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01.12.2017 | Research article | Ausgabe 1/2017 Open Access

BMC Cancer 1/2017

Effect of myeloid differentiation primary response gene 88 on expression profiles of genes during the development and progression of Helicobacter-induced gastric cancer

Zeitschrift:
BMC Cancer > Ausgabe 1/2017
Autoren:
Ivonne Lozano-Pope, Arnika Sharma, Michael Matthias, Kelly S. Doran, Marygorret Obonyo
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12885-017-3114-y) contains supplementary material, which is available to authorized users.
Abbreviations
(NF)-κB
Nuclear factor kappa B
Apoa1
Apolipoprotein A-1
Atp4a
ATPase H+/K+ alpha subunit
B2m
β 2 -microglobulin
Cd74
CD74 antigen
Chil4
Chitinase-like 4
DAVID
Database for Annotation, Visualization and Integrated Discovery
Gbp2
Guanylate binding protein 2
Gif
Gastric intrinsic factor
GO
Gene ontology
Ido1
Indoleamine2, 3- dioxygenase 1
IFN
Interferon
IL-1/IL-8
Interleukin-1/-8
INS-GAS
Insulin-gastrin transgenic gastric mice
KEGG
Kyoto Encyclopedia of Genes and Genomes
MDS
Multi dimensional scaling
MIF
Migration inhibitory factor
Muc5ac
Mucin 5 AC
Rnf213
Ring finger protein 213
STAT3
Signal transducer and activator of transcription 3
TLR
Toll like receptor
Ubd
Ubiquitin d

Background

Gastric cancer is one of the most common causes of cancer-related death worldwide with an estimated 738,000 deaths each year [1]. Recently, H. pylori was recognized as the foremost cause of gastric cancer [27]. With an estimated half of the world’s population being infected, Helicobacter infection contributes significantly to the worldwide gastric cancer burden [7, 8]. Recognition of the factors leading up to the development and progression towards gastric cancer are critical in determination of cancer pathology. H. pylori-induced gastric carcinogenesis involves a multistep progression from normal gastric mucosa to superficial gastritis, chronic gastritis, atrophic gastritis, metaplasia, dysplasia, and finally gastric carcinoma [8, 9]. Molecular events associated with disease progression to gastric malignancy have not been elucidated. Considerable amount of confirmatory evidence shows that host immune response to H. pylori is crucial in determining gastric cancer predisposition [1012]. We have previously shown that a key signal transduction adaptor protein, myeloid differentiation primary response gene 88 (MyD88), regulates Helicobacter-induced gastric cancer progression in a mouse model of gastric cancer [13]. We demonstrated that H. felis-infected MyD88 deficient (Myd88 −/−) mice exhibited severe gastric pathology and an accelerated progression to gastric dysplasia compared to wild type (WT) mice [13] However, the MyD88-dependent gene responsible for this pathology were not described.
MyD88 is a key adaptor molecule that is crucial in mediating innate immune signals from members of the toll-like receptor (TLR) and interleukin-1 (IL-1)/IL-18 families leading to downstream activation of nuclear factor (NF)-κB [1416]. Consistent with involvement in these inflammatory pathways, MyD88 signaling has been associated with cancer progression, which stems from the understanding that inflammation is linked to cancer promotion [17, 18]. Studies on the role of MyD88 cancer progression have been the subject of recent intense investigations. However, the data are contradictory, which indicate that the role of MyD88 in the development and progression of inflammation-associated cancers is complex [19]. Several studies using genetic or chemical carcinogenesis models involving Myd88 deficient mice have shown MyD88 to either promote [2027] or suppress [13, 2834] tumor development. The complex role of MyD88 in carcinogenesis is best typified by studies in colon cancer models [22, 24, 29, 35] showing contradictory roles in the same tissue. The mechanistic basis for these opposing observation is still not fully understood and could be due to many factors including, the type of inflammation, the extent of tissue damage, and immune response elicited [35]. Further, the MyD88 dependent genes in this accelerated progression to dysplasia remain unknown. Therefore, this study was performed to identify potential genes involved in the accelerated progression of gastric cancer.

