ORIGINAL RESEARCH

published: 09 February 2021

doi: 10.3389/fgene.2021.627464

Knockdown of RIPK2 Inhibits

Proliferation and Migration, and
Induces Apoptosis via the NF-κB
Signaling Pathway in Gastric Cancer
Qian Yang 1,2,3 , Shan Tian 1 , Zhengru Liu 1,2,3 and Weiguo Dong 1,2*
1
Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, China, 2 Key Laboratory of Hubei Province
for Digestive System Disease, Wuhan, China, 3 Central Laboratory, Renmin Hospital of Wuhan University, Wuhan, China

RIPK2 is a 62 kDa protein and a member of the receptor interacting protein kinases
(RIPK) family. It was previously demonstrated that RIPK2 might play a role in promoting
malignant tumor progression; however, the precise function of RIPK2 in the onset and
progression of gastric cancer (GC) remains unclear. In the current study, we investigated
the role of RIPK2 in GC. First, we explored the expression levels of RIPK2 in multiple
Edited by:
cancers, including GC, using a bioinformatics approach. We constructed the RIPK2-
Obul Reddy Bandapalli, associated protein-protein interaction network using the search tool for the retrieval
Hopp Children’s Cancer Center
of interacting genes/proteins for gene ontology and Kyoto encyclopedia of genes
Heidelberg (KiTZ), Germany
and genomes analysis. Next, we compared the RIPK2 expression levels between GC
Reviewed by:
Kumar Varun, cells and normal gastric mucosal epithelial cell (GES-1) using reverse transcription
European Molecular Biology quantitative PCR analysis. We downregulated the expression of RIPK2 in GC cells to
Laboratory Heidelberg, Germany
Yanqiang Li,
determine the effects of RIPK2 on cell growth, migration, and apoptosis. Finally, we used
Harvard Medical School, western blotting to investigate the RIPK2 downstream signaling pathway involved in the
United States
regulation of GC progression. Our results showed that RIPK2 was overexpressed in
*Correspondence:
various tumor tissues, including GC, compared to non-cancer tissues. Moreover, RIPK2
Weiguo Dong
[email protected] expression was significantly upregulated in all four GC cell lines (MGC-803,SGC-7901,
HGC-27 and AGS) comparing the GES-1 cells. Silencing of RIPK2 suppressed GC cell
Specialty section:
growth by inhibiting migration, and inducing apoptosis through the nuclear factor-κB
This article was submitted to
Cancer Genetics, (NF-κB) signaling pathway. In summary, we demonstrate that RIPK2 plays an important
a section of the journal role in modulating GC cell proliferation, migration, and apoptosis through the NF-κB
Frontiers in Genetics
signaling pathway. Therefore, RIPK2 functions as a potential oncogene. We believe that
Received: 09 November 2020
Accepted: 13 January 2021 RIPK2 can be used as a candidate biomarker, as well as a diagnostic tool, and the
Published: 09 February 2021 therapeutic target for GC.
Citation:
Keywords: RIPK2, gastric cancer, proliferation, migration, apoptosis, NF-κB signaling
Yang Q, Tian S, Liu Z and Dong W
(2021) Knockdown of RIPK2 Inhibits
Proliferation and Migration,
Abbreviations: GC, gastric cancer; DEGs, differentially expressed genes; GO, gene ontology; BP, biological process; CC,
and Induces Apoptosis via the NF-κB cellular component; MF, molecular function; KEGG, Kyoto encyclopedia of genes and genomes; PPI, protein-protein
Signaling Pathway in Gastric Cancer. interaction; STRING, search tool for the retrieval of interacting genes; TCGA, The cancer genome atlas; TIMER, tumor
Front. Genet. 12:627464. immune estimation resource; ROC, receiver operating characteristic; AUC, area under the ROC curve; HPA, human protein
doi: 10.3389/fgene.2021.627464 atlas; AV, annexin V FITC; PI, propidium iodide.

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Yang et al. Role of RIPK2 in GC

