Cell lines and cell culture
The human HCC cell line SMMC-7721 was obtained from the Cell Bank of the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The PLC/PRF/5, HepG2, SK-HEP-1, and Hep3B2.1-7 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The Huh7 cell line was obtained from the Riken Cell Bank (Tsukuba, Japan). The MHCC-97L and MHCC-LM3 cell lines were kindly provided by the Liver Cancer Institute, Zhongshan Hospital of Fudan University (Shanghai, China). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) at 37 °C in a 5% CO2 incubator. Standard transient transfections for all cell lines were conducted using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions.
RNA extraction and quantitative real-time polymerase chain reaction
Total RNA was extracted from tissues and cells using TRIzol reagents (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA was reversely transcribed with the Prime Script RT Reagent Kit (Perfect Real Time) (TaKaRa Biotechnology, Dalian, China). Polymerase chain reaction (PCR) analysis was performed using specific primers for the Id4 gene: forward, 5′-GTGCGATATGAACGACTGCT-3′, and reverse, 5′-CAGGATCTCCACTTTGCTGA-3′. The expression levels were normalized using human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal control: forward, 5′-AGAAGGCTGGGGCTCATTTG-3′, and reverse, 5′-AGGGGCCATCCACAGTCTTC-3′.
Protein isolation and Western blotting
After specific treatments, proteins (20 µg) were separated using 12% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred to nitrocellulose membrane by an electroblotting Bradford assay, according to the manufacturer’s instructions (Sigma-Aldrich). The anti-Id4 monoclonal antibody (sc-365656, 1:100) and anti-C/EBPβ (CCAAT/enhancer-binding protein β) polyclonal antibody (sc-150, 1:400) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the β-actin antibody (A3854, 1:10,000) was purchased from Sigma-Aldrich.
Patient samples
Twenty-seven human HCC tissue samples were obtained from the Qidong Liver Cancer Institute (Qidong, Jiangsu, China). Tumor tissues and adjacent non-tumor tissues were used to detect the Id4 mRNA and protein levels by real-time PCR and Western blotting. All procedures were performed under consensus agreements and in accordance with the China Ethical Review Committee.
Immunohistochemical analysis
Fifty-seven human HCC tissue specimens were collected from patients who underwent surgical treatment at Qidong Liver Cancer Institute or at the First Affiliated Hospital of Zhejiang University (Hangzhou, Zhejiang, China). The 57 HCC patients, including 52 males and 5 females (mean age 45.0 years, ranging from 21.0 to 70.0 years), were followed up from November 21, 2001 to November 3, 2010. No patient received preoperative chemotherapy or radiotherapy. Informed consent was obtained from all patients, and the study was approved by the Ethics Committee of Fudan University.
Anti-Id4 polyclonal antibody (sc-491) was purchased from Santa Cruz Biotechnology. Immunohistochemical (IHC) analysis and signal evaluation were performed according to our previously described procedures [15]. All the HCC tissue slides were observed and photographed using an Axioskop 2 microscope (Carl Zeiss, Oberkochen, Germany). The IHC results were determined according to both staining intensity and the percentage of positive cells as described previously [15].
Plasmid constructs for overexpression of Id4 and C/EBPβ and RNA interference of Id4
The full-length human Id4 and C/EBPβ open reading frame (ORF) were respectively generated and cloned into the lentiviral vector pWPLX (Addgene, Cambridge, MA, USA) at the BamHI and EcoRI sites. The primers of Id4 ORF used for cloning and testing were as follows. Forward: 5′-GGATCCATGAAGGCGGTGAGCCCG-3′; reverse: 5′- GAATTCTCAGCGGCACAGAATGCT-3′. The primers of C/EBPβ (LAP1) ORF used for cloning and testing were as follows. Forward: 5′-CGCGGATCCATGCAACGCCTGGTGGCCT-3′; reverse: 5′-CCGGAATTCCTAGCAGTGGCCGGAGGAG-3′. We ordered two small-interfering RNAs (siRNAs) and two short-hairpin RNAs (shRNAs) targeting Id4, which were synthesized and constructed, respectively, by the GenePharma (Shanghai, China). The shId4 and shNC sequence (siId4-1, 5′-GCACGUUCAUAAACAUUCUTT-3′; siId4-2, 5′-CCCAACAAGAAAGUCAGCATT-3′; and siNC, 5′-TTCTCCGAACGTGTCACGT-3′) were cloned into the lentiviral vector pLVTHM (Addgene, Cambridge, MA, USA) to construct pLVTHM-shId4 and pLVTHM-shNC. To verify the effect of overexpression or gene silencing, real-time PCR and Western blotting were performed.
Colony formation assays
For colony formation assays, 500 SMMC-7721, 2000 MHCC-97L, and 3000 Huh7 cells per well were seeded in 6-well plates and cultured at 37 °C for 10–14 days. Then, the cells were fixed in 10% formaldehyde for 20 min and stained for 30 min with Giemsa solution (Sigma-Aldrich). Each measurement was performed in triplicate, and the experiments were each conducted three times.
Cell growth assay
Cell proliferation analyses were performed using a WST-8 Cell Counting Kit-8 (CCK-8, Ruian Biotech, Shanghai, China). Cells (800 for SMMC-7721, 2000 for MHCC-97L, and 1500 for Huh7) suspended in DMEM (100 μL) with 10% fetal bovine serum were seeded in 96-well plates and incubated. After 24 h, 10 μL of CCK-8 solution was added to each well, and the cultures were incubated at 37 °C for 2 h. Absorbance was measured at 450 nm for 7 days. The relative absorbance value was calculated, and the absorbance value measured on the first day was used as a control. Each measurement was performed in triplicate, and the experiments were each conducted three times.
Tumor xenograft assay
Six- to eight-week-old male BALB/c (nu/nu) mice were housed and treated under specific pathogen-free conditions at the Experimental Animal Center of Shanghai Jiaotong University School of Medicine (Shanghai, China). They were randomly divided into groups (eight mice per group) and maintained under standard conditions according to institutional animal guidelines. SMMC-7721-Id4 cells and their pWPXL vector control (SMMC-7721-pWPXL) cells (2 × 106 cells per mouse) were separately injected subcutaneously into the right flank of nude mice. After 5 weeks, the mice were euthanized, and the xenograft tumors were weighted.
Statistical analysis
Data were analyzed using SPSS 13.0 software (IBM Corporation, New York, NY, USA). Results are presented as mean ± standard deviation and compared using Student’s t test. The overall survival was calculated from the 4th month after hepatectomy to the date of death or the last follow-up. Univariate survival analysis was performed according to the Kaplan–Meier method, and differences in survival curves were assessed with the log-rank test. P values less than 0.05 were considered statistically significant.
