Significance And Biological Function Of AQP5, CCL20and NDRG2in Hepatocellular Carcinoma With Bile Duct Tumor Thrombi

Hepatocellular Carcinoma with Bile Duct Tumor Thrombi (HCCBDT) is a subtype ofHepatocellular carcinoma (HCC), and it is of worse prognosis compared with HCCwithout Bile Duct Tumor Thrombi (BDT), and appeared to have more invasion, moremetastasis, and more tumor recurrence. There are approximately5000～38700newpatients per year in china, and the number increases year by year.The1,3, and5years survival rates of HCCBDT are respectively40-70%,20-50%,10-30%. The reason that HCCBDT possesses so bad prognosis may be as follows: First, ithas no specific serological markers for diagnosis, and when BDT occurred in smallbranches of bile duct, it would be of missed diagnosis. Second, it appears to have moreinvasion, more metastasis, and more tumor recurrence.Before started our study, we found that there were some genes expressed differentlybetween the tumor and BDT in HCCBDT, and then we chose three genes, which had moresignificant difference and been reported to have more relationship with prognosis, toresearch.Part I: The significance and biological fuction of AQP5in HCCBDTAQP5is a member of aquaproin family, and it has been reported to have relation withbreast cancer, Colorectal cancer, Lung cancer, Gastric cancer, Ovarian cancer,and Chronicmyelogenous leukemia (CML), and AQP5might promote proliferation, invasion andmetastasis of tumor cells.This study found that the mRNA level of AQP5was higher in5BDT tissues thancorresponding tumor tissues (P＜0.027) by Real-time PCR detection, and in3specimenswhich had corresponding paracancerous tissues, AQP5mRNA level was highest in BDT,while lowest in paracancerous tissues, but it was not of significance (P=0.064). Whendetected the protein level of AQP5in tissue slices of84patients by immunohistochemistry,it suggested that it was higher in tumors than paracancerous tissues (P＜0.001). In samepatients, the AQP5level was highest in BDT, and lowest in paracancerous tissue(P＜0.001). And when analyzed with Clinical datas of HCCBDT patients, we found that thelevel of AQP5was an independent risk factor for the overall survival time of patients.(Therelative risk was1.366)We down-regulated the level of AQP5by transfecting siRNA-AQP5into three HCCcell lines (Huh7, HepG2and PVTT) in this study. We named cells transfectedsiRNA-AQP5as interference group, while siRNA-N as control group. Compared with control group, both mRNA and protein level of AQP5declined in interference group,accompanied with down-regulation of MMP2,MMP9,ERK1/2. And we also found thatthe ability of growth and invasion of tumor cells declined obviously after RNA interferenceof AQP5.Part II: The significance and biological fuction of CCL20in HCCBDTCCL20is an important member of chemokines, and belongs to CC subfamily, and itsreceptor is CCR6. CCL20can be expressed in activated Monocytes, T lymphocytes,Dendritic cells(DC),or Endothelial cells. CCR6, receptor of CCL20, is mainly expressed inliver, lung, and lymphoid tissues. CCL20's expression can be induced by Tumor necrosisfactor α (TNFα), Interleukin1β(IL-1β), IL-17, Ligand of CD40, Interferon γ (IFN-γ), andso on, and it plays a important role in promoting the growth and migration (especiallymigration to liver) of malignant tumors.This study found that the mRNA level of CCL20was higher in5BDT tissues thancorresponding tumor tissues (P=0.048) by Real-time PCR detection, and in3specimenswhich had corresponding paracancerous tissues, CCL20mRNA level was highest in BDT,while lowest in paracancerous tissues (P=0.048). When detected the protein level ofCCL20in tissue slices of84patients by immunohistochemistry, it suggested that it washigher in tumors than paracancerous tissues (P＜0.001). In same patients, the CCL20levelwas highest in BDT, and lowest in paracancerous tissue (P＜0.001). when analyzed withClinical datas of HCCBDT patients, we found that the level of CCL20was an independentrisk factor for the overall survival time of patients.(The relative risk was1.310)We down-regulated the level of CCL20by transfecting siRNA-CCL20into threeHCC cell lines (Huh7, HepG2and PVTT) in this study. We named cells transfectedsiRNA-CCL20as interference group, while siRNA-N as control group. Compared withcontrol group, both mRNA and protein level of CCL20declined in interference group,accompanied with down-regulation of MMP2,MMP9,ERK1/2. And we also found thatthe ability of growth and invasion of tumor cells declined obviously after RNA interferenceof CCL20.Part III: The significance and biological fuction of NDRG2in HCCBDTNDRG2is an important member of NDRG (N-Myc downstream-regulated gene)family, up-expressed in some Neurodegenerative diseases, and it has been reported to be acandidate tumor-suppressor for many cancers. Now it has been known that NDRG2hasrelation with HCC, Pancreatic cancer, Glioma, Prostate cancer, Colorectal cancer, and so on, and it is down-expressed in these malignant tumors. It was considered that there werenegative correlation between the level of NDRG2and the degree of tumor malignancy,but positive correlation between the level of NDRG2and prognosis of patients.This study found that the mRNA level of NDRG2was lower in5BDT tissues thancorresponding tumor tissues (P=0.002) by Real-time PCR detection, and in3specimenswhich had corresponding paracancerous tissues, NDRG2mRNA level was highest inparacancerous tissues, while lowest in BDT (P=0.014).We up-regulated the level of NDRG2by transfecting NDRG2overexpression vectorinto three HCC cell lines (Huh7, HepG2and PVTT) in this study. We named cellstransfected overexpression vector as overexpression group, while empty vector as controlgroup. Compared with control group, both mRNA and protein level of NDRG2arised inoverexpression group, accompanied with down-regulation of MMP2,MMP9,ERK1/2.And we also found that the ability of growth and invasion of tumor cells were declinedobviously after overexpression of NDRG2(P＜0.05).In summary, through our study we found that: AQP5and CCL20were overexpressedin HCCBDT, and they could promote the Proliferation and invasion of tumor cells. To becontrast, NDRG2was down-expressed in HCCBDT, and it could suppress the proliferationand invasion of tumor cells. Results above might provide us a reference for prognosticjudgment or Gene therapy in HCCBDT.