INTRODUCTION MATERIALS AND METHODS

Gastric cancer (GC), the third most common malignancy, RIPK2 Expression in Human Cancers
is a primary cause of tumor-related deaths worldwide (Bray
Using Pan-Cancer Analysis
et al., 2018). The existing first-line therapeutic approaches for
RIPK2 mRNA expression levels in various solid tumors were
GC, such as surgery and chemotherapy, are still limited in
identified using the oncomine (Rhodes et al., 2004)1 and tumor
their ability to improve the prognosis. Early detection and
immune estimation resource (TIMER) (Li et al., 2017)2 databases.
treatment for GC may reduce pain and have a vital impact
In TIMER, we used the DiffExp module to analyze the differential
on the potential for complete recovery. But unfortunately,
expression between tumor and adjacent normal tissues for RIPK2
majority of GC patients often diagnosed at Stage II or
across all the cancer genome atlas (TCGA)3 .
III (Choi et al., 2015, 2019). The pathogenesis of GC is
diverse, involving multiple genes and cellular pathways. Despite
numerous efforts to study the underlying mechanisms of RIPK2 Expression in Gastric Cancer
development in GC, many details regarding these aspects To evaluate the expression of RIPK2 in GC, we used three
remain unclear. So far, esophagogastroduodenoscopy is a independent databases: TCGA (April, 2020), national center for
sensitive clinical screening method for GC; however, due to the biotechnology information- (NCBI) gene expression omnibus
invasiveness of the examination, poor patient compliance, and (GEO) (Barrett et al., 2013)4 [GSE19826 (Wang et al., 2012),
psychological fear, its application is underutilized. Currently, GSE79973 (Jin et al., 2017), and GSE33335 (Cheng et al., 2013)],
targeted cancer therapies serve as a critical cornerstone of and oncomine. We used receiver operating characteristic (ROC)
precision medicine. Thus, development of the targeted therapy curves to assess the diagnostic value of RIPK2. The protein
has required the identification of good targets, and these specific expression level of RIPK2 was investigated using the human
molecular targets should play a critical role in regulating protein atlas (Uhlen et al., 2017) (HPA)5 database.
cancer cell growth, apoptosis, and survival. On the other
hand, biochemical markers could be used as non-invasive Identification of RIPK2-Associated
diagnostic tools; therefore, it is imperative to investigate Proteins and Functional Enrichment
novel potential molecular markers for the diagnosis and Analysis
treatment of GC. The construction of the RIPK2-associated protein network was
Receptor interacting serine/threonine kinase 2 (RIPK2, carried out using search tool for the retrieval of interacting
also known as RIP2, RICK, and CARD3), which is known genes/proteins (STRING) (Szklarczyk et al., 2015)6 (interaction
for its role in immunity and inflammation, is a potent score > 0.9); stringApp (a plugin of Cytoscape, v3.7.2)
activator of nuclear factor κB (NF-κB) (McCarthy et al., 1998; software was used to visualize the network. The gene ontology
Park et al., 2007). Activation of RIPK2 may subsequently (GO) (Ashburner et al., 2000) and Kyoto encyclopedia of
activate IκB kinases (IKKs), followed by the translocation genes and genomes (KEGG) (Kanehisa, 2002) analyses for
of the NF-κB dimers (e.g., p65/p50) into the nucleus to RIPK2-associated proteins were applied using the database for
trigger the transcription of target genes (Hoesel and Schmid, annotation, visualization and integrated discovery (DAVID)
2013). Previous studies have investigated the importance of (Huang et al., 2007)7 ; P < 0.05 was identified as a significance
RIPK2 in inflammatory pathogenesis and (auto) immune threshold. The molecular functions of the genes were visualized
diseases, such as multiple sclerosis (Shaw et al., 2011), using Sangerbox8 .
pancreatitis (Duggan et al., 2017) and inflammatory bowel
disease (Chen et al., 2019). Therefore, RIPK2 might play
Validation Based on Human GC Samples
a vital role in overactive inflammatory responses observed
To further verify the data from GEO, TCGA, and oncomine,
in various diseases, including cancer. Our previous study
we detected the RIPK2 mRNA expression using quantitative
demonstrated that RIPK2 was overexpressed in colorectal cancer
reverse transcription- (qRT) PCR. Human GC samples (13 GC
(CRC), and, in combination with Fusobacterium nucleatum,
tissues and 13 matched non-tumor tissues) were obtained from
was involved in the regulation of CRC metastases (Chen
the Renmin Hospital of Wuhan University (Wuhan, China).
et al., 2020). In addition, RIPK2 played a crucial role
Informed consent was provided by all participants, and the
in the formation and progression of oral squamous cell
research protocol was approved by the Institutional Review Board
cancer (Wang et al., 2014), inflammatory breast cancer
of the Renmin Hospital of the Wuhan University.
(Zare et al., 2018). Nevertheless, the role of RIPK2 in GC
remains unclear. 1
https://www.oncomine.org/
In the current study, we demonstrated that RIPK2 was 2
https://cistrome.shinyapps.io/timer
overexpressed both at mRNA and protein levels in GC tissues. In 3
https://portal.gdc.cancer.gov/
addition, we further investigated the biological effects of RIPK2 4
http://www.ncbi.nlm.nih.gov/geo
on GC cell proliferation, apoptosis and migration. These findings 5
https://www.proteinatlas.org/
indicate that RIPK2 plays a vital role in GC cell tumorigenicity 6
http://string-db.org
and proliferation by regulating NF-κB signaling and suggest that 7
http://david.ncifcrf.gov
RIPK2 could be a potential target for GC therapy. 8
http://www.sangerbox.com/tool

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Yang et al. Role of RIPK2 in GC

