The expression of Wnt/β-cantenin signaling pathway related protein in Kazak esophageal squamous cell carcinoma and its prognostic significance

Background Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors. Kazak in Xinjiang is a high incidence of esophageal cancer in China. It is known that the occurrence of ESCC is related to the abnormal activation of Wnt/β-cantenin signaling pathway, but the regulatory mechanism of its abnormal activation is not clear. This study aims to study the role of Wnt/β-cantenin signaling pathway related proteins in the development of Kazakh esophageal cancer. Methods: Methods In this study, 226 paraffin tissues and 29 fresh esophageal squamous carcinoma tissues were included. Immunohistochemistry and RT-PCR were used to detect Wnt/β-cantenin signaling pathway related proteins (Wnt2, β-cantenin, GSK3, The expression of suivivin in ESCC and its corresponding normal esophageal mucosa of Kazak and Han nationality, and the relationship between the expression of each protein and the clinicopathological characteristics and prognosis of Kazak ESCC were analyzed. Results We found that there was a significant difference between Han nationality and age (p = 0.038) and lymph node metastasis (p = 0.043). The proportion of Kazak patients under 60 years old was higher than that of Han nationality, indicating that the age of Kazak ESCC patients was lower; the proportion of Han nationality with lymph node metastasis was higher, indicating that the recurrence and progression rate was higher; the expression of Wnt2 in ESCC was related to TNM (p= 0.012), I = 9.8%, II = 52.9%, III = 29.4%, IV = 7.8%; the expression of β- Catenin was significantly different in the depth of invasion (p = 0.032), postoperative radiotherapy or chemotherapy (p = 0.049); the expression of GSK3β in ESCC was related to TNM (p = 0.013), in which I = 13.2%, II = 51.5%, III = 29.4%, IV = 5.9%.The invasion (p= 0.001) and radiotherapy and chemotherapy (p < 0.001) can be used as independent factors to affect the Overalll survivalof patients with ESCC; TNM (p= 0.017), nerve invasion (p= 0.028), hematogenous metastasis (p=0.048), radiotherapy and chemotherapy (p = 0.008) and Survivin (p = 0.0029) were all independent influence shadows of squamous cell carcinoma in patients with ESCC. There was no difference between Kazak and Han in Overalll survival(p = 0.929) and progression free survival (p = 0.845). GSK3βand no significant ESCC.

