Whole transcriptome sequencing identifies increased expression of HKDC1 in Helicobacter pylori-associated gastric cancer

To identify target proteins highly expressed in gastric cancer tissues and associated with H. pylori, whole transcriptome sequencing was initially performed on AGS cells co-cultured with H. pylori ATCC43504, alongside control cell lines, to generate the dataset (RPKM). In addition, transcriptome data for AGS cells co-cultured with ATCC26695 from the GEO database (GSE108305) and with ATCC49503 (GSE70394) were incorporated. From these datasets, 11 genes significantly upregulated after co-culture, collectively defined as AGS(HP), were identified through intersecting data based on a significance threshold (|logFC| > 2.0, p < 0.01). Following this, the transcriptomes of 8 paired human gastric cancer and adjacent non-cancerous tissues were sequenced, producing another dataset (TVSN). This dataset was further intersected with the transcriptome data from 80 paired gastric cancer and adjacent tissues in the GEO database (GSE122401), filtered at the same significance threshold (|logFC| > 2.0, p < 0.01), leading to the identification of 210 significantly upregulated genes, collectively termed Tissue. Tissue was then intersected with AGS(HP), resulting in the identification of two candidate molecules, HKDC1 and MMP1 (Fig. 1a). Both genes exhibited marked upregulation in both gastric cancer tissues and H. pylori co-cultured cells, suggesting their potential role in H. pylori-mediated gastric carcinogenesis. Given that MMP1 has been extensively studied in H. pylori-related gastric cancer, HKDC1 was selected as the focus for further investigation in this study.

To validate these sequencing findings, an analysis of HKDC1 expression in gastric cancer was conducted using the TCGA database (Fig. 1b). The results demonstrated a significantly elevated expression of HKDC1 in gastric cancer tissues compared to normal tissues. The ROC curve analysis showed an AUC of 0.753, indicating a high level of accuracy (Fig. 1c), consistent with the transcriptomic data trends.

Fig. 1

Whole transcriptome sequencing and TCGA database analysis to identify candidate molecules for Helicobacter pylori-associated gastric cancer. (a) Venn diagram depicting the overlap between sequencing results from H. pylori-co-cultured AGS cells and gastric cancer/adjacent tissues, identifying three candidate genes: HKDC1, TNFAIP3, and MMP1. (b) TCGA database analysis of HKDC1 expression in gastric cancer. (c) ROC curve analysis for HKDC1

HKDC1 expression is increased in Helicobacter pylori-associated gastritis

To investigate the differential expression of HKDC1 in H. pylori-associated gastritis, CagA-positive H. pylori strains ATCC26695, ATCC43504, and HPWT were co-cultured with normal gastric epithelial cells (GES-1). Cellular proteins were then extracted at four time points (0, 4, 8, and 12 h) to assess the time-dependent expression of HKDC1 using Western blot analysis. The results revealed that HKDC1 expression increased progressively in GES-1 cells with prolonged H. pylori co-culture, compared to the control group at 0 h. The presence of the CagA protein, a marker of H. pylori infection, confirmed successful infection of the cells (Fig. 2a) [20]. To further validate the association between HKDC1 and the progression of gastritis, IHC was performed to examine the expression levels of HKDC1 and E-cadherin—an epithelial marker of EMT(Epithelial-Mesenchymal Transition)—in non-atrophic gastritis, atrophic gastritis, and intestinal metaplasia. HE staining and IHC results indicated that during the Correa cascade of gastritis progression [7], HKDC1 expression increased with the severity of gastritis, while E-cadherin expression decreased correspondingly (Fig. 2b, c). Since E-cadherin is a key molecule in the EMT pathway [19], these results suggest that HKDC1 promotes gastritis progression and may drive gastric cancer development through EMT signaling.

To elucidate the role of HKDC1 in gastritis progression at the animal model level, C57BL/6J mice were used for gastritis modeling (Fig. 2d). Following H. pylori gavage, mice in modeling groups 1 and 2 were euthanized at 12 and 36 weeks, respectively, to analyze gastritis development and related protein expression. HE staining revealed that group 1 exhibited chronic non-atrophic gastritis, characterized by inflammatory cell infiltration, while group 2 displayed pre-cancerous lesions such as intestinal metaplasia. The presence of H. pylori infection was confirmed by the expression of the HP protein (Fig. 2f). Western blot and IHC analyses demonstrated that HKDC1 expression increased in conjunction with the degree of gastric mucosal damage, while E-cadherin expression was inversely correlated with HKDC1 expression and the severity of gastritis (Fig. 2e, g, S1a). These results highlight HKDC1 as a critical molecule in the progression of gastritis and its potential role in mediating gastric cancer through the EMT pathway.