Cell Culture were as follows: anti-RIPK2 (1:1,000, 15366-1-AP; Proteintech,

All cell lines were obtained from the Key Laboratory of Hubei Wuhan, China), anti-Cleaved-caspase 3 (1:1,000, #9661S; CST,
Province for Digestive System Disease (Wuhan, Hubei, China). United States), anti-Bcl-2 (1:1,000, #2872T; CST, United States),
GES-1 (gastric mucosal epithelial cell line) and GC cell lines SGC- anti-Bax (1:1,000, 50599-2-Ig; Proteintech, Wuhan, China),
7901, MGC-803, AGS, and HGC-27 were cultured in DMEM/F- anti-NF-κB P65 (1:1,000, #8242T; CST, United States), anti-
12 medium (SH30023; HyClone, United States) containing 10% p-NF-κB P65 (Ser536) (1:1,000, #3033; CST, United States),
fetal bovine serum (FBS, sijiqing, Hangzhou, China) at 37◦ C in a anti-IκBα (1:1,000, #4814T; CST, United States), anti-p-IκBα
humidified atmosphere of 5% CO2 . (Ser32) (1:1,000, #2859; CST, United States). Anti-GAPDH
(1:10,000; AP0063; Bioworld, Nanjing, China) antibody was
used as a loading control. Then, the membranes were
Cell Transfection
washed three times with Tris-buffered saline-tween 20 (TBST),
Cells were plated overnight to grow to 40–50% confluence at
and incubated with species-specific secondary antibodies for
the time of transfection. siRNAs targeting the human RIPK2
1.5 h at room temperature. After washing with TBST three
gene (siRNA-RIPK2) and non-targeting negative control siRNA
times, the membranes were then detected with enhanced
(siRNA-NC) were purchased from Suzhou GenePharma Co., Ltd.
chemiluminescence reagent (Servicebio Technology Co., Wuhan,
(Shanghai, China). The siRNAs were as follows: si-RNA-RIPK2:
Chnia) and were observed by the Bio- Rad ChemiDoc R

sense, 50 -CAAUAUGACUCCUCCUUUATT-30 , antisense, 50 -

Touch Imaging System (BioRad, Hercules, United States).
UAAAGGAGGAGUCAUAUUGTT-30 ; non-targeting siRNA
The image J software was used to analyze the results,
(siRNA-NC): sense, 50 -UUCUCCGAACGUACGUTT-30 ,
and all protein expression levels were assessed relative to
antisense, 5 -ACGUGACACGUUCGGAGAATT-30 . Lipid-based
0
GAPDH expression.
transient transfections were performed using Lipofectamine 6000
Transfection Reagent (Biyuntian, Shanghai, China) according
to the manufacturer’s instructions. Six hours later, the culture Cell Viability Assay
medium was replaced with fresh medium containing 10% FBS. HGC-27 and AGS cells (2,000 cells per well) were seeded
After 48–72 h of transfection, fluorescence, quantitative reverse into 96-well plates and transfected with siRNA-RIPK2 and
transcription- (qRT) PCR and western blotting were used to siRNA-NC. Cell Counting Kit-8 (CCK-8, Biyuntian, Shanghai,
assess the transfection efficiency. China) reagent 10µl was added to each experimental well
after 0, 24, 48, and 72 h, respectively. Cell proliferation rates
were measured using a microplate reader (BD Biosciences,
RT-qPCR
United States) at 450 nm.
Total RNA was extracted using Trizol reagent (15596-026;
Invitrogen, Carlsbad, CA, United States) and then reverse-
transcribed complementary DNA (cDNA) was synthesized using Clone Formation Assay
a PrimeScriptTM RT reagent Kit with gDNA Eraser (RR047A; Twenty-four hours after AGS and HGC-27 cells were transfected
Takara, Shiga, Japan), following the manufacturer’s instructions. with siRNA-RIPK2 or siRNA-NC, infected cells (300 cells per
The conditions of RT-PCR were 37◦ C for 15 min, 85◦ C for well) were incubated in six-well plates. The transfected cells
5 s, and held at 4◦ C. Then, the cDNA was diluted four times were cultured at 37◦ C for 13 days. Then, the cells were fixed
with nuclease-free water, and the RIPK2 mRNA expression with methyl alcohol for 10 min and stained with 4% crystal
(sense: 50 - GAATCATGTGGATCCTCTCAGC-30 ; anti-sense: 50 - violet solution. The colonies in each group were washed before
TGATTTCCAGGACAGTGATGC-30 ) was detected by real-time counting. ImageJ software (ImageJ 1.52a, United States) was used
PCR using SYBR Premix Ex TaqTM (RR420A, Takara). The
R to analyze and quantify the number of colonies.
reaction conditions were as follows: 95◦ C for 30 s, followed by
40 cycles at 95◦ C for 5 s, and 60◦ C for 30 s in 7900 real-time Analysis of Apoptosis
PCR system (Applied Biosystems, CA, United States). GAPDH AGS and HGC-27 cells were transfected as described in section
(sense: 50 -CATCATCCCTGCCTCTACTGG-30 ; anti-sense: 50 - “Cell Transfection” and cultured for another 24 h to perform
GTGGGTGTCGCTGTTGAAGTC-30 ) was used as the internal the apoptosis analysis. The cells were harvested and washed
control. The relative RIPK2 mRNA expression was calculated with ice-cold PBS for twice, and then the cell concentration was
using the 2−11CT method. adjusted to 1 × 106 cells/ml. Next, cells were stained with 5 µl
Annexin V-FITC (Biyuntian, Shanghai, China) for 10 min. And,
Western Blotting the cells were centrifuged at 1,000 × g for 5 min to remove
Total proteins were extracted from transfected AGS and the supernatant, and then suspended in binding buffer. Finally,
HGC-27 cells. Western blotting was performed according to the cells were stained with 10 µl propidium iodide (Biyuntian,
standard protocols and as previously described (Li et al., Shanghai, China) for 5 min in the dark and analyzed by flow
2019). Cell lysates were separated by 10–12% sodium dodecyl cytometry (BD Biosciences) to evaluate the rate of apoptosis.
sulfate- polyacrylamide gel electrophoresis (SDS- PAGE) and
transferred to a nitrocellulose membrane. After blocking with Wound-Healing Assay
5% non-fat milk for 1 h, the membranes were incubated with Cells were transfected and cultured to 90% confluence in a
primary antibodies at 4◦ C overnight. Primary antibodies used six-well plate. Then, wounds were scratched with a sterile

