Data-independent acquisition (DIA) mass spectrometry reveals related proteins involved in the occurrence of early intestinal-type gastric cancer

doi:10.21203/rs.3.rs-3129410/v1

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Data-independent acquisition (DIA) mass spectrometry reveals related proteins involved in the occurrence of early intestinal-type gastric cancer

https://doi.org/10.21203/rs.3.rs-3129410/v1

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Background: The identification of proteins involved in the occurrence of early intestinal-type gastric cancer (EIGC) may provide valuable insights into the pathogenesis of intestinal gastric cancer.

Methods: Data-independent acquisition mass spectroscopy (DIA-MS) was utilized to identify the differential protein between 10 cases of EIGC and atrophic gastritis with intestinal metaplasia (NGC). The expressions of IPO4, TBL1XR1, p62/SQSTM1, PKP3, and CRTAP were verified by immunohistochemistry (IHC) in 20 EIGC samples, 17 gastric low-grade intraepithelial neoplasia (LGIN), and 21 healthy controls. The prognostic values of the five genes were validated in the transcriptome data by survival analysis.

Results: A total of 4,028 proteins were identified using DIA-MS, with fold change> 1.5 times as a significant difference, and a total of 177 different proteins were screened. Among them, 113 proteins were significantly up-regulated in EIGC tissues, and 64 proteins were significantly down-regulated in EIGC tissues. IHC results showed that proteins IPO4, TBL1XR1, p62/SQSTM1, PKP3, and CRTAP were highly expressed in the cytoplasm of EIGC and LGIN, which was consistent with the quantitative results of DIA-MS. Among them, p62/SQSTM1 may undergo nuclear-cytoplasmic transfer. The expression of noncancerous gastric mucosa was different from LGIN and EIGC, while LGIN was similar to EIGC. The five protein coding genes were associated with intestinal-type gastric cancer survival and differentially expressed in different stages.

Conclusion: The study successfully identified differentially expressed proteins between EIGC and NGC, which may provide valuable insights into the mechanism of intestinal-type gastric cancer. Additionally, the study highlights the risk of some LGIN developing into invasive gastric cancer, which warrants further attention.

Pathway

proteome

pathogenesis

molecular mechanisms

diagnosis

biomarker

Gastric cancer, also known as stomach cancer, is a common type of cancer that affects the lining of the stomach. It accounts for approximately 5% of all cancer cases worldwide (Ferlay et al., 2015). Gastric cancer is often diagnosed at an advanced stage, with lymph node metastasis present in up to 50% of patients at initial presentation (Ajani et al., 2013). The disease is also known for its insidious onset, making it difficult to detect in its early stages (Fujiyoshi et al., 2021). These factors contribute to the high mortality rate associated with gastric cancer, with a 5-year overall survival rate of only 30–40% (Wagner et al., 2017). Treatment options for gastric cancer include surgery, chemotherapy, and radiation therapy, but the effectiveness of these treatments depends on the stage of the disease at diagnosis (Lordick et al., 2022). According to Lauren's classification, gastric cancer is divided into diffuse-type and intestinal-type gastric cancer (IGC) (Lauren, 1965). IGC is characterized by a clear duct structure, with tumor cells that are columnar or cuboid, similar in structure to intestinal cancer (Correa, 2013). The etiology and pathogenesis of IGC differ from those of diffuse-type gastric cancer, and its incidence is significantly higher. The occurrence of IGC involves multiple steps, including normal gastric mucosa, chronic gastritis, atrophic gastritis, intestinal metaplasia, dysplasia, and intestinal gastric cancer (Correa, 2013). The histopathological diagnosis of early gastric cancer has been approached differently by Japanese and Western pathologists (Sekine et al., 2016). In general, Japanese pathologists diagnose early gastric cancer based on cell atypia and tissue structure atypia, without the need for infiltrating conditions. In contrast, Western pathologists believe that conditions for infiltration are necessary (Raftopoulos et al., 2012). As a result, the Japanese definition of early gastric cancer places greater emphasis on the concept of early-stage. Therefore, the definition of well-differentiated gastric cancer in Japan is broader, including well-differentiated gastric cancer, high-grade intraepithelial neoplasia (HGIN) in the WHO classification, and even some low-grade intraepithelial neoplasia (LGIN) (Schlemper et al., 2001). Early lesions can be removed endoscopically, which is more cost-effective and has a higher survival rate.

