Differential expression and significance of Stat3 and IL-17 in gastritis and gastric intraepithelial neoplasia and gastric cancer

Background: The role of Stat3 and IL-17 in the development of gastric cancer was investigated by detecting Stat3 activation level and IL-17 expression in gastritis, GIN and gastric cancer. Methods : (1) We collected 70,137 patients who underwent gastroscopy at the First Affiliated Hospital of Shihezi University Medical School from November 2007 to December 2015, coded and screened all the diagnostic results. (2) We used immunohistochemical to detect differences in phosphorylated Stat3 and IL-17 expression in gastritis, GIN, and gastric cancer. (3) Kruskal-Wallis H test compared IL-17 or p-Stat 3 expression in gastric cancer, GIN and gastritis; Non-parametric test compared difference of IL-17 expression and p-Stat3 activation level between randomized and disease progression groups indifferent gastric diseases. Results: (1)The results of immunohistochemistry showed that as the degree of disease increased, the staining increased too; (2) There were differences in Stat3 activation level and IL-17 expression between gastritis, GIN and gastric cancer tissues (P<0.001). With the disease increasingly serious, the Stat3 activation level and IL-17 expression are higher; (3) The ROC of p-Stat3 is the largest (0.923, P < 0.001) and the ROC of IL-17 is 0.844 (P < 0.001); (4) The activated Stat3 and IL-17 both are the risk factors of gastric cancer. Conclusions: (1) With the disease increasingly serious, the Stat3 activation level and IL17 expression are higher. (2) There is a high correlation between activated Stat3 and IL17 expression, both of which are risk factors for gastric cancer.

3 390,182 deaths occur in China, accounting for 44.1% and 49.9% of the world, respectively, and the morbidity and mortality rank second in malignant tumors [1,2]. In recent years, with the advancement of science and technology and the people's awareness of health, the incidence and mortality of gastric cancer have been controlled. However, this phenomenon is not optimistic.
Signal transducer and activator of transcription 3 (Stat 3) is a protein with dual functions of signal transduction and transcriptional regulation. Under normal circumstances, the body's Stat 3 exists in the cytoplasm in a non-activated state. When it is stimulated by the external environment, it can be activated into p-Stat3, and then enters the nucleus to bind to DNA, participate in the formation of tumor microenvironment by regulating downstream proinflammatory cytokines and factors that promote tumor growth and proliferation [3]. In recent years, many studies have shown that highly activated Stat 3 is found in various malignant tumor cells such as breast cancer, cervical cancer, prostate cancer, and oral squamous cell carcinoma. p-Stat3 can up-regulate the expression of downstream genes such as vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX-2) and metalloproteinases (MMPs), inhibit tumor cell apoptosis, promote tumor cell proliferation and neovascularization, as well as tumor cell infiltration and metastasis [4] Among the cytokines induced by p-Stat3, Th17 cells and neutrophil secretion in the CD4+ T cell subset are the main sources of leukocyte 17 (Interleukin-17, IL-17). As a proinflammatory factor, IL-17 can not only promote tumor growth and metastasis, up-regulate the production of pro-angiogenic factors, but also play a clear role in tumors by enhancing the local T cell-associated immune response in tumors [5]. Some studies have shown that IL-17 expression enhanced in malignant tumors such as liver cancer, breast cancer, colorectal cancer, and lung cancer [6,7,8]. Some studies have shown that the expression levels of IL-17 in cancer tissues and serum of patients with gastric cancer are significantly 4 higher than those in the normal groups, and IL-17A and IL-17F can be expressed in gastric cancer tissues and normal gastric mucosa tissues [9].
According to the Cell magazine, unless the microbe interacts with the immune system, the microbe itself cannot independently promote tumor growth. The bacterial metabolites can cause changes in the immune system, which in turn promotes the development and progression of tumors, and changes in the immune system can counteract microbes and thus affect tumors.Pathogen-Associated Molecular Patterns(PAMPs) can affect immune cells through Toll-like receptors and produce the inflammatory factor IL-17, which in turn activates the NF-κB/Stat3 signaling pathway and ultimately affects tumor progression [10].
Some studies showed that IL-17 affects the progression of chronic inflammations and tumors by promoting the release of various inflammatory factors, angiogenesis and cell proliferation [11].
The early symptoms of gastric cancer patients have no obvious specificity, which makes the diagnosis of early gastric cancer difficult, and it is easy to miss diagnosis and misdiagnosis. Therefore, how to improve the early diagnosis rate of gastric cancer, enhance the curative effect and prolong the survival time of patients is a difficult problem in clinical practice. By understanding the research status at home and abroad, we found that most scholar's research focuses on the changes of cytokines in different signaling pathways of different individual gastric cancers, and few people have studied the dynamic changes of gastric cancer progression in the same individual. Therefore, our study collected and analyzed a large number of conventional gastroscope, Based on the number of patients' visits, the study population was divided into a single gastroscope group (54,846 cases) and gastroscope group (9,501 cases). We screened 36 patients with disease progression in multiple gastroscope populations, 21 of whom had intact paraffin tissue(Supplementary Table 1). We randomly selected gastritis (30 cases), intraepithelial neoplasia (30 cases) and gastric cancer (40 cases) from a single gastroscope group. A total of 100 cases were used as randomized control group, and 75 cases were reviewed as the final experimental sample (Supplementary Table 1).
All the cases were not treated with surgery, radiotherapy or chemotherapy.

