Abstract

Background

   Gastric cancer (GC), a prevalent aggressive form of tumor, imposes a
   significant burden in terms of morbidity and mortality. Prolyl
   4-hydroxylase, alpha polypeptide I (P4HA1), a key enzyme in collagen
   synthesis, comprises two identical alpha subunits and two beta
   subunits. Studies on the expression and impact of P4HA1 in GC cells are
   limited.

Methods

   The expression and prognosis of P4HA1 in GC were analyzed using
   bioinformatics. To confirm the P4HA1 level in GC tissues and cells,
   Western blot (WB) and RT-qPCR experiments were conducted. The signaling
   pathways related to P4HA1 in GC were examined using the DAVID database.
   Moreover, the expression of P4HA1 was downregulated by transfecting GC
   cell lines (HGC-27 and SGC-7901) with siRNA technology. Furthermore, GC
   proliferation, migration, and invasion were detected via plate cloning,
   CCK-8, and Transwell assays. The epithelial-mesenchymal transition
   (EMT) genes (E-cadherin, N-cadherin, Vimentin) and the stemness marker
   CD44 protein expression in GC cells were detected using WB. The
   sphere-forming ability of GC cells was analyzed using a sphere-forming
   assay to determine the effect of P4HA1.

Results

   Bioinformatics and experimental analyses demonstrated that P4HA1
   expression was extensively detected in GC tissues and cells, and
   strongly related to a poor prognosis for GC. In vitro studies
   demonstrated that P4HA1 suppression hindered the proliferation,
   migration, and invasion of GC cells and suppressed EMT characteristics.
   Both sphere-forming and WB assays revealed that the sphere-forming
   potential of GC cells and the level of CD44 protein decreased after
   knocking down the expression of P4HA1, indicating that suppression of
   P4HA1 could inhibit the stemness of GC cells.

Conclusion

   The study concluded that P4HA1 has the potential to be expressed
   substantially in GC tissues and cells and is capable of enhancing the
   proliferation, metastasis, and stemness of GC.

Supplementary Information

   The online version contains supplementary material available at
   10.1007/s12672-025-02337-1.

   Keywords: Gastric cancer, Proline 4-hydroxylase 1, Proliferation,
   Migration, Stemness

Introduction

   Globally, GC is the fifth most prevalent invasive tumor, with the third
   highest mortality rate [[36]1]. In China, it has now risen to the third
   position among the most aggressive cancers and has become the second
   most prevalent cause of cancer-associated death [[37]2]. Presently,
   imaging and surgical treatment are the primary diagnostic and
   therapeutic procedures for GC patients.

   Collagen (Col) has a crucial role in the tumor microenvironment (TME),
   contributing to cancer fibrosis, enhancing tumor tissue rigidity, and
   metastasis, and modulating tumor immunity. Collagen
   prolyl-4-hydroxylase (P4HA) is a key enzyme for preserving the Col
   stability. However, the dysfunctional status of P4HA has been related
   to several pathological events, such as the development and advancement
   of tumors [[38]3]. It has three isoforms, including P4HA1, P4HA2, and
   P4HA3, with P4HA1 being the most common [[39]4]. P4HA1 is highly
   expressed in various types of tumors and can be used as a target for
   potential therapeutic intervention, such as clear cell renal cell
   carcinoma [[40]5], microglial cell carcinoma [[41]6], colorectal cancer
   [[42]7], esophageal carcinoma [[43]8, [44]9], and nasopharyngeal
   carcinoma [[45]10]. Conversely, there is limited data relating to the
   expression and functionality of P4HA1 in GC. Here, the current
   study conducted experiments and used online databases to identify the
   expression, prognosis, and associated indexes of P4HA1 in GC tissues.
   The study also developed a P4HA1 knockdown model and observed its
   suppressive effects on the proliferation, migration, invasion, and
   stemness of GC cell lines via loss-of-function experiments to
   demonstrate that P4HA1 can enhance the development of GC cells and
   provide a possibility for their growth. The focus was to elucidate the
   ability of P4HA1 to enhance the development of GC cells and to identify
   new targets for the detection and therapeutics of GC.