Gastric cancer can benefit from the high throughput capabilities of proteomics method, data-independent acquisition (DIA), which offers unbiased and comprehensive protein quantitation of tissue samples (Ludwig et al., 2018). Quantitative proteomics can accurately examine and differentiate molecular markers that are diagnostic or prognostic (Domon and Aebersold, 2006). Proteomics techniques have been used to study gastric cancer using various specimens, including cell lines, serum, tissues, and more (Balluff et al., 2011;Kuramitsu et al., 2010;Liu et al., 2014). Currently, most researchers use surgically resected freshly advanced gastric cancer tissues as specimens (Aquino et al., 2012;Zhou et al., 2016). However, selecting fresh early-stage gastric cancer tissue can better reflect the biological changes that occur during this critical stage. Early gastric cancer is located in the gastric mucosa and submucosa, and biopsy forceps can obtain the gastric mucosa and part of the submucosa tissue. The protein profile obtained using gastroscopic biopsy specimens can distinguish gastric cancer from normal specimens (Kim et al., 2010).

The present study employs gastroscopic biopsy forceps to obtain fresh tissue and utilizes DIA mass spectrometry technique to screen for differential proteins. The unique aspect of this experiment lies in the selection of two crucial stages of atrophic gastritis with intestinal metaplasia and early intestinal gastric cancer (EIGC). We hypothesized that subtle changes in proteins during these two closely linked stages may play a pivotal role in carcinogenesis. The differential proteins are important for the investigation of the mechanism underlying EIGC. Furthermore, the study provides a protein perspective to support one side of the dispute between Japanese and Western pathologists.

Patients

Ten patients diagnosed with EIGC were recruited at Lihuili Hospital of Ningbo Medical Center between December 2019 and October 2020. Patients with a history of cancer treatment or severe chronic diseases were excluded from the study. The patient's demographic and clinical information is presented in Table 1. The experimental protocol was approved by the Ethics Committee of Ningbo Medical Center Lihuili Hospital (KY2019PJ055). Informed consent was obtained from all participating patients or their family members.

Table 1

Clinical data of 10 patients with early intestinal-type gastric cancer
No.	Sex	Age	Pathological classification	H. pylori infection
1	Woman	68	Well-differentiated adenocarcinoma	Positive
2	Woman	65	Well-differentiated adenocarcinoma	Positive
3	Man	59	Medium-differentiated adenocarcinoma	Positive
4	Man	71	Medium-differentiated adenocarcinoma	Positive
5	Man	61	Well-differentiated adenocarcinoma	Positive
6	Man	66	High-grade intraepithelial neoplasia	Positive
7	Man	63	Well-differentiated adenocarcinoma	Positive
8	Man	61	Medium-differentiated adenocarcinoma	Positive
9	Man	56	Medium-differentiated adenocarcinoma	Positive
10	Man	80	Well-differentiated adenocarcinoma	Positive

Histopathological diagnosis

The diagnosis of gastric cancer was typically based on a combination of endoscopic observations and biopsy histopathological results. In cases where EIGC was suspected, we carefully observed the lesion during gastroscopy to determine whether it is located in the mucosal background of atrophic gastritis with intestinal metaplasia. This approach was used to ensure accurate diagnosis and appropriate treatment planning for patients with this condition (Kaise et al., 2009). Biopsy procedures were performed by two experienced endoscopists. Tissue samples was obtained from both gastric cancer tissue and adjacent tissue located 2 cm from the edge of the tumor. The collected tissue samples were then fixed, embedded, and stained with Hematoxylin-Eosin for histopathological analysis. The diagnosis of intestinal metaplasia was based on the updated Sydney System (Dixon et al., 1996), while the diagnosis of intestinal-type gastric cancer was based on the Lauren classification (Lauren, 1965). The staging of intestinal-type gastric cancer was confirmed as early stage based on the histopathological results of specimens obtained through endoscopic submucosal dissection (Japanese Gastric Cancer, 2011). The histopathological interpretation was conducted by experienced pathologists in a blinded manner to ensure objectivity and accuracy. Additionally, biopsy tissue samples were quickly rinsed with pre-cooled physiological saline to remove surface blood and mucus, and then placed in marked cryotubes for rapid freezing in a liquid nitrogen tank. The entire process was completed within 2 minutes to ensure the integrity of the tissue samples. The cryopreserved samples were then stored in a -80 ℃ refrigerator for subsequent DIA-MS quantitative analysis. These standardized procedures were employed to ensure the reliability and reproducibility of the experimental results.