Results

Gene expression and analysis

Prior to differential gene analysis, all data from 23,015 genes with a standard deviation of less than 0.1 were used for multiple dimensional scaling (MDS) analysis (Fig. 1) to verify that Myd88 −/− and WT samples were differentiated according to gene expression in each sample with a relative p-value. Each sample is represented with distance between each one reflecting their approximate degree of correlation [36]. All genes included in the analysis had a minimum standard deviation of less than 0.1. The analysis showed that all uninfected mice were clustered together irrespective of genetic background or time point. For infected mice, WT and Myd88 −/− mice clustered distinctively separate indicating differential expression of their genes.
Statistical analysis of all 23,015 genes that went through the filtering process identified a total of 286 genes in WT and 4,151 in Myd88 −/− mice in response to H. felis infection with more genes differentially expressed at 47 than 25 weeks (Table 1). Comparing the number of upregulated genes between Myd88 −/− and WT at 47 weeks post infection, there were more upregulated genes (1140) in Myd88 −/− mice compared to WT mice (189 genes). A similar trend was observed for upregulated genes at 25 weeks and for downregulated genes at both time points, with more genes differentially expressed in Myd88 −/− than WT mice in response to H. felis infection. The number of differentiated genes at each time point in comparison to uninfected controls is illustrated in Fig. 2. Most genes overlapped between time points, however, there were a substantial number of genes that were unique to each set of time points that were differentially regulated (Fig. 2).
Table 1
Summary of microarray-based analysis of DEGs
Strain
UP
DOWN
TOTAL
WT 25 weeks
7
3
10
WT 47 weeks
189
87
276
Myd88 −/− 25 weeks
1031
958
1989
Myd88 −/− 47 weeks
1140
1022
2162
Number of differentially regulated genes at 25 weeks and 47 weeks in Myd88 −/− and WT mice infected with H. felis compared to their matched uninfected controls. In total 23,015 genes were analyzed in both mouse backgrounds. Totals depict number of DEGs, both up and downregulated
Scatterplot depiction of differentially expressed genes shows significantly (P < 0.05) up- and downregulated genes in Myd88 −/− mice in response to H. felis infection (Fig. 3). A majority of genes were altered after 25 weeks of H. felis infection. Some of the new additional genes at 47 weeks post-H. felis infection included the ring finger protein 213 (Rnf213) and Furin, which have been reported to be involved in angiogenesis [37, 38] and cancer progression [39], respectively indicating their role in advanced stages of cancer progression.

