Introduction

Gastric cancer is the most common malignant tumor of the digestive tract in men. With the change of people’s living habits in recent years, such as irregular diet and disordered work and rest, the incidence of gastric cancer is increasing year by year. Its mortality ranks the fourth in the world, only after lung cancer, colorectal cancer and liver cancer. Gastric cancer has become a disease affecting the quality of life and even the health of patients worldwide.1 Gastric cancer can be resulted from a variety of factors. Helicobacter pylori (HP) infection, age, high salt intake, and a diet low in fruits and vegetables are all risk factors for gastric cancer. Surgery is one of the main treatment methods for gastric cancer. The application of neoadjuvant chemotherapy before or after surgery has received increasing attention, and some patients will undergo surgery after neoadjuvant chemotherapy. However, due to the insidious onset of early gastric cancer, most patients are in the middle and advanced stages when diagnosed, and miss the best treatment time. Even if they can be treated in time, patients still have a high recurrence rate and a poor prognosis.2 Vascular endothelial growth factor (VEGF) and microvessel density (MVD) are important indicators for clinical diagnosis of gastric cancer. VEGF can increase the permeability of microvessels and induce neovascularization. However, there are many factors affecting the expression of VEGF and MVD with low specificity.3 Mucin (MUC) is a glycoprotein with high molecular weight, which can be divided into membrane-bound type and secretory type. Among them, MUC1 and MUC2 are mainly expressed in the stomach.4 It has been found that MUC plays an important role in the normal physiological function and lubrication of the stomach. It is rich in a variety of amino acids, and its secretion level may affect the adhesion, immune recognition, and metastasis of tumor cells.5 It has been reported that MUC shows significantly abnormal expression in the occurrence and development of gastric cancer.6 Jade family PhD index factor 1 (JADE1) is a tumor-related gene, with anti-proliferation and apoptosis-promoting effects. Von Hippel-Lindau (VHL) is a tumor suppressor gene that plays a role in tumor development through the VHL-hypoxia inducible factor (HIF)-dependent pathway, which can control the stability of the cell cycle genome and cell death. It has been found that JADE1 can interact with a variety of genes such as VHL, and then participate in the occurrence and development of diseases.7 The expression of JADE1 has been discovered to be significantly abnormal in a variety of malignant tumors such as renal cancer, colorectal cancer and other malignant tumors.8 The pathogenesis of gastric cancer is complex, and HP infection is an important cause of gastric cancer. The previous study has found that HP infection can induce the expressions of a variety of gastric cancer related factors.9 MUC2 and JADE1 were selected for this study based on the following considerations. Firstly, as a glycoprotein widely expressed in epithelial cells, MUC2 is involved in the renewal and differentiation of epithelial cells and the maintenance of the integrity of epithelial cells, which is closely related to the occurrence and development of a variety of cancers.10 Secondly, the expression of JADE1 is found to be significantly abnormal in renal clear cell carcinoma cells, and it can interact with a variety of genes to participate in the occurrence and development of diseases.11 Therefore, we hypothesized that MUC2 and JADE1 might also play an important role in the pathogenesis of gastric cancer. Based on these findings, the aim of this study is to investigate the relationship between the expression of MUC and JADE1 in gastric cancer tissues and HP infection as well as the depth of tumor infiltration. We hope our results can help clinicians more effectively grasp the diagnostic value of MUC and JADE1 as markers in HP infection and pathogenesis of gastric cancer, and help to further evaluate the patient’s condition.

Materials and MethodsGeneral Materials

The surgical specimens of 132 patients with gastric cancer diagnosed and treated in our hospital from March 2018 to May 2019 were selected as the research subjects. According to the results of immunohistochemical staining, the subjects were divided into MUC2 negative group (n=43), MUC2 positive group (n=89), JADE1 negative group (n=36) and JADE1 positive group (n=96) based on immunohistochemical staining results. There were 79 males and 53 females, aged from 27 to 75 years, with the average age of (50.58 ± 7.26) years. Criteria inclusion: (1) All subjects met the criteria for the diagnosis and treatment of gastric cancer,12 and were confirmed by pathological examination; (2) The subjects without endoscopic surgery, radiotherapy or chemotherapy before participating in the study; (3) All subjects signed the informed consent form and actively participated in the study; (4) The study was approved by the Ethics Committee of our hospital; (5) All subjects were able to cooperate with relevant research contents. Exclusion criteria: (1) The subjects combined with other malignant tumors; (2) The subjects with obviously abnormal liver and kidney function or heart function; (3) Pregnant or lactating subjects; (4) The subjects who were unable to cooperate with the study and poor compliance; (5) The subjects accompanied by mental disorders; (6) The subjects complicated with infectious diseases. The selection process of general materials was shown in Figure 1.

Figure 1 Selection process of general materials.