Materials and methods

Cells and reagents

   Human GC cell lines (HGC-27, SGC-7901) and gastric mucosal normal cells
   GES-1 were procured from the Cell Bank of Shanghai Chinese Academy of
   Sciences, (Shanghai, China). P4HA1 small interferences were constructed
   by Shanghai (Jimma Biotech). P4HA1, N-cadherin, E-cadherin, Vimentin,
   CD44, and β-actin (Proteintech), RPMI-1640 medium (Gibco), BCA protein
   quantification kit (Beyotime Biotechnology, China), B27, CCK-8 kit
   (Biosharp), Transwell chambers (Corning). HRP-linked goat anti-rabbit
   secondary antibody (LiankeBio, China). Growth factors (EGF and bFGF)
   were procured from (PeproTech, USA), and fetal bovine serum (FBS)
   (ExCell, Uruguay).

Cell culture

   Cells were grown in RPMI-1640 medium with 10% FBS and 1%
   penicillin–streptomycin. The culture conditions were set at 37 ℃, with
   a 5% CO[2] incubator. The exhausted medium was changed after two days.
   The cells were allowed to grow to > 80% for transmigration or to
   continue for further experiments.

Gastric cancer tissue samples

   GC surgical tissue: Eight pairs of fresh GC lesions and respective
   normal gastric mucosa tissues (around 5 cm from the tumor peripheries)
   were collected intraoperatively (within 1 h after gastrectomy) from the
   First Affiliated Hospital of Bengbu Medical University for GC. To
   remove surface blood stains, all tissue samples were washed with
   saline, cut with scissors, and placed into sterile and enzyme-free EP
   tubes. These tubes were promptly frozen and kept at − 80 ℃ for future
   WB analysis of P4HA1. Tissue wax block specimen: GC radical surgery
   resection specimens were routinely formalin fixed and sent to
   pathology, routinely taken for paraffin embedding, dry and preserved at
   room temperature, according to the patient's pathology number, the GC
   tissues and corresponding paracarcinoma tissues wax blocks were
   retrieved from the Department of Pathology, and were used for
   immunohistochemical staining of P4HA1.The project was reviewed and
   approved by the Ethics Committee of the First Affiliated Hospital of
   Bengbu Medical University, and all subjects signed an informed consent
   form.

Immunohistochemical staining

   Pathological wax blocks were cut into 4 μm thin slices using a
   pathology sectioning machine, and the slices were placed in an oven at
   65 ℃for 1 h. Xylene was deparaffinized and then alcohol was
   sequentially hydrated, and citrate buffer was used to repair the
   antigen. 5% BSA or closed serum was added dropwise to the slices and
   incubated in a thermostat at 37 ℃for 30 min. PBS was used in place of
   primary antibody for immunostaining blanks, non-immunized goat or
   rabbit serum was used for negative controls, and lung tissue was used
   for positive controls. The primary and secondary antibodies were
   incubated overnight and then DAB was used to develop the color,
   followed by restaining using hematoxylin.

Bioinformatics analysis

The cancer genome atlas (TCGA)

   The TCGA database ([46]https://portal.gdc.cancer.gov/) is a free
   website providing cancer genomics databases. The data of RNA-seq was
   obtained from TCGA-STAD for 410 GC and 36 neighboring normal tissue
   samples using the R package “TCGAbiolinks” in R (v 4.3.3).
   “Adjusted p < 0.05 and fold change > 1.0” were defined as the
   thresholds for the screening of the differential expression of mRNAs.
   The “DESeq-2” package was used to detect differentially expressed genes
   (DEGs). All the relevant clinical data was retrieved from TCGA-STAD and
   used for the OCLR algorithm constructed by Malta et al. [[47]11]. This
   algorithm was used to determine mRNAsi and evaluate the correlation
   between P4HA1 and stemness score.