Fig. 2

HKDC1 promotes the progression of gastritis. (a) Western blot analysis of HKDC1 protein levels in GES-1 cell lines co-cultured with H. pylori strains ATCC26695, ATCC43504, and HPWT. (b and c) Immunohistochemical (IHC) detection of HKDC1 and E-cadherin expression in non-atrophic gastritis, atrophic gastritis, and intestinal metaplasia tissues. (d) Schematic diagram of a mouse model of H. pylori infection (gastritis). (e) Western blot analysis of HKDC1 and E-cadherin expression in gastric tissues of mice 12 and 36 weeks post-H. pylori infection. (f) Representative images of gastric mucosa in mice with gastritis, assessed by HE staining, and H. pylori expression detected by immunohistochemistry. (g) IHC analysis of HKDC1 and E-cadherin expression in normal and chronic gastritis gastric mucosa

HKDC1 is upregulated in gastric cancer and predicts poor prognosis

The expression of HKDC1 in gastric cancer was subsequently examined. Protein and mRNA levels of HKDC1 were evaluated in various gastric cancer cell lines (SGC-7901, AGS, MGC-803, HGC-27, MKN45) using Western blot analysis and real-time quantitative fluorescent PCR. The analysis revealed a marked upregulation of HKDC1 expression at both the protein and mRNA levels in these gastric cancer cell lines compared to the normal gastric epithelial cell line GES-1 (Fig. 3a, c). To further confirm these findings, HKDC1 expression was assessed in human gastric cancer tissues and their matched adjacent non-tumor tissues using Western blot analysis, qRT-PCR, and IHC. Consistent with prior results, HKDC1 expression was significantly elevated in gastric cancer tissues compared to adjacent normal tissues (Fig. 3b, d, e).

Fig. 3

HKDC1 expression is upregulated in gastric cancer and predicts poor prognosis. (a) Western blot analysis of HKDC1 expression in GES-1 and gastric cancer cell lines. (b) Western blot analysis of HKDC1 expression in gastric cancer tissues and paired adjacent tissues. (c and d) qRT-PCR analysis of HKDC1 mRNA levels in cell lines and gastric cancer tissues with paired adjacent tissues. (e) IHC analysis showing differential HKDC1 expression in gastric cancer and paired paracancerous tissues. (f) Representative image of HKDC1 immunohistochemistry. (g) Kaplan-Meier survival curves showing the correlation between HKDC1 expression and overall survival in patients with gastric cancer. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Table 1 Clinicopathological parameters in gastric cancer patientsTable 2 Univariate and multivariate survival analysis between clinicopathological parameters and HKDC1 expression and gastric cancer prognosisThe upregulation of HKDC1 expression is induced by Helicobacter pylori

To elucidate the role of H. pylori in inducing HKDC1 expression, AGS and MKN45 gastric cancer cell lines were co-cultured with two standard CagA-positive H. pylori strains (ATCC26695 and ATCC43504) as well as a wild-type strain isolated from fresh gastric tissues by our group. Cellular proteins were extracted at 0, 4, 8, and 12 h of co-culture, following a time gradient, and HKDC1 expression was detected via Western blot analysis at each time point. The presence of the CagA protein confirmed successful H. pylori infection. The results showed a significant, time-dependent increase in HKDC1 expression in all tested gastric cancer cell lines—AGS (Fig. 4a, b) and MKN45 (Fig. S2a)—compared to controls without H. pylori. Further analysis of HKDC1 expression was conducted on both H. pylori-positive and H. pylori-negative gastric cancer tissues using Western blot analysis and RT-PCR. Samples were collected from tumor and adjacent normal tissues of 32 patients. The results revealed that HKDC1 expression was significantly higher in H. pylori-positive tumors compared to those lacking infection (Fig. 4c, d), suggesting that H. pylori enhances HKDC1 expression in gastric cancer cells.