Immunohistochemical Staining

Gastric cancer tissues and adjacent normal tissues were routinely made into paraffin sections, and then were subjected to dry, dewaxing, hydration, antigen retrieval, block and membrane broken. MUC2 and JADE1 antibody diluents were prepared, then added to each tissue section in turn, and incubated at 4 °Covernight. Subsequently, the tissue sections were cleaned by addition of phosphate buffer solution (PBS), followed by addition of horseradish peroxidase secondary antibody to each tissue section in turn and incubation at 37 °C for 60 min. The sections were washed with PBS, and then being dehydration and transparent after DAB for color development and hematoxylin re-staining. The sections were sealed with resin, and observed under microscope.

The staining results were considered as positive expression when the tissues were stained as brown yellow and yellow. To be specific, positive expression: both staining intensity and the proportion of positive cells were considered as (≥10%); Negative expression: no staining or the staining intensity and the proportion of positive cells were less than 10%. The staining scores of MUC2 and JADE1 were evaluated using a semi-quantitative scoring system: Staining intensity: 0 points for colorless, 1 point for light yellow, 2 points for brown yellow, and 3 points for tan. Proportion of positive cells: The score was based on the proportion of positive cells in total cells, 0 points for 0% to 5%, 1 point for 6% to 25%, 2 points for 26% to 50%, 3 points for 51% to 75%, and 4 points for 76% to 100%. Final score: The staining intensity score was multiplied with the proportion of positive cells score to obtain the final score. According to the final score, the staining results were divided into four grades: negative (−, 0–2 points), weak positive (+, 3–4 points), positive (++, 5–8 points) and strong positive (+++, 9–12 points).

Collection of Clinical Pathological Data

The clinicopathological data of the patients were collected, including the age (≤ 55 years old, > 55 years old), sex (male, female), tumor size (≤ 4cm, > 5cm), location of onset (gastric antrum, gastric body, cardia), degree of differentiation (low differentiation, medium and high differentiation), HP infection (yes, no) (The surface and crypt of gastric mucosal tissue were stained with Warthin-Starry silver staining.13 HP infection was considered positive if it was black dot, curved rod or short rod, otherwise it was negative), lymph node metastasis (yes, no), depth of tumor invasion (T1+T2, T3+T4) (Staging was performed according to the International Association for Cancer Control TNM staging scheme)14 and Lauren typing (diffuse type, mixed type, intestinal type).

Follow-up

Follow-up using telephone interview and outpatient examination was conducted for 36 months, once every 3 months until 12 months and once every 6 months thereafter.

Outcome Measures Detection of the expression of MUC2 and JADE1: The expressions of MUC2 and JADE1 in tissues were detected by immunohistochemistry. The relationship between MUC2 and JADE1 expression in gastric cancer tissues and clinicopathological features was analyzed. The relationship between MUC2 and JADE1 expression and the survival and prognosis of patients: Kaplan-Meier survival curve was used to analyze the relationship between the expression of MUC2 and JADE1 and the survival and prognosis of patients. In the survival analysis, the selected cancer tissue samples were all from the central region of the tumor and did not include the tissue edge part to avoid the influence of the edge effect on the results. Diagnostic value: The diagnostic value of MUC2 and JADE1 alone or in combination for gastric cancer was analyzed using receiver operating characteristic (ROC) curve. Statistical Analysis

According to different observation indicators and data, SPSS 21.0 statistical software was used to analyze the data. Sex, the relationship between the expression of MUC2 and JADE1 with clinicopathological features and other enumeration data were expressed in [cases (%)], and compared using the χ2 inspection. Age and other measurement data were tested by normal distribution, and all were consistent with the normal distribution. The measurement data were expressed in the form of mean ± standard deviation (SD), and compared using t-test between two groups. P<0.05 was regarded as statistically significant.

ResultsExpression of MUC2 and JADE1 in Gastric Cancer and Non-Tumor Tissues

The positive expression rates of MUC2 and JADE1 in gastric cancer tissues were 67.42% (89/132) and 72.73% (96/132), respectively. The positive expression rates of MUC2 and JADE1 in adjacent normal tissues were 22.73% (30/132) and 27.27% (36/132), respectively. The expressions of MUC2 and JADE1 in gastric cancer tissues were significantly higher than that in adjacent normal tissues (P<0.05). MUC2 was mainly expressed in the cytoplasm and JADE1 was mainly expressed in the cytoplasm and cell membrane of gastric cancer tissues, in which the staining was brownish yellow (Figure 2).

Figure 2 Expression of MUC2 and JADE1 in gastric cancer and adjacent normal tissues. (A) Expression of MUC2 in gastric cancer tissues; (B) Expression of MUC2 in adjacent normal tissues; (C) Expression of JADE1 in gastric cancer tissues; (D) Expression of JADE1 in adjacent normal tissues.

