The expression and clinical significance of serine hydroxymethyltransferase2 in gastric cancer

Differential expression of SHMT2 in multiple human tumours

Pancancer analysis was conducted on the expression of SHMT2 mRNA in a variety of human tumour tissues and precancerous tissues by using the UALCAN database (http://ualcan.path.uab.edu), which is a comprehensive, user-friendly and interactive web resource for analysing cancer OMICS data (Chandrashekar et al., 2022).

SHMT2 gene expression differences

First, differential expression of the SHMT2 gene was carried out from the module of TCGA and GTEX modules of the Assistant for Clinical Bioinformatics (http://www.aclbi.com) (Izzi, Davis & Naba, 2020). In addition, the difference in gene expression was analysed by the UALCAN database.

Survival and clinical data correlation analysis in GC

Prognostic analysis was implemented through the Kaplan-Meier plotter online tool (http://kmplot.com/analysis) and the 214096_s_at data set was selected to analyse and draw the survival curves of three indices including overall survival rate (OS), first progression survival time (FP) and second progression survival time (PPS).The Kaplan-Meier plotter is able to assess the correlation between gene expression and survival in multiple tumour samples. Sources for databases include TCGA, GEO and EGA (Lanczky & Gyorffy, 2021).

Immunohistochemical staining and scoring

Human gastric cancer tissue samples were purchased from Shanghai Outdo Biotech Company (No. HStma180su16), in which 101 cancer tissues and 80 normal tissues had complete clinical case data. Since the clinical cases had been screened, all the original data generated from the immunohistochemistry experiment using the microarray will be included in the scope of this statistical analysis. The paraffin-embedded tissue microarray was dewaxed and hydrated by fresh dimethylbenzene and ethanol. The antigen was repaired by the microwave repair method using 0.1 M sodium citrate buffer. After blocking the antigen, the tissues were incubated with anti-SHMT2 (#33443s, CST, 1:100) at 4 °C overnight. The sections were incubated with secondary antibodies for 15 min. The experimental data were interpreted by two experienced pathologists using the integral system. The staining intensity score was as follows: 0-negative, 1-weakly positive, 2-positive and 3-strongly positive; The score of the positive staining rate was as follows: 1-(0 ~ 25%), 2-(26 ~ 50%), 3-(51 ~ 75%), 4-(76 ~ 100%). The total score was the product of the staining intensity score and staining positive rate (Guo et al., 2021). A score <6 was defined as low SHMT2 expression, and a score ≥6 was defined as high SHMT2 expression.

Correlation analysis between SHMT2 and Ki-67, TP53, CDH1, and PROM1 in GC

The microarray also included the expression information of Ki-67, TP53, CDH1, and PROM1. The correlation analysis was performed by the correlation model of GEPIA (http://gepia.cancer-pku.cn). The Pearson test method was used to analyse the paired gene expression correlation. GEPIA is an interactive web for analysing the RNA sequencing expression data that includes single gene analysis, cancer type analysis and multiple gene analysis plates and can be used for the analysis of differential gene expression and survival analysis (Tang et al., 2017).

Relationship between SHMT2 gene and numerous pathways, MSI, and TMB

The correlation between the SHMT2 gene and pathway scores was analysed by Spearman correlation. All the analysis methods were implemented by the Assistant for Clinical Bioinformatics.

Cell lines and cell culture

The human cell lines GES-1, AGS, MGC-803, HGC-27, and MKN-45 were obtained from the Chinese Academy of Sciences, Science Bank of the Typical Culture Collection (Shanghai, China). The cell lines were cultured in DMEM (Biological Industries, Beit Haemek, Israel) supplemented with 10% foetal bovine serum (FBS, Gibco, Waltham, MA, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin (#P1400, Solarbio, Beijing, China), 37 °C, 5% CO2 incubator in a closed-culture environment.

Western blot

Total cell lysates were extracted with RIPA buffer (#R0010, Solarbio, Beijing, China). The protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking for 1 h, the PVDF membranes was infiltrated at four degrees overnight with anti-SHMT2 (1:1,000, #33443S, CST, San Antonio, TX, USA) and anti-beta actin (1:2,500, 66009-1-Ig, Proteintech, Wuhan, China). The PVDF membrane was washed with TBST three times and incubated in a diluent containing goat anti-mouse or goat anti-rabbit IgG (1:10,000, RS23710 and RS23720, Immunoway, USA) at room temperature for 1 h. Next, the membrane was detected by a near-infrared imaging system (Image-Quant LAS 3000, General Electric Co., Fairfield, CT, Boston, MA, USA). The relative protein expression was analysed with ImageJ software.

Statistical analysis

SPSS statistics 20.0, and GraphPad Prism 8 were used for statistical analysis and plotting. The chi-square test was used to analyse the difference in SHMT2 expression in tumour and precancerous tissue and the correlation of SHMT2 expression with clinical indices. The correlation between SHMT2 expression and prognosis was analysed by Kaplan-Meier survival analysis. The variables with statistical significance in univariate analysis were analysed by Cox multivariate regression analysis. The difference in quantitative data between the two groups was tested by Student’s t test. P < 0.05 was considered statistically significant.

Ethics approval statement

The study was approved by the ethics committee of Shanghai Outdo Biotech Co. Ltd. (SHXC2021YF01) and observed the Declaration of Helsinki.

