Expression and function analysis of CRABP2 and FABP5, and their ratio in esophageal squamous cell carcinoma

2.1 Expression of CRABP2 in esophageal cancer and normal esophageal tissues

The basic clinical information of tumor cases, such as demographic data, treatment process, clinical stage, tumor pathology, survival status, and any other related information (mRNA, microRNA, copy number, mutation, protein, methylation information, etc.) was collected from TCGA (The Cancer Genome Atlas http://cancergenome.nih.gov/) and GEO (https://www.ncbi.nlm.nih.gov/gds/) database. According to TGGA, the expression of CRABP2 mRNA in EC tissues and normal tissues was analyzed using GEPIA.

2.2 The expression status of different stages of EC patients

The expression of the CRABP2 gene in different stages of EC was evaluated by the GEPIA database.

2.3 Survival analysis

The relationship between the CRABP2 gene expression level and the prognosis of patients with EC was analyzed by the GEPIA database.

2.4 Protein interaction network

Related genes and a scatter diagram were derived from the GEPIA database. The CRABP2 gene analysis of protein–protein, genetic interactions, pathways, and protein co-expression networks were dependent on the Genemania database.

2.5 Gene function

WebGestalt is an online website for gene function enrichment analysis. To explore the involved cell components, biological processes, and biological functions, the key genes interacting with CRABP2 were analyzed by WebGestalt.

2.6 Tissue microarray (TMA)

TMAs were constructed by the Department of Pathology, The First Affiliated Hospital of Xinjiang Medical University, and technical supports were provided by Shanghai Outdo Biotech Company (Shanghai, China). The TMA contained a total of 240 ESCC patients and 240 matched normal tissues. Paraffin-embedded tissue in each core was obtained from the non-necrotic area of the carcinoma and/or noncarcinoma foci.

2.7 Patients

This study included a retrospective series of 240 patients with ESCC and treated at the First Affiliated Hospital of Xinjiang Medical University from January 2008 to June 2019. The diagnoses of all cases were confirmed by an expert pathology review (SF). The inclusion criteria were as follows: (1) patients with ESCC; (2) patients who did not receive radiotherapy or chemotherapy before surgery; (3) the esophagus was the primary lesion site; and (4) patients of Han or Kazakh ethnicity. The exclusion criteria included the following: (1) patients with adenocarcinoma of the esophagus; (2) patients received radiotherapy or chemotherapy before surgery; (3) patients with tumor metastasis to the esophagus; (4) other ethnic groups, including Uyghurs, Mongolians, etc.; (5) patients who died during the operation and in hospital; and (6) patients with distant metastasis before operation.

According to the above criteria, 172 cases of esophageal squamous cell carcinoma (including 101 cases of Han nationality and 71 cases of Hazak nationality) were randomly collected, including their clinicopathological data: age (<60, ≥60), gender (male, female), tumor location (upper, middle, lower), tumor size (<3 cm, ≥3 cm), differentiation degree (well, moderate, poor), lymph node metastasis (yes, no), vascular invasion (yes, no), nerve invasion (yes, no), hematogenous metastasis (yes, no), AJCC (according to the eighth version of esophageal cancer released by American Joint Committee on Cancer [AJCC] and Union for International Cancer Control [UICC] in 2017, I + II, III + IV), depth of invasion (Mucosa, Muscularis, Full thickness), and postoperative treatment (chemotherapy/radiotherapy, no) (Table 1). The patients were clearly informed about the operation, material extraction, pathological examination, and experimental purpose.

All the selected cases were patients with immersive esophageal squamous cell carcinoma without high-grade intraepithelial neoplasia and carcinoma in situ. According to 2020 CSCO esophagus cancer diagnosis and treatment guidelines, endoscopic mucosal resection can be considered for T1a tumors (involving mucosa but not submucosa), while esophagectomy can be used for T1b or deeper tumors. There was no T1a in the 172 patients and they were all T1b–T4. Radical resection of esophageal carcinoma and lymph node dissection was performed in all the selected patients, followed by esophageal reconstruction with gastroesophageal anastomosis.

It took about 4 months including experiment design, reagents purchase, data organization, regular follow-up, pre-experiment, formal experiment, result interpretation, statistical analysis, and completion of the first draft (from May 2020 to the end of August). Our follow-up time ended in July 2020 through inquiring about the medical records and telephone calls. Overall survival (OS) was calculated as the time interval between the date of surgery and death or last follow-up. Progression-free survival (PFS) was determined from the date of surgery to tumor recurrence or the last follow-up. The time range of OS and PFS is 1–108. The median follow-up time of OS was 28 months while the median follow-up time of PFS was 20 months.

1. Ethics approval and consent to participate: This study was approved by the ethics committee of the First Affiliated Hospital of Xinjiang Medical University. A Signed written informed consent was obtained from all participants before the study.