Analysis of differentially expressed genes

Tables 2 and 3 show a list of the top 50 up- and downregulated genes in Myd88 −/− mice at 25 and 47 weeks post-H. felis infection compared to uninfected controls. The most highly upregulated gene during H. felis infection in Myd88 −/− mice included Chitinase-like (chil4), which is involved in tissue remodeling and wound healing [4042]. Many of the upregulated genes in both 25 weeks and 47 weeks post-H. felis infection involved genes in the H2 Complex (murine major MHC), particularly the class I heavy chains, H2-K and H2-D. The light chain for this MHC complex consists of the β 2 -Microglobulin (B2m) [43]. MHC class II antigen presentation including the CD74 antigen (Cd74) was another gene that was upregulated in response to H. felis infection. High expression of Cd74 has been linked to chronic inflammation and carcinogenesis in the gastrointestinal tract [44]. Another highly expressed gene was Indoleamine 2,3-Dioxygenase 1 (Ido1), which is suggested to play a role in immune tolerance and high expression in colorectal cancer and is correlated with a poor clinical outcome (reviewed in [45]. The entire list of altered genes in response to infection with H. felis including those in WT mice have been submitted and can be uploaded as an excel file in Additional files 1: Table S1 (inf vs uninfectd charts.xlsx).
Table 2
Top 50 most differentially expressed annotated genes in H. felis-infected Myd88 −/− mice at 25 weeks compared to uninfected controls
Symbol
Gene Name
LogFC
Adj. P.Val
Chil4
chitinase-like 4
5.77
4.05E-04
Bpifb1
BPI fold containing family B, member 1
4.90
1.97E-04
Cd74
CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated)
4.28
4.93E-08
H2-Ab1
histocompatibility 2, class II antigen A, beta 1
4.09
2.35E-07
Ido1
indoleamine 2,3-dioxygenase 1
3.88
1.39E-04
H2-DMa
histocompatibility 2, class II, locus DMa
3.34
1.29E-05
H2-DMb2
histocompatibility 2, class II, locus Mb2
3.33
2.22E-06
Igtp
interferon gamma induced GTPase
3.29
1.54E-04
H2-Eb1
histocompatibility 2, class II antigen E beta
3.28
2.51E-06
Gbp2
guanylate binding protein 2
3.27
3.49E-05
Sftpd
surfactant associated protein D
3.26
3.73E-07
Psmb8
proteasome (prosome, macropain) subunit, beta type 8 (large multifunctional peptidase 7)
2.92
3.46E-06
H2-K2
histocompatibility 2, K region locus 2
2.78
2.64E-07
H2-K1
histocompatibility 2, K1, K region
2.64
1.04E-06
B2m
beta-2 microglobulin
2.59
3.46E-06
Pkp4
plakophilin 4
2.54
2.04E-06
H2-DMb1
histocompatibility 2, class II, locus Mb1
2.52
3.46E-06
Ubd
ubiquitin D
2.51
2.90E-04
H2-Aa
histocompatibility 2, class II antigen A, alpha
2.47
2.15E-05
Irgm2
immunity-related GTPase family M member 2
2.42
9.04E-05
H2-Q7
histocompatibility 2, Q region locus 7
2.32
2.22E-06
Cxcl9
chemokine (C-X-C motif) ligand 9
2.30
6.23E-05
Irf1
interferon regulatory factor 1
2.23
7.54E-05
Serpina3g
serine (or cysteine) peptidase inhibitor, clade A, member 3G
2.22
5.85E-06
H2-M2
histocompatibility 2, M region locus 2
2.21
5.85E-06
Casp1
caspase 1
2.17
1.86E-04
H2-D1
histocompatibility 2, D region locus 1
2.17
1.07E-06
Ear2
eosinophil-associated, ribonuclease A family, member 2
2.16
6.91E-04
H2-T23
histocompatibility 2, T region locus 23
2.07
5.85E-06
Psmb9
proteasome (prosome, macropain) subunit, beta type 9 (large multifunctional peptidase 2)
2.05
1.54E-04
Psmb10
proteasome (prosome, macropain) subunit, beta type 10
2.03
5.50E-05
Sgk1
serum/glucocorticoid regulated kinase 1
−2.01
3.48E-04
Apoa4
apolipoprotein A-IV
−2.31
2.33E-05
Ttr
transthyretin
−2.37
2.15E-05
Fabp3
fatty acid binding protein 3, muscle and heart
−2.37
1.61E-04
Muc5ac
mucin 5, subtypes A and C, tracheobronchial/gastric
−2.41
7.14E-04
Smim24
small integral membrane protein 24
−2.61
3.46E-06
Lpl
lipoprotein lipase
−2.75
8.29E-05
Pdia2
protein disulfide isomerase associated 2
−3.08
3.10E-05
Pnliprp1
pancreatic lipase related protein 1
−3.18
5.76E-04
Gm5771
predicted gene 5771
−3.36
3.53E-04
Hamp2
hepcidin antimicrobial peptide 2
−3.43
1.59E-04
Pnliprp2
pancreatic lipase-related protein 2
−3.44
7.47E-04
Rnase1
ribonuclease, RNase A family, 1 (pancreatic)
−3.78
5.55E-04
Try5
trypsin 5
−4.17
7.45E-04
Gm5409
