Uridine-cytidine kinase 2 as a novel potential prognostic and therapeutic biomarker in gastric cancer

Abstract

Background

Gastric cancer (GC) is one of the most prevalent and lethal malignancies worldwide, with its high mortality rate attributed to late diagnosis, limited prognostic stratification tools, and a lack of targeted therapeutic options. This study aimed to explore the biological functions and molecular mechanism of uridine-cytidine kinase 2 (UCK2) in GC to identify powerful diagnostic, prognostic, and therapeutic biomarkers for GC.

Methods

We systematically analyzed the expression pattern of UCK2 across multiple datasets, including The Cancer Genome Atlas (TCGA) GC cohort, Gene Expression Omnibus (GEO) datasets, and a prospectively collected cohort of GC patients from Department of Gastric Surgery, Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) (comprising matched tumor and normal adjacent tissues). We then examined the biological roles of UCK2 in vitro and in vivo. A downstream gene expression analysis was performed to explore the potential mechanism of UCK2.

Results

The results showed that UCK2 expression was higher in the GC tissues than in the non-tumor tissues in TCGA and GEO datasets, and in the cohort of patients from our center. The high expression of UCK2 was closely correlated with more malignant features, such as an advanced pathological T (pT) stage (P=0.02), pathological N (pN) stage (P=0.03), and larger tumor size (P=0.01). The univariate and multivariate Cox regression analyses showed that UCK2 expression level was an independent prognostic factor (hazard ratio: 1.524, 95% confidence interval: 1.039–2.234, P=0.03). Functionally, UCK2 down-regulation or knockout significantly attenuated the proliferation, colony formation, and motility capacity of GC cells in vitro. Meanwhile, the silencing of UCK2 inhibited xenograft tumor growth in vivo. Mechanistically, the knock-down/knock-out of UCK2 might decrease the expression levels of the p-AKT, cyclin D1, and cyclin E1 proteins in GC cells, leading to G1/S phase arrest.

Conclusions

Our findings established UCK2 as a novel and clinically valuable biomarker, providing a potential tool for improving the diagnosis and prognosis evaluation of GC, and highlighted its potential as a therapeutic target for future anti-tumor strategies.

Highlight box.

Key findings

• This study identified uridine-cytidine kinase 2 (UCK2) as a novel biomarker with utility for both the diagnosis and prognosis of gastric cancer (GC).

What is known, and what is new?

• Previous research suggests that UCK2 plays a potential oncogenic role in malignant tumors, including lung cancer, hepatocellular carcinoma, breast cancer, and colorectal cancer. Key aspects of UCK2 in GC, including its clinical significance, potential biological roles, and molecular mechanism, remain poorly understood.

• Our findings found UCK2 promoted GC cell proliferation and metastasis. Further, we revealed that UCK2 altered the gene expression profile and influenced the cell cycle via the AKT pathway in GC.

What is the implication, and what should change now?

• UCK2 could serve as a novel diagnostic and prognostic biomarker for GC. UCK2 was found to promote the proliferation and motility capacity of GC cells. Before entering the clinical phase, our findings on UCK2 need to be validated in larger clinical cohorts, and further in-depth mechanistic research on UCK2 needs to be conducted.

Introduction

Gastric cancer (GC) is a highly heterogeneous and complex disease, characterized by abnormal sustained proliferation and enhanced metastatic ability. Treatments for GC, such as radical gastrectomy, chemotherapy, targeted therapy, and immunotherapy, have advanced rapidly and can significantly improve the prognosis of patients; however, challenges such as cancer therapy resistance and immune-related adverse events remain (1-3). Thus, the mechanism of tumor progression needs to be identified urgently, and novel diagnostic, prognostic, and therapeutic biomarkers need to be established.

The metabolic reprogramming of tumors, especially the abnormal reprogramming of nucleic acid-related metabolism, is considered a hallmark in the occurrence and development of tumor cells. Cancer cells undergo metabolic reprogramming to satisfy energetic and biosynthetic requirements that support their sustained proliferation and metastasis (4). Consequently, the mechanism of action of several anti-cancer drugs, including 5-fluorouracil (5-FU), is focused on nucleic acid-related metabolism (RNA/DNA metabolism) (5).

Targeted therapy and immunotherapy have become new options for the treatment of GC; however, chemotherapy containing platinum-based drugs and fluoropyrimidines, which is considered the backbone of targeted therapy and immunotherapy, also plays a crucial role in its treatment (6). A detailed understanding of the modification in the nucleic acid-related metabolism of GC and the screening of key biomarkers could provide novel insights into the treatment of GC.