Relationship Between the Expression of MUC2 and JADE1 and Clinicopathological Features in Gastric Cancer

The expressions of MUC2 and JADE1 in gastric cancer tissues were significantly different in patients with different tumor size, differentiation degree, HP infection, lymph node metastasis, depth of tumor invasion and Lauren classification (P<0.05). The expressions of MUC2 and JADE1 in gastric cancer tissues were closely related to tumor size, differentiation degree, HP infection, lymph node metastasis, depth of tumor infiltration and Lauren typing (P<0.05, Table 1).

Table 1 Relationship Between Expression of MUC2 and JADE1 With Clinic-Pathological Features in Gastric Cancer [n (%)]

Univariate Analysis of 3-year Survival Rate of Patients with Gastric Cancer

The followed up time was 0.9~36 months, and the median survival time was 23.5 months. The 1-year survival rate was 100.00% (132/132), and the 3-year survival rate was 90.91% (120/132). Univariate analysis showed that tumor size, differentiation degree, lymph node metastasis, depth of tumor infiltration, expression of MUC2 and JADE1 were the influencing factors of 3-year survival rate of patients with gastric cancer (P<0.05, Table 2).

Table 2 Univariate Analysis of 3-year Survival Rate in Patients With Gastric Cancer

Cox Regression Analysis of Risk Factors Affecting the 3-year Survival Rate of Patients with Gastric Cancer

Multivariate Cox regression analysis showed that tumor size, differentiation degree, lymph node metastasis, depth of tumor infiltration, and MUC2 and JADE1 expression were risk factors for the 3-year survival rate of patients with gastric cancer (Table 3).

Table 3 Cox Regression Analysis of Risk Factors Affecting the 3-year Survival Rate of Patients With Gastric Cancer

Relationship Between the Expression of MUC2 and JADE1 in Gastric Cancer Tissue and the Survival and Prognosis of Patients

The patients were followed up for 36 months, and the Kaplan-Meier survival curve was established. The analysis showed that the survival rate of patients with negative expression of MUC2 and JADE1 was significantly lower than that of patients with positive expression of MUC2 and JADE1 (P<0.05, Table 4 and Figure 3).

Table 4 Differences Among Various Factors

Figure 3 The relationship between the expression of MUC2 and JADE1 in gastric cancer tissue and the survival and prognosis of patients. (A) Survival curve of different expression of MUC2; (B) Survival curve of different expression of JADE1.

Analysis of the Diagnostic Value of MUC2 and JADE1 in Gastric Cancer Tissue Alone or in Combination in Gastric Cancer

Based on the ROC curve, the area under the curve of MUC2 in gastric cancer tissues alone in detection of gastric cancer was 0.774, with the sensitivity and specificity of 72.46% and 80.03%, respectively. The area under the curve of JADE1 in gastric cancer tissues alone in detection of gastric cancer was 0.796, with the sensitivity and specificity of 82.14% and 76.48%, respectively. The area under the curve of the combined detection of the two was 0.918, with the sensitivity and specificity of 91.34% and 89.57%. The results indicated that the combined detection of MUC2 and JADE1 had a high diagnostic value in the pathogenesis of gastric cancer (Table 5 and Figure 4).

Table 5 Analysis of the Diagnostic Value of MUC2 and JADE1 in Gastric Cancer Tissue Alone or in Combination in the Diagnosis of Gastric Cancer

Figure 4 ROC curve analysis.

Discussion

Surgery is one of the main treatment methods for gastric cancer. Especially for patients with early gastric cancer, a high cure rate can be achieved by radical resection of the diseased tissue, but there are still some patients with poor long-term survival and prognosis after surgery.15 The pathogenesis of gastric cancer is relatively complex, and it is believed that it may be the result of the comprehensive effect of genetics, HP infection, environment and other factors.16 Early diagnosis of gastric cancer plays an important role in improving the survival rate and prognosis of patients. No previous study has reported the relationship between the expression of MUC and JADE1 and HP infection. The aim of this study was to investigate the relationship between the expression of MUC and JADE1 in gastric cancer tissue and HP infection as well as the depth of tumor infiltration, so as to provide relevant theoretical learning basis for clinicians to evaluate the condition of patients in time and provide effective treatment.