predicted pseudogene 5409
−4.44
4.89E-05
Try4
trypsin 4
−4.87
4.39E-05
Try10
trypsin 10
−5.08
2.69E-06
Amy2a5
amylase 2a5
−6.44
2.22E-06
Chia1
chitinase, acidic 1
−6.68
8.93E-05
Genes with no known annotated name were excluded from the analysis. Genes were considered to be statistically significant when a threshold adjusted p value < 0.05 and Log FC > 2 were reached
Table 3
Top 50 most differentially expressed annotated genes in H. felis-infected Myd88 −/− mice at 47 weeks compared to uninfected controls
Gene Symbol
Gene Name
LogFC
Adj. P.Val
Chil4
chitinase-like 4
4.97
1.24E-03
Cd74
CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated)
4.18
7.41E-08
Bpifb1
BPI fold containing family B, member 1
4.02
6.43E-04
H2-Ab1
histocompatibility 2, class II antigen A, beta 1
3.96
4.14E-07
Ido1
indoleamine 2,3-dioxygenase 1
3.78
1.44E-04
Igtp
interferon gamma induced GTPase
3.29
1.25E-04
Sftpd
surfactant associated protein D
3.24
4.86E-07
H2-DMb2
histocompatibility 2, class II, locus Mb2
3.20
4.43E-06
Psmb8
proteasome (prosome, macropain) subunit, beta type 8 (large multifunctional peptidase 7)
3.20
1.71E-06
Gbp2
guanylate binding protein 2
3.15
4.53E-05
H2-DMa
histocompatibility 2, class II, locus DMa
3.13
2.17E-05
H2-Eb1
histocompatibility 2, class II antigen E beta
3.05
6.39E-06
Ubd
ubiquitin D
2.85
8.22E-05
B2m
beta-2 microglobulin
2.81
4.14E-07
H2-K2
histocompatibility 2, K region locus 2
2.58
7.37E-07
H2-Aa
histocompatibility 2, class II antigen A, alpha
2.56
1.39E-05
Irgm2
immunity-related GTPase family M member 2
2.53
5.62E-05
H2-DMb1
histocompatibility 2, class II, locus Mb1
2.47
5.72E-06
H2-K1
histocompatibility 2, K1, K region
2.40
3.89E-06
Serpina3g
serine (or cysteine) peptidase inhibitor, clade A, member 3G
2.30
4.90E-06
Psmb9
proteasome (prosome, macropain) subunit, beta type 9 (large multifunctional peptidase 2)
2.30
4.92E-05
Psmb10
proteasome (prosome, macropain) subunit, beta type 10
2.29
1.49E-05
Pigr
polymeric immunoglobulin receptor
2.29
2.37E-05
Oasl2
2′-5′ oligoadenylate synthetase-like 2
2.24
6.52E-04
H2-Q7
histocompatibility 2, Q region locus 7
2.22
4.90E-06
Casp1
caspase 1
2.20
1.33E-04
Irf1
interferon regulatory factor 1
2.17
8.33E-05
C3
complement component 3
2.15
4.52E-05
Cxcl9
chemokine (C-X-C motif) ligand 9
2.14
1.04E-04
Pkp4
plakophilin 4
2.11
1.28E-05
Rnf213
ring finger protein 213
2.08
1.59E-04
H2-D1
histocompatibility 2, D region locus 1
2.04
2.78E-06
Ear2
eosinophil-associated, ribonuclease A family, member 2
2.04
9.57E-04
Sst
somatostatin
−2.01
1.25E-04
Smim24
small integral membrane protein 24
−2.11
3.12E-05
Muc5ac
mucin 5, subtypes A and C, tracheobronchial/gastric
−2.23
1.14E-03
Lpl
lipoprotein lipase
−2.32
3.50E-04
Sgk1
serum/glucocorticoid regulated kinase 1
−2.33
8.22E-05
Ckb
creatine kinase, brain
−2.35
1.02E-04
Gif
gastric intrinsic factor
−2.36
3.50E-04
Cox7a1
cytochrome c oxidase subunit VIIa 1
−2.49
1.06E-04
Ttr
transthyretin
−2.57
9.85E-06
Dpcr1
diffuse panbronchiolitis critical region 1 (human)
−2.57
1.05E-03
Fabp3
fatty acid binding protein 3, muscle and heart
−2.61
6.23E-05
Atp4a
ATPase, H+/K+ exchanging, gastric, alpha polypeptide
−2.95
6.99E-04
Apoa1
apolipoprotein A-I
−3.13
1.23E-03
Hamp2
hepcidin antimicrobial peptide 2
−3.45
1.25E-04
Pdia2
protein disulfide isomerase associated 2
−3.57
7.21E-06
Amy2a5
amylase 2a5
−4.09
1.74E-04
Chia1
chitinase, acidic 1
−5.25
6.66E-04
Genes with no known annotated name were excluded from the analysis. Genes were considered to be statistically significant when a threshold adjusted p value < 0.05 and Log FC > 2 were reached
STRING summary networks depicting protein- protein interactions among the top differentially expressed genes (DEGs) for both up- and downregulated genes in Myd88 −/− mice are shown in Figs. 4 and 5 for 25 and 47 weeks, respectively. Thicker lines connecting the genes indicate a stronger association between the genes. A confidence score of at least 0.70 (high) was used. One of the key central nodes in the top DEGs in Myd88 −/− mice at both 25 and 47 weeks post-infection was guanylate-binding protein 2 (Gbp2), which is considered a potential marker for esophageal squamous cell carcinoma [46].