2. Patient consent for publication: Not applicable.

2.8 Immunohistochemistry

Rabbit monoclonal anti-CRABP2 antibody (ab211927, Abcam, dilution, 1:2,000), rabbit monoclonal anti-FABP5 antibody (ab255276, Abcam, dilution,1:5,000) were purchased for the immunohistochemistry test.

Deparaffinization/hydration of TMAs was performed as follows: sliced specimens were incubated at 58–65°C for 1 h, On the day before the experiment, tissue chips were put into the 37°C oven overnight, softening the wax layer that covered the tissue chip. After dewaxing and dehydration, the tissue chip was put in a boiling EDTA repair solution (pH 9.0) and boiled for 15 min; After 30 min of cooling at room temperature, endogenous peroxidase was added and incubated for 20 min at room temperature. After rinsing with phosphate-buffered saline three times, the CRABP2 and FABP5 antibodies were put in the 37°C oven for 1 h. After cleaning, the goat antirabbit secondary antibody (PV-6001) was placed in a 37°C oven for 30 min. Finally, they were DAB stained solution to brown. Each experiment included a positive control slide. For negative control, the primary antibody was replaced with PBS. Both were located in the cytoplasm or nucleus.

The microscopic examination of each point of the tissue microarray was performed at the same incident light intensity and compensation intensity. Five high-power fields (×400) were selected at random. The evaluation was based on a semi-quantitative method described as follows: Staining index = percentage of positive cells × staining strength; the rule of staining intensity score: 0 point (Negative), 1 point (Light brown), 2 points (Brown), and 3 points (Dark brown). The rule of stained positive cells scores: 0 points (0–10%), 1 point (11–25%), 2 points (26–50%), 3 points (51–75%), and 4 points (76–100%). Above all, the staining index calculations were 0, 1, 2, 3, 4, 6, 8, 9, 12, respectively. For CRABP2, we define it as high expression if the total score is >1 point; otherwise, we define it as low expression. For FABP5, we define it as high expression if the total score is >6 points; otherwise, we define it as low expression. The total score of the tissue chip was independently completed by two pathologists who had no knowledge of the patient’s clinical case data. All scoring differences were resolved through discussion.

2.9 Statistical methods

Statistical analyses were performed by SPSS 26.0 (SPSS Inc, Chicago, IL, USA). The characteristics of the ESCC patients were compared using the χ ² test. OS and progression-free survival (PFS) were assessed using the Kaplan–Meier method and the log-rank test. Multivariate analysis was carried out using the Cox proportional hazard regression model. p < 0.05 was considered statistically significant (χ ² test was used for univariate analysis, p < 0.15).

3.1 Results of differential expression of CRABP2 genes in ESCC and normal tissues in the database

Through the online analysis of the GEPIA website, in Figure 1a, we compared the expression levels of CRABP2 mRNA in 286 cases of EC and 182 cases of esophageal normal tissues. It was found that the expression level in esophageal normal tissues was significantly higher than that in ESCC (p < 0.01).

Figure 1

(a) Analysis of the expression level of CRABP2mRNA in esophageal cancer tissues and normal esophageal tissues based on the GEPIA database (red indicates esophageal cancer tissues, gray indicates normal esophageal tissues, and the difference is statistically significant). (b) The relationship between CRABP2 and tumor staging.

Based on the analysis of the GEPIA database, Figure 1b shows that the expression level of CRABP2 in EC tissues with stages Ⅰ–IV was not statistically significant (p > 0.05).

We used the GEPIA database to analyze the correlation between CRABP2 different expression levels and the OS and PFS of 182 EC patients. The patients were divided into the CRABP2 high expression group (n = 91) and the CRABP2 low expression group (n = 91) according to the median expression. The results were not statistically significant (Figure 2a and b).

Figure 2

(a) Analysis of the overall survival of patients with esophageal cancer with different expression levels of CRABP2 in the GEO database. (b) Analysis of the disease free survival of patients with esophageal cancer with different expression levels of CRABP2 in the GEO database.

3.2 Prediction and functional analysis of the CRABP2 protein interaction network and GO analysis

Using Genemania analysis, we screened a total of 20 CRABP2 interacting proteins: FABP5, CSTA, CSTB, BNIPL, S100A9, S100A8, SBSN, IL1RN, TMEM40, TMEM79, RHCG, RAET1E, CLIC3, ACER1, SLURP1, ENDOU, NLRX1, EVPL, SCNN1B, and PPL, and the interaction relationship included physical interaction, co-expression, sharing protein domains, and co-localization. The scatter plot of the correlation between CRABP2 and each gene is shown in Figure 3. The interactive network is shown in Figure 4.

