DANCR Acts as a Diagnostic Biomarker and Promotes Tumor Growth and Metastasis in Hepatocellular Carcinoma

Materials and Methods

Patients and samples. Tissue samples were collected from patients with HCC who underwent partial liver resection at Department of Hepatobiliary and Pancreatic Surgery, the Affiliated Hospital of Qingdao University, from June 2014 to August 2016. Plasma samples were collected from HVs and patients with CHB, cirrhosis and HCC before therapy. Patients with CHB or cirrhosis were diagnosed and selected randomly at Department of Infectious Diseases ranged from June 2015 to August 2016. HCC, CHB and cirrhosis were confirmed by laboratory tests, imaging and histological techniques. The clinicopathological characteristics of patients and HVs are listed in Table I. This work was carried out in accordance with the Declaration of Helsinki for experiments involving humans. This study was also approved by the Ethics Committee of Affiliated Hospital of Qingdao University (application number: 20140105) and all participants provided their informed consent to participate in this study.

Cell culture. HCC cell lines SMMC-7721 and HCCLM3 were obtained from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) in a humidified atmosphere of 5% CO₂ at 37°C.

Quantitative reverse transcription-PCR (qRT-PCR). Total RNAs of tissues or plasma were extracted using Trizol (Invitrogen, Carlsbad, CA, USA). For the extraction of RNA from plasma, 1 pmol SV40 miRNA mimic (Qiagen, Valencia, CA, USA) per 200 μl plasma was added as spike-in RNA for normalization. After reverse transcription, cDNA was amplified by using SYBR-Green Premix (Takara, Otsu, Japan). The expression of DANCR, vimentin, cyclin D1 (CCND1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC) in tissues and cell lines were normalized to the expression of β-actin. The data were analyzed by delta Ct method. The primers used in this study are listed in Table II.

Transfection and luciferase reporter assay. siRNAs targeting DANCR and negative control (NC) were purchased from GenePharma Company (Shanghai, China). Oligonucleotides were transfected into cells by Hiperfect transfection reagent (Qiagen). TOP/FLASH reporter gene including β-catenin-binding sites was obtained from Millipore Corporation (Billerica, MA, USA). The reporter constructs were transfected into cells using Lipofectamine 3000 (Invitrogen). Luciferase activity assay was conducted using Dual Luciferase Assay System (Promega, Madison, WI, USA). pRL-TK plasmid (Promega) was used to normalize the transfection efficiency.

Cell proliferation assays. Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) was used to evaluate cell proliferation. Briefly, cells were seeded into a 96-well plate and transfected with corresponding oligonucleotides. At different time points, 10 μl CCK-8 solution was added into each well of the plate. The plates were incubated at 37°C for 1 h and the absorbance at 450 nm was measured. The OD450 value reflected the cell viability.

Transwell invasion assays. Cell invasion assays were conducted using 24-well transwell chambers with 8.0-μm pore size polycarbonate membrane (Corning Incorporated, Corning, NY, USA). Cells were seeded on the top side of the membrane pre-coated with Matrigel (BD, Franklin Lakes, NJ, USA). After incubation in a humidified atmosphere of 5% CO₂ at 37°C, cells in the upper chamber were removed with cotton swabs. Invaded cells on the lower membrane surface were fixed with methanol and stained with 5% crystal violet. The cells were counted under a Leica DMI4000B microscope (Leica, Heidelberg, Germany).

HCC xenograft mouse model. DANCR-knockdown lentivirus and the NC lentivirus were purchased from Genechem corporation (Shanghai, China). HCCLM3 cells (5×10⁶) infected with the corresponding lentivirus were injected subcutaneously to the posterior flank of the BALB/c nude mice purchased from the Shanghai Institute of Materia Medica (Shanghai, China). One to two weeks later, the subcutaneous tumors were implanted into the livers of new nude mice. About 4 weeks after implantation, the mice were euthanized and the tumors were removed. The tumor volumes were calculated as follows: tumor volume (mm³) = (length × width²)/2. The lungs of mice were fixed with formaldehyde and the fixed tissues were serial sectioned and stained with hematoxylin-eosin. Then lung metastatic nodules were observed under a Leica DMI4000B microscope (Leica). All animal studies were conducted in the Animal Institute of Qingdao University according to the protocols approved by the Medical Experimental Animal Care Commission of Qingdao University (application number: QDFYMAL-2015122503).

Western blot. Total proteins were extracted from cells transfected with indicated siRNAs using RIPA lysis buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a 0.45 μm polyvinylidene fluoride membrane (Millipore). The following primary antibodies were used in the immunoblotting assays: β-actin and β-catenin (Cell Signaling Technology, Danvers, MA, USA).

Statistical analysis. Statistical analysis was performed using the SPSS program (version 18.0; Chicago, IL, USA). Data are presented as the mean±standard deviation (SD). Statistical significance was calculated by Student's t-test, χ² test or one-way ANOVA followed by Newman-Keul's test. Pearson analysis was used in correlation analysis. Receiver operating characteristic (ROC) and the area under the ROC curve (AUC) was applied to evaluate the diagnostic value and the Youden index was applied to determine the optimal cutoff points of DANCR and alpha fetoprotein (AFP) values in the diagnosis of HCC. The statistical significance of differences between two AUCs was calculated and compared by using Delong's algorithm. A value of p<0.05 was considered statistically significant.