Functional enrichment analysis of differentially expressed genes (DEGs)

To gain insights into the biological meaning and function of the differentially expressed genes, enrichment analysis was performed using the database for annotation, visualization and integrated discovery (DAVID) online analytical tools [4749]. Annotation according to tissue expression, molecular function, cellular component and biological processing was done using Gene Ontology (GO) [49]. Enrichment analysis was performed to identify pathways, processes and gene categories that are over-represented in the list of DEGs compared to the mouse genome. GO clustering analysis for biological processes showed that responses related to immune system processes were the most upregulated enriched process in Myd88 −/− mice at both 25 and 47 weeks in response to H. felis infection (Fig. 6a and c). For molecular functions, antigen and protein complex binding were the most enriched processes (Fig. 6b and d). Downregulated enriched processes in Myd88 −/− mice in response to H. felis infection are presented as supplementary data (Additional files 2: Figure S1). A summary of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation shown in Fig. 7 revealed that the most enriched pathway was antigen processing and presentation. This pathway shared the majority of its genes with the other major pathways and completely engulfed the other pathways by sharing more than 90% of the genes annotated. A breakdown of up- and downregulated KEGG pathway at 25 and 47 weeks in Myd88 −/− mice in response to H. felis infection is presented as supplementary data (Additional files 3: Figure S2).