Tissue samples

To conduct DIA-MS analysis, a total of 20 tissue samples were collected and divided into two groups: 1) early intestinal-type gastric cancer samples (EIGC, n = 10), and 2) intestinal metaplasia samples (NGC, n = 10). This approach was employed to enable a comparative analysis of the proteomic profiles of these two distinct tissue types.

Filter-aided sample preparation (FASP) digestion procedure

Equal quantities of proteins were extracted from each tissue sample and incorporated into SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0). The detergent, DTT, and other low-molecular-weight components were subsequently removed using UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0) through repeated ultrafiltration using Microcon units with a 10 kD cutoff. To block reduced cysteine residues, 100 µL of iodoacetamide (100 mM IAA in UA buffer) was added to each sample, which was then incubated in darkness for 30 minutes. The filters were washed with UA buffer three times and 25 mM NH₄HCO₃ buffer twice. The protein suspensions were then digested with 4 µg trypsin (Promega) in 40 µL 25 mM NH₄HCO₃ buffer overnight at 37°C, and the resulting peptides were collected as a filtrate. The peptides from each sample were desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL, Sigma), concentrated by vacuum centrifugation, and reconstituted in 40 µL of 0.1% (v/v) formic acid. The peptide content was estimated by UV light spectral density at 280 nm using an extinction coefficient of 1.1 of 0.1% (g/L) solution that was calculated based on the frequency of tryptophan and tyrosine in vertebrate proteins.

The digested pool peptides underwent fractionation into 10 fractions using the High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific Pierce™). Each fraction was subsequently concentrated through vacuum centrifugation and reconstituted in 15 µL of 0.1% (v/v) formic acid. The collected peptides were then desalted using C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL, Sigma) and reconstituted in 40µL of 0.1% (v/v) formic acid. To correct the relative retention time differences between runs, iRT-Kits (Biognosys) were utilized with a volume proportion of 1:3 for iRT standard peptides versus sample peptides.

Data dependent acquisition (DDA) mass spectrometry assay

For the generation of the DDA library, all fractions were injected into a Q Exactive HF-X mass spectrometer (Thermo Scientific), which was connected to an EASY-nLC 1200 chromatography system (Thermo Scientific). The peptide was initially loaded onto an EASY-Spray™ C18 Trap column (Thermo Scientific, P/N 164946, 3 µm, 75 µm×2 cm), followed by separation on an EASY-Spray™ C18 LC Analytical Column (Thermo Scientific, ES802, 2 µm, 75 µm×25 cm) using a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min over 90 min. The MS detection method employed was positive ion, with a scan range of 300–1800 m/z. The resolution for the MS1 scan was 60000 at 200 m/z, with a target of automatic gain control (AGC) set at 3×10⁶ and a maximum ion injection time (IT) of 25 ms. Dynamic exclusion was set at 30.0 s. Each full MS-SIM scan was followed by 20 ddMS2 scans, with a resolution for MS2 scan set at 15000, AGC target at 5×10⁴, maximum IT at 25 ms, and the normalized collision energy at 30 eV.

Data independent acquisition (DIA) mass spectrometry assay

The peptides from each sample were subjected to LC-MS/MS analysis in the DIA mode by Shanghai Applied Protein Technology Co., Ltd. The DIA cycle consisted of one full MS-SIM scan, and 30 DIA scans was performed to cover a mass range of 350–1800 m/z. The SIM full scan resolution was set at 120,000 at 200 m/z, with an AGC of 3×10⁶ and a maximum IT of 50 ms in profile mode. The DIA scans were set at a resolution of 15,000, with an AGC target of 3×10⁶ and Max IT auto. The normalized collision energy was set at 30 eV. The runtime was 90 min, with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min. Quality control (QC) samples, which were pooled from an equal aliquot of each sample in the experiment, were injected with DIA mode at the beginning of the MS study and after every 6 injections throughout the experiment to monitor the MS performance.

