Int J Clin Exp Pathol 2017;10(3):2496-2509 www.ijcep.com /ISSN:1936-2625/IJCEP0044430 Original Article shRNA-mediated silencing of PKHD1 gene promotes proliferation, migration and invasion of human intrahepatic cholangiocarcinoma HuCCT-1 cells Su Lin1, Cheng He1, Min-Fang Wang1, Yin-Lian Wu1, Jian Lin2, Yi Liu3, Yue-Yong Zhu1 1Liver Research Center, 2Department of Hepato-Biliary-Pancreatic Surgery, The First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian Province, China; 3Department of Gastroenterology, Huashan Hospital of Fudan University, Shanghai, China Received November 16, 2016; Accepted January 7, 2017; Epub March 1, 2017; Published March 15, 2017 Abstract: Intrahepatic cholangiocarcinoma (ICC), a type of cholangiocarcinoma, is characterized by insidious onset and lack of typical clinical symptoms at early onset, and the lack of effective treatments results in a poor prognosis. Identification of novel biomarkers and treatment targets is therefore of great significance to improve the survival for ICC patients. Polycystic kidney and hepatic disease 1 (PKHD1), a gene responsible for autosomal recessive poly- cystic kidney disease (ARPKD), has been linked to cancers, and mutation of the PKHD1 gene may cause abnormal proliferation and differentiation of bile duct epithelial cells. However, the role of the PKHD1 gene in the biological be- haviors of ICC cells remains unknown until now. The present study was therefore designed to examine the effects of PKHD1 knockdown on the proliferation, migration and invasion of human ICC HuCCT-1 cells and investigate the un- derlying mechanisms. We transfected a lentiviral vector LV3-PKHD1 that contained the short hairpin RNA (shRNA)- mediated silencing of the PKHD1 gene into HuCCT-1 cells, while GFP lentiviral vector LV3NC-transfected cells served as negative controls. The cell proliferation, migration and invasion were measured, and the ultrastructure of primary cilium was observed using scanning electron microscopy (SEM). The expression of PI3K/Akt signaling proteins was determined with Western blotting. qRT-PCR assay determined down-regulation of PKHD1 mRNA expression and Western blotting analysis revealed reduced FPC expression in HuCCT-1 cells post-transfection with LV3-PKHD1, which validated the effective silencing of the PKHD1 gene in HuCCT-1 cells. Wound scratch assay showed that the LV3-PKHD1 transfected HuCCT-1 cells had a greater healing ability of the scratch than the LV3NC-transfected and nontransfected cells at 24 and 48 h, and CCK-8 assay revealed that the LV3-PKHD1 transfected HuCCT-1 cells exhibited a greater proliferative ability than the LV3NC-transfected and nontransfected cells (P < 0.01). In addition, Transwell migration assay showed significantly more LV3-PKHD1 transfected HuCCT-1 cells penetrating through the Transwell chamber than the LV3NC-transfected and nontransfected cells (P < 0.01), and Transwell invasion assay revealed more LV3-PKHD1 transfected HuCCT-1 cells crossing the Matrigel than the LV3NC-transfected and nontransfected cells (P < 0.01). Moreover, Western blotting assay detected significant up-regulation of PI3K, Akt, p-Akt, and NF-κB expression in LV3-PKHD1 transfected HuCCT-1 cells as compared to that in the LV3NC-transfected and nontransfected cells (P < 0.05), and SEM displayed shorter length, less number and lower distribution density of primary cilium on the surface of LV3-PKHD1 transfected HuCCT-1 cells relative to LV3NC-transfected cells. The results of this study demonstrate that the silencing of the PKHD1 gene promotes the proliferation, migration and invasion of human ICC HuCCT-1 cells via the PI3K/Akt signaling pathway. Keywords: Intrahepatic cholangiocarcinoma, PKHD1, proliferation, migration, invasion, PI3K/Akt signaling, gene silencing Introduction only to hepatocellular carcinoma (HCC) in all hepatobiliary malignancies and comprises Cholangiocarcinoma is a primary bile tract approximately 3% of all gastrointestinal tumors malignant tumor that originates from the bile [2]. This malignancy is found to be highly preva- duct epithelial cells [1]. The incidence of chol- lent in people at ages of approximately 70 angiocarcinoma is estimated to rank second years, with a bit higher incidence in men than in PKHD1 silencing promotes HuCCT-1 cell proliferation, migration and invasion women [2]. Epidemiological data show an ongo- invasion of human ICC HuCCT-1 cells and inves- ing rise in both the incidence and mortality of tigate the underlying mechanisms, so as to pro- cholangiocarcinoma worldwide [3]. vide new insights into the screening of novel targets for the treatment of ICC. Cholangiocarcinoma is anatomically classified into three groups, intrahepatic, perihilar, and Materials and methods distal [4]. Intrahepatic cholangiocarcinoma (ICC) originates from the intrahepatic bile duct Cell lines and culture and its branch to the interlobular ductile, which is characterized by insidious onset and lack of Human ICC HuCCT-1 cell lines were purchased typical clinical symptoms at early onset [5-7]. from Riken (Kyoto, Japan), and GFP lentiviral