Discussion

Gastric cancer develops and progresses through a stepwise sequence of events from inflammation to atrophy, metaplasia, dysplasia, and finally to gastric cancer [50]. We previously demonstrated using a mouse model of gastric that mice deficient in MyD88 signaling exhibited dramatic pathology and an accelerated progression to gastric neoplasia in response to H. felis infection [13]. In the present study, we used microarray gene expression analysis to identify the genes involved in this progression to gastric neoplasia. Although previous studies have investigated differential gene expression in mice stomachs in response to Helicobacter infection, most have focused on H. pylori [5153], which does not result in neoplastic changes in mice [54, 55]. The few studies that have examined gene expression profiles in mouse model of gastric cancer have used the insulin-gastrin (INS-GAS) transgenic gastric cancer mouse model [56, 57]. These mice have been shown to spontaneously develop gastric cancer even in the absence of Helicobacter infection [58]. We have previously reported that Myd88 −/− mice do not exhibit abnormal pathology in the absence of Helicobacter infection [13]. The global transcriptional profiling of mouse gastric tissue identified a large number of significant differentially expressed genes in H. felis-infected Myd88 −/− mice compared to H. felis-infected WT mice. The most over expressed gene in Myd88 −/− mice during H. felis infection at 25 weeks was Chil4. Chitinase like proteins (CLPs) have been studied in relation to other cancers yet little has been investigated in relation to gastric cancer except for our present study and a couple other studies [57, 59]. Upregulation of CLPs has been shown in a number of human cancers including brain, bone, breast, ovaries, lung, prostate, colon, thyroid, and liver [41, 60]. For gastric cancer studies, chitinase protein 3 like 1 (Chil1) was upregulated in INS-GAS mice infected with H. felis, [57]. In our present study, Chil1 was not upregulated in response to H. felis infection. However, in addition to upregulation of Chil4, another CLP, Chil3 was also significantly over expressed in H. felis-infected mice at both 25 weeks (p = 0.03) and 47 weeks (p = 0.01) (gene not listed in Tables 2 and 3, only the top 50 are listed). An abundant over expression of Chil1, Chil4 as well as Chil3 has been reported in early preneoplastic stage in the epidermis [61]. Overall, CLPs have been implicated to play a role in chronic inflammation, tissue remodeling, and wound healing [40]. Up-regulation of genes involved in tissue remodeling is noteworthy because chronic inflammation and subsequent damage to the gastric epithelium has been suggested to play an important role in cancer development and progression [62]. During chronic inflammation, the resulting prolonged tissue damage creates a loss of control over normal tissue repair mechanisms resulting in persistent hyper-tissue repair, which is accompanied with sustained proliferation [63] and ultimately advancing to precancerous lesions. Lost tissue is then replaced with stem and progenitor cells that are under a continuous stimulus of proliferation, leading to the accumulation of replacement cells with dysregulated and altered signaling pathways [63]. Further, studies investigating associations between chronic inflammation, tissue repair and carcinogenesis highlight the potential of these cellular changes in inducing both pro-oncogenic and tumor suppressor pathways [62, 6467]. Our study, in addition to the work done by Li et al. [59] and Takaishi and Wang [57] show a need for further investigation into the role of CLPs in gastric carcinogenesis.
Other upregulated genes included Cd74, B2m, and interferon (IFN) induced genes such as GTPases (interferon gamma induced GTPase, lgtp, immune mediated GTPase family M member 2, lrgm2), Guanylate binding protein 2 (Gbp2), and transcription factor interferon regulatory factor 1 (Irf1). Cd74 or invariant chain (Ii) protein is a chaperone molecule responsible for regulating antigen presentation of MHC II molecules. It has been linked to chronic inflammation and carcinogenesis in the gastrointestinal tract [44]. Further, Cd74 was also shown to play a role as a receptor for migration inhibitory factor (MIF), a molecule reported to have pro-carcinogenic effects on gastric epithelial cells [68]. IFNs are known to activate signal transducer and activator of transcription 3 (STAT3) [69, 70] signaling leading to epithelial proliferation and inhibition of apoptosis [71, 72]. Currently not much is known about IFNs in gastric cancer. Ubiquitin D (Ubd), which is associated with progression of colon cancer [73], was also significantly expressed genes in Myd88 −/− in response to H. felis infection.
For downregulated genes, the significantly expressed ones included, ATPase H+/K+ transporting, alpha subunit (Atp4a), Atp4b, Mucin 5 AC (Muc5ac), apolipoprotein A-1 (Apoa1), and gastric intrinsic factor (Gif). The genes, Atp4a and Atp4b encode gastric H+/K + − ATPase alpha and beta subunits, respectively. Gastric H+/K + − ATPase alpha and beta subunits are expressed in parietal cells [74] and their loss has been associated with gastric dysplasia [58]. We observed downregulation of Atp4a and Atp4b in response to infection with H. felis, which may represent a loss of parietal cells that has been shown to precede gastric dysplasia. These results are in line with those observed in another fast progressing gastric cancer model involving the use of INS-GAS mice [58]. Muc5ac, which encodes gastric M1 mucin [75] has been reported to play a role in gastric carcinogenesis [76] was also downregulated in response to infection with H. felis in Myd88 −/− mice. Progression of gastric lesions has been reported to be associated with the gradual decrease in expression of Muc5ac [57, 7779] followed by the transformation of the gastric epithelium [80] resulting in gastric dysplasia. Another highly downregulated gene we found in Myd88 −/− mice infected with H. felis was Apoa1, which was also reported to be downregulated in a fast progressing gastric cancer mouse model [57]. Proteomics approach in human gastric cancer also showed downregulation of Apoa1 [81], but its role in gastric carcinogenesis is unknown. In the lung, downregulation of Apoa1 was associated with an increased risk of lung cancer [82]. Data from Apoa1-deficient mice suggest antitumorigenic properties of Apoa1 via modulation of the immune system [83]. Gastric intrinsic factor (Gif), another downregulated gene is secreted by parietal cells and is required for Vitamin B12 absorption [84]. Concomitantly, the downregulation of Gif results in vitamin B12 deficiency (pernicious anemia). Gastric intrinsic factor was downregulated in H. felis-infected Myd88 −/− mice at both 25 and 47 weeks post-infection. A previous study using INS-GAS mice infected with H. felis also reported downregulation of Gif [57]. In human gastric cancer, Gif was one of the genes found by SAGE analysis to be downregulated [85]. Further, studies have shown an increased risk of gastric cancer in pernicious anemic patients [86].
We found a number of new genes in our fast progressing gastric mouse model, i.e., Myd88 −/− mice infected with H. felis. The genes included up- and downregulated genes, which had not been previously linked to Helicobacter-related gastric carcinogenesis including BPI fold containing family B member 1 (Bpifb1) and proteasome subunit beta 8 (Psmb8). These genes have been linked to cancer-related processes including, apoptosis and in some cases other cancers as well as prognosis indicators [87, 88]. Psmb8 was shown to be significantly up regulated in cancers such as bladder, breast, kidney, lung, uterine, and head and neck [89]. A recent study by Kwon, et al. [90], which was published during the writing of our manuscript reported that Psmb8 may be a potential marker for prognosis in gastric cancer. Bpifb1 may be involved in the innate immune response particularly in response to bacterial exposure. The protein encoded for by Bpifb1 binds bacterial lipopolysaccharide (LPS) as well as modulates the cellular response to LPS [91]. Bpifb1 has been found to be overexpressed in mucous cells of salivary gland tumors of papillary cystadenocarcinoma [87]. Future studies using a gastric culture organoid system will validate these genes and some of the important novel genes we identified for their role in rapid progression of Helicobacter-induced gastric cancer.