Human uridine-cytidine kinase 2 (UCK2) belongs to the uridine-cytidine kinase (UCK) family, which comprises two members: UCK1 and UCK2. As the rate-limiting enzymes in pyrimidine salvage synthesis, UCKs catalyze the phosphorylation of uridine and cytidine, converting them into uridine monophosphate and cytidine monophosphate, respectively (7). However, in humans, the catalytic efficiency of UCK2 is significantly higher than that of UCK1 (8).

Some research suggests that UCK2 may play an oncogenic role in malignant tumors (9-13). Research has shown that UCK2 is upregulated and UCK2 enzyme activity is increased in lung cancer (9), hepatocellular carcinoma (10), breast cancer (11), bladder cancer (12), and colorectal cancer (13). Further research has identified that the mechanism underlying UCK2-mediated tumor progression could be related to the enzyme’s metabolic and non-metabolic roles. It may be that UCK2 accelerates the salvage pathway of nucleotide synthesis via the metabolic pathway, which is crucial for maintaining the tumor cell division cycle.

In colorectal cancer, the inhibition of the UCK2 enzyme led to the down-regulation of 18S ribosomal RNA expression and induced cell-cycle arrest in the G0/G1 phase, ultimately leading to cancer cell death (13). However, other studies have reported that UCK2 plays non-metabolic roles. For example, in hepatocellular carcinoma, UCK2 promotes tumor cell metastasis by activating the EGFR-AKT pathway rather than the metabolic pathway (10). Currently, the clinical significance, potential biological function, and molecular mechanism of UCK2 in GC are not yet fully understood.

In this study, we examined the relationship between clinical characteristics and UCK2 expression, which could serve as a novel diagnostic and prognostic biomarker. We also investigated the biological functions of UCK2 in promoting tumor cell proliferation and metastasis, and preliminarily explored its underlying molecular mechanism. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1165/rc).

Methods

Patients and tissue samples

A total of 144 GC tissues and 138 matched non-tumor tissues served as the tissue specimens in this study. All the tissues were obtained from patients with GC who underwent curative gastrectomy at the Department of Gastric Surgery, Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) from August 2004 to December 2007. Tissue microarrays (TMAs; Cat. No. T14-501, TMA1-3, Shanghai Outdo Biotech Co., Ltd., Shanghai, China) were constructed from these tissue specimens by Shanghai Outdo Biotech Co., Ltd. (Shanghai, China). All the enrolled patients underwent gastrectomy without prior neoadjuvant therapy, and their clinicopathological features are summarized in Table 1. Follow-up assessments were conducted every 3–6 months, with data collection completed in September 2012. The median follow-up period was 34.0 months (range, 2–75 months).

Table 1. Correlation analysis of UCK2 expression level.

Characteristics	Low expression (IHC score ≤4)	High expression (IHC score >4)	P value
Gender			0.83
Male	52	48
Female	22	22
Age			0.14
<60 years	45	34
≥60 years	29	36
pT stage			0.02*
pT2–3	15	5
pT4	59	65
pN stage			0.03*
pN0–2	47	32
pN3	27	38
Tumor location			0.66†
Up 1/3	12	13
Middle 1/3	6	9
Low 1/3	42	33
≥2/3	14	15
Tumor size			0.01*
≤4 cm	33	17
>4 cm	41	53
Borrmann type			0.61†
I	1	0
II	17	12
III	40	39
IV	16	19
Lauren type			0.18
Diffuse	41	43
Intestinal	16	19
Mixed	17	8

^†, Fisher’s exact test; *, P<0.05. IHC, immunohistochemistry; pN, pathological N; pT, pathological T; UCK2, uridine-cytidine kinase 2.

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All the experiments related to the tissue specimens and the clinical data were approved by the Institutional Research Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) (No. bc2019087), and informed consent was obtained from all individual participants.

Immunohistochemistry (IHC) staining

IHC staining was used to detect UCK2 expression. Briefly, deparaffinized paraffin-embedded tissue sections underwent 30-minute heating for antigen retrieval, followed by incubation overnight at 4 ℃ with anti-UCK2 antibody (1:200; Cat. No. 10511-1-AP, Proteintech, Wuhan, China). After incubation with a secondary antibody for 30 min, the sections were stained with a diaminobenzidine (DAB) staining kit. UCK2 expression was scored based on intensity (0 = negative; 1 = weak; 2 = moderate; 3 = strong) and positive cell frequency (0 =<5%; 1 =5–25%; 2 =26–50%; 3 =51–75%; 4 =>75%). Each final IHC score was obtained by multiplying the intensity score with the frequency score of the positive cells. Each specimen was independently scored by two pathologists, and if any differences arose, the average of the IHC scores was used.