Since the currently available diagnostic tools vector LV3NC (target sequence: TTCTCCGAAC- show a low sensitivity and there is a lack of GTGTCACGT) transfected HuCCT-1 cells were specific biomarkers, most cases with ICC are provided by the Shanghai GenePharma Biotech identified as a late stage or having severe com- Company (Shanghai, China). The lentiviral vec- plications upon definite diagnosis [8]. Surgical tor LV3-PKHD1 (target sequence: AAGCAGT- resection and liver transplantation remain the CCAAATCCAGGACC) that contained the short most effective approaches for the treatment of hairpin RNA (shRNA)-mediated silencing of the ICC; however, surgical therapy has a low com- PKHD1 gene was constructed in vitro by the plete resection rate, which achieves 10% to Shanghai GenePharma Biotech Company 30% 5-year survival rate post-surgery [9], and (Shanghai, China), and the LV3-PKHD1 was there is a huge lack of matched liver donors for transfected into HuCCT-1 cells to construct sta- liver transplantation which suffers from a great bly transfected HuCCT-1 cells with low PKHD1 deal of money [10-12]. Screening and identifi- expression. Both vectors contained GFP. All cell cation of novel biomarkers for early diagnosis lines were maintained in the Dulbecco’s modi- and prognosis prediction is therefore of great fied Eagle’s medium (DMEM; Gibco, Carlsbad, significance to improve the survival in ICC CA, USA) supplemented with 10% fetal bovine patients, which is urgently needed. serum (FBS; Gibco, Carlsbad, CA, USA), 1.5 mM L-glutamine (Hyclone; Logan, UT, USA), 100 U/ Polycystic kidney and hepatic disease 1 (PK- ml penicillin(Hyclone; Logan, UT, USA) and 100 HD1), a gene responsible for autosomal reces- μg/ml streptomycin (Hyclone; Logan, UT, USA) sive polycystic kidney disease (ARPKD), is at 37°C containing 5% CO2. encoded by the membrane-associated recep- tor-like protein fibrocystin/polyductin (FPC), Wound scratch assay which is composed of 4074 amino acids [13- 15]. PKHD1 gene is expressed at a high level in Six culture inserts (Ibidi GmbH; Munich, Ger- the kidney and at lower levels in the liver and many) were placed in the center of a 24-well pancreas [16-18], while the PFC protein is plate (Corning Inc.; Corning, NY, USA), and were mainly distributed on the primary cilia and api- divided into 3 groups, of two inserts for each cal membrane in the bile duct, which is involved group. Log-phase, LV3NC-transfecteds and LV3- in the maintenance of the normal tubular struc- PKHD1-transfected HuCCT-1 cells were seeded ture of the intrahepatic bile duct epithelial cells onto culture inserts at a density of 3.5 × 104 [19]. Mutation of the PKHD1 gene has been cells in each insert, respectively. Cells were cul- shown to cause abnormal proliferation and dif- tured overnight at 37°C in a saturated humidity ferentiation of bile duct epithelial cells [20], incubator containing 5% CO2 (Thermo Fisher and the morphological defect of primary cilium Scientific; Cleveland, OH, USA). Then, the cul- and defective cilium-mediated intracellular sig- ture inserts were gently removed with a sterile nal transduction may lead to cell cycle abnor- forceps, and a scratch with 500 μm in width mality and benign cell over-proliferation, result- was produced. Each well that contained an ing in cyst formation and enlargement [21, 22]. insert was slowly transferred with approximate- However, the role of the PKHD1 gene in the bio- ly 500 μl fresh medium through the plate wall, logical behaviors of ICC cells remains unknown and the changes of scratches in the culture until now. The present study was therefore plate was observed once every 4 to 6 h under designed to examine the effects of PKHD1 an inverted microscope (Zeiss; Oberkochen, knockdown on the proliferation, migration and German), and photographed. 2497 Int J Clin Exp Pathol 2017;10(3):2496-2509 PKHD1 silencing promotes HuCCT-1 cell proliferation, migration and invasion CCK-8 assay let solution for 20 min, washed with clean water and photographed. Log-phase, LV3NC-transfecte and LV3-PKHD1- transfected HuCCT-1 cells were harvested, Scanning electron microscopy (SEM) digested with 0.25% trypsin (Hyclone; Logan, UT, USA) and prepared into single-cell suspen- Sterile coverslips measuring 0.5 cm × 0.5 cm sions. Cells were seeded onto 96-well plates at were transferred to 24-well plates. Log-phase, a density of 2000 cells in each well and incu- LV3NC-transfected and LV3-PKHD1 transfect- bated overnight. Then, each cell-contained well ed-HuCCT-1 cells were digested with 0.25% was transferred quickly with 10 μl CCK-8 solu- trypsin and prepared into single-cell suspen- tion along the plate wall, and gently shook for sions. Cells were then seeded onto 24-well 4 completely even mixture. Following 2 h incuba- plates at 2 × 10 cells per well and incubated tion, the absorbance was measured at 260 nm overnight at 37°C containing 5% CO2. When with a NanoDrop2000 UV-Vis spectrophotome- cells were adherent to the plate wall, they were ter (Thermo Fisher Scientific; Cleveland, OH, rinsed gently with PBS to remove the dead USA).