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Yang et al. Role of RIPK2 in GC

pipette tip and cells were incubated in the medium without RESULTS
FBS. Distance migrated by the cells was recorded at 0 and
24 h after wound scratching. Microscopy images detected mRNA Expression Levels of RIPK2 in
cells that migrated into the wound area. Three independent
Pan-Cancer
experiments were performed.
To determine the differences in RIPK2 mRNA expression in
tumor and adjacent normal tissues, RIPK2 expression levels were
Cell Migration Assays analyzed using the oncomine database over a cancer-wide range.
To assess cell migration, the transwell migration assay was This analysis showed that the RIPK2 had a higher expression
performed. Transfected cells were seeded in the top chambers in gastric, breast, esophageal, colorectal, head and neck, liver,
containing 1% FBS, while medium containing 20% FBS was kidney, as well as pancreatic cancers, myeloma and sarcoma,
added to the lower chambers. After 24 h, the migrated compared to that in the respective normal tissues (Figure 1A).
cells on the bottom part of the membrane were fixed with In addition, RIPK2 expression was low in head and neck, and
70% methanol and stained with 0.1% crystal violet. The kidney cancers, and leukemia in some datasets. Supplementary
cells from the top of the membrane were carefully removed, Table 1 summarizes the details of RIPK2 expression in different
while the migrated cells were quantified in three random types of cancers.
microscopic fields. To verify the expression level of RIPK2 in human cancers,
we analyzed RIPK2 expression based on TCGA data by
Statistical Analysis TIMER. As shown in Figure 1B, RIPK2 was differentially
Statistical analyses were carried out using R version 3.6.1 and expressed in tumor tissues compared to that in the adjacent
GraphPad Prism7 (GraphPad software, Inc., San Diego, CA, normal tissues. RIPK2 was significantly upregulated in BLCA
United States). All experiments were repeated at least thrice. (bladder urothelial carcinoma), BRCA (breast invasive
The data are presented as mean ± SEM. Data were analyzed carcinoma), ESCA (esophageal carcinoma), COAD (colon
using either a two-tailed Student’s t-test, or a two-way ANOVA adenocarcinoma), CHOL (cholangiocarcinoma), HNSC (head
with Sidak’s multiple comparisons test. Statistical significance and neck cancer), STAD (stomach adenocarcinoma), READ
was set at P < 0.05 (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, (rectum adenocarcinoma), KIRC (kidney renal clear cell
∗∗∗∗ P < 0.0001). carcinoma), LUAD (lung adenocarcinoma), UCEC (uterine

FIGURE 1 | RIPK2 expression levels in cancers. (A) Differential expression of RIPK2 in tumor tissues compared with normal tissues in oncomine. Number in each
cell indicates the number of datasets; red indicates that RIPK2 is upregulated in corresponding cancer, blue indicates that RIPK2 is downregulated in corresponding
cancer. (B) Human RIPK2 expression levels in different types of tumors from TCGA data in TIMER. *P < 0.05, **P < 0.01, ***P < 0.001. TCGA, the cancer genome
atlas; TIMER, tumor immune estimation resource.