TFF1, typically expressed in normal gastric epithelial cells, exhibits reduced levels in gastric cancer and other malignancies, potentially contributing to gastric carcinogenesis through various mechanisms [21,22,23]. To further examine H. pylori’s effect in an animal model, TFF1-KO mice were orally gavaged with H. pylori for 36 weeks before being euthanized for analysis. Western blot analysis and IHC were employed to assess protein expression in the stomachs of these mice (Fig. 4e). HE staining revealed that precancerous lesions, such as atrophic gastritis and low-grade endomatous metaplasia, were present in the control group, while the modeling group developed high-grade endomatous metaplasia or intramucosal carcinoma. The presence of H. pylori proteins served as a reliable indicator of infection (Fig. 4f). These groups were used to model the progression from precancerous lesions to gastric cancer. IHC analysis demonstrated a significant increase in HKDC1 expression in the H. pylori-modeled gastric cancer group compared to the precancerous lesion group. Conversely, E-cadherin expression was significantly reduced and inversely correlated with HKDC1 levels (Fig. 4g). Collectively, these findings confirm that H. pylori promotes gastric carcinogenesis.

In TFF1-KO mice and induces HKDC1 expression in a mouse model of gastric cancer.

Fig. 4

H. pylori enhances HKDC1 expression in gastric cancer. (a and b) Western blot analysis of HKDC1 expression in AGS cell line models infected in vitro with Helicobacter pylori strains ATCC26695, ATCC43504, and HPWT. (c and d) qRT-PCR and Western blot assays were used to measure HKDC1 expression in H. pylori-negative and -positive tissues. (e) Schematic diagram of the TFF1-KO mouse model (gastric cancer) infected with H. pylori. (f) Pathological changes in the gastric mucosa of TFF1-KO mice were assessed by HE staining, and H. pylori expression was detected by immunohistochemistry. (g) Immunohistochemical (IHC) analysis of HKDC1 and E-cadherin expression in the gastric mucosa of precancerous lesions and gastric cancer tissues in mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Knockdown of HKDC1 impedes the oncogenic characteristics of gastric cancer

To elucidate the role of HKDC1 in driving the malignant phenotype of gastric cancer cells, lentiviral transfection was utilized to knock down and overexpress HKDC1 in the SGC-7901, HGC-27, and AGS cell lines, which exhibit differential HKDC1 expression. The transfection efficiency was validated, and two specific sequences (shHKDC1#1 and shHKDC1#2) were selected for further functional assays (Fig. 5a). EdU incorporation and colony formation assays demonstrated a marked reduction in proliferation and clonogenic capacity in SGC-7901 and HGC-27 cells following HKDC1 knockdown, as compared to the control group (Fig. 5b, S3a, b). In contrast, HKDC1 overexpression significantly elevated these parameters in AGS cells (Fig. 5c). Additionally, Transwell assays indicated that HKDC1 knockdown led to diminished migration and invasion capabilities in SGC-7901 and HGC-27 cells, as evidenced by fewer perforated membranes and impaired scratch closure (Fig. 5d, S3c). Conversely, HKDC1 overexpression in AGS cells enhanced both migration and invasion, as reflected by increased membrane perforations and improved scratch closure (Fig. 5e). Collectively, these results underscore the potential of HKDC1 inhibition as an effective strategy to suppress the malignant phenotype in gastric cancer cells.

Fig. 5

Effect of HKDC1 on the malignant phenotype of gastric cancer. (a) Western blot assay confirming the transfection efficiency of HKDC1 lentivirus. (b and c) EdU and colony formation assays assessing the impact of HKDC1 overexpression or knockdown on the proliferation of gastric cancer cells. (d and e) Wound healing and Transwell assays evaluating the effects of HKDC1 knockdown and overexpression on the invasion and migration of gastric cancer cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Overexpression of HKDC1 promotes tumorigenicity of gastric cancer cells in vivo

Following the confirmation of HKDC1’s functional effects on gastric cancer cells in vitro, its influence on tumorigenesis was further examined in vivo using a subcutaneous tumor model in nude mice. Tumors formed by stable HKDC1 overexpressing cells were consistently larger and heavier than those formed by control GC cells, suggesting that HKDC1 overexpression can effectively promote tumor growth in vivo. In addition, the volume and weight of subcutaneous tumors were significantly reduced after treatment with Galunisertib (Fig. 6a). Tumor sections were then embedded and processed for IHC staining to assess the Ki67 index, a marker of cell proliferation, between the experimental groups. The results showed a significant increase in both tumor weight and volume in the HKDC1 overexpression group compared to the control group (Fig. 6b, c). These data suggest that HKDC1 has a role in promoting the proliferation of gastric cancer cells in vivo, and this effect is eliminated by Galunisertib.