Xiantao academic platform

   The expression and prognosis of P4HA1 in GC, as well as the association
   between the levels of P4HA1 and various clinical characteristics, were
   analyzed via the Xiantao Academic Platform
   ([48]https://www.xiantaozi.com), an online platform that can analyze
   TCGA data online.

Interactive analysis of gene expression profiles

   The GEPIA2 database ([49]http://gepia2.cancer-pku.cn) [[50]12] is a
   free online platform that provides extensive RNA sequencing data for
   cancer. GEPIA2 was used to evaluate the correlation between P4HA1 and
   stem cell markers.

Kaplan–Meier plotter database

   Kaplan–Meier plotter online database ([51]https://kmplot.com/analysis/)
   [[52]13] is a website that facilitates gene survival analysis. In this
   study, survival analysis was conducted to evaluate the levels of P4HA1
   in STAD.

Gene set enrichment analysis

   The top 50 genes interacting with P4HA1 were obtained via the String
   ([53]http://string-db.org) [[54]14] database and the top 50 genes
   interacting with P4HA1 were extracted via Spearman correlation analysis
   (|r|≥ 0.5) in the data from the GC of cBioPortal
   ([55]http://www.cbioportal.org/) [[56]15]. Intersect the genes obtained
   from the String database with the cBioPortal database to obtain the
   intersected genes, and the cBioPortal database was used to conduct a
   correlation analysis of P4HA1 with the overlapped genes. A heatmap of
   the correlation between P4HA1 and the overlapped genes in pan-cancer
   was examined in the TIMER2.0 database ([57]http://timer.cistrome.org/)
   [[58]16]. The DAVID website ([59]https://david.ncifcrf.gov/summary.jsp)
   [[60]17] was used to integrate 100 genes, remove duplicate genes, and
   conduct gene ontology (GO) and Kyoto Encyclopedia of Genes (KEGG)
   pathway analyses.

Transfection

   Cells (HGC-27 and SGC-7901) were transfected with siRNA-P4HA1
   synthesized by Gemma Bio Ltd. using a Lip2000 transfection reagent.
   Experiments were grouped into si-NC and si-P4HA1, with the following
   interference sequences.

   si-NC (F):5′-UUCUCCGAACGUGUCACGUTT-3′,
   si-NC(R):5′-ACGUGACACGUUCGGAGAATT-3′. si-P4HA1
   (F):5′-GGCCUAUACAGAAGCAGAUTT-3′, si-P4HA1
   (R):5′-AUCUGCUUCUGUAUAGGCCTT-3′.

CCK-8 assay and plate cloning assay for cell proliferation

   CCK-8 assay: The cells to be tested were trypsinized and washed with
   PBS for 3 times, a single cell suspension was prepared and seeded into
   a 96-well plate, 2 × 10^3 cells were counted per well, repeat three
   times per well, 10 μl of CCK-8 reagent was added at 0, 24, 48 and 72 h,
   respectively, and the absorbance value OD[450 nm] was detected after
   incubation in a CO[2] incubator for 4 h. Plate cloning assay: Cells
   from both groups were enzymatically digested and collected using
   centrifugation to obtain a single-cell suspension. The cell suspension
   was seeded into 6-well plates at a density of 2 × 10^3/well, with 2 ml
   culture medium in each well. Cells were regularly observed and the
   media was changed timely. After two weeks, the cells were placed with
   4% paraformaldehyde (PFA) for 15 min fixation, stained with crystal
   violet for 15 min, and then counted after being rinsed with PBS.

Transwell assay

   Cells were digested by trypsin and resuspended to prepare a single-cell
   suspension. The upper chamber of the Transwell was filled with 200 μl
   of basal medium with 2 × 10^4 cells/well, while the lower chamber was
   filled with 750 μl of FBS-containing medium. For the invasion test,
   matrix gel was added to the upper chamber and then washed with PBS.
   After that, 4% PFA was used to fix the cells that passed through the
   upper chamber. They were stained with crystal violet for 15 min and
   then counted under the inverted microscope after the chambers had
   dried.