University of Alabama at Birmingham Cancer (UALCAN) database analysis

The Cancer Genome Atlas (TCGA) patient data were analyzed using the UALCAN data online analysis tools (https://ualcan.path.uab.edu/analysis.html) (14). The expression levels of UCK2 were examined in 415 GC tissues and 34 normal tissues.

Gene Expression Profiling Interactive Analysis (GEPIA) data analysis

Data from TCGA and Genotype-Tissue Expression (GTEx) databases were analyzed using the online Gene Expression Profiling Interactive Analysis (GEPIA) tool (http://gepia.cancer-pku.cn/index.html) (15). The expression levels of UCK2 were examined in 408 GC tissues and 211 normal tissues.

GEO data analysis

The expression levels of UCK2 were also examined in the GSE27342, GSE63089, and GSE33335 datasets, which were obtained from the Gene Expression Omnibus (GEO) database. Additionally, the GEO data of 592 GC patients (datasets: GSE14210, GSE15459, GSE22377, GSE29272, and GSE51105) were analyzed using the Kaplan-Meier Plotter online tool (http://kmplot.com/analysis/index.php?P=background) (16).

Cell lines

Four GC cell lines, including KATO III, SNU-1, NCI-N87, and AGS, were obtained from the American Type Culture Collection (Manassas, VA, USA). Three GC cell lines, including MKN-45, HGC-27, and BGC-823, and the immortalized human gastric epithelial cell line (GES-1) were obtained from the Cancer Research Institute of Beijing, Beijing University, China. All the GC and GES-1 cell lines, excluding the KATO III and AGS cell lines, were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS). The KATO III cell line was cultured in Iscove’s Modified Dulbecco’s Medium (Gibco, Carlsbad, CA, USA) supplemented with 20% FBS. The AGS cell line was cultured in F12 Nutrient Mixture medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS. All the cell lines were incubated at 37 ℃ in a humidified setting consisting of 5% carbon dioxide and 95% air.

RNA extraction and quantitative polymerase chain reaction (qPCR) analyses

Trizol reagent (Invitrogen, Waltham, USA) was used to extract total RNA from the cell lines. Complementary DNA (cDNA) was then generated using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., Nanjing, China) in accordance with the manufacturer’s instructions. For the quantitative real-time qPCR analysis, gene expression levels were measured using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China). The following primers (5' to 3') were used: UCK2 (forward), 5'-GCCCTTCCTTATAGGCGTCAG-3'; UCK2 (reverse), 5'-CTTCTGGCGATAGTCCACCTC-3'; GAPDH (forward), 5'-CTCCTCCACCTTTGACGCTG-3'; GAPDH (reverse), 5'-TCCTCTTGTGCTCTTGCTGG-3'.

Western blot analyses

Total protein extraction was performed using radioimmunoprecipitation assay buffer supplemented with protease and a phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, USA). A 20-µg aliquot of the extracted protein was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto a nitrocellulose membrane. The following primary antibodies were used: anti-UCK2 (Proteintech, Cat. No. 10511-1-AP, 1:1,000), anti-β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:1,000), anti-cyclin D1 (Proteintech, Cat. No. 60186-1-Ig, 1:1,000), anti-cyclin-E1 (Proteintech, Cat. No. 11554-1-AP, 1:1,000), anti-AKT (1:1,000; CST, #9272, Danvers, USA), and anti-P-AKT (CST, #4060,1:1,000). The original images of all the results are presented in Figure S1.

shRNA plasmid construction and the CRSPR/Cas9 technique

The vector pSIH-H1-puro was provided by Professor Zhihua Liu, National Cancer Center/Cancer Hospital, Beijing, China. The specific sequences corresponding to the short hairpin RNA (shRNA) were as follows: 5'-CAAAGAAATCACTGAAGGGAA-3' (shUCK2 #1), and 5'-GATAGCTTCTACCGTGTCCTT-3' (shUCK2 #2). The plasmid lentiCRISPR-V2 (#52961) was provided by Professor Weihua Fu, Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China. Two specific guide RNA sequences were selected for cloning into the lentiCRISPR-V2 plasmid (target sequences: single guide RNA (sgRNA) #1, 5'-TACTGTCTATCCCGCAGACG-3', and sgRNA #2, 5'-GTAGCCGTTGAGACAGCCAT-3').

The empty pSIH1-H1-puro vector and lentiCRISPR-V2 vector were used as negative controls. Lentivirus production was achieved by co-transfecting the HEK293T cells with the specified lentiviral vectors, along with psPAX2 (Plasmid #12260) and pMD2.G (Plasmid #12259). After 12 h, the transfection medium was replaced with complete medium, and the recombinant lentiviruses were harvested 48 and 72 h later.