MUC is a glycoprotein widely expressed in epithelial cells, which can participate in the renewal and differentiation of epithelial cells and maintain the integrity of epithelial cells. In addition, it also has a certain protective and lubricating effect. MUC2 is a subtype of MUC, and is mainly expressed in goblet cells of the intestine. MUC2 is not expressed in gastric mucosa under normal condition, but its expression is significantly increased upon gastric cancer. MUC2 may participate in the recognition and adhesion between cells.17 Evaluation of MUC2 expression in gastric cancer patients revealed that MUC2 was more frequently detected in multiple histologically negative lymph nodes.18 Additionally, relevant data show that MUC2 is not expressed in normal lymph nodes, but specifically expressed in perigastric lymph nodes of patients with pT1 gastric cancer, suggesting that the expression of MUC2 has certain sensitivity and specificity. It is helpful to detect lymph node micrometastasis of gastric cancer, and is conducive to early detection and minimally invasive surgery.19 The study has also showed that different concentrations of MUC2 antisense oligonucleotides have a significant inhibitory effect on the growth of SGC7901 cells.20 Thus, the expression of MUC2 protein may play an important role in the biological behavior of gastric cancer. In addition, it has been reported that the expression level of MUC2 is significantly up-regulated in colorectal cancer tissues, and the upregulation of MUC2 is an indicator of high treatment resistance and poor prognosis in patients with colorectal cancer.21 In the present study, the results showed that the expression level of MUC2 in gastric cancer was significantly increased, and its expression level was closely related to tumor size, differentiation degree, HP infection, lymph node metastasis, depth of tumor infiltration and Lauren’s classification. Moreover, the survival rate of patients with positive expression of MUC2 was significantly lower than that of patients with negative expression. This result was similar to previous studies in other tumor types, which showed that MUC2 expression was closely related to the degree of differentiation, tumor stage, and lymph node metastasis of gastric cancer, and that the 5-year survival rate of MUC2-positive gastric cancer patients after surgery was significantly higher than that of MUC2-negative patients.22

JADE1, a member of the JADE protein family, can promote the massive acetylation of histone 4 (H4) by interacting with endogenous histone acetyltransferase (HAT). It has been found that the JADE1S and JADE1L isoforms of JADE1 also seem to play different cellular roles, and that the JADE1 protein is able to directly regulate the process of cell cycle.23 JADE1 is an important regulator of cytokinesis. It has been reported that JADE1 can also act as an E3 ubiquitin ligase by targeting β-Catenin for proteasomal degradation, thus regulating the proliferation, differentiation and distant metastasis of tumor cells.24 The study of the role of JADE1 in pancreatic cancer showed that the expression level of JADE1 in pancreatic tissue was significantly increased, in which JADE1 might regulate phosphatidylinositol 3-kinase/protein kinase B/rapamycin target protein (PI3k/AKT/mTOR) signaling pathway, thereby affecting the development of renal cell carcinoma.8 The results of this study showed that the expression level of JADE1 was highly increased in gastric cancer tissues, and its expression level was closely related to the clinicopathological data and the prognosis of patients. The results were similar to those of Seidl et al25 in other tumor types, in which multivariate Cox regression analysis showed that positive expression of JADE1 was one of the independent risk factors for poor prognosis of patients with gastric cancer, and the HR value was 2.141, indicating that the prognosis of patients with positive expression of JADE1 was poor. These results suggested that the high expression of JADE1 in gastric cancer might be closely related to the poor prognosis of patients, and it could be used as a potential biomarker to evaluate the prognosis of gastric cancer patients.

HP is a pathogen isolated from gastric mucosa and has been classified as a class I carcinogen. Previous studies have found that HP infection is closely related to the occurrence and development of chronic gastritis, gastric cancer and other diseases.24,26 HP may cause inflammation of gastric mucosa for a long time, and bind to the specific receptors of normal gastric mucosal epithelial cells through a variety of adhesion factors. In addition, HP can also damage the normal gastric mucosal barrier by secreting urinary hormones, and then cause the gastric mucosa to develop into gastric cancer. The study has determined the depth of tumor invasion and regional lymph node involvement are determined by detecting HP infection and endoscopic ultrasonography, and the results showed that HP might affect the occurrence and development of gastric cancer by regulating the Wnt/β-catenin pathway and inducing the expression of angiogenic factors.27 The results of this study showed that MUC2 and JAD1 in gastric cancer tissue were closely related to HP infection and tumor infiltration depth, and the combined detection of MUC2 and JAD1 had important value in predicting the incidence of gastric cancer.

In conclusion, the expressions of MUC2 and JADE1 in gastric cancer tissue are significantly increased, and their expressions are closely related to tumor size, differentiation degree, HP infection, lymph node metastasis, depth of tumor infiltration, Lauren’s classification and the survival prognosis of patients. Combined detection of MUC2 and Jade1 has high value in the diagnosis of gastric cancer.

Data Sharing Statement

All data, models, and code generated or used during the study appear in the submitted article.

Ethics Approval and Consent to Participate

This research was approved by the Ethics Review Committees of The General Surgery Of Lanxi People’s Hospital and conducted according to the Declaration of Helsinki.

Consent for Publication

The patients participating in the study all agree to publish the research results.

Funding

There is no funding to report.

Disclosure

The authors declare that they have no conflicts of interest in this work.

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