Fig. 6

Subcutaneous tumor formation experiment in nude mice. (a) Representative images of nude mice and subcutaneous tumors. (b) Immunohistochemical staining of subcutaneous tumors from two groups (Ki67). (c and d) Tumor volume growth curve and tumor weight measurements. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

HKDC1 promotes EMT in gastric cancer by activating TGF-β1/smad2 signaling pathway

In the study above, we have established the critical role of HKDC1 in the transition from gastritis to gastric cancer, particularly through the classical Correa cascade. Immunohistochemical analysis of gastritis and gastric cancer tissues further revealed a progressive decline in E-cadherin expression, an epithelial marker indicative of EMT(Epithelial-Mesenchymal Transition), correlating with increasing severity of gastric injury and gastritis development. Notably, this decline in E-cadherin was inversely correlated with elevated HKDC1 expression. These observations led to the hypothesis that HKDC1 may drive gastric cancer progression through EMT pathway activation. As TGF-β1 is a well-established key inducer of EMT across various cancers, its role in this process was explored.

TGF-β1 expression levels were initially examined via Western blot analysis in the normal gastric epithelial cell line GES-1 and the gastric cancer cell lines AGS and MKN45, following co-culture with H. pylori for different durations. Results revealed a time-dependent increase in TGF-β1 expression, consistent with previously reported trends for HKDC1 (Fig. 7a, b; S4a).

Further investigation into EMT-related protein expression was conducted in gastric cancer cell lines stably transfected for either HKDC1 overexpression or knockdown. Overexpression of HKDC1 in AGS cells led to elevated TGF-β1 expression and decreased E-cadherin levels, accompanied by an upregulation of N-cadherin, Vimentin, and Snail (Fig. 7c). Conversely, HKDC1 knockdown in SGC-7901 and HGC-27 cells resulted in a marked downregulation of TGF-β1 and EMT-related pathway proteins (Fig. 7c, S4b). These data suggest that HKDC1 overexpression promotes both TGF-β1 and EMT-related proteins expression, though the specific mechanisms linking HKDC1 to EMT via TGF-β1 remain unclear.

To elucidate the involvement of TGF-β1 in EMT, experiments were conducted using Galunisertib (LY2157299), a TGF-β1 inhibitor, and recombinant TGF-β1 protein in lentivirally transfected cell lines. After 8 h of incubation with 5 µM Galunisertib, total cellular proteins were extracted, and the levels of smad2, p-smad2, and EMT-related proteins were assessed. A key feature of EMT is the downregulation of E-cadherin, which destabilizes adhesion junctions, balanced by an upregulation of N-cadherin. Additionally, SNAIL proteins—SNAIL1 and SNAIL2 (SLUG)—are pivotal in activating EMT during development, fibrosis, and cancer progression [19]. Therefore, E-cadherin, N-cadherin, Vimentin, and Snail were selected as EMT markers.

The results demonstrated that, following Galunisertib treatment, p-smad2, N-cadherin, TGFβ1, Vimentin, and Snail expression levels were downregulated, while E-cadherin expression increased in both the Vector and Control groups, with HKDC1 expression remaining unchanged (Fig. 7d). Conversely, treatment of SGC-7901 and HGC-27 cell lines with 10 ng/ml recombinant TGF-β1 protein for 12 h reversed the HKDC1 knockdown-induced downregulation of p-smad2, N-cadherin, TGFβ1, Vimentin, and Snail, while also restoring the upregulation of E-cadherin (Fig. 7d and d). These results indicate that TGF-β1 promotes EMT progression by enhancing smad2 phosphorylation levels.

Fig. 7

TGF-β1 promotes EMT in gastric cancer via p-smad2 activation. (a and b) Expression of TGF-β1 in GES-1 and AGS cells co-cultured with H. pylori. (c) Expression of TGF-β1 and EMT-related proteins in AGS cell lines overexpressing HKDC1 and in SGC-7901 cell lines with HKDC1 knockdown. (d) Inhibition of the EMT pathway and p-smad2 expression following the addition of Galunisertib in SGC-7901 cells with HKDC1 knockdown, and activation of the EMT pathway with the addition of TGF-β1 recombinant protein in AGS cells overexpressing HKDC1