Expression Of PBK/TOPK In Human Normal And Tumor Tissues And Its Clinical Significance In Cholangiocarcinoma

BackgroundThe increased expression of PBK/TOPK is associated with some malignant tumors, but PBK/TOPK expression and its function in primary liver cancer have not been studied. In our early study, it was found that PBK/TOPK was expressed only in normal bile duct but not in hepatocyte. In this study, we analyzed the expression of PBK/TOPK in hepatic primary cancer and explored its role in cholangiocarcinoma biology.Objectives1. To study the distribution of PBK/TOPK Protein in human normal and tumor tissues by immunohistochemsitry, and to know if it could be as a tumor marker; 2. To study PBK/TOPK expression in liver tissue, to reveal if PBK/TOPK expression was related to cholangiocarcinoma grade and to the patient prognosis; 3. To study PBK/TOPK physiological function in cholangiocarcinoma cell line. Methods1. The expression of PBK/TOPK was detected in normal tissues, including 19 types of normal human tissue and tumor tissues including, 19 types of tumors by using tissue chip assay and EnVision immunohistochemical staining. 2. 117 (74 cholangiocarcinomas, 33 hepatocellular carcinomas and 10 normal liver tissues) samples were prepared from paraffin-embedded surgical specimen. PBK/TOPK protein was detected by immunohistochemical staining and the relationship between PBK/TOPK and Ki67 labeling index and the tumor grade were assessed. The normal bile duct staining was used to set the score intensity and Image-Pro Plus 4.5 software was used to count and to analyze the immunostaining of PBK/TOPK and Ki67. 3. Among the 57 patients with surgical specimens of cholangiocarcinoma, clinical data for 24 patients were attained. The patients'survival time with cholangiocarcinoma and the relationship between PBK/TOPK and survival time were analyzed with the Kaplan–Meier method. 4. The mRNA and protein levels of PBK/TOPK in cholangiocarcinoma (QBC939) cells and other cell lines were examined by RT-PCR and Western Blot, and eukaryotic expression vector for PBK/TOPK was constructed. 5. The suppressive effect of PBK/TOPK with small interfere RNA in cholangiocarcinoma cell line was studied by detecting cell cycle. 6. QBC939 cells were stimulated with EGF and the alteration of mRNA and protein were detected; and cell cycle of the cells was examined by FCM analysis.Results1. PBK/TOPK was expressed in 8 normal tissues and special cells, such as the sweat glands of scalp, renal distal tubular epithelial cells, esophageal mucous gland, pancreatic ductal epithelial cells and biliary epithelium. High level expression of PBK/TOPK protein was observed especially in breast carcinoma, thyroid carcinoma, and cervical cancer.2. We found PBK/TOPK was usually expressed in normal bile duct epithelial cells, and much more expressed in cholangiocarcinomas (68/74) but never expressed in hepatocytes and hepatocellular carcinomas.Statistically, PBK/TOPK expression was significantly different between cholangiocarcinoma and hepatocellular carcinoma(P<0.001). There was no positive correlation between the expression of Ki67 and PBK/TOPK (P=0.286). A correlation between the expression of PBK/TOPK and the histopathological grading of the tumor was also not found(P=0.67).3. The median survival time was 8 months for13 patients with low expression of PBK/TOPK and 14 months for 11 patients with high expression of PBK/TOPK. Low expression of PBK/TOPK was predictive of poor survival of the patients with cholangiocarcinoma (P=0.013). It was also found that the median survival for the patients was related with the differentiation of cholangiocarcinoma (P=0.011) and the gender (P=0.009).4. We detected PBK/TOPK mRNA and Protein for by RT-PCR and Western blotting in cholangiocarcinoma cell line. PBK/TOPK gene was cloned from the total RNA of QBC939 cells by RT-PCR, and their sequences were identical to the report in GenBank. The full length eukaryotic expression vector pcDNA3.1+PBK/TOPK was constructed.5. After the stimulation with EGF (20ng/ml) for about 2h, the mRNA of PBK/TOPK in QBC939 cells began to increase and followed by the increased protein. Then we analyzed the cell cell cycle of QBC939 cells after the stimulation with EGF and found that the cell number of G1 phase remained stable but the cell number of G2/M period reduced with that of S phase increased.6. After the transfection of the siRNA into QBC939 cells, PBK/TOPK expression was decreased compared with the negative control. No significant difference was observed in the cell cycle profile between PBK/TOPK the transfected cells and the negative control.ConclusionsPBK/TOPK is expressed in several normal tissues and some special cells,and certain carcinoma tissues strongly expressed PBK/TOPK. As PBK/TOPK was usually expressed in normal bile duct cell and most of human cholangiocarcinomas, but never in hepatocyte and hepatocelllular carcinomas, it indicates that PBK/TOPK protein could serve as a potential diagnostic marker to make the differential diagnosis of CC from HCC. Furthermore, the low expression of PBK/TOPK is predicative of poor survival in CC patients. EGF stimulation can enhance the expression of PBK/TOPK in cholangiocarcinoma cells but suppression of PBK/TOPK in cholangiocarcinoma cells with siRNA did not affect its cell cycle. The data indicate that PBK/TOPK may not be a key protein in cell cycle control for cholangiocarcinoma cells. However, the exact biologic function of PBK/TOPK in both normal bile duct epithelial cells and in cholangiocarcinoma cells needs to be further investigated in our future research work..