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Yang et al. Role of RIPK2 in GC

corpus endometrial carcinoma), THCA (thyroid carcinoma), The results of the ROC analysis were as follows: GSE19826 (area
LIHC (liver hepatocellular carcinoma), and PRAD (prostate under the ROC curve (AUC) = 0.930, 95% confidence interval
adenocarcinoma) compared to that in their respective normal (CI): 0.798–1.063, p = 0.0003); GSE79973 (AUC = 0.9, 95% CI:
tissues. Meanwhile, RIPK2 expression was significantly lower 0.7141–1.086, p = 0.0025); GSE33335 (AUC = 0.98, 95% CI:
in LUSC (lung squamous cell carcinoma) compared to that in 0.953–1.009, p < 0.0001) (Figure 2D). Moreover, RIPK2 also
the normal tissues. showed higher mRNA expression in GC tissues in the oncomine
database (median rank = 392.0, p = 5.55E-5) (Figure 2E), and
RIPK2 Was Consistently Upregulated in RIPK2 protein expression in GC was also increased compared to
that in normal gastric tissues (Figure 2F).
GC
The mRNA expression of RIPK2 was highly overexpressed in
GC tissues compared to that in the normal gastric tissues based GO and KEGG Analysis of RIPK2-Related
on TCGA-stomach tumor database (Figures 2A,B) (A: 375 GC Proteins
tissues, 32 normal gastric tissues; B: 28 GC tissues and paired Previous studies showed that RIPK2 plays a role in tumor
non-tumor tissues) and GEO (Figure 2C) (GSE19826: 12 GC progression; however, these observations were limited to
tissues, 12 matched non-cancer gastric tissues; GSE79973: 10 carcinomas of the breast (Zare et al., 2018), bladder (Zhang
GC tissues and paired adjacent tissues; GSE 33335: 25 GC and and Chin, 2014) and colon (Chen et al., 2020). To date,
25 normal gastric tissues). In addition, the RIPK2 diagnostic there is no clear evidence of the role of RIPK2 in GC. Thus,
value for GC was determined using the ROC curve analysis. cytoscape stringApp was used to construct the protein-protein

FIGURE 2 | RIPK2 expression in gastric cancer. (A,B) Expression level of RIPK2 in TCGA gastric tumor and normal tissues (A: Tumor = 375, Normal = 32; B:
Tumor = 28, Paired non-tumor tissues = 28; ∗∗∗∗ P < 0.0001; two-tailed t-test). (C) RIPK2 expression was upregulated in gastric cancer tissues compared to that in
the normal gastric tissues; based on the GSE19826 (∗∗∗∗ P < 0.0001, two-tailed t-test), GSE79973 (∗∗ P < 0.001, two-tailed t-test) and GSE33335 (∗∗∗∗ P < 0.0001,
two-tailed t-test). (D) ROC curve analysis of the RIPK2 mRNA expression levels in the GC datasets from the GEO database: GSE19826 (AUC = 0.93), GSE79973
(AUC = 0.9), GSE33335 (AUC = 0.98). (E) Oncomine database was applied to verify the increased expression of RIPK2 in various subtypes of gastric cancer tissues.
(F) RIPK2 protein expression was higher in tumor tissues compared to normal tissues based on HPA database (Normal: id 2,471; male; 65 years old; staining
negative; tumor: id 2,473; male; 59 years old; staining: moderate; intensity: medium; quantity: 75%; location: cytoplasm/membrane). ROC, receiver operating
characteristic; AUC, area under the ROC curve; HPA, human protein atlas; GC, gastric cancer.

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Yang et al. Role of RIPK2 in GC

interaction (PPI) network of RIPK2 and its protein partners. investigate the pathway that might be involved in RIPK2-
Next, a network of proteins that were tightly associated with mediated gastric cancer progression, GO and KEGG pathway
RIPK2 was established (Figure 3A). There were 11 nodes and analyses were performed. GO analysis of these proteins in the
43 edges (mean node degree is 7.82, mean local clustering PPI network showed that RIPK2 was closely related to the
coefficient is 0.908, and PPI enrichment p-value is 9.25E-13) regulation of apoptosis, production of cytokines, and immune
in this network. The 10 predicted RIPK2-interacting proteins response. The KEGG pathway analysis indicated that RIPK2
included NOD1 (nucleotide-binding oligomerization domain was associated with NOD-like receptor and IκB/NF-κB signaling
containing 1), NOD2, CYLD (CYLD lysine 63 deubiquitinase), pathway (Figure 3B).
BIRC3 (baculoviral IAP repeat containing 3), TRAF6 (TNF
receptor associated factor 6), CARD6 (caspase recruitment
domain family member 6), IKBKG (inhibitor of nuclear factor Inhibition of RIPK2 Suppressed GC Cell
kappa B kinase regulatory subunit), NGFR (nerve growth factor Proliferation
receptor), MYD88 (MYD88 innate immune signal transduction To determine the molecular function of RIPK2 in GC, we
adaptor), and BIRC2 (baculoviral IAP repeat containing 2). examined RIPK2 mRNA expression levels in GC tissues and
The analysis of these genes will be done in the future. To four GC cell lines. As expected, RIPK2 was significantly

FIGURE 3 | Identification of predicted interacting proteins of RIPK2 and the molecular function analysis. (A) The PPI network that was closely associated with RIPK2.
(B) GO and KEGG analysis of the predicted proteins interacting with RIPK2. BP, biological process; CC, cellular component; MF, molecular function; PPI,
protein–protein interaction; GO, gene ontology; KEGG: Kyoto encyclopedia of genes and genomes.