The expression of Wnt/β-cantenin signaling pathway related protein in Kazak esophageal squamous cell carcinoma and its prognostic significance CURRENT  Methods: Methods In this study, 226 paraffin tissues and 29 fresh esophageal squamous carcinoma tissues were included. Immunohistochemistry and RT-PCR were used to detect Wnt/β-cantenin signaling pathway related proteins (Wnt2, β-cantenin, GSK3, The expression of suivivin in ESCC and its corresponding normal esophageal mucosa of Kazak and Han nationality, and the relationship between the expression of each protein and the clinicopathological characteristics and prognosis of Kazak ESCC were analyzed. Results We found that there was a significant difference between Han nationality and age (p = 0.038) and lymph node metastasis (p = 0.043). The proportion of Kazak patients under 60 years old was higher than that of Han nationality, indicating that the age of Kazak ESCC patients was lower; the proportion of Han nationality with lymph node metastasis was higher, indicating that the recurrence and progression rate was higher; the expression of Wnt2 in ESCC was related to TNM (p= 0.012), I = 9.8%, II = 52.9%, III = 29.4%, IV = 7.8%; the expression of β-Catenin was significantly different in the depth of invasion (p = 0.032), postoperative radiotherapy or chemotherapy (p = 0.049); the expression of GSK3β in ESCC was related to TNM (p = 0.013), in which I = 13.2%, II = 51.5%, III = 29.4%, IV = 5.9%.The mRNA expression of β-cantenin and GSK3β in ESCC was significantly higher than that in normal esophageal mucosa (p < 0.001), but there was no significant difference in Wnt2 and survivin. Lymph node metastasis (p=0.009), nerve Introduction Esophageal squamous cell carcinoma(ESCC) is one of the most common upper gastrointestinal cancers in the world. The mortality and incidence rate is eighth and sixth [1] in the world respectively. The distribution of ESCC in China is obviously different from that in the region. The Kazakh people in Xinjiang are the most prevalent ethnic groups in China. The incidence rate is as high as 155.9/10 million, far higher than the national average of 14.95/10 million. The incidence rate of ESCC in Han nationality living in the same area is only 13/10 10000, and its mortality is 2.3 times higher than the national average level [2,3] .In addition, Kazak ESCC is a high incidence disease in the region, but there are few studies on Wnt pathway and ESCC. Therefore, it is of practical significance for the clinical treatment of esophageal squamous carcinoma of Kazak nationality in Xinjiang to study the related genes in the process of occurrence and development of esophageal squamous carcinoma of Kazak nationality in Xinjiang and clarify its mechanism of action and explore the potential specific therapeutic target.
Wnt/β-cantenin pathway is the classic pathway of Wnt signaling pathway, and it also plays a major role. Wnt signaling pathway includes multiple oncogenes (β-cantenin, CtBP, c-myc, CyclinD1, survivin, etc.) and tumor suppressor genes (GSK3β, APC, wtx1, Axin, etc.), and the structural connection between the related proteins of this pathway is based on the regulation of oncogenes and tumor suppressor genes. As early as 2012, Katrin hoffmeyer published an article on science entitled Wnt/β-cantenin signaling regulates telomerase of stem cells and cancer cells, laying a foundation for discovering the mechanism of Wnt/βcantenin signaling pathway in tumor [4] .β-cantenin, is the key protein of Wnt pathway. βcantenin, (β-cantenin,) was first found as a kind of adhesion factor. Later, it was found that β-cantenin, is a multifunctional protein, whose function is mainly to mediate intercellular adhesion and participate in gene expression. It widely exists in various types of cells, such as endothelial cells, fibroblasts, osteoblasts, and participate in the proliferation, differentiation and apoptosis of these cells It has played an important regulatory role [5] . The genes regulating the transfer of β-cantenin, from cell membrane to cytoplasm and nucleus belong to the upstream genes of the signal pathway, such as Wnt2, LRP5 / 6, APC, GSK, CK1, Axin; the genes regulated by β-cantenin, in the nucleus belong to the downstream genes, such as c-myc, CyclinD1, survivin, VEGF. When Wnt is activated abnormally, its signal can inhibit the function of regulatory complex, and then enhance the stability of β-cantenin, and cause its shift. Therefore, we chose Wnt2 and GSK3βas the upstream genes, survivin as the research objects.
Wnt2 is a ligand protein secreted by tumor fibroblasts, secreted in a paracrine manner, which can activate the Wnt/β-cantenin pathway [6] . The main function of GSK3βis to promote the phosphorylation and degradation of β-cantenin,. The expression of GSK3βis significantly down regulated in tumor tissues, and the function of phosphorylated βcantenin, is weakened [7] . Survivin (encoding gene BIRC5) is a dual functional protein, which can be used as a key inhibitor of apoptosis (IAP) and a key regulator of cell cycle progression. The overexpression of survivin often occurs in various human cancers, and the increase of survivin is related to poor clinical prognosis, tumor recurrence and drug resistance. Because of its selective expression in tumors rather than normal tissues, survivin is considered to be an attractive target for cancer treatment [8] .
The purpose of this study is to study the correlation between Wnt/β-cantenin, signal pathway related differential protein and clinicopathological parameters in Kazakh esophageal squamous carcinoma, and to explore whether it can be used as a marker for metastasis and prognosis evaluation of esophageal squamous carcinoma, so as to lay a foundation for the future study of target gene therapy and prognosis monitoring of Kazakh esophageal squamous carcinoma.