Pelleting experiment

   Cells were trypsinized, counted, and then cultured in ultra-low
   adsorption 6-well plates. Approximately 2 × 10^3 cells/well were added
   with FBS-free suspension culture medium. The size and number of cell
   spheres were observed and counted after 7 days. The serum-free medium
   comprised DMEM/F12 basal medium with B27 (10 µl/ml), EGF (20 ng/ml),
   bFGF (10 ng/ml), 2 ml of the growth medium/well, and 1 ml of
   rehydration solution. The cells were observed to aggregate into spheres
   within 2–3 days.

Gene expression analysis via RT-qPCR

   The total content of RNA was isolated from the FastPure Cell/Tissue
   Total RNA Isolation Kit (Vazyme, Nanjing, China) and reverse
   transcribed to cDNA. A total of 1 µg of RNA was used for
   RT-qPCR analysis via the ChamQ Universal SYBR qPCR Master Mix Kit
   (Vazyme, Nanjing, China). P4HA1-(F):5′-TGATGTGTCTGCAGGAGGAG-3′,
   P4HA1-(R):5′-CCACGTTCAT-GGAGCCATTT-3′,
   β-actin-(F):5′-AAGGCTGTGGGCAAGG-3′,β-actin-(R):5′-TGGAGGAGTGGGTGTCG-3′.

   PCR amplification was conducted in 20 µl reactions at 95 ℃ for 5 min,
   followed by 40 cycles of 95 ℃ (10 s), 60 ℃ (30 s), 95 ℃ (15 s), 60 ℃
   (60 s), and 95 ℃ (15 s) on a LightCycler 96 real-time PCR System
   (Roche, Basel, Switzerland). The readings were calculated via the
   2^−△△CT method.

Western blotting

   The tissues and cells were trypsinized and disrupted using a lysis
   buffer (RIPA: PMSF = 100: 1) on ice. The suspension was shaken every
   10 min thrice. The content of protein was measured via the BCA
   quantification method. Protein mixtures were then run in SDS-PAGE and
   then transferred onto a PVDF membrane. The membrane was kept with milk
   for 1 h blocking at 25 ℃ and then kept with phosphorylated antibody
   BSA, followed by primary antibodies P4HA1, E-cadherin, N-cadherin,
   Vimentin, CD44, β-actin (1:2000) for 24 h at 4 ℃ at shaking condition.
   The next day, the membrane was kept with a secondary antibody at 25 ℃
   for 2 h and washed thrice with TBST. Finally, the membrane was
   activated using ECL, and the protein bands were visualized.

Statistical analysis

   Data were statistically analyzed and processed via Excel, Image J, and
   GraphPad Prism 8 software. Each experiment was repeated thrice. The
   independent samples t-test was used to examine the variations between 2
   groups. To analyze comparisons between multiple groups, a one-way ANOVA
   was used, with a significance criteria of p < 0.05.

Results

Overexpression of P4HA1 in GC tissues and cell lines

   Data obtained by TCGA-STAD yielded 4518 DEGs, of which 2287 were
   downregulated and 2231 were upregulated in gene expression (including
   P4HA1) (Fig. [61]1A). P4HA1 mRNA expression was enhanced in GC tissues
   relative to healthy tissues, as demonstrated by the Xiantao Academic
   Platform (Fig. [62]1B–C). All of these analyses represented that P4HA1
   was abundantly expressed in GC tissues. To validate the levels of P4HA1
   in GC tissues, eight pairs of GC and related paracancerous tissues were
   collected from the clinic. The WB and immunohistochemistry assays
   results indicated that P4HA1 expressed highly in GC tissues in
   comparison to paracancerous tissues (Fig. [63]1D–E). RT-qPCR findings
   demonstrated that P4HA1 was abundantly found in GC cell lines relative
   to GES-1 (Fig. [64]1F).

