Worldwide, bladder cancer is the common cause of cancer incidence and mortality in the urology system, with an approximately 549000 new cases and 200000 deaths in 2018 [1]. Urothelial carcinoma is the leading type of bladder cancers. However, the risk of recurrence and progression of infiltrating urothelial carcinoma of bladder (IUCB) remains high [2]. Thyroid hormone receptor interacting protein 13 (TRIP13) which was originally identified as a protein interacting with human papillomavirus E1 protein belongs to AAA + ATPase superfamily. TRIP13 which is located on chromosome 5p15.33 encodes 432 amino acids. It has been demonstrated that TRIP13 plays an important role in spindle assembly checkpoint pathway and meiotic recombination [3]. Accumulating researcher had demonstrated that overexpression or amplification of TRIP13 should play an important role in many human cancers, such as thyroid cancer [3], colorectal cancer [4], hepatocellular cancer [5], prostate cancer [6], and ovarian cancer [7].
Epithelial-mesenchymal transition (EMT) is characterized that epithelial cells loses their epithelial characteristics, such as cell junctions, morphology, and polarity, acquires mesenchymal characteristics, such as motility and invasiveness. Zinc finger E-box binding homebox 1 (ZEB1), an important EMT-activators, which regulates EMT by repression of epithelial-cadherin (E-cad) expression [8, 9]. ZEB1 is also involved in many human tissue differentiations, such as bone, smooth muscle, and neural tissue. Overexpression of ZEB1 could cause EMT process in various human cancers, such as colorectal cancer, lung cancer, breast cancer, liver cancer, and pancreatic cancer [8–12].
E-cad which can regulate epithelial cell-cell and epithelial cell-stromal cell adhesion belongs to transmembrane glycoprotein superfamily. Down- or lost-expression of E-cad may weaken the adhesion of cell-cell, resulting in the cells easy to move and cells deformation, which leads to cells infiltrate and metastasize. Down- or lost-expression of E-cad is a pivotal role in EMT process and promotes EMT process in various human epithelial cancer.
Overall, accumulating evidence have indicated that expression of TRIP13, ZEB1, and E-cad is closely linked to cancer infiltration and metastasis. However, the correlations between expression of these proteins and IBC have not been widely reported. The purpose of this study is to investigate the expression of these proteins in IUCB and analyze the association among expression of these proteins each other.
Methods
150 cases of IUCB’s patients who were diagnosed in the Department of Pathology of the First Affiliated Hospital of Bengbu Medical College, from January 2013 to December 2015 were considered as experimental group. 150 cases of corresponding “normal bladder mucosa tissues” which came from the same patients were considered as control group. IUCB’s patients who received neoadjuvant chemoradiation treatment or any other anti-cancer treatment were excluded. This study was approved by the ethical committee of Bengbu Medical College and carry out according to the guidelines issued of the Declaration of Helsinki. All IUCB’s specimens were obtained with patients writing consent. Clinicopathological characteristics, demography, and follow-up data of patients were collected at the same time. Overall survival (OS) time was calculated from patients’ surgery date to his or her death date or December 2020. Tumor-node-metastasis (TNM) stages were evaluation by the 8th edition of the guidelines issued by American Joint Committee on Cancer (AJCC). Specific clinicopathological characteristics see Table 1.
Table 1
Patients characteristics | Frequency (n) | Percentage (%) |
Age (years) | | |
< 60 | 92 | 61.3 |
≥ 60 | 58 | 38.7 |
Gender | | |
Male | 93 | 62.0 |
Female | 57 | 38.0 |
Location | | |
Side | 72 | 48.0 |
Posterior | 46 | 30.7 |
Top | 23 | 15.3 |
Triangle | 9 | 6.0 |
Size (cm) | | |
< 2.0 | 87 | 58.0 |
≥ 2.0 | 63 | 42.0 |
Smoking | | |
No | 75 | 50.0 |
Yes | 75 | 50.0 |
Alcohol | | |
No | 66 | 44.0 |
Yes | 84 | 56.0 |
Tumor stages | | |
T1 | 45 | 30.0 |
T2 | 83 | 55.3 |
T3 | 22 | 14.7 |
Lymph node metastasis stages | | |
No | 136 | 90.7 |
Yes | 14 | 9.3 |
TNM stages | | |
Ⅰ | 46 | 30.7 |
Ⅱ | 76 | 50.7 |
Ⅲ | 28 | 18.7 |
Immunohistochemistry
Immunohistochemical staining was performed in accordance with the Elivision™ Plus detection kit instructions. All tissue specimens were fixed in 10% buffered formalin solution and embedded in paraffin, and then cut continuous 4-µm-thick slices. All slices were deparaffinized and dehydrated with xylene and alcohol, then rinsed with phosphate buffer solution (PBS, pH 7.2) for 10 min. Methanol containing 3% hydrogen peroxide solution was used endogenous peroxidase activity of tissues block. 95 ℃ citrate buffer (pH 6.0) for 30 min was used antigen repair. Subsequently, rinsed with PBS several times, all slices were blocked with goat serum for 20 min, then incubated with rabbit polyclonal antibody against human TRIP13, ZEB1, and mouse monoclonal antibody against human E-cad at 4℃ overnight. Reagent A and reagent B were added in sequence, and then developed in diaminobenzidine (DAB) substrate solution and re-dyed with hematoxylin.
Evaluation of immunohistochemical staining
Ten fields randomly at high-power-field (HPF) from different areas of every slice was selected. Two pathologists who were blind on all patients (such as clinicopathological, demography, and follow-up) data independently explained immunostaining results. Immunostaining results was assessed in accordance with the product of immunostaining extent and intensity [13]. For IBC tissues that were positive expression of TRIP13, ZEB1, and E-cad, an average of the result of each slice was taken. When score ≥ 3 was considered positive result in this study.
Statistical analysis
Relationships between clinicopathological characteristics and TRIP13, ZEB1, and E-cad expression were used Chi-square test or Fisher’s exact test. Associations among TRIP13, ZEB1, or, E-cad expression were used Spearman’s test. The effects of TRIP13, ZEB1, and E-cad expression on OS were defined using Kaplan-Meier method with Log-rank test for univariate analysis. Multivariate analysis was used COX regression model. SPSS 19.0 software for Window was used statistical analysis. When P < 0.05 was considered statistically significant.