Cell Counting Kit 8 (CCK-8) assay

To assess the roles of UCK2 in GC cell viability and proliferation, the CCK-8 assay was used. AGS and HGC27 cells that had been stably transfected with shRNA/sgRNA plasmids were seeded into 96-well plates as part of the experimental setup. Three independent biological replicates were performed.

Colony formation assay

Stable transfected cells (300 cells per well) were seeded in a six-well plate and incubated for 1 week. First, the colonies were stained with crystal violet solution. Subsequently, the colonies containing >50 cells each were counted. Three independent biological replicates were performed.

Transwell assay

The role of UCK2 in regulating the motility and metastatic capacity of GC cells was determined by Transwell assay. Cell invasion assays with Matrigel (100 µL of serum-free medium with 2.6 µL of Matrigel; BD Biosciences, San Jose, CA, USA) and migration assays without Matrigel (8.0 µm pore size; Corning Costar, Cambridge, MA) were performed using 24-well Transwell chambers. Approximately 100 µL of serum-free medium with 4×10⁵/mL stable transfected cells were seeded into the upper chamber, while a medium with 20% FBS as a chemoattractant was added into the lower chamber. After 24 h of incubation, the non-invaded cells were removed using cotton swabs. The cells on the underside of the chambers were fixed in methanol for 30 min. The invaded cells were then stained with crystal violet solution for 2 min. Three independent biological replicates were performed.

In vivo tumor model

A subcutaneous tumorigenesis model was established to examine the role of UCK2 in the proliferation of GC cells in vivo. Subcutaneous injections of AGS cells—stably transfected with the control plasmid or sgRNA1/2 plasmid, at a density of 2×10⁶ cells in 0.1 mL of phosphate-buffered saline—were administered to the dorsal flanks of 5-week-old male Balb/c nude mice (n=6). Tumor volume (mm³) was estimated weekly by measuring the longest and the shortest diameters of the tumor, and calculated as follows: volume = (shortest diameter)² × (longest diameter) ×0.5.

Next, an intraperitoneal implant transfer model was established to examine the role of UCK2 in the motility of GC cells in vivo. The AGS cells (2×10⁶ cells in 0.1 mL of phosphate-buffered saline) stably transfected with control plasmid or sgRNA1/2 plasmid were separately intraperitoneally injected into the male Balb/c nude mice (n=3). After 60 days of rearing, the mice were sacrificed by carbon dioxide anesthesia and underwent laparotomy. The implant metastases in the small intestine were carefully identified.

The mice were randomly treated using the random number table method. The mice were housed in specific pathogen free (SPF) conditions. The data acquisition and analysis were performed using a double-blind method. Three independent biological replicates were performed.

All the animal experiments were conducted in accordance with the Tianjin Medical Experimental Animal Care guidelines. The animal protocols were approved by the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) Co., Ltd. (protocol No. YSY-DWLL-2024431).

TCGA database gene set enrichment analysis

The samples were divided into high- and low-expression groups based on their UCK2 expression values in TCGA dataset. Differentially expressed genes (DEGs) between the high- and low-expression groups were identified based on the following cut-off values: |log₂fold change| >0.58 and P<0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the identified DEGs were then conducted.

RNA-sequencing and analysis

After the AGS cells were stably transfected with shUCK2 #1 or the control vector, total RNA extraction was performed using TRIzol reagent (Invitrogen). RNA integrity was subsequently analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RNA libraries were sequenced on the Illumina Novaseq^TM 6000 platform by LC Bio Technology Co., Ltd. (Hangzhou, China).

Statistical analysis

All the statistical analyses were conducted using SPSS version 24.0. The categorical data corresponding to the clinicopathological characteristics were analyzed using the Chi-squared (χ²) test, and the continuous variables were analyzed using the Student’s t-test; the data are expressed as the mean ± standard deviation. Overall survival (OS) was analyzed using the Kaplan-Meier method (significance was determined by the log-rank test). Univariate and multivariate Cox regression models were used to compute the hazard ratios (HRs) and 95% confidence intervals (CIs) for OS. A two-tailed P<0.05 was considered statistically significant.

Results

High UCK2 expression in GC tissues predicted a worse prognosis

To explore the expression level of UCK2 in the GC tissues, we analyzed the different expression levels between the GC tissues and non-tumor tissues in TCGA dataset, TCGA with GTEx dataset, and GEO dataset. UCK2 was significantly more highly expressed in the GC tissues than the non-tumor tissues in all the datasets, including TCGA dataset (P<0.001, Figure 1A), TCGA with GTEx dataset (P<0.001, Figure 1B), and the GSE27342/63089/33335 datasets (all P<0.001, Figure 1C).