2.Immunohistochemistry
In this study, 229 cases of paraffin embedded ESCC and normal mucosa were made into tissue chips by immunohistochemical technique. Paraffin section was dewaxed and hydrated, 3% hydrogen peroxide blocked endogenous peroxidase, and citric acid (pH = 6) was put into boiling water at 100 ℃, respectively, for antigen repair, calf serum blocked non-specific antigen, and rat anti-human antibody (Wnt2, survivin, GSK3β, β-canteninwas added) was put into the incubator at 37 ℃ for 60 minutes, and then added into the second general antibody of rabbit and mouse respectively. It was put into the oven at 37 ℃ for 30 minutes, and the results were observed under the microscope. The dyeing intensity was divided into 0 (non staining), 1 (weak staining), 2 (medium staining) and 3 (strong staining). The number of positive cells was 0 (< 5%), 1 (5-25%), 2 (26-50%) and 3 (> 50%) respectively. If the multiplier is less than 6, the expression level is defined as "low expression"; otherwise, it is defined as "high expression". Wnt2 is expressed in the stroma of ESCC; β-cantenin, is expressed in the cell membrane of ESCC; suivin is expressed in the nucleus of ESCC, and GSK3β is expressed in the cytoplasm of ESCC. At the bottom of the tube, a small amount of RNA precipitate can be seen. Discard the supernatant, add 1 ml of 75% ethanol, and shake. 7500 × g, centrifuged at 4 ℃, 10 min.

RT-PCR
Discard the supernatant, carefully suck the residual liquid with a pipette, and dry at room temperature for 5-10 min. The precipitate was dissolved in 20 µ L DEPC water, 1.5 µ l total RNA concentration was measured in nanodrop and the purity was calculated by od260 / od280. The configured reverse transcription system was put into the PCR instrument, and the reaction conditions were set at 42 ℃ for 60 minutes and 72 ℃ for 5 minutes. After the reverse transcription, the cDNA was stored at − 20 ℃ for standby. SYBR green dye and 7500 real-time PCR system were used to detect the expression of target gene in fresh tissue. β -acting as a reference gene. All reactions were carried out three times under the following conditions: activation of the polymerase for 2 minutes at 50 ℃, initial denaturation for 10 minutes at 94 ℃, followed by 40 denaturation for 15 s at 94 ℃, annealing and elongation for 30 s at 60 ℃. The mRNA expression of the target gene was calculated by ΔΔct. Table 1 the primer sequence of RT-pCR  (Table 2), we found that there was a significant difference between nationality and age (p = 0.038).The percentage of Kazak patients younger than 60 years old is higher than that of Han, which indicates that the age of Kazak esophageal cancer patients is lower.It was found that there was a significant difference in the incidence of lymph node metastasis(p = 0.043) between ethnic groups. The percentage of Kazak esophageal cancer patients with lymph node metastasis was lower than that of Han patients, suggesting that the probability of Kazak patients with lymph node metastasis was lower than that of Han patients.

2.Wnt/β-cantenin related protein expression in ESCC
As the key protein of Wnt/β-cantenin signaling pathway, Wnt2 is expressed in the cytoplasm of ESCC ( Fig. 1. A), with a positive rate of 45.1% (Table 3), and in the normal mucosa ( Fig. 1. B). The results show that the expression of Wnt2 in ESCC is significantly different in TNM (p = 0.012), among which I = 9.8%, II = 52.9%, III = 29.4%, IV = 7.8%. Table 3  Table 3 The relationship between the expression of Wnt2, -Catenin, survivin and GSK3 and the clinicopathological parameters of ESCC β-cantenin is mainly expressed in the cell membrane of ESCC (Fig. 1C), and its positive expression rate is 69.4%, which is significantly higher than that of 30.6% of ESCC (Table 3). It is negative in normal esophageal mucosa (Fig. 1D). It was found that the expression of β-Catenin in ESCC was significantly different in patients with different depth of invasion (p = 0.032), and there was also a difference in the expression of β-Catenin between patients who received radiotherapy or chemotherapy after operation.
Survivin is mainly expressed in the nucleus of esophageal cancer ( Fig. 1. E), and it is negative in normal esophageal mucosa ( Fig. 1. F), but the positive expression rate is 36.2% in ESCC. There was a significant difference between the expression of Survivin and the depth of infiltration (p = 0.027).
There was no positive expression of Survivin in the mucosa, and 50% in the muscular layer and the whole layer (Table 3).
GSK3βwas mainly expressed in cytoplasm ( Fig. 1. G) and was negative in esophageal mucosa ( Fig. 1.   H). In ESCC, the positive rate of GSK3β was 29.7%, much lower than the negative expression rate of 70.3%, which was significantly different from TNM stage (p = 0.013), in which I = 13.2%, II = 51.5%, III = 29.4%, IV = 5.9% (Table 3). It was considered that the earlier the stage, the higher the expression of GSK3β.
The results showed that the expression of survivin was positively correlated with Wnt2 (r = -0.148, p = 0.026).However, the expression of β-Catenin and GSK3β protein was positively correlated with Wnt2 protein expression(r = 0.156,p = 0.019;r = 0.277 p < 0.001), and there was no significant correlation between other related proteins (Table 4).