Mass spectrometry data analysis

The Spectronaut™ 14.4.200727.47784 (Biognosys) software was used to search the FASTA sequence database for DDA library data. The database was downloaded from the website http://www.uniprot.org, and the iRT peptides sequence was added (Biognosys, iRT Kit). The parameters were set as follows: trypsin was used as the enzyme, with a maximum of 2 missed cleavages. Carbamidomethyl (C) was set as the fixed modification, while oxidation (M) and acetyl (Protein N-term) were set as dynamic modifications. All reported data were based on 99% confidence for protein identification, as determined by a false discovery rate (FDR) of ≤ 1%. FDR was calculated by FDR\(=\frac{\text{N}\left(\text{d}\text{e}\text{c}\text{o}\text{y}\right)\times 2}{\text{N}\left(\text{d}\text{e}\text{c}\text{o}\text{y}\right)+\text{N}\left(\text{t}\text{a}\text{r}\text{g}\text{e}\text{t}\right)}\times 100\text{%}\). The spectral library was constructed by importing the original raw files and DDA searching results into Spectronaut Pulsar X ^TM 12.0.20491.4 (Biognosys).

The DIA data was analyzed using Spectronaut™ 14.4.200727.47784, which searched the spectral library constructed as described above. The main software parameters were set as follows: the retention time prediction type was dynamic iRT, interference on MS2 level correction was enabled, and cross-run normalization was enabled. All results were filtered based on a Q value cutoff of 0.01, which is equivalent to an FDR of ≤ 1%.

Immunohistochemistry

We selected 21 normal gastric mucosal wax blocks (NGC), including 11 cases of intestinal metaplasia and 10 cases of chronic gastritis. Additionally, we selected 17 LGIN wax blocks and 20 EIGC wax blocks (including HGIN and intramucosal carcinoma) for immunohistochemical (IHC) detection. The following primary antibodies were used: anti-TBL1XR1 (1:200; Proteintech, Wuhan, CHN; 55312-1-AP; RRID:AB2881304), anti-p62/SQSTM1 (1:200; Proteintech, Wuhan, CHN; 18420-1-AP; RRID:AB10694431), anti-PKP3 (1:400; Proteintech, Wuhan, CHN; 18338-1-AP; RRID:AB2164281), anti-CRTAP (1:400; Proteintech, Wuhan, CHN; 15724-1-AP; RRID:AB2261089), and anti-IPO4 (1:400; Proteintech, Wuhan, CHN; 11679-1-AP; RRID:AB_2127677). The intensity of the IHC staining and the percentage of positive cells was evaluated by two pathologists. The percentages of positive tumor cells were scored as 0 (< 5%), 1 (6–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). For statistical evaluation, slides with a final score of ≥ 3 were considered positive by multiplying the quantity and intensity scores; otherwise, they were considered negative.

Statistical analysis

The difference between the experimental groups was analyzed using the chi-square test. All analyses were performed using SPSS24.0 software (SPSS Inc., Chicago, IL, USA). The relative expression level of differential protein of IHC data was tested using the chi-square test. The correlation of IHC with clinical-pathological characteristics was tested using Fisher's exact test. The significance of changes in protein abundance was calculated using the non-parametric Student's t-test and multiple test correction using Bonferroni. A P-value of ≤ 0.05 was considered significant. Gene level survival analysis was performed using the Kaplan-Meier Plotter tool (Szasz et al., 2016). GC stage specific gene expression analysis was executed using the UALCAN database (Chandrashekar et al., 2022).