Fig. 1.

   [65]Fig. 1
   [66]Open in a new tab

   P4HA1 expression in GC tissues and cells. A Volcanic maps of DEGs based
   on TCGA database analysis. B, C P4HA1 level in unpaired and paired GC
   and paracancerous tissues analyzed by Xiantao Academic Platform based
   on TCGA database. D Protein expression of P4HA1 in eight pairs of GC
   and paracancerous tissues via WB assay. E Expression of P4HA1 in GC
   tissues and paracancerous tissues by immunohistochemistry assays. F
   P4HA1 mRNA expression in GC cell lines and GES-1 cells via RT-qPCR
   analysis. **p < 0.01, ***p < 0.001

Correlation of P4HA1 level in GC tissues with survival and clinical
characteristics of GC patient

   Various databases were used to investigate the relationship between
   P4HA1 expression and the prognosis of GC patients. The Kaplan–Meier
   online database analysis demonstrated that high P4HA1 expression was
   related to a worse prognosis of GC tissues (Fig. [67]2A). As per the
   TCGA database, the Log-rank test was employed to evaluate the
   association between P4HA1 level and survival time in GC patients. The
   results indicated that P4HA1 overexpression in GC patients was
   associated with reduced overall survival (OS) and disease-specific
   survival (DSS) (Fig. [68]2B). After that, receiver operating
   characteristic (ROC) curves were obtained, and the findings depicted
   that the 1-year area under the curve (AUC) was 0.943, suggesting that
   P4HA1 in GC is a better predictive gene (Fig. [69]2C). To further
   determine the significance of P4HA1 expression, the correlation between
   P4HA1 level and different clinical characteristics was investigated in
   GC tissues via the Xiantao Academic Platform. The findings depicted
   that the expression of P4HA1 elevated with increasing age
   (Fig. [70]2D). Due to the high TNM stage, the overexpression of P4HA1
   was substantially associated with the TNM stage (Fig. [71]2E–G).
   Furthermore, P4HA1 expression was higher in both pathological and
   tissue stages than in the normal stage (Fig. [72]2H–[73]I).

Fig. 2.

   [74]Fig. 2
   [75]Open in a new tab

   Association between P4HA1 level, prognosis of GC patients, and
   different pathologic features. A Kaplan–Meier online database to
   examine the relationship between P4HA1 expression and prognosis in GC
   patients. B The association between P4HA1 expression and OS and DSS in
   GC patients was evaluated based on the TCGA database via the Xiantao
   Academic Platform. C ROC curve of the prognostic value of P4HA1 in GC
   tissues. D–I P4HA1 expression in GC patients was evaluated based on the
   TCGA database and different clinical characteristics D gender, E
   T-staging, F N-staging, G M-staging, H histologic grading, and I
   pathological staging. **p < 0.01, ***p < 0.001

Enrichment analysis of P4HA1-related genes in cancer

   To understand the relationship between P4HA1 and related genes in GC,
   the top 50 genes binding to P4HA1 were screened from the String
   database. The cBioPortal database was used to screen the top 50 genes
   that were co-expressed with P4HA1. The intersection of these genes
   yielded four genes: ALDOA, ERO1A, LDHA, and PLOD1 (Fig. [76]3A). P4HA1
   showed a positive association with these four genes in GC and different
   tumors (Fig. [77]3B–C). To examine the possible mechanism of P4HA1 in
   gastric carcinogenesis, a total of 100 acquired genes with duplicates
   were removed for GO and KEGG pathway analyses in the DAVID database.
   The GO analysis revealed that P4HA1 is involved in various biological
   processes including collagen fiber organization, collagen metabolic
   processes, ECM organization, and response to hypoxia (Fig. [78]3D–F).
   KEGG pathway analysis depicted that P4HA1 was related to protein
   digestion and absorption, HIF-1 signaling pathway, proline and arginine
   metabolism, and ECM-receptor interaction (Fig. [79]3G). Collectively,
   all of the above analyses suggest that P4HA1 may be a valuable
   regulator in GC.