Experimental methods
All tissue specimens were fixed in 10% neutral formalin, embedded in paraffin, and 6 sectioned thickness serially at 4um. Immunohistochemical staining was performed using the Envision two-step method. Specific antibodies: p-Stat 3 antibody (purchased from Abcam, ab76315, sourced from rabbit anti-human antibody, working concentration was 1:300, buffer was EDTA), IL-17 antibody (purchased from Abcam, ab79056, sourced from rabbit anti-human antibody, working concentration was 1:300, buffer was EDTA). The rest of the reagents were purchased from the Company of Beijing Zhongshan Jinqiao Biological.
Breast cancer tissues stained positive with known p-Stat 3 and kidney cancer tissues positive for IL-17 staining were used as positive controls, respectively; PBS solution was used as a negative control.

Results determination
The results of the sectioning were judged by two pathologists using a double-blind method. The cytoplasm of IL-17 was positively expressed as light yellow to brown granules, and the nuclei of p-Stat3 was positively expressed as light yellow to brown granules. Ten different fields of view were randomly selected under a 400x optical microscope, and 100 cells of the same type were counted in each field of view.

Statistical analyses
Statistical analysis was performed using the SPSS statistical software package (version 17.0) Kruskal-Wallis H test was adopted to compare the expression of IL-17 or p-Stat 3 in gastric cancer, intraepithelial neoplasia and gastritis; In random group and disease progression group, non-parametric test was used to compare the difference of IL-17 expression and p-Stat3 activation levels in different gastric diseases; Spearman rank correlation analysis was used to compare IL-17 expression and p-Stat3 activation level; Univariate logistic regression analysis of gender, age, IL -17 expression and p-Stat3 activation level were used to judge the hazards of gastric cancer; All P values were bilateral, and difference with P < 0.05 was considered statistically significant.

Active p-Stat3 and IL-17 levels were tested using immunohistochemistry (IHC) assay in gastritis, GIN and GC
A positive stain for p-Stat3 was defined as light yellow or yellow-brown stain observed in the nucleus, IL-17 with positive stain light yellow or yellow-brown is mainly localized in the cytoplasm. As shown in Figure 1, the coloration of degree and area were gradually increased in gastritis, GIN and GCdegree of p-Stat3 activation and IL-17 levels.

The degree of p-Stat3 activation and the expression of IL-17 in gastritis, GIN and GC
In the random group and the progression group of Table 1, the degree of p-Stat3 activation showed that the strong positive expression rate in the three groups between gastritis, GIN and GC gradually increased with the severity of the disease, and the difference was statistically significant (random group χ 2 =52.99, P<0.001; progression group χ 2 =28.57, P<0.001), and the degree of p-Stat3 activation was the highest in gastric cancer tissues. There were differences statistically significant of the expression of IL-17 levels in both group, and the expression rate was also increased with disease severity among the gastritis, GIN and GC. the random group χ 2 =45.29 P <0.001 the progress group χ 2 =24.02 P <0.001 , the higher expression rate of IL-17 appeared on the gastric cancer between the both groups ( Table 2) .

The differences between p-Stat3 activation and IL-17 expression in random and progression groups in different diseases
In Table 3

Using ROC curve to analyze the ability of each index to predict gastric cancer
The area under the showss the ability of each indicator to predict gastric cancer. The larger the area under the curve is, the stronger the predictive abilityis. The Yoden index is a comprehensive index for evaluating sensitivity andspecificity. Correspondingly, the larger the area under the curveis, the higher the Yoden indexis. In Fig. 3, the area under thecurveof p-Stat3 is the largest (0.923, P < 0.001), the maximum value of the Yoden index is 0.707, and the cut-off value is 5. The area under thecurveofIL-17 line is0.844( P < 0.001), the maximum value of the Yoden index is 0.615, and the cut-off value is 7. Age and gender did not show significant predictive power in this study.