The expression of SHMT2 is upregulated in many tumours including GC

First, we analysed the difference in SHMT2 expression levels between human tumours and normal tissue through the UALCAN database. The results showed that compared with normal tissues, SHMT2 is upregulated in many human tumours including GC (Fig. 1A). Subsequently, the difference in SHMT2 expression between CG and precancerous tissues was detected by using the TCGA and UALCAN databases. The results showed that SHMT2 was elevated in GC compared to normal tissues, (P < 0.001; Figs. 1B and 1C). In addition, WB results showed that the expression of SHMT2 protein in AGS (P = 0.013), MGC-803 (P = 0.001), HGC-27 (P = 0.001), and MKN-45 (P = 0.03) cells increased, compared with that in GES-1 cells (Fig. 1D).

High expression of SHMT2 is positively correlated with tumour size and TNM stage

The tissue chip contains 101 cancer tissues and 80 normal tissues. The high expression percentages of SHMT2 in cancer and normal tissues were 56.4% and 38.75%, respectively (P = 0.018). The chi-square test showed that the expression of SHMT2 was correlated with tumour size (P = 0.034) and TNM stage (P = 0.042), but not with age, sex, pathological grade, vessel invasion, or lymph node metastasis (Table 1).

SHMT2, vessel invasion and M stage are independent factors for GC

Univariate analysis showed that SHMT2 (HR = 1.910 (1.180–3.090), P = 0.008), tumour size (HR = 2.038 (1.259–3.298), P = 0.004), pathological grade (HR = 2.363 (1.082–5.162), P = 0.031), vessel invasion (HR = 4.164 (2.366–7.327), P < 0.001), T stage (HR = 3.320 (1.209–9.116), P = 0.020), M stage (HR = 5.094 (2.370–10.950), P < 0.001), TNM stage (HR = 2.379 (1.392–4.067), P = 0.002) were prognostic factors in patients with gastric cancer. The above factors were included in the multivariate Cox regression model for analysis (Table 2). SHMT2 was considered an independent prognostic risk factor (HR = 1.683 (1.014–2.792), P = 0.044). In addition, vessel invasion and M stage were also considered (HR = 3.777 (2.105–6.780), P < 0.001; HR = 4.196 (1.890–9.315), P < 0.001).

High expression of SHMT2, T stage, M stage, pathological grade, tumour size, vessel invasion and TNM stage are associated with poor prognosis

To clarify the effect of high expression of SHMT2 on the OS and PPS of patients with GC, a dataset (214096_s_at) was selected by Kaplan-Meier plotter. Compared with the low expression group, the OS, FP and PPS of patients with high expression of SHMT2 were significantly lower with the median survival times of 40 months and 25.9 months, 24.53 months and 13.2 months, 11.2 months and 8.1 months, respectively (Fig. 2B; HR = 1.27 (1.05–1.53), P = 0.012; HR = 1.31 (1.07–1.6), P = 0.0087; HR = 1.3 (1–1.68), P = 0.047).

The results of IHC staining revealed that the high expression rates of SHMT2 in precancerous tissues and cancer tissues were 38.75% and 56.44%, respectively (Fig. 2A). Then, Kaplan -Meier survival analysis was performed in combination with the clinical data of the patients. The results indicated that factors such as TNM stage (χ² = 10.810, log-rank P = 0.001), high expression of SHMT2 (χ² = 7.279, log-rank P = 0.007), T stage (χ² = 6.181, log-rank P = 0.019), M stage (χ² = 21.903, log-rank P < 0.001), pathological grade (χ² = 5.006, log-rank P = 0.025), tumour size (χ² = 8.876, log-rank P = 0.005), and vessel invasion (χ² = 29.205, log-rank P < 0.001) were associated with poor prognosis (Figs. 2C–2I).

SHMT2 is closely related to MKI67, TP53, CDH1, and PROM1

Furthermore, we analysed the correlation between SHMT2 expression and TP53, MKI67, VEGFR, PROM1, and E-cadherin in GC. The results showed significant differences in the expression of TP53, VEGFR, PROM1 and SHMT2 expression (P < 0.05, Table 3). In addition, the correlation between SHMT2 and the above indicators was analysed through GEPIA. The results showed that the expression of SHMT2 was significantly correlated with MKI67, TP53, CDH1, and PROM1 (Figs. 3A–3D; P < 0.001, R = 0.6; P < 0.001, R = 0.49; P < 0.001, R = 0.38; P < 0.001, R = 0.15).

The SHMT2 gene is involved in multiple signalling pathways

To further demonstrate that SHMT2 may promote the occurrence and development of GC and affect the prognosis of patients via certain pathways, we searched the relevant signalling pathways involved in the SHMT2 by Assistant for Clinical Bioinformatics. The results showed that the expression of the SHMT2 gene was closely related to tumour proliferation, DNA repair, the G2M checkpoint, MYC-targeted genes and DNA replication (Figs. 4A–4E; P = 2.85e−32, CI [0.48–0.63]; P = 8.19e−39, CI [0.53–0.67]; P = 2.7e−39, CI [0.54–0.67]; P = 8.29e−45, CI [0.58–0.70]; P = 6.76e−32 CI [0.54–0.67]).