Fig. 3.

   [80]Fig. 3
   [81]Open in a new tab

   Enrichment analysis of P4HA1 interacting or co-expressed genes. A The
   top 50 genes screened from the String database were intersected with
   the top 50 genes screened from the cBioPortal database. B P4HA1
   correlation analysis with ALDOA, ERO1A, LDHA, and PLOD1 in GC. C TIMER2
   database analysis of P4HA1 correlation heatmap with ALDOA, ERO1A, LDHA,
   PLOD1 D–F GO analysis of P4HA1 reciprocal or co-expressed genes D BP, E
   CC, F MF. G KEGG analysis of P4HA1 reciprocal or co-expressed genes

Suppression of P4HA1 expression inhibited GC proliferation

   To further observe the impact of P4HA1 on the function of GC cells, GC
   cell lines were transfected via siRNA technology. Both WB and RT-qPCR
   assays depicted that protein and mRNA expression of P4HA1 in the
   knockdown group (si-P4HA1 group) considerably decreased relative to the
   control group (si-NC group) (Fig. [82]4A–B). The findings of the CCK-8
   assay revealed that P4HA1 suppression could inhibit the proliferation
   of GC cells (Fig. [83]4C). The findings of plate cloning demonstrated
   that the P4HA1 suppression could suppress the clone formation potential
   of GC cells (Fig. [84]4D). These results suggest that inhibition of
   P4HA1 expression inhibits the proliferation of GC cells, while
   overexpression of P4HA1 has the opposite effect (Supplementary
   Fig. 7C-D).

Fig. 4.

   [85]Fig. 4
   [86]Open in a new tab

   Expression of P4HA1 enhances GC proliferation. A, B Expression of P4HA1
   after transfection of GC cell lines using siRNA technology. C Detection
   of GC proliferation via the CCK8 assay. D Detection of colony formation
   ability of GC cells via plate cloning assay. **p < 0.01, *** p < 0.001

P4HA1 suppression inhibits GC migration and invasion and the level of
EMT-related genes

   The findings of the Transwell assay demonstrated that the number of
   migrating and invading GC cells crossing the chamber membrane was
   substantially decreased in the P4HA1 knockdown group in comparison to
   the control group, suggesting that the GC migration and invasion can be
   suppressed by the P4HA1 suppression (Fig. [87]5A–B). Conversely,
   overexpression of P4HA1 was able to promote metastasis of GC cells
   (Supplementary Fig. 7E). Moreover, WB results indicated that the
   decreased expression of E-cadherin and enhanced expression of
   Vimentin and N-cadherin (EMT characteristics of GC cells), were
   inhibited by the P4HA1 suppression (Fig. [88]5C).

Fig. 5.

   [89]Fig. 5
   [90]Open in a new tab

   P4HA1 expression promotes GC migration and invasion and EMT. A, B
   Detection of GC migration and invasion ability via Transwell assay. C
   Detection of E-cadherin, N-cadherin, and Vimentin protein expression in
   GC cells via WB. *p < 0.05, **p < 0.01, ***p < 0.001

P4HA1 knockdown suppresses GC stemness

   Considering the close association between P4HA1 and CSCs, the stemness
   score of P4HA1 was evaluated based on the clinical data of GC in TCGA
   database. The analysis showed that P4HA1 was significantly and
   positively correlated with stemness scores (Fig. [91]6A). Both WB and
   sphere-forming assays revealed that silencing of P4HA1 expression
   suppressed the level of CD44, a stemness marker in GC (Fig. [92]6B) and
   sphere-forming ability of GC cells (Fig. [93]6C). Moreover, the
   correlation between P4HA1 and GC stemness markers was detected by the
   GEPIA2 database, and the results depicted that P4HA1 was strongly
   associated with multiple GC stemness markers (SOX2, CD133, LGR5, SALL4,
   CD44) (Fig. [94]6D). These analyses suggest that P4HA1 is involved in
   the development of CSCs in GC.