Logistic analysis of risk factors for gastric cancer
According to the cut-off value calculated by ROC as the critical value, the value of the cutoff value is greater than the critical value of the risk of gastric canceror equal, and is less than the critical valueregardedas the low risk group of gastric cancer. According to the results in Table 4 The results of immunohistochemistry in this study showed that p-Stat3 was activated in gastritis tissue with little or no obvious expression; p-Stat3 was expressed in the nucleus of gastric glandular cells in intraepithelial neoplasia; In gastric cancer tissues, the degree of activation inp-Stat3 is stronger than that of gastric epithelial neoplastic tissue, and the coloration area is larger. This phenomenon exists in both randomized and disease-promoting populations. In the randomized group, the positive expression rates of activated p-stat3 in gastritis, gastric intraepithelial neoplasia and gastric cancer tissues were 23.8%, 85.7% and 92.3%, among which the strong positive expression rates were 4.8%, 42.9% and 69.2%.In the disease progression group, the positive rate of p-Stat3 was 17.6% in gastritis, 93.3% in gastric epithelial tumors, and 100% in gastric cancer tissues.
The strong positive expression rates were 5.9%, 13.3%, and 81.8%. The positive expression rate of p-Stat3 in the randomized group and the disease progression group gradually increased with the progression of the disease, and the difference was significant (P<0.001), suggesting that the abnormal activation of p-Stat3 may be closely related to the occurrence of gastric cancer.
In human gastric cancer and colorectal cancer studies, high-density Th17 cells are associated with increased angiogenesis in invasive tumors [12,13]. Tumors with high levels of IL-17mRNA , the number of vascular endothelial cells and infiltrating neutrophils was significantly higher than the low one [12]. All above suggest that IL-17 high expression is involved in tumor angiogenesis and promotes tumor growth. There are many ways of tumor metastasis. The common ones are blood and lymphatic metastasis. IL-17 in nonsmall cell lung cancer tumors can induce the production of VEGF-C and VEGF-D, which is associated with increased lymphangiogenesis and poor prognosis [14]. The expression levels of IL-17 in serum and cancer tissues of patients with gastric cancer were significantly higher than those in the normal group. What's more, IL-17A and IL-17F could also promote the proliferation of gastric cancer cell lines [15,16]. With the progression of the lesion, the positive expression rate of IL-17 is gradually increased in cervical control tissues, CIN1, CIN2, CIN3 and cervical squamous cell carcinoma tissues [17], which can be inferred that IL-17 is in tumor growth, metastasis and angiogenesis play a role.
The results of immunohistochemistry in randomized and advanced populations of this 11 study confirmed that IL-17 expression was low in gastritis tissues; IL-17 is only expressed in the cytoplasm of gastric glandular cells of intraepithelial neoplasia, and its staining intensity is significantly deeper than that of gastritis; In gastric cancer tissues, IL-17 expression was observed in the cytoplasm of gastric cancer cells, monocytes and vascular epithelial cells. Compared with the intraepithelial neoplasia tissue, the expression level of IL-17 was significantly increased in gastric cancer tissues, and the coloration was deeper and the area was larger, which indicated that both the random group and the disease progression population existed. In the randomized group, the positive expression rates of IL-17 in gastritis, gastric intraepithelial neoplasia and gastric cancer were respectively 76.2%, 100%, and 100%, respectively, and the strong positive expression rates were respectively 19.0%, 89.2%, and 84.6%. In the disease progression group, the positive expression rates of IL-17 in gastritis, gastric intraepithelial neoplasia and gastric cancer were respectively 88.2%, 100%, and 100%, and the strong positive expression rates were respectively 17.6%, 80.0%, and 100%. The positive expression rate of IL-17 in the randomized group and the disease progression group gradually increased with the progression of the lesion, and the difference was significant at different stages (P<0.001).
By further comparing the expression of IL-17 in gastritis, gastric intraepithelial neoplasia and gastric cancer tissues in the randomized and advanced groups, the results showed no significant difference between the two groups (P>0.05). It is suggests that the abnormal expression of IL-17 may be an early event of gastric cancer, which plays a role in promoting the progression from gastritis to gastric intraepithelial neoplasia to the development of gastric cancer. In the early stage of gastritis, the immune system secretes a small amount of IL-17. It synergizes with various cytokines to amplify the inflammatory response, and damages the gastric mucosa leading to disease progression.With the progression of the disease, the injured gastric tissue further recruits the autoimmune system and the adaptive immune system secretes more IL-17, which induces the mutation of cell damage gene and leads to the occurrence of tumor. This is a common phenomenon and does not produce different results due to individual differences.
Th17 differentiation is impaired in CD4+ T cells of Stat3-deficient mice, whereas overexpression of p-Stat3 increases IL-17 production [18,19]. Stat3 is highly activated and IL-17 is highly expressed in colon and breast cancer [20,21]. In this study, IL-17 expression, Stat3 activation was highly positively correlated with disease severity. As the disease progresses, the degree of Stat 3 activation is low when IL-17 is low, and the level of Stat 3 is also increased when IL-17 is high. Both are consistent in gastric tissue and are expressed in gastric cancer. The highest, followed by intraepithelial neoplasia, the lowest gastritis.

Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.

Ethics approval and consent to participate
Ethical approval was obtained from the Institutional Ethics Review Board (IERB) of the First Affiliated Hospital of School of Medicine, Shihezi University (No. 2018-067-01). The IERB waived the need for patient consents due to anonymous analyses of the data and confidentiality and anonymity in the handling and publication of patients' tissues.
Standard University Hospital Guidelines in accordance with the Declaration of Helsinki were followed in this study.