mRNA expression of Wnt/β-cantenin p a t h w a y r e l a t e d p r o t e i n s i n E S C C
RT-pCR was used to detect the mRNA expression of Wnt / β -cantenin pathway key protein in 29 fresh tissues of ESCC and matched fresh tissues of normal esophageal mucosa. The results showed that the mRNA expression of Wnt 2 (p = 0.752) (Fig. 2. A) in ESCC was not significantly different from that in normal mucosa. The mRNA expression of β-cantenin, (p < 0.001) (Fig. 2. B) and GSK3β (p < 0.001) (Fig. 2. D) was significantly different in esophageal cancer and normal esophageal mucosa (Table 5).Survivin mRNA expression in ESCC was higher than that in normal mucosa, but there was no statistical significance (p = 0.097) (Fig. 2. C).
At the same time, we found that the mRNA expression of β-Catenin and survivinin ESCC was positively correlated with the expression of GSK3β, among which β-Catenin (r = 0.723, p < 0.001), survivin (r = 0.425, p = 0.022), were all positively correlated (Table 6).  (Table 7). There was no significant difference in Overalll survivaltime (P = 0.929) and progression free survival time (P = 0.845) between Kazakh and Han ESCC patients (Fig3). Lymph node metastasis (P = 0.009), nerve invasion (P = 0.001) and radiotherapy and chemotherapy (P < 0.001) can be used as independent factors to affect the Overalll survivalof patients with ESCC. The overall survival of patients with lymph node metastasis, nerve invasion and no radiotherapy and chemotherapy is shorter (Fig 4). TNM stage (P = 0.017), nerve invasion (P = 0.028), hematogenous metastasis (P = 0.048), radiotherapy and chemotherapy (P= 0.008) and Survivin (P = 0.0029) were all independent influence shadows of squamous non progression stage in patients with esophageal squamous carcinoma (Fig 5), which could affect the occurrence and development of patients with esophageal squamous carcinoma. In TNM stage, patients in stage I and stage II did not show disease progression for a longer time than those in stage III and stage IV, patients without nerve infiltration, blood metastasis and postoperative radiotherapy and chemotherapy had a longer progression free survival period, and patients with survivin positive had a shorter recurrence time than those with negative.