Fig. 6.

   [95]Fig. 6
   [96]Open in a new tab

   P4HA1 expression promotes stemness in GC cells. A Correlation analysis
   between P4HA1 and stemness score. B Detection of CD44 protein level in
   GC cells via WB. C Detection of the sphere-forming ability of GC cells.
   D Correlation analysis of P4HA1 with stemness in GC by GEPIA2 database.
   **p < 0.01

Discussion

   Research has shown a strong association between tumor growth,
   metastasis, and TME [[97]18]. The extracellular matrix (ECM), a key
   component of the TME, has been identified as playing a crucial role in
   tumor progression and resistance to immunotherapy [[98]19]. The current
   focus in cancer therapy depends on the invention of new drugs that
   target the ECM and the invention of novel treatment options [[99]20].
   This ECM can be altered by an excessive deposition of collagen, which
   is an essential part of the ECM. Collagen may promote the invasion and
   dissemination of malignant tumors [[100]21].Therefore, novel
   therapeutic strategies and small-molecule inhibitors targeting collagen
   synthesis are being developed for cancer treatment. As a key enzyme in
   collagen biosynthesis, P4HA1 mediates excessive collagen deposition and
   progression in the TME [[101]22]. Recent studies have demonstrated that
   P4HA1 acts as a critical regulator of immune cell function to suppress
   tumor progression, suggesting that targeting P4HA1 or its inhibitors
   may represent a promising strategy to counteract tumorigenesis
   [[102]23]. As previously mentioned, P4HA1 is overexpressed in multiple
   tumor types and promotes tumor progression. However, there are no
   reports of P4HA1's role in GC. This study used the online TCGA revealed
   that P4HA1 was remarkably expressed in GC tissues in comparison to
   normal tissues. To confirm this finding, eight pairs of GC and
   respective paracancerous tissues were collected from the clinics. The
   results of the WB and immunohistochemistry assays confirmed that GC
   tissues showed a substantially higher expression of P4HA1 than
   paracancerous tissues. The expression of P4HA1 in GC cells was
   considerably higher than that in normal gastric mucosa cell lines, as
   demonstrated by the RT-qPCR experiment. This suggested that P4HA1 was
   extensively expressed in GC cells and tissues. Bioinformatics analysis
   further determined the correlation between the survival prognosis of GC
   patients and the expression of P4HA1. The results indicated that the OS
   rate of GC patients decreased as the P4HA1 level increased. In order to
   understand the relationship between P4HA1 and proteins in GC, we
   screened 50 genes from String and cBioPortal databases for each gene
   that binds to or co-expresses with P4HA1, and took the intersection to
   obtain four genes: ALDOA, ERO1A, LDHA, and PLOD1, with which P4HA1
   showed significant positive correlation in GC as well as in a variety
   of tumours. ALDOA, ERO1A, LDHA, and PLOD1 have been shown to be highly
   expressed in GC, capable of serving as poor prognostic markers
   [[103]24–[104]29], and closely associated with glycolysis
   [[105]30–[106]32], mTOR signalling pathway [[107]33], hypoxia
   [[108]34], and EMT [[109]27]. GO analysis of 100 genes associated with
   P4HA1 with duplicate values removed for enrichment analysis in cancer
   revealed that these genes are mainly involved in biological processes
   including collagen fibre organisation, collagen metabolic processes,
   extracellular matrix organisation, and response to hypoxia.The KEGG
   pathway enrichment analysis of P4HA1-related genes in cancer revealed
   that P4HA1 was correlated with protein digestion and absorption,
   proline and arginine metabolism, HIF-1 signaling pathway, and
   ECM-receptor interaction. These findings imply that P4HA1 may have a
   major regulatory function in GC.