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Yang et al. Role of RIPK2 in GC

overexpressed in 13 GC tissues (Figure 4A). And, RIPK2 analysis. HGC-27 and AGS cells were transfected with siRNA-
expression was upregulated in MGC-803, SGC-7901, HGC-27, RIPK2 and the non-targeting siRNA (siRNA-NC). Fluorescence,
and AGS cells, compared to that in GES-1 cells (Figure 4B). western blotting and qRT-PCR assays were used to determine
Therefore, AGS and HGC-27 cells were selected for further the transfection efficiency of these two cell lines (Figures 4C–J).

FIGURE 4 | RIPK2 expression in gastric cancer tissues and cells. (A)The mRNA expression levels of RIPK2 in GC tissues (∗∗∗ P < 0.001, versus normal gastric
tissues; two-tailed t-test). (B) The mRNA expression levels of RIPK2 in GES-1 and GC cells (∗ P < 0.5, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001 versus GES-1; two-tailed t-test).
Analysis of the transfected efficacy of RIPK2 knockdown in AGS and HGC-27 cells: (C,D) Bright-field images and the corresponding fluorescence images of
transfected AGS and HGC-27 cells. (E–J) Expression levels of RIPK2 were examined after siRNA-RIPK2 transfection in AGS and HGC-27 cells by qRT-PCR and
Western blotting (∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001 versus siRNA-NC; two-tailed t-test). NC, negative control; GC, gastric cancer.

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Yang et al. Role of RIPK2 in GC

To investigate the effect of RIPK2 downregulation on GC cell AGS and HGC-27 cell lines compared to that in the control
growth, AGS and HGC-27 cells were transfected with siRNA- (siRNA-NC) (Figures 6A–D). In addition, western blotting
RIPK2, and cell proliferation was measured using the CCK8 analysis was showed that anti-apoptotic protein Bcl2 was
assay. Our results demonstrated that the downregulation of decreased in both RIPK2-silenced AGS and HGC-27 cells, while
RIPK2 significantly inhibited the proliferation of AGS and the expression of apoptotic proteins Bax and cleaved caspase-
HGC-27 cells (Figures 5A,B). The knockdown of RIPK2 also 3 were increased (Figures 6E–H). These data showed that the
significantly decreased the number of colonies compared to that downregulation of RIPK2 promoted apoptosis in GC cells.
in siRNA-NC transfected cells, as determined by the colony
formation assay (Figures 5C–F). CCK8 cell proliferation and
colony formation assays both showed that decreased expression Inhibition of RIPK2 Decreases the
of RIPK2 suppressed the proliferation of GC cells. Migration Capacity of GC Cells
To investigate whether RIPK2 silencing affected GC cell
migration, we performed wound healing and transwell assays.
Inhibition of RIPK2 Induced Apoptosis of It was shown that the wound closure of siRNA-RIPK2-
GC Cells transfected cells was suppressed compared to that in both AGS
This suppression of cell proliferation prompted us to investigate (Figures 7A,C-left) and HGC-27 (Figures 7B,D-left) control
the effect of RIPK2 silencing on apoptosis. Apoptosis of GC cells. Furthermore, the transwell migration assay indicated
cells was evaluated using annexin V-FITC/PI flow cytometry. that the number of migrated AGS (Figures 7C-right, E) and
Silencing of RIPK2 increased the rate of apoptotic cells in HGC-27 cells (Figures 7D-right, F) was significantly reduced

FIGURE 5 | Knockdown of RIPK2 inhibited GC cell proliferation. (A,B) Cell counting kit-8 (CCK8) assays shows that downregulation of RIPK2 suppresses growth of
AGS and HGC-27 cells (**P < 0.01, ****P < 0.0001 versus siRNA-NC; two-way ANOVA). (C–F) In the colony formation assay, silencing of RIPK2 decreases the
colony number in AGS and HGC-27 cells (****P < 0.0001 versus siRNA-NC; two-tailed t-test). NC, negative control; GC, gastric cancer.

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Yang et al. Role of RIPK2 in GC

FIGURE 6 | Knockdown of RIPK2 induced apoptosis. (A–D) Apoptotic rates were measured by Annexin V/PI staining and analyzed by an image flow assay in AGS
and HGC-27 cells (****P < 0.0001 versus siRNA-NC; two-tailed t-test). (E–H)Western blot analysis of Bcl2, Bax and cleaved-caspase 3 protein levels in AGS and
HGC-27 cells with downregulation of RIPK2 (*P < 0.05, ***P < 0.001, ****P < 0.0001 versus siRNA-NC; two-tailed t-test). AV, annexin V FITC; PI, propidium iodide;
siRNA-NC, NC, negative control; GC, gastric cancer.