Figure 1.

High UCK2 expression is closely correlated with more advanced TNM staging and a worse clinical prognosis. (A-C) UCK2 was differently expressed in the GC tissues compared to the non-tumor tissues in TCGA (A), TCGA with GTEx (B), and GEO (C) datasets. (D) A survival analysis was performed using the GEO datasets. (E) IHC staining was performed to detect UCK2 expression. (F) IHC grades were examined. (G) A Kaplan-Meier analysis was conducted based on different UCK2 expression levels to assess the OS of the GC patients. (H) Forest plot showing the independent predictors of prognosis through a Cox regression model. Data are expressed as the mean ± standard deviation unless otherwise stated. * P<0.05, *** P<0.001. CI, confidence interval; GC, gastric cancer; GEO, Gene Expression Omnibus; GTEx, Genotype-Tissue Expression; HR, hazard ratio; IHC, immunohistochemistry; OS, overall survival; pN, pathological N; TCGA, The Cancer Genome Atlas; TNM, tumor-node-metastasis; UCK2, uridine-cytidine kinase 2.

We also investigated the relationship between UCK2 expression level and the survival outcomes of GC patients in the GEO dataset using the Kaplan-Meier Plotter website tool. The results showed a higher UCK2 expression level was associated with poorer survival in 592 GC patients (HR =1.48, P<0.001, Figure 1D). These results suggest that UCK2 may play a critical role as a proto-oncogene in GC.

To further confirm the high expression and prognostic significance of UCK2 in GC, TMAs comprising 144 GC tissues and 138 adjacent non-tumor tissues collected at Department of Gastric Surgery, Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) were stained using a UCK2 antibody (Figure 1E). The IHC scores of UCK2 were found to be significantly more elevated in the GC tissues than the adjacent non-tumor tissues (Figure 1F). Subsequently, the 144 GC patients from our center were divided into the UCK2 high-expression group and the UCK2 low-expression groups based on their UCK2 IHC scores. Our results suggested a potential correlation between the UCK2 expression levels and OS of these GC patients (Figure 1G).

We further analyzed the correlations between the clinical factors and the expression level of UCK2. As shown in Table 1, the patients in the UCK2 high-expression group exhibited an advanced pT stage (P=0.02), pN stage (P=0.03), and larger tumor size (P=0.01) compared with those in the UCK2 low-expression group. In addition, univariate and multivariate survival analyses were conducted to verify the prognostic value of UCK2 (Table 2). The Cox regression analysis revealed that the UCK2 expression level (HR: 1.524, 95% CI: 1.039–2.234, P=0.03) and pN stage (HR: 1.621, 95% CI: 1.115–2.356, P=0.01) were independent prognostic predictors (Figure 1H). These results indicated that UCK2 plays critical roles in GC, and thus could serve as a novel prognostic biomarker.

Table 2. Univariate and multivariate Cox proportional hazard models for overall survival.

Variables	Univariate analysis			Multivariate analysis
	HR (95% CI)	P		HR (95% CI)	P
Gender (female vs. male)	1.388 (0.942–2.046)	0.09		–	–
Age (≥60 vs. <60 years)	1.477 (1.020–2.138)	0.03*		–	–
pT stage (pT4 vs. pT2–3)	0.985 (0.579–1.676)	0.95		–	–
pN stage (pN3 vs. pN0–2)	1.709 (1.180–2.475)	0.005**		1.621 (1.115–2.356)	0.01*
Tumor location
Middle 1/3 vs. up 1/3	1.028 (0.518–2.038)	0.93		–	–
Low 1/3 vs. up 1/3	0.759 (0.454–1.269)	0.29		–	–
≥2/3 vs. up 1/3	1.023 (0.563–1.857)	0.94		–	–
Tumor size	1.553 (1.045–2.308)	0.02*		–	–
Borrmann type
II vs. I	0.371 (0.049–2.788)	0.33		–	–
III vs. I	0.349 (0.048–2.551)	0.30		–	–
IV vs. I	0.441 (0.059–3.275)	0.42		–	–
Lauren type
Intestinal vs. diffuse	1.090 (0.690–1.721)	0.71		–	–
Mixed vs. diffuse	1.616 (0.991–2.636)	0.055		–	–
UCK2 expression level (high vs. low)	1.623 (1.110–2.371)	0.01*		1.524 (1.039–2.234)	0.03*

*, P<0.05; **, P<0.01. CI, confidence interval; HR, hazard ratio; pN, pathological N; pT, pathological T; UCK2, uridine-cytidine kinase 2.