Figure 3

Correlation analysis between CRABP2 and each gene.

Figure 4

Genemania analysis of CRABP2 interaction proteins.

It is found that these proteins are mainly involved in biological regulation, multicellular biological processes, and responses to stimuli. CRABP2-related genes are located in a variety of cell components, including cell membrane, extracellular, cell vesicles, and so on. Molecular functions include binding proteins, participating in iron ion binding, etc. (Figure 5).

Figure 5

Analysis of biological processes, cell components, and molecular functions involved in the interaction with CRABP2 protein.

3.3 Clinicopathologic characteristics

The demographic data of the 172 patients with ESCC included in the study and the pathological characteristics are summarized in Table 1. The median age of patients at the time of diagnosis was 63 years (35–83 years). The patients were followed up for a mean of 28 months (range 1–108), and 130 (75.5%) patients died during the follow-up period.

3.4 Expression of CRABP2 and FABP5 in ESCC and normal esophageal tissues

We detected CRABP2 and FABP5 in both adjacent normal esophageal mucosa epithelium and ESCC tissues by IHC. The representative IHC images of CRABP2 and FABP5 are presented in Figure 6.

Figure 6

Immunohistochemical detection of the expression of CRABP2 and FABP5 in esophageal cancer and normal esophageal tissues. (a and b) CRABP2 is highly expressed in the normal esophageal squamous epithelium and esophageal squamous cell carcinoma; (c) low expression of CRABP2 in esophageal squamous cell carcinoma; (d and e) FABP5 is highly expressed in the normal esophageal squamous epithelium and esophageal squamous cell carcinoma; and (f) low expression of FABP5 in esophageal squamous cell carcinoma.

CRABP2 was localized in cell nuclei and cytoplasm of ESCC and normal esophageal mucosa. Among 172 cases of the normal esophageal mucosa, CRABP2 was high in all patients. The high expression rate of CRABP2 in the normal esophageal mucosa was 100% (172/172). For the cancer specimens, CRABP2 high expression was observed in 75.6% (130/172) and the difference was statistically significant (Table 2). Moreover, the degree of expression was different, and the staining and scoring of CRABP2 in normal tissues of the esophagus were stronger, while those in ESCC were weaker. This proves that CRABP2 is a downregulated protein.

FABP5 was localized in cell nuclei and cytoplasm of ESCC and normal esophageal mucosa. Among 172 cases of the normal esophageal mucosa, 162 were high expression and 10 were low expression. The high expression rate of FABP5 in normal esophageal mucosa was 94.1% (162/172). For the cancer specimens, FABP5 high expression was observed in 58.7% (101/172) and the difference was statistically significant (Table 2). The staining and scoring of FABP5 in normal tissues of the esophagus were stronger, while those in ESCC were weaker. FABP5 is a downregulated protein in cancer tissue, too.

3.5 Analysis of association between the expression of CRABP2 and FABP5 proteins and clinicopathological characteristics in ESCC

Table 3 reveals that the expression of CRABP2 was not associated with age, gender, degree of differentiation, invasion depth, vascular invasion, nerve invasion, hematogenous metastasis, and postoperative treatment in ESCC (p > 0.05).

The expression of FABP5 was correlated with the lymph node metastasis (p = 0.032), invasion depth (p = 0.041), and AJCC stage (p = 0.013), while no association was seen with age, gender, ethnicity, vascular invasion, nerve invasion, hematogenous metastasis, and postoperative treatment in ESCC (p > 0.05).

We also analyzed the relationship between the CRABP2 and FABP5 ratio and clinical parameters. The ratio was correlated with ethnicity (p = 0.001), nerve invasion (p = 0.031), and postoperative treatment (p = 0.038), while no association was seen with age, gender, tumor location, tumor size, invasion depth, AJCC, lymph node metastasis, vascular invasion, and hematogenous metastasis in ESCC (p > 0.05).

3.6 The relationship between CRABP2 and FABP5

The pairwise correlation among CRABP2, FABP5, and the ratio of the two proteins was evaluated by the Spearman rank correlation test.Table 4 shows that CRABP2 had a positive correlation with FABP5 (r = 0.156, p = 0.041). There was an positive correlation between the expression of CRABP2 and the ratio (r = 0.334, p = 0.000). Meanwhile, FABP5 had a negative correlation with the ratio (r = −0.269, p = 0.000). The above data suggest a correlation between CRABP2 and FABP5. Moreover, the result is similar to Genemania analysis, that is CRABP2 expression was positively correlated with FABP5 (p = 4.3 ×10⁻¹², r = 0.48). CRABP2 and FABP5 may play different roles in ESCC.

In conclusion, we conclude that CRABP2 and FABP5 are low expressed in ESCC, and CRABP2 expression is positively correlated with FABP5 expression. The expression and relationship between the two proteins may be related to tumor progression in ESCC.