   To understand the comprehensive impact of P4HA1 on the biological
   functions of GC cells, siRNA technology was used to transfect GC cells.
   The proliferation, migration, and invasion potential of these cells
   were inhibited by the suppression of P4HA1, as demonstrated by CCK8,
   plate cloning, and Transwell assays. Moreover, EMT is a critical
   prerequisite for the formation of migratory phenomena in different
   cancer types, as it can modify the structure and functional behavior of
   cancer cells to mimic mesenchymal cell types [[110]35, [111]36]. The
   EMT process in cancer progression has been closely associated with
   collagen biosynthesis and deposition [[112]37]. In renal cell
   carcinoma, an elevated level of P4HA1 enhances proliferation,
   migration, invasion, and EMT in renal cell carcinoma cells. In ovarian
   cancer, P4HA1, as a potent diagnostic marker and therapeutic option,
   can inhibit migration, penetration, and EMT in the peritoneal cavity of
   ovarian cancer cells [[113]38]. In glioblastoma, P4HA1 has the
   potential to accelerate glioblastoma cells migration and penetration by
   promoting EMT in the hypoxic microenvironment [[114]39]. In this study,
   the WB experiments displayed that P4HA1 knockdown inhibited EMT
   characteristics, suggesting that P4HA1 may regulate GC cell biology via
   EMT modulation.

   Cancer stem cells (CSCs) were initially recognized in hematological
   cancers [[115]40]. They are distinguished by their specific cell
   surface epitopes, capacity to self-renew, retain tumor metastasis, and
   use multidrug efflux pumps. Their clinical significance originates from
   their intrinsic therapeutic resistance, which allows them to initiate
   tumor regrowth after effective treatments with chemotherapy,
   radiotherapy, or targeted therapies [[116]41]. Various studies have
   shown the involvement of P4HA1 in the regulation of CSCs. The ectopic
   expression of P4HA1 in pancreatic cancer resulted in an elevation in
   the stemness of pancreatic cancer cells, enhanced sphere formation, and
   elevated levels of cancer stemness-associated proteins (OCT4, SOX2, and
   NANOG) [[117]42]. In lung adenocarcinoma, P4HA1 enhances cell stemness
   and cisplatin resistance and may provide a novel target for lung cancer
   therapy [[118]43]. In glioblastoma, P4HA1 promotes angiogenesis in
   polymorphic by driving the transformation of stem cell-like cells into
   tumor-inner cells [[119]39], suggesting that P4HA1 is closely involved
   in the formation of CSCs. The findings of the TCGA data revealed a
   substantial positive association between P4HA1 and stemness score. In
   GC CD44 is a classical and reliable marker for gastric cancer stem
   cells (GCSCs) [[120]44]. Furthermore, cells overexpressing CD44 have
   various CSCs characteristics, such as self-renewal, EMT capacity, and
   resistance to chemotherapy and radiotherapy [[121]45]. This study found
   that the suppression of P4HA1 inhibited CD44 expression in respective
   cells, and also suppressed the sphere-forming potential in respective
   cells. Finally, the GEPIA2 database revealed that P4HA1 was markedly
   and positively associated with GC stemness markers (CD133, SOX2, SALL4,
   LGR5, CD44). These findings indicate that P4HA1 is implicated in the
   formation of CSCs in GC.

   Our findings indicate that P4HA1 is highly expressed in GC and promotes
   GC cell proliferation and metastasis, potentially through EMT
   regulation. Additionally, we observed that P4HA1 influences GC cell
   stemness. However, limitations of this study include insufficient
   clinical samples for validation and the need for further mechanistic
   exploration.

Conclusion

   The study concluded that P4HA1 is substantially overexpressed in GC.
   Its knockdown effectively hinders the growth, migration, invasion, and
   stemness of GC cells. Therefore, targeting P4HA1 could potentially
   serve as a novel treatment approach for GC treatment.

Supplementary Information

   [122]Supplementary material 1. ^(8MB, tif)

Acknowledgements