Proteome profiles of early intestinal-type gastric cancer

The identification of proteins in cancer and adjacent tissues from 10 patients with EIGC was conducted using DIA. The samples were divided into two groups, namely the EIGC group and the NGC group, and were numbered 1–10. The numbers of identified and quantified peptides and proteins were similar across 20 samples (Fig. 1A). A total of 4,028 proteins were identified, among which 3,819 proteins were identified in both groups (Fig. 1B). Principal component analysis (PCA) showed that within-group samples are clustered with each other (Fig. 1C). Overall, these results suggest the reproducibility of the proteome data.

Differential protein analysis

The criteria used for differential protein screening were fold change ≥ 1.5 and P value ≤ 0.05. A total of 177 differentially expressed proteins were identified, including 113 up-regulated proteins and 64 down-regulated proteins (Fig. 1D). The top 10 most significantly up- and down-regulated proteins includes TBL1XR1, PRPF8, MPHOSPH10, NEK7, p62/SQSTM1, AGPS, MYO5B, PTP4A2, CNBP, and EIF2AK2 (Fig. 1E). A hierarchical clustering algorithm was used to group the samples. Based on the differential proteins, the EIGC group and the NGC group were effectively separated (Fig. 1F). This indicates that the differential proteins can represent the protein expression characteristics of EIGC and NGC.

Functional enrichment analysis

Gene Ontology (GO) analysis was performed to characterize the differential proteins (Ashburner et al., 2000). The number of differentially expressed proteins was counted at the GO Cellular Component level (Fig. 2A). The enrichment of GO Biological Process was analyzed for up- and down-regulated respectively. The up-regulated proteins were enriched with rRNA metabolism and defense against fungus (Fig. 2B), while the down-regulated proteins were enriched with muscle system and branched-chain amino acid metabolism (Fig. 2C). All the differential proteins were enriched with myofibril, lysosome, and extracellular matrix (Fig. 2D).

Weighted gene co-expression network analysis

The quantified proteins were analyzed by WGCNA to detect expression patterns across all samples. The important parameter of network is power, which was set at 16 and resulted in a scale-free network (Fig. 3A). The genes were clustered based on their expression pattern similarity, and then partitioned into modules (Fig. 3B). A total of 16 gene co-expression modules were identified, among which modules M2 and M9 were positively and negatively associated with EIGC (Fig. 3C). The top enriched KEGG pathways of modules M2 and M9 were spliceosome and focal adhension (Fig. 4D, E). Among the enriched pathways, nucleocytoplasmic transport was up-regulated and bacterial invasion of epithelial cells was down-regulated, suggesting the aberrant protein location and epithelial in EIGC. The highly connected hub proteins in the two modules include WDR36 and ACTA2.

Protein interaction analysis

Cytoscape was used to construct a protein interaction network for the differentially expressed proteins (Fig. 4A). Degree analysis showed that POLR2A and DDX18 had the highest connection with other differential proteins. Notably, IPO4 and p62/SQSTM1were found to form highly aggregated protein-interaction networks with several other proteins (Fig. 4B, C), indicating their importance in the early gastric cancer. These key nodes may play a crucial role in the disease development.

Immunohistochemical verification of differentially expressed proteins

IHC was performed to analyze the expression of the five proteins in the NGC, LGIN, and EIGC groups (Fig. 5). These five proteins varies significantly among the NGC, LGIN, and EIGC groups. The pairwise comparison revealed significant differences between the NGC group and the LGIN group, as well as between the NGC group and the EIGC group. However, no significant difference was observed between the LGIN and EIGC groups (Table S1).

Prognostic analysis of differentially expressed proteins

To check if the identified differential proteins are prognostic, 477 intestinal-type gastric cancer microarray samples from NCBI GEO were analyzed. All the five genes were prognostic for patients overall survival (Fig. 6A). All the genes were differentially expressed in stage 1 samples compared to non-cancer tissues except for PKP3 (Fig. 6B). However, proteins TBL1XR1 is favorable and IPO4, SQSTM1, PKP3, and CRTAP are unfavorable for overall survival in intestinal-type gastric cancer. Therefore, these proteins may play different roles in EIGC development.