UCK2 promoted GC cell viability and proliferation

To further examine the biological role of UCK2 in GC, we first evaluated its expression level in a series of GC cell lines and an immortalized human gastric epithelial cell line, GES-1. As Figure 2A,2B show, our results demonstrated that most GC cell lines exhibited significantly elevated messenger RNA (mRNA) and protein expression levels of UCK2, respectively, compared to the GES-1 cell line.

Figure 2.

UCK2 expression levels in GC cell lines and the establishment of silenced UCK2 cell lines. (A,B) The expression levels of UCK2 in the GC cell lines were detected by qPCR (A) and western blot (B). (C,D) Stable cell lines expressing shRNA plasmids were constructed, and their characterization was confirmed by qPCR (C) and western blot (D). (E) Stable UCK2-knocked-out gastric cell lines were also established using the CRSPR/Cas9 techniques. UCK2 expression was examined by western blot. Data are expressed as the mean ± standard deviation unless otherwise stated. ***, P<0.001. GC, gastric cancer; NC, negative control; qPCR, quantitative polymerase chain reaction; shRNA, short hairpin RNA; UCK2, uridine-cytidine kinase 2.

The human UCK2 shRNA expression plasmids were then stably transfected into the UCK2 highly expressed cell lines, AGS and HGC27, to block the expression of UCK2. Stable cell lines with the shRNA plasmid were established, and examined by qPCR (Figure 2C) and western blot (Figure 2D). In addition, stable UCK2-knocked-out gastric cell lines, AGS and HGC27, were also established, using the clustered regularly interspaced short palindromic repeats (CRSPR)/Cas9 techniques (Figure 2E).

The CCK-8 assay results showed that the knock-down of UCK2 impaired the viability and proliferation of the GC cells in the AGS and HCG27 cell lines (Figure 3A). Similarly, the down-regulation of UCK2 inhibited the colony formation capacity of the GC cells (Figure 3B). We also examined the stable UCK2-knocked-out gastric cell lines, AGS and HGC27. The knock-out of UCK2 inhibited the survival and proliferation abilities of the GC cells as demonstrated in both the CCK-8 assay (Figure 3C) and colony formation assay (Figure 3D). The results showed that UCK2 effectively promoted GC cell proliferation in vitro.

Figure 3.

The down-regulation of UCK2 attenuated GC cell viability and proliferation. (A,B) The knock-down of UCK2 impaired GC cell viability and proliferation in the AGS and HCG27 cells as shown by CCK-8 (A) and colony formation (crystal violet staining; magnification, XXXXXX) (B) assays. (C,D) The knock-out of UCK2 inhibited the survival and proliferation ability of the GC cells as demonstrated by CCK-8 (C) and colony formation (crystal violet staining; magnification, XXX) (D) assays. (E) AGS cells with control or sgRNA plasmids were subcutaneously injected into the male Balb/c nude mice to establish an in vivo xenograft model (n=6). (F-H) Image showing tumor volumes (F), tumor growth curves (G), and tumor weight (H) for each group. Data are expressed as the mean ± standard deviation unless otherwise stated. **, P<0.01; ***, P<0.001. GC, gastric cancer; NC, negative control; OD, optical density; SEM, standard error of the mean; sgRNA, single guide RNA; shRNA, short hairpin RNA; UCK2, uridine-cytidine kinase 2.

To examine the ability of UCK2 to promote the proliferation of GC cells in vivo, an in vivo xenograft model was established by subcutaneously injecting the male Balb/c nude mice with AGS cells that had been transfected with control plasmids or sgRNA plasmids. The tumor volumes of the mice were measured every 5 days. As shown in Figures 3E-3G, the knock-down of UCK2 significantly decreased the tumor growth rate, compared with that of the control lines. Similarly, an analysis of the weights of the harvested tumor masses revealed that UCK2 promoted the viability and proliferation of the GC cells (Figure 3H). These results showed that UCK2 promoted the viability and proliferation of the GC cells both in vitro and in vivo.

UCK2 enhanced GC cell motility and metastasis

Given our finding that the UCK2 expression level was significantly correlated with the pN stage, we then explored the role of UCK2 in the migration and metastasis of GC cells. The Transwell assays showed that the knock-down of UCK2 significantly inhibited the migration (Figure 4A) and invasion (Figure 4B) ability of the GC cells. Similarly, we also examined the stable UCK2-knocked-out gastric cell lines, AGS and HGC27. The migration assay (Figure 4C) and invasion assay (Figure 4D) results showed that the knock-out of UCK2 significantly impaired the motility of the GC cells.