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Yang et al. Role of RIPK2 in GC

FIGURE 7 | Knockdown of RIPK2 suppressed GC cell migration. (A,B) Representative images or wound healing assay, AGS and HGC-27 at 0 and 24 h,
respectively (***P < 0.01 versus siRNA-NC; two-tailed t-test). (C,D left) Relative migration distance was analyzed in AGS and HGC-27 cells, respectively. (C,D right)
The number of migrated cells was analyzed in AGS and HGC-27 cells, respectively. (E,F) Representative images of the migrated AGS and HGC-27 cells transfected
with siRNA-RIPK2 or siRNA-NC (***P < 0.01, ****P < 0.0001 versus siRNA-NC; two-tailed t-test). GC, gastric cancer; NC, negative control.

in siRNA-RIPK2 compared to that in the siRNA-NC group. the role of RIPK2 in cancer development has been investigated,
These results suggested that RIPK2 plays a role in cell especially in colorectal cancer (Chen et al., 2020), its function
migration in GC cells. in GC is not yet clear. Only one study has suggested that the
carriers of RIPK2 single nucleotide polymorphism rs16900627
NF-κB Signaling Is Regulated by RIPK2 minor allele may have a higher risk for the progression of gastric
in GC cancer (Ota et al., 2018).
The NF-κB pathway is critical for tumor formation and A major goal of cancer research is to identify the alterations
development. We predicted that the IκB-α/NF-κB pathway might driving tumorigenesis (Hu et al., 2018). In our study, first,
play a role in the function of RIPK2 in GC (Figure 3). The western we explored RIPK2 expression levels in cancers based on
blotting analysis showed that the phosphorylation of P65 and the oncomine and TCGA databases. As expected, RIPK2 was
IκB-α was decreased in the RIPK2-silenced AGS and HGC-27 overexpressed in various types of cancer tissues compared
cells (Figures 8A–D). These findings suggest that the observed with corresponding normal tissues. Next, we evaluated the GC
regulation of proliferation, apoptosis, and migration in GC cells microarray data from three independent online public databases,
in response to RIPK2 knockdown, could be mediated via the and found that both RIPK2 mRNA and protein expression levels
NF-κB pathway. were significantly upregulated in GC tissues compared to that in
normal tissues. Combined with the ROC analysis, our findings
suggest that RIPK2 expression levels have the potential to become
DISCUSSION a novel diagnostic biomarker for GC.
To elucidate the role of RIPK2 in GC, we investigated
RIPK2 is a serine/threonine/tyrosine kinase with a carboxy- the RIPK2-associated proteins using PPI network analysis.
terminal caspase activation and recruitment domain (Zare et al., We found that the genes identified during this analysis were
2018), which has been identified as a oncogene participating in implicated in the modulation of apoptosis, immune response
several process involved in the development of tumors. Although and cytokine production and, therefore, could be contributing

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Yang et al. Role of RIPK2 in GC

FIGURE 8 | Knockdown of RIPK2 suppressed the NF-κB signaling pathway. (A,B) AGS cells and (C,D) HGC-27 cells were transfected with siRNA-NC or
siRNA-RIPK2 and the protein levels of NF-κB (P65), phosphorylated P65, IκBα, and phosphorylated IκBα were measured by western blot (∗ P < 0.05, ∗∗ P < 0.01,
∗∗∗ P < 0.001 versus siRNA-NC; two-tailed t-test). NC, negative control. ns, not significant.

to tumor (especially inflammatory carcinoma) formation and proinflammatory bacterium, can facilitate ulcerative colitis and
development. For example, NOD-like receptors are involved promote colorectal cancer metastasis via upregulation of RIPK2
in innate immunity and the formation of inflammasomes, expression (Chen et al., 2019, 2020). In addition, increased RIPK2
and can activate NF-κB, inflammatory caspases, and autophagy activity contributing to inflammatory breast cancer pathogenesis
(Velloso et al., 2019). NOD1 and NOD2 are sensors of different and aggressiveness (Zare et al., 2018). Thus, we hypothesize that
bacterial peptidoglycan components. It has been previously RIPK2 might also serve as a biomarker for GC progression. With
shown that the activation of NOD1 promotes colon cancer regard to the current study, to explore the role of RIPK2 in GC
metastases (Jiang et al., 2020); inhibition of RIPK2 can delay cells, HGC-27 and AGS cells were selected to perform loss-of-
NOD signaling to decrease the production of inflammatory function experiments. Our results demonstrated that silencing of
cytokine (Nachbur et al., 2015), so RIPK2 could regulate the RIPK2 significantly decreased the proliferative capacity of AGS
NOD signaling to control the occurrence and development and HGC-27 cells. These findings are consistent with previous
of inflammation-related tumors. Collectively, these genes were results showing that RIPK2 may play a vital role in the cancer cell
predicted based on the neighborhood, co-expression, database, growth (Wu et al., 2012; Zhang and Chin, 2014).
experiment data, co-occurrence, and text mining. Further work Apoptosis and proliferation are two important cellular
is required to provide insight into the association of each gene processes involved in the development of cancer (Fesik, 2005).
with respect to RIPK2 in GC. Our results demonstrated that silencing RIPK2 significantly
RIPK2 has been shown to affect cell growth and, therefore, increased the apoptosis rate in both AGS and HGC-27 cells.
may have a significant effect on cell viability and biological Moreover, we found that knockdown of RIPK2 induced apoptosis
function (McCarthy et al., 1998). Two important study was related to regulate the Bcl2 family expression. The Bcl2
demonstrated that Fusobacterium nucleatum, a well-recognized protein family is a large family of apoptosis modulating proteins