We for the first time profiled the proteome of EIGC. The selection of appropriate cases for comparison is a key highlight of this study. We selected 10 cases of early-stage gastric cancer and compared them with the background mucosa of atrophic intestinal metaplasia. The inclusion of severe dysplasia and carcinoma in situ, which are considered carcinoma by Japanese standards, in the HGIN group provides a comprehensive representation of the initial biological characteristics of intestinal-type gastric cancer before the cancer cells infiltrate the interstitium. Biopsy tissue obtained by endoscopy is more accurate, and the tissue extracted by the biopsy clamp can precisely meet the depth of early gastric cancer (Fujiyoshi et al., 2021). Although the definition of early gastric cancer differs between the West and Japan, LGIN is considered a neoplastic lesion in both regions. This comparison between the two groups is a crucial step in the study of intestinal-type gastric cancer and is more likely to yield key information regarding the process of gastric cancer development.

Furthermore, the 10 cases of EIGC selected for this study were all in the state of H. pylori infection, rather than gastric cancer after sterilization. Selecting gastric cancer after sterilization for comparison may not accurately represent the characteristics of the early stages of intestinal-type gastric cancer. Therefore, studying early gastric cancers infected with H. pylori is of great significance.

The use of DIA-MS technology in this study is another highlight. This technology is capable of detecting a wide range of proteins and is particularly useful for screening and identifying differential proteins in a relatively short period of development of EIGC (Kitata et al., 2022). The identification of potential biomarkers for early detection and diagnosis of intestinal-type gastric cancer is of great significance and may lead to the development of new therapeutic interventions.

The expressions of TBL1XR1, IPO4, p62/SQSTM1, PKP3, and CRTAP in NGC, LGIN, and EIGC groups were quantitatively localized using immunohistochemistry. The results indicated that the expressions of these proteins were significantly up-regulated in LGIN and EIGC groups. These findings were consistent with the DIA-MS quantitative analysis results. However, it is important to note that the sample size needs to be expanded in future, and more accurate quantitative methods, such as western blot or Parallel Reaction Monitoring quantitative technology, should be employed to verify the expression of differential proteins. Additionally, it is necessary to include other non-gastric cancer groups to identify these differential proteins with early gastric cancer specificity.

IPO4 is responsible for transporting histones H3 and H4 from the cytoplasm to the nucleus for chromatin assembly. Dysregulation of histone assembly can lead to altered gene expression, DNA replication, and repair, ultimately contributing to cancer development. Our IHC results showed that IPO4 was highly expressed in the cytoplasm of EIGC, suggesting that the nucleoprotein transport process may be hindered. Previous studies have also implicated IPO4 in cancer development, with upregulation of IPO4 observed in non-small cell lung cancer and gastric cancer. Knockdown of IPO4 inhibited the proliferation and migration of gastric cancer cells, indicating that IPO4 may drive the occurrence of gastric cancer (Xu et al., 2019). TBL1XR1 is an important regulatory protein that controls gene activation and repression mediated by nuclear receptors. Overexpression of TBL1XR1 has been linked to cellular epithelial-mesenchymal transition (EMT) and gastric cancer cell lung and lymphatic metastasis and is associated with poor prognosis (Wang et al., 2018). Our IHC results showed that TBL1XR1 was highly expressed in EIGC, with localization in the nucleus according to the Human Protein Atlas (HPA). However, our IHC results showed that TBL1XR1 was mainly localized in the cytoplasm. Further research is needed to explore the role of TBL1XR1 in EIGC and its potential as a therapeutic target (Lu et al., 2021). These IHC results were consistent with the WGCNA results, which suggest the up-regulated nucleocytoplasmic transport pathway. Autophagy plays a critical role in maintaining cell survival and inducing cell death, and its dysregulation has been linked to the development of gastric cancer. Our IHC results showed that p62/SQSTM1 expression in EIGC tissue was mainly in the cytoplasm. p62/SQSTM1 is a stress-inducible intracellular protein known to regulate cell survival and cell death (Komatsu et al., 2012). The occurrence of gastric cancer is closely related to H. pylori infection. H. pylori can persist in the stomach by destroying autophagic cells to escape autophagic cell clearance (Raju et al., 2012). Long-term exposure of gastric mucosal epithelial cells to the vacuolar toxin vacA of H. pylori will damage autophagy cells, increase the level of reactive oxygen species, and overexpression of the protein p62/SQSTM1, which promotes chronic inflammatory damage to the gastric mucosa and eventually leads to the occurrence of early gastric cancer (Tang et al., 2012). We also identified the differential protein PKP3, which is one of the armadillo-related proteins. Our IHC results showed that PKP3 was highly expressed in the cytoplasm of EIGC. However, previous studies have found that the expression level of PKP3 in gastric cancer tissue is down-regulated, and the low expression of PKP3 is related to the number of lymph node metastasis and the stage of gastric cancer, suggesting a poor prognosis (Demirag et al., 2011). Finally, CRTAP is negatively correlated with the expression of miR-200c, which can inhibit TGF-β/BMP signaling and promote epithelial gene expression (Perdigao-Henriques et al., 2016). Our IHC results showed that CRTAP was up-regulated in the cytoplasm of EIGC. Overall, our findings suggest that these differential proteins may be involved in the occurrence and progression of EIGC. Further research is needed to confirm these findings and explore their potential as therapeutic targets.

