Suppression of osteopontin inhibits chemically induced hepatic carcinogenesis by induction of apoptosis in mice

Previous clinical reports have found elevated osteopontin (OPN) levels in tumor tissues to be indicative of greater malignancy in human hepatocellular carcinoma (HCC). However, the role of OPN on carcinogenesis and its underlying mechanism remain unclear. In the present study, we investigated the oncogenic role of OPN in diethylnitrosamine (DEN)-induced hepatic carcinogenesis in mice. The overall incidence of hepatic tumors at 36 weeks was significantly lower in OPN knockout (KO) mice than in wild-type (WT) mice. Apoptosis was significantly enhanced in OPN KO mice, and was accompanied by the downregulation of epidermal growth factor receptor (EGFR). In the in vitro study, OPN suppression also led to lower mRNA and protein levels of EGFR associated with the downregulation of c-Jun in Hep3B and Huh7 human HCC cells lines, which resulted in increased apoptotic cell death in both cell lines. Moreover, a positive correlation was clearly identified between the expression of OPN and EGFR in human HCC tissues. These data demonstrate that the OPN deficiency reduced the incidence of chemically induced HCC by suppressing EGFR-mediated anti-apoptotic signaling. An important implication of our findings is that OPN positively contributes to hepatic carcinogenesis.


Immunohistochemical staining for OPN and TUNEL assay
In order to perform immunohistochemical staining for OPN in human and mouse tissue, replicate sections of paraffin-embedded liver tissue were mounted on silicon-coated slides, dewaxed, and rehydrated, and antigen retrieval was then performed by heating at 100°C for 20 minutes in a 0.01 M citrate buffer (pH 6.0). The inactivation of endogenous peroxidase and blocking of non-specific protein binding were relieved utilizing hydrogen peroxide and serum-free protein block solution (DakoCytomation, Glostrup, Denmark). The slides were incubated with anti-mouse or-human OPN antibody (1:100; R&D Systems, Minneapolis, MN, USA). After incubation with the primary antibody, slides were stained using the indirect labeling streptavidin avidin-biotin technique with 3-3'-diaminobenzene as a substrate.
We also performed IHC for proliferating cell nuclear antigen (PCNA) using anti-PCNA antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) in order to investigate whether OPN depletion affected cell proliferation.
Quantitation of immunoreactivity was performed using an H-scoring system, in which scores were calculated based on the intensity and number of positive cells according to the equation: Score = (3 × % intensely positive) + (2 × % moderately positive) + (1 × % weakly positive).
Apoptotic cell death in mouse tissue samples and human HCC cell lines was determined through the terminal deoxynucleotidyl transferase dUTP nickend labeling (TUNEL) assay using the Fluorecein FragEL DNA Fragmentation Detection kit (Calbiochem, Darmstadt, Germany).

Generation of OPN knockdown cell lines
The human HCC cell lines Hep3B and Huh7 were purchased from the Korean Cell Line Bank (KCLB; Seoul, Korea). Hep3B and Huh7 cells were separately cultured in Dulbecco's modified Eagle medium (Gibco, Grand Island, NY, USA) or RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS; Gibco), 1% penicillin, and streptomycin (Invitrogen Biotechnology, Grand Island, NY, USA). For the stable knockdown of the Spp1 gene in the Hep3B and Huh7 cell lines, a lentiviral vector-mediated short-hairpin RNA (shRNA) construct targeting the human Spp1 gene (Sigma-Aldrich, St. Louis, MO, USA) with the pLKO.1-puro enhanced green fluorescent protein control vector (Sigma) were produced from accession number NM_000582. The lentivirus was generated by the cotransfection of the shRNA-expressing vector and packaging vectors (Addgene) into 293Ts cell through Lipofectamine™ 2000 (Invitrogen Biotechnology). After transfection for 48 hours, virus-containing supernatant was collected, filtered using a 0.45-μm filter, assessed for titer values, and used for viral transduction with 10 μg/ mL of polybrene. Cells were selected over the course of three days through treatment with 2 μg/mL of puromycin after viral transduction. Knockdown efficiency and OPNrelated molecular changes were confirmed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and western blotting.

OPN transfection
cDNA containing the coding region of human OPN-a, which was subcloned into the expression vector pDest-490, was a gift from Xin Wang (Addgene plasmid # 17590) [1]. Hep3B or Huh7 cells were transfected with 500 ng of pDest490-OPN-a or pDest490 control vector using Lipofectamine™ 2000 (Invitrogen Biotechnology) according to the manufacturer's instructions. At the appropriate time point after transfection, the cells were harvested to perform an Annexin V assay or a western blot analysis.

RNA extraction, RT-PCR, and quantitative realtime RT-PCR
The total RNA from Hep3B and Huh7 cells was extracted using the RNeasy Plus Mini kit (Qiagen, Hilden, Germany), and 500 ng of total RNA from each sample was reverse-transcribed using the QuantiTect Reverse Transcription kit (Qiagen) and analyzed by quantitative real-time RT-PCR using the Rotor-Gene SYBR Green PCR kit (Qiagen) with specific primers. The amplification and quantitation of target genes were performed using the Rotor-Gene Q and the manufacturer-provided software (Qiagen). The amount of the target gene was calculated using mRNA encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. The amplified target genes were also loaded on 2% agarose gel. The primer pairs for human genes were F: 5'-GCC AAG GCA CGA GTA ACA AGC-3' and R: 5'-AGG GCA ATG AGG ACA TAA CC-3' for EGFR, F: 5'-TGA AAC GAG TCA GCT GGA TGA CCA-3' and R: 5'-GCT CTC ATC ATT GGC TTT CCG CTT-3' for OPN and F: 5'-GAG TCA ACG GAT TTG GTC G-3' and R: 5'-TGG AAT CAT ATT GGA ACA TGT AAA C-3' for GAPDH.