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Yang et al. Role of RIPK2 in GC

that regulate the mitochondrial-mediated intrinsic pathway and RIPK2 expression inhibited the GC cell growth and migration,
includes anti-apoptotic proteins and pro-apoptotic proteins induced apoptosis by suppressing the NF-κB signaling. Our
such as Bcl-2 and Bax (Delbridge et al., 2016). In our study, results suggest that RIPK2 could be a novel therapeutic target
western blotting showed that the expression level of apoptotic for GC and provide new insights into the potential mechanism
protein Bax, as well as cleaved caspase-3, were increased, while for the treatment of GC.
anti-apoptotic protein Bcl2 expression was decreased. It was
suggested that RIPK2 is responsible for the suppression of
apoptosis in human GC. DATA AVAILABILITY STATEMENT
Tumor cell migration is an important step and prerequisite
for tumor invasion. Our previous research indicated that RIPK2 The original contributions presented in the study are included
facilitated the colorectal cancer cells migration and invasion via in the article/Supplementary Material, further inquiries can be
inducing epithelial-mesenchymal transition (Chen et al., 2020). directed to the corresponding author/s.
Thus, we performed transwell and wound healing assays to
determine whether RIPK2 influenced the migration of GC cells.
The results showed that silencing of RIPK2 significantly inhibited ETHICS STATEMENT
the AGS and HGC-27 cells migration which suggested that
RIPK2 promoted cell migration behavior in GC cells. The studies involving human participants were reviewed
It is well known that NF-κB plays an essential role in and approved by the Institutional Review Board
cell growth, differentiation, apoptosis, invasion, and metastases of the Renmin Hospital of Wuhan University. The
(Dolcet et al., 2005; Scholz and Taylor, 2013). The KEGG pathway patients/participants provided their written informed consent
analysis indicated that the IκB-α/NF-κB signaling pathway may to participate in this study.
function downstream of RIPK2 in GC. Since RIPK2 has sequence
homology to RIP, a known activator of NF-κB (McCarthy
et al., 1998), we investigated a possible role of RIPK2 in NF-
AUTHOR CONTRIBUTIONS
κB activation in GC. In the present study, silencing of RIPK2 QY was responsible for the conception and design of
was found to suppress IκBα by phosphorylation at Ser32 sites, bioinformatic analysis, experiments, and original draft
and the phosphorylation activity of NF-κB (P65) was positively preparation. ST was responsible for data collection and
regulated by RIPK2. Therefore, our study indicate that targeting statistical analysis. ZL was responsible for data curation.
RIPK2 could provide a potential strategy for GC therapy through WD was responsible for providing experimental funds and
deactivating IκBα/NF-κB signaling. technical guidance. All authors have read and approved the
In summary, data from GEO, TCGA, and oncomine datasets, final manuscript.
as well as comprehensive bioinformatics analyses, allowed us
to identify RIPK2 as a candidate gene for GC diagnosis. Our
results demonstrated that RIPK2 was significantly overexpressed ACKNOWLEDGMENTS
in GC tissues. We also showed that RIPK2 was involved in
cell proliferation, apoptosis, and migration, while knockdown of We would like to thank Editage (www.editage.cn) for English
RIPK2 had a suppressive effect on tumorigenesis in GC cells, by language editing.
downregulating the NF-κB activation.
However, our study has limitations: First, our cohort of
patients with GC from our hospital was relatively small. SUPPLEMENTARY MATERIAL
Secondly, we only investigated silencing of RIPK2 in vitro studies.
These aspects need to be addressed in future work. The Supplementary Material for this article can be found
In conclusion, this study demonstrated that RIPK2 was online at: https://www.frontiersin.org/articles/10.3389/fgene.
significantly upregulated in GC tissues. Furthermore, silencing of 2021.627464/full#supplementary-material

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Nachbur, U., Stafford, C. A., Bankovacki, A., Zhan, Y., Lindqvist, L. M., Fiil, Copyright © 2021 Yang, Tian, Liu and Dong. This is an open-access article distributed
B. K., et al. (2015). A RIPK2 inhibitor delays NOD signalling events yet under the terms of the Creative Commons Attribution License (CC BY). The use,
prevents inflammatory cytokine production. Nat Commun. 6:6442. doi: 10. distribution or reproduction in other forums is permitted, provided the original
1038/ncomms7442 author(s) and the copyright owner(s) are credited and that the original publication
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(2018). Association between receptor interacting serine/threonine kinase 2 distribution or reproduction is permitted which does not comply with these terms.

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