Discussion
The main treatment of esophageal cancer is surgical resection, but it is very difficult to treat those who relapse or progress. Therefore, it is of great significance to study the potential biomarkers of ESCC, reveal its potential mechanism, and find a potential effective targeted drug to improve the prognosis of ESCC patients. In recent years, a large number of studies have suggested that Wnt/βcantenin signaling pathway plays an important role in the occurrence and development of esophageal cancer, so we aimed to find out the role of its key proteins.
Wnt2/β-cantenin signaling pathway plays an important role in regulating the growth and differentiation of cell cycle during the growth and differentiation of tissue and embryo, and also plays an important role in the development of many malignant tumors, such as esophageal cancer [9] , breast cancer [10] , cervical cancer [11] . Wnt2, β-cantenin, GSK3β and Survivin are key proteins of Wnt pathway, which play an important role in regulating cell proliferation, inhibiting cell differentiation and cell cycle.
In this study, we found that there is a certain relationship between the age of ESCC patients and ethnic groups. As a carcinogen, Wnt2 can be detected to increase expression in many cancers and participate in tumor progression. It has also been found that Wnt2 can promote the growth of NSCLC cells by activating Wnt/β-cantenin signaling pathway [12] . In human colorectal cancer (CRC), Wnt2 selectively increases in cancer-related fibroblasts (CAF), leading to increased invasion and metastasis. It is mainly associated with the expression of Wnt2, which leads to the increase of angiogenesis, vascular density and tumor volume [13] . In rectal cancer, Wnt2 is associated with tumor invasion depth, lymph node metastasis, TNM stage, vascular invasion and recurrence [14] . We found that Wnt2 was highly expressed in esophageal cancer, and it was related to TNM stage and lymph node metastasis. High TNM stage or lymph node metastasis was the positive rate of Wnt2, so it was considered that Wnt2 could promote the development of ESCC.
Survivin is a member of the inhibitor apoptosis gene family [15] . The gene may be related to the aggressive behavior in many types of cancer, such as ovarian [16] , bladder cancer [17] , esophageal [18], [19] and so on. In our study, we found that the high expression of survivin was related to the depth of invasion, and the positive expression of Survivin in patients with esophageal cancer invading into the muscular layer and the whole layer was higher than that in patients only invading into the mucosal layer, suggesting that survivin can be used as a predictor of invasion progress.
Moreover, we found that survivin is related to the progression free survival of esophageal cancer patients. The progression free survival of patients with positive survivin expression is shorter than that of patients with negative survivin expression. Therefore, we consider survivin as an independent prognostic factor of esophageal squamous cancer patients. At the same time, it has also been found that the high expression of Survivin in ESCC is related to the degree of tissue differentiation and invasion, and the prognosis of patients with high expression of survivin ESCC is relatively poor [18] .It is consistent with our conclusion. The expression of Wnt2 was negatively correlated with the expression of survivin. However, the actual expression rate of survival in esophageal cancer tissue is higher than that in normal mucosa tissue. The reason may be that although the expression rate is higher than that in normal tissue, it may be because the sample size is not enough to lead to the low positive rate, resulting in the phenomenon of negative correlation with Wnt2 expression.
GSK3β has been found to be a tumor suppressor gene in many tumors. FAK / Pyk2 can activate Wnt/ β-cantenin signal transduction by phosphorylating GSK3β, and DAX1 can promote the growth and tumorigenicity of cervical cancer cells by phosphorylating GSK3β;mir-135a can activate Wnt/βcantenin, signaling pathway by down regulating GSK3 β expression, thus accelerating EMT of bladder cancer cells, promoting invasion and migration [22] . In this study, GSK3β was found to be low expression in ESCC, and TNM stage was related, but it was not found that GSK3β had an impact on the prognosis of esophageal cancer. However, in 29 cases of ESCC, the mRNA expression of GSK3β was higher than that of normal mucosa, and the mRNA expression ofβ-cantenin, survivin and GSK3βwere correlated with each other. Considering that they were Wnt signal proteins, there was a relationship of mutual regulation.
β-cantenin, is the key protein of Wnt signaling pathway. It is reported that acly stabilizes β-cantenin, protein through interaction, promotes the transport of CTNNB1 from cytoplasm to nucleus, and then promotes the transcription activity of β-cantenin, and the migration and invasion ability of colon cancer cells [23] . this study found that the expression of β-cantenin, in ESCC was high, which was related to the depth of invasion, radiotherapy and chemotherapy, and was related to the expression of Wnt2 at the protein level. At the transcription level, it was found that the expression of β-cantenin, mRNA was positively correlated with the expression of GSK3β. In Ishiguro Hideyuki's study, it was also found that β-cantenin, was associated with TNM stage and lymph node metastasis in esophageal cancer patients, and was co expressed with CDH1 [24] . It is suggested that there may be a co expression relationship between β-cantenin and Wnt2, GSK3β.
In conclusion, Wnt2,β-cantenin and Survivin are highly expressed in Wnt/β-cantenin signaling pathway in ESCC. Wnt2 is related to TNM stage and lymph node metastasis. β-cantenin, is related to depth of invasion and radiotherapy and chemotherapy. Survivin is related to depth of invasion. All   Overalll survival analysis and progression free survival in Kazak and Han ESCC patients.

Figure 4
The overall Survival analysis chart of prognsis of ESCC affected by clinicopathological and protein factors.

Figure 5
The progression-free-survival analysis chart of prognsis of ESCC affected by clinicopathological and protein factors.