding factors such as the patient's state of mind, economic
status, and family environment. Taken together, we identified VGLL3 as a novel
prognostic biomarker for GC.
To further elucidate the underlying molecular mechanisms, we analyzed the PPI
network of VGLL3 through the STRING database, and found that the Hippo pathway
was significantly enriched. In addition, the identified upstream molecules of VGLL3
are YAP1[6], TEAD1, WWTR1, VGLL4, and the downstream molecules are POU1F1,
TEAD3, AKAP11, SYT17, CHMP28, and TEAD4. Interestingly, the expression level of
VGLL3 was related to ERBBE2 (HER2) mutation, although the relevant mechanistic
connection is unclear.
To conclude, VGLL3 is highly expressed in GC and an independent risk factor, and
further studies are needed to determine its underlying mechanism. Nevertheless,
VGLL3 is a novel biomarker for GC prognosis and a potential therapeutic target for
this malignancy.
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Table 1 Characteristics of gastric cancer patients according to vestigial like family member 3 expression status
Characteristic Cases High expression Low expression P-value
Age (yr)
≤60 97 47 50 0.548
>60 75 32 43
Gender
Female 101 43 58 0.369
Male 71 36 35
Differentiation
Well 84 40 44 0.779
Poor and moderate 88 39 49
T stage
T1/2 91 36 55 0.105
T3/4 81 43 38
N stage
N0/1 95 36 59 0.028
N2/3 77 43 34
TNM stage
I-II 119 48 71 0.041
III-IV 53 31 22
HER2
Negative 89 32 57 0.010
Positive 83 47 36
Table 2 Univariate and multivariate analyses of factors associated with overall survival
Characteristic
Univariate analysis Multivariate analysis
Hazard ratio 95%CI P-value Hazard ratio 95%CI P-value
Age (yr) 0.83 0.55-1.26 0.389
Gender (Male vs Female) 1.31 0.87-1.96 0.194
Differentiation (Well vs Poor and moderate) 0.81 0.54-1.22 0.304
T stage (T3/4 vs T1/2) 1.16 0.77-1.73 0.484
N stage (N2/3 vs N0/1) 0.94 0.63-1.42 0.778
TNM stage (III/IV vs I/II) 1.27 0.82-1.97 0.283
HER2 (Positive vs Negative) 1.19 0.79-1.8 0.392
VGLL3 (High vs Low) 1.66 1.09-2.55 0.019 1.66 1.09-2.55 0.019
VGLL3: Vestigial like family member 3.
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Figure 3
Figure 3 Prognostic value and putative molecular mechanism of vestigial like family member 3 in gastric cancer. A-D: HER2 mutation status detected by
FISH (A and C) and IHC (B and D) in gastric cancer (GC) tissues; E and F: In situ vestigial like family member 3 (VGLL3) status in GC tissues; G: Kaplan-Meier
survival analysis on patients stratified by VGLL3 expression status; H: Prognostic nomogram for gastric cancer based on VGLL3 expression status and clinical
features; I: Time-dependent receiver operating characteristic curves for the combination of VGLL3-based prognostic score and clinico-pathological variables; J:
Protein-protein interaction network of VGLL3 based on STRING database.
ARTICLE HIGHLIGHTS
Research background
Gastric cancer (GC) is the most prevalent gastrointestinal tract malignancy. The prognosis of GC
patients remains relatively poor. It is urgent to explore prognostic markers for GC.
Research motivation
There are insufficient reports about the correlation between VGLL3 and GC.
Research objectives
The aim of the present study was to explore the expression pattern and clinical significance of
VGLL3 in GC.
Research methods
It was found that VGLL3 would be a potential prognostic marker by bioinformatics analysis. To
validate the in silico data, the authors identified the expression of VGLL3 in GC patient samples
by immunohistochemistry and evaluated clinical outcomes.
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Zhang LH et al. VGLL3 is a novel prognostic biomarker for gastric cancer
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Research results
Analysis of the ONCOMINE and GEPIA databases showed that VGLL3 was significantly up-
regulated in GC tissues, and associated with the tumor TNM stage. In addition, the high VGLL3
expression group had a significantly worse prognosis compared to the low expression group, as
per both GEPIA and ONCOLNC. The bioinformatics results were validated by the significantly
higher VGLL3 mRNA and protein levels in the GC tissues compared to the adjacent normal
tissues in a cohort of 30 GC patients. Furthermore, high in situ expression of VGLL3 protein was
associated with more advanced N and TNM stages and HER2 mutation in a cohort of 172
patients. Kaplan-Meier analysis showed that the high VGLL3 expression group had a worse
prognosis compared to the low VGLL3 expression group. Multivariate analysis showed that
VGLL3 expression status was an independent risk factor for prognosis. In addition, the
prognostic risk model nomogram showed that VGLL3 was the most important indicator, with an
AUC of 0.613 for 3-year survival and 0.706 for 5-year survival. Finally, the protein interaction
network analysis revealed that VGLL3 is likely involved in the Hippo signaling pathway.
Research conclusions
VGLL3 is overexpressed in GC tissues and associated with a poor prognosis, indicating its
potential as a novel prognosis biomarker and therapeutic target for GC.
Research perspectives
The present study suggested that VGLL3 is a novel prognostic biomarker for GC, and the
significance of VGLL3 as a promising therapeutic target for GC is highlighted.
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