IHC was used to compare protein expression among three validation groups, revealing significant differences between NGC and LGIN, as well as between NGC and EIGC. However, no significant difference was observed between LGIN and EIGC. The Japanese criteria for diagnosing gastric cancer are based on biological properties, whereas Western pathologists rely on the location of tumor cells. According to the Japanese concept, a mucosal lesion exhibiting the same cytological and structural atypia as an adenocarcinoma invading the submucosa should be considered an adenocarcinoma. Therefore, some LGIN should be treated similarly to early gastric cancer. The LGIN wax blocks selected for validation in this study were preoperatively diagnosed as early gastric cancer according to Japan creteria. The differences in diagnostic criteria between Japan and Western countries reflect the emphasis placed by Japanese pathologists on using morphological features to predict poor clinical outcomes, rather than relying solely on evidence of stromal invasion. The identification of LGINs that require treatment similar to early-stage stomach cancer is an ongoing focus of this research.

The study identified prognostic proteins for EIGC. Five of the differential proteins were validated in pathological section. The proteins are differentially expressed during the disease initiation. The study provides candidate protein biomarkers for EIGC diagnosis and treatment.

Acknowledgements We are grateful for the technical support from Shanghai Applied Protein Technology Co. Ltd. This study was funded by the Zhejiang Provincial Natural Science Foundation of China (Nos. LGF19H030006 and LQ20H030001), Ningbo Science and Technology Project (No. 2019C50100), and Ningbo Clinical Medicine Research Center Project (No. 2019A21003).

Author contributions Hua Ye and Hong Li: conceived and designed the experiments. Liangshun Zhang and Feng Xu: collected samples, performed the proteome analysis, and analysed the data. Hongna Lu and Xianwen Dong: data analysis and plotting figures. Zhiqiang Gao and Qiaosu Zhao: performed the WGCNA and transcriptome data analysis. Ting Weng: survival analysis. Hong Li: drafting the manuscript. Hua Ye: project administration and revised the manuscript. All the authors participated in the manuscript revision.

Data availability statement The datasets analyzed in the study are available in public repositories at NCBI GEO (https://www.ncbi.nlm.nih.gov/geo/).

Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval The experimental protocol was approved by the Ethics Committee of Ningbo Medical Center Lihuili Hospital (KY2019PJ055). Informed consent was obtained from all participating patients or their family members.

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No competing interests reported.

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Data-independent acquisition (DIA) mass spectrometry reveals related proteins involved in the occurrence of early intestinal-type gastric cancer

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Abstract

Figures

Introduction

Materials and methods

Patients

Histopathological diagnosis

Tissue samples

Filter-aided sample preparation (FASP) digestion procedure

Data dependent acquisition (DDA) mass spectrometry assay

Data independent acquisition (DIA) mass spectrometry assay

Mass spectrometry data analysis

Immunohistochemistry

Statistical analysis

Results

Proteome profiles of early intestinal-type gastric cancer

Differential protein analysis

Functional enrichment analysis

Weighted gene co-expression network analysis

Protein interaction analysis

Immunohistochemical verification of differentially expressed proteins

Prognostic analysis of differentially expressed proteins

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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