Figure 4.

UCK2 enhances GC cell motility and metastasis. (A,B) Transwell assays showed that the knock-down of UCK2 inhibited the migration (A) and invasion (B) ability of the GC cells. Crystal violet staining; scale bar: XXX. (C,D) The knock-out of UCK2 significantly impaired the motility of GC cells as demonstrated by the migration assays (C) and invasion (D) assays. Crystal violet staining; scale bar: XXX. (E) A tumor peritoneal metastasis mouse model was established by the intraperitoneal injection of AGS cells with control or sgRNA plasmids into male Balb/c nude mice (n=3). The abdominal tumor metastatic sites (red arrows) are presented. Data are expressed as the mean ± standard deviation unless otherwise stated. **, P<0.01; ***, P<0.001. GC, gastric cancer; NC, negative control; sgRNA, single guide RNA; shRNA, short hairpin RNA; UCK2, uridine-cytidine kinase 2.

To further investigate the role of UCK in cell motility in vivo, a tumor peritoneal metastasis mouse model was established via the intraperitoneal injection of AGS cells with control or sgRNA plasmids into the male Balb/c nude mice. On the 60th day, all the mice were sacrificed to examine abdominal tumor metastasis. As Figure 4E shows, the knock-out of UCK2 significantly decreased the number of solitary metastatic lesion of the GC cells in the small intestines of the mice. These results indicated that UCK2 also played a key role in the motility and metastasis of the GC cells in vitro and in vivo.

UCK2 altered the gene expression profile and influenced the cell cycle via the AKT pathway in GC

To further explore the potential molecular mechanisms of UCK2, we analyzed TCGA data and divided the patients into high- and low-expression groups based on the UCK2 expression level. Among the 19,564 genes screened, 648 DEGs showed significant differences (Figure 5A).

Figure 5.

UCK2 altered the gene expression profile and regulated the cell cycle in GC. (A) Patients in TCGA database were divided into high- and low-expression groups based on their UCK2 expression levels. Volcano plots were the generated from the mRNA-sequencing analysis of the two groups. (B,C) KEGG (B) and GO (C) enrichment analyses were performed on all the DEGs in TCGA dataset. (D,E) RNA sequencing was used to examine the mRNA profile of the silenced-UCK2 AGS cells and the non-silenced control AGS cells, and a total of 1,014 DEGs were identified (|log₂FC| ≥1 and P<0.05). After conducting the mRNA-sequencing analysis, we generated volcano plots (D) and conducted the KEGG enrichment analysis (E) based on the resulting data. (F) Western blotting was performed to analyze the roles of UCK2 in AKT, cyclin D1, and cyclin E1 expression. (G) AKT kinase inhibitor (HY-10249A from MCE, HY) was used to treat the GC cells. AKT kinase inhibitor (HY) blocked the effect of UCK2 knock-down/knock-out on the p-AKT, cyclin D1, and cyclin E1 expression. The red box marks the signaling pathway and its downstream signals, which were the key focus of the study. DEGs, differentially expressed genes; FC, fold change; GC, gastric cancer; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; mRNA, messenger RNA; NC, negative control; TCGA, The Cancer Genome Atlas; UCK2, uridine-cytidine kinase 2.

GO and KEGG enrichment analyses were then conducted of all the DEGs. The results revealed that 47 DEGs were significantly enriched (Figure 5B), which suggests that UCK2 might regulate the cell-cycle pathway and thus be involved in cell proliferation and death. Similarly, the GO enrichment analysis showed that UCK2 play crucial roles in chromosome segregation, nuclear division, and DNA replication (Figure 5C). Together, these results suggest that UCK2 might enhance GC cell viability and motility by sustaining and promoting the cell cycle.

RNA-sequencing was then performed to compare the mRNA profiles between the UCK2-knocked-down AGS cells and their control counterparts. Using the thresholds of a |log₂fold change| ≥1 and a P value <0.05, the results showed that 1,014 DEGs exhibited significant expression alterations (Figure 5D). Figure 5E shows the results of the KEGG pathway enrichment analysis of these DEGs, which suggested that the AKT pathway might be the crucial downstream pathway of UCK2.

Given the above findings, we speculated that UCK2 might promote the cell cycle and cell proliferation via the AKT pathway. To test this hypothesis, the AKT, p-AKT, cyclin D1, and cyclin E1 proteins were detected by western blot assay. As Figure 5F shows, the down-regulation of UCK2—whether by knock-down or knock-out—significantly decreased the expression levels of the p-AKT, cyclin D1, and cyclin E1 proteins.