3.7 Association clinical features in ESCC with patient survival

In univariate analysis, death and recurrence were regarded as dependent variables, and 16 factors were regared as arguments, such as the gender, ethnicity, degree of differentiation, depth of invasion, AJCC stage, lymph node metastasis, nerve invasion, hematogenous metastasis, CRABP2 expression, etc. In order to avoid missing some important factors, p = 0.15 was setted in univariate analysis. The result showed that the gender (p = 0.119), ethnicity (p = 0.118), differentiation of degree (p = 0.000), depth of invasion (p = 0.117), AJCC stage (p = 0.028), lymph node metastasis (p = 0.140), hematogenous metastasis (p = 0.020), nerve invasion (p = 0.045), and CRABP2 expression (p = 0.079) were the risk factors for OS of patients with ESCC (Table 5). Additionally, the differentiation of degree (p = 0.018), AJCC stage (p = 0.013), lymph node metastasis (p = 0.067), hematogenous metastasis (p = 0.094), and CRABP2 expression (p = 0.152) were the risk factors for PFS of patients with ESCC (Table 5).

In Cox proportional multivariate analysis, death was defined as a dependent variable, and nine factors were defined as arguments such as the gender, ethnicity, differentiation of degree, depth of invasion, AJCC stage, lymph node metastasis, hematogenous metastasis, nerve invasion, CRABP2 expression, etc. Results of all the significant variables and patient survival are shown in Table 6. We found that nerve invasion (β = 0.522, HR: 1.685, 95% CI: 1.095–2.592, p = 0.018) was a risk factor for OS of ESCC (Table 6). In addition, compared with poorly differentiated, moderately differentiated (β = −0.381, HR: 0.683, 95% CI: 0.445–1.051, p = 0.083) and well differentiated (β = −0.977, HR: 0.376, 95% CI: 0.205–0.692, p = 0.002) were protective factors for the OS of ESCC. However, the p-value of CRABP2 was close to 0.05 and CRABP2 expression (β = −0.405, HR: 0.667, 95% CI: 0.440–1.011, p = 0.056) was probably protective factor for OS of ESCC in Table 6. Furthermore, recurrence was regarded as a dependent variable, and five factors such as differentiation of degree, AJCC stage, lymph node metastasis, hematogenous metastasis, and CRABP2 expression were regarded as arguments. The result of all the significant variables and patient survival are shown in Table 7. We found that compared with poorly differentiated, moderately differentiated (β = −0.525, HR: 0.592, 95% CI: 0.388–0.902, p = 0.015) and well differentiated (β = −0.958, HR: 0.383, 95% CI: 0.217–0.679, p = p = 0.001) were protective factors for the PFS of ESCC. Hematogenous metastasis (β = 0.800, HR: 2.225, 95% CI: 1.074–4.608, p = 0.031) was also found to be a risk factor affecting PFS. However, the invasion depth, AJCC stage, lymph node metastasis, and CRABP2 expression were not risk factors or protective factors for PFS of ESCC as shown in Table 7.

The Kaplan–Meier method was used to continuously analyze OS and PFS of ESCC patients with clinical parameters.

The results showed that the OS of patients with high expression of CRABP2 has a longer OS than the negative CRABP2 (p = 0.025) (Figure 7a). The OS of poorly differentiated patients was significantly shorter than that of patients with well-differentiated patients (p = 0.000) (Figure 7b). The OS of patients with early esophageal cancer (AJCC stage I and II) was better than that of patients with III and IV (p = 0.014) (Figure 7c). For patients with lymph node metastasis and nerve invasion, their OS time was also shorter (p = 0.016 and p = 0.025) (Figure 7d and e).

Figure 7

Kaplan–Meier survival curves for patients. (a) The overall survival of patients in ESCC with the expression of CRABP2; (b) the overall survival of patients in ESCC with a degree of differentiation; (c) the overall survival of patients in ESCC with AJCC; (d) the overall survival of patients in ESCC with lymph node metastatic; (e) the overall survival of patients in ESCC with nerve invasion; (f) progression-free survival of patients in ESCC with a degree of differentiation; (g) progression-free survival of patients in ESCC with AJCC; (h) progression-free survival of patients in ESCC with lymph node metastatic; and (i) progression-free survival of patients in ESCC hematogenous metastatic.

Besides, the PFS with poorly differentiated patients was significantly shorter than that of patients with well-differentiated patients (p = 0.001) (Figure 7f). The patients with AJCC stage I and II was longer than that in AJCC stage III and IV (p = 0.001) (Figure 7g). For patients with lymph node metastasis and hematogenous metastasis, their PFS time was also shorter (p = 0.002 and p = 0.009) (Figure 7h and i).