Conclusions

In this study, we have identified genes that are involved in the rapid progression of Helicobacter-induced gastric cancer that are also potentially regulated by MyD88. The identification of these important genes could potentially serve as targets for disease prevention. In addition, we show that our model is a useful mouse model system to identify genes involved in gastric cancer progression.

Methods

Animals

Six- to ten- week-old wild type (WT) and MyD88 deficient (Myd88 −/−) mice in the C57BL/6 background were used in this study. WT mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Myd88 −/− mice were from our breeding colony originally provided by Dr. Akira (Osaka University, Japan). All mice were housed together before infection with H. felis and for the duration of the study. The Institutional Animal Care and Use Committee at the University of California, San Diego, approved all animal procedures and performed using accepted veterinary standards.

Bacterial growth conditions

Helicobacter felis, strain CS1 (ATCC 49179) was purchased from American Type Culture Collection (Manassas, VA). H. felis was routinely maintained on solid medium, Columbia agar (Becton Dickinson, MD) supplemented with 5% laked blood under microaerophilic conditions (5% O2, 10% CO2, 85% N2) at 37 °C and passaged every 2–3 days as described previously [13, 92]. Prior to mouse infections, H. felis was cultured in liquid medium, brain heart infusion broth (BHI, Becton Dickinson) supplemented with 10% fetal calf serum and incubated at 37 °C under microaerophilic conditions for 48 h. Spiral bacteria were enumerated using a Petroff-Hausser chamber before infections.

Mouse infections

A well-characterized cancer mouse model, which involves infecting C57BL/6 mice with H. felis (strain CS1), a close relative of the human gastric pathogen H. pylori was used in this study. Mice were inoculated with 109organisms in 300 μL of BHI by oral gavage three times at 2-day intervals as previously described [13, 92]. Control mice received BHI only. At 25 and 47 weeks post-infection, mice were euthanized and the stomachs removed under aseptic conditions and processed for assessment of gene expression.

RNA extraction and oligonucleotide microarray hybridization

Total RNA was extracted from gastric tissue obtained from H. felis-infected and uninfected WT and Myd88 −/− mice. Stomach tissue sections of 12 mice i.e., two uninfected controls and four infected WT (6) and MyD88 −/− (6) per time point (25 and 47 weeks) were analyzed. RNA was extracted from each section using the RNeasy miniprep kit (Qiagen) according to the manufacturer’s instructions followed by digestion with DNase 1 to remove genomic DNA. RNA concentration was determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Inc., Waltham, MA). Double stranded cDNA and biotin-labeled cRNA were synthesized following the recommended Illumina protocol. Integrity of purified cRNAs was assessed on an Agilent 2100 Bioanalyzer prior to hybridization. 1.5 μg of labeled cRNA was hybridized to MouseWG-6 v2 Expression BeadChips genome wide arrays, which analyzes 25,600 transcripts (Illumina, San Diego, CA) using recommended Illumina reagents and protocols.

Identification of differentially expressed genes

Probe profiles (each row corresponding to a given probe and different columns for each sample) were exported from Genome Studio v1.8 (Illumina). The resultant tab-delimited file (Additional file 4, Probe_Profile_f.txt) was used as input for the Bioconductor lumi v2.18 R package (http://​bioconductor.​org/​packages/​release/​bioc/​html/​lumi.​html) [93]. Sample information is provided in Additional file 5 (sampleInfotxt.txt). Following quality assessment [density plot of intensity, cumulative density (CD), MA (transformed data onto log ratios and mean average) and pairwise MDS plots], the data were transformed using the vst (variance-stabilizing transformation) algorithm [94] then normalized using the Robust Spline Normalization (rsn) algorithm [94]. A second round of quality control of the normalized data was done to ensure data quality. After normalization genes with minimal variance across samples and genes that were not expressed in any sample (determined by detection calls) were removed prior to differential gene expression using linear models as implemented in limma [95]. We compared gene expression profiles of uninfected and infected mice at 25 and 47 weeks post infection. All mice used were on the C57BL/6 background. In addition, after normalization, significantly expressed genes were identified through volcano plot generation on the R - platform. Genes exhibiting statistically significant differential mean expression values (p < 0.05) were subjected to hierarchical clustering.