Further to confirm the role of the AKT pathway in GC, the AKT kinase inhibitor (HY-10249A from MedChemExpress, XXX, XXX) was used to treat the GC cells. As shown in Figure 5G, the AKT kinase inhibitor (HY-10249A) blocked the effects of UCK2 knock-down/knock-out on p-AKT, cyclin D1, and cyclin E1 expression. These results suggest that UCK2 might enhance malignant phenotypes in GC via the AKT pathway.

In summary, our study identified UCK2 as a novel prognostic biomarker. UCK2 exerts a regulatory effect on GC cells by enhancing their viability and motility, and this effect was confirmed both in vitro and in vivo. Further, we investigated the potential molecular mechanisms of UCK2, and found that UCK2 might influence the cell cycle via the AKT pathway, which is essential in the proliferation and motility of GC cells.

Discussion

In this study, we investigated the clinical significance and prognostic value of UCK2 in GC. Our analysis revealed that high UCK2 expression is an independent prognostic risk factor, and is strongly associated with more advanced pathological tumor-node-metastasis (pTNM) stages and worse survival outcomes. We provided sufficient clinical and experimental evidence identifying UCK2 as the potential oncogene in GC.

Tumor cells exhibit high heterogeneity. This study examined the biological function of UCK2 in GC cells. The experimental evidence showed that the down-regulation of UCK2 both in vitro and in vivo attenuated the proliferative activity and motile capacity of the GC cells. Similar results have also been reported for several other types of cancers, including lung cancer (9,17), breast cancer (11), and hepatocellular carcinoma (10). Wu et al. (17) also showed that poorer clinicopathological features (e.g., a higher TNM stage, earlier recurrence, and a poorer prognosis) were related to high UCK2 expression.

Research has shown that silencing UCK2 suppresses the proliferation and migration of lung cancer cells (17). Shen et al. reported a close relationship between a poor prognosis and higher UCK2 expression, and suggested that UCK2 could serve as a promising prognostic biomarker in breast cancer (11). In hepatocellular carcinoma, UCK2 was found to have both metabolic and non-metabolic roles in tumor cells (10). Thus, UCK2 might promote proliferation in a metabolic manner, while non-catalytically facilitating cell metastasis. The non-catalytical mechanism might be due to the UCK2-AKT signaling pathway.

Research has shown that UCK2 interacts with EGFR to suppress EGF-induced EGFR ubiquitination and degradation, activating EGFR-AKT signaling (10). However, the specific mechanism by which UCK2 functions in GC has not yet been fully elucidated. In this study, we showed that silencing UCK2 weakened cyclin D1 and E1 expression via the AKT pathway, which is crucial for maintaining the cell cycle of tumor cells, especially in the G1/S phase. However, further research needs to be conducted to examine the regulation of UCK2 enzymes in the tumor cell cycle via metabolic or non-metabolic pathways.

In terms of drug therapy, UCK2 may be an effective target or biomarker for tumor treatment due to its key roles in pyrimidine salvage synthesis (18). Some cytotoxic ribonucleoside analogs, such as 5-FU (19) and RX-3117 (fluorocyclopentenyl cytosine) (20), were designed to interfere with nucleotide metabolism in tumor cells. Previous studies reported a close association between the expression level of UCK2 and the objective response to cytotoxic ribonucleoside analogs. Przybyła et al. reported that high UCK2 expression could serve as a prospective biomarker for 5-FU sensitivity in colorectal cancer (21). Further, El Hassouni et al. reported a close relationship between UCK2 expression and the response to RX-3117 in pancreatic cancer cells (22). These findings suggest that the UCK2 expression level may reflect key changes in the metabolic reprogramming of tumor cells, and thus could provide an optional approach for the treatment of GC.

Conclusions

This study identified UCK2 as a novel diagnostic and prognostic biomarker for GC. UCK2 was shown to promote the proliferation and motility capacity of GC cells. The down-regulation of UCK2 was shown to induce cell-cycle arrest.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All experiments related to the tissue specimens and the clinical data were approved by the Institutional Research Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) (No. bc2019087) and informed consent was obtained from all individual participants. All animal experiments were conducted in accordance with the Tianjin Medical Experimental Animal Care guidelines. Meanwhile, the animal protocols were approved by the Institutional Animal Care and Use Committee of Yi Shengyuan Gene Technology (Tianjin) Co., Ltd. (protocol number YSY-DWLL-2024431).

Data Sharing Statement

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DOI: 10.21037/tcr-2025-1165