Enrichment analysis and pathway generation

To analyze KEGG pathway enrichment, official gene symbols for differentially expressed genes were submitted to DAVID (Database for Annotation, Visualization and Integrated Discovery) (http://​david.​abcc.​ncifcrf.​gov). This tool allows for identification of over expression within a set of genes as compared to the entire genome of a specific animal [47]. In addition, it allows for the demonstration of Gene Ontology (GO) terms as well as pathway and functional processes (molecular functions, biological expression, and cellular expression) enrichment. Functionally enriched gene networks, KEGG Pathways and ontology terms were identified. The mouse, Mus musculus complete genome was used as background genes. All functionally annotated genes presented in gene networks or pathways had p values < 0.05.

Gene network analysis

Gene networks were built using data on protein-protein interactions from EMBL STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database (http://​string-db.​org). This search tool is used to identify interactions correlated to expression data and/or literature citations among other criteria [96]. All gene connections created using STRING had a combined confidence score higher than 0.7 as previously described [97], Garcia-Alonso, 2014 #613}.

Statistical analysis

Statistics were done on the R platform using the Limma package from Bioconductor. To control for multiple testing, the False Discovery Rate (FDR) method was used with a cutoff for statistical significance of P values of < 0.05 and a log fold expression of 2. Differentially expressed genes were determined at 25 and 47 weeks after removing background differences from both Myd88 −/− and WT mice by comparing infected to uninfected mice in the same background.

Acknowledgements

The authors thank the Biomedical Genomics Laboratory (BIOGEM), UCSD for microarray analysis.

Funding

This work was supported in part by grants from the National Institutes of Health (NIH) U54CA132384, U54CA132379, R21CA188752, and R21AI115273-02.

Availability of data and materials

All data analyzed during this study are included in this published article and as supplementary material.

Authors’ contributions

MO and KD conceived and designed the study; AS carried out the experiments; IL performed database searches; IL and MM analyzed the microarray data; MO helped draft the manuscript; IL wrote the manuscript and was edited by MO. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Animals were studied under a research protocol approved by the Institutional Care and Use Committee (IACUC) of the University of California, San Diego.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://​creativecommons.​org/​licenses/​by/​4.​0/​), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated.
Zusatzmaterial
Additional files 1:Table S1. Excel file: Data of upregulated and downregulated genes in Myd88 −/− and WT mice at 25 and 47 weeks. (XLSX 4782 kb)
12885_2017_3114_MOESM1_ESM.xlsx
Additional files 2:Figure S1. Downregulated biological processes and molecular functions in Myd88 −/− mice. Enriched Go terms are shown at both 25 (A, B) and 47 weeks (C, D). Biological processes are depicted in figures A and C while molecular functions are depicted in B and D. These functions were identified using STRING functional annotation tool. Relative scores were calculated using the number of genes found within each process relative to the total number of genes entered into the annotation tool. The top 20 Biological Processes are shown. (PPTX 482 kb)
12885_2017_3114_MOESM2_ESM.pptx
Additional files 3:Figure S2. KEGG Pathways for both up and downregulated Genes at 25 and 47 weeks in Myd88 −/− mice. KEGG pathway analysis of up- and downregulated genes in Myd88 −/− mice at 25 (Additional files 3: Figure S2a and b) and 47 weeks (Additional files 3: Figure S2c and d). Significantly enriched pathways ( p < 0.05) are presented in each pie graph. (PPTX 1888 kb)
Additional files 4: Text File: Probe Profile of Microarray Data used in analysis of gene expression. (TXT 30459 kb)
12885_2017_3114_MOESM4_ESM.txt
Additional files 5: Text file: Sample Info file listing name and ID of each mouse used in the analysis. (TXT 533 bytes)
12885_2017_3114_MOESM5_ESM.txt
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