Histidine-rich glycoprotein function in hepatocellular carcinoma depends on its N-glycosylation status, and it regulates cell proliferation by inhibiting Erk1/2 phosphorylation.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality. Significantly downregulated histidine-rich glycoprotein (HRG) during the dynamic stages (WB, WB7, and WB11) of neoplastic transformation of WB F344 hepatic oval-like cells was screened out by iTRAQ labeling followed by 2DLC-ESI-MS/MS analysis. HRG expression was significantly lower in HCC tissues. HRG overexpression in Huh7 and MHCC-97H hepatoma cell lines led to decreased cell proliferation, colony-forming ability, and tumor growth, and increased cell apoptosis. HRG could inhibit cell proliferation via the FGF-Erk1/2 signaling pathway by reducing Erk1/2 phosphorylation. On the other hand, the functional expression of HRG was also dependent on the glycosylation status at its N-terminal, especially at the glycosylation site Asn 125. The glycosylation of HRG may play a key competitive role in the interaction between HRG and heparin sulfate for binding bFGF and activating the FGF receptor. These findings provide novel insights into the molecular mechanism of HRG in HCC.


IMMUNOHISTOCHEMISTRY ANALYSIS ON TISSUE MICROARRAYS
Tissue array containing 75 HCC samples (Shanghai OUTDO Biotech, China), and mouse anti-HRG monoclonal antibody (R&D Systems, Inc. US) were used in this study.
The process of immunohistochemistry was according to the protocol of HRG antibody. Briefly, paraffin sections were first deparaffinized and then hydrated. After microwave antigen retrieval, as required, endogenous peroxidase activity was blocked with incubation of the slides in 3% H 2 O 2 , and non-specific binding sites were blocked with 10% rabbit serum. After serial incubation with primary antibody (1:400) and secondary antibody, the sections were developed in diaminobenzidine solution under a microscope and counterstained with hematoxylin. Negative control slides omitting the primary antibodies were included in all assays.

SIRNA TRANSFECTION
Three pairs of predesigned siRNA oligonucleotide against HRG were purchased from Biomics. The sequences of siRNA were listed in Supporting Table 1. Three pairs of siRNA for each target were mixed respectively and transfected into Huh7 cells and MHCC-97H cells at the final concentration of 50 nM using Lipofectamine 2000 (Invitrogen) as manufacturer's instruction. The culturing medium was changed to complete medium after 12 h of transfection and the cells were cultured for another 48 h until detection of gene knockdown.

SOFT AGAR COLONIZATION
For clone formation assay, 1ml of sterilized 0.6% low melting point (LMP) agarose (Sigma) in complete medium was added to one well of 6-well plate first. After the medium became solid gel, 1ml of 0.4% LMP agarose in complete medium with 1000 Huh7 cells and MHCC-97H cells was added on top of the base gel. After culturing for 14 days, clone (>50 cells) numbers were assessed microscopically. All experiments were performed in triplicate.

WESTERN BLOT ANALYSIS
Equal amounts of total proteins (20 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membrane using a Bio-Rad SemiDry apparatus. The membrane was blocked by 5% milk or 2% BSA at room temperature for 1 h. Then, the membrane was incubated with specific primary antibody with suitable dilution at 4°C overnight. The used primary antibodies and their dilution information were listed in Supporting Table 2. After 3 times of 10 min washing by TBST, the membrane was further incubated with HRP-conjugated secondary antibodies (Bio-Rad) at room temperature for 1 h, and then washed again by TBST for 3 times of 10 min. ECL prime Western Blotting Detection Reagents (GE) and ChemiDoc XRS+ system (Bio-Rad) were used to visualize the bands on membrane.

IMMUNOFLUORESCENCE MICROSCOPY
For Immunofluorescence staining, cells grown on glass coverslip were fixed in 4% paraformaldehyde and permeabilized using 0.5% Triton X-100. Then cells were incubated with the primary antibody overnight at 4°C. After thorough washing, cells were then incubated with Alexa-Fluor 555 anti-mouse IgG or anti-rabbit IgG (1:100 dilution, Cell Signaling Technology, Danvers, MA). Finally, cells were washed and stained with DAPI. Images were captured using a Leica fluorescence microscope.

QUANTITATIVE RT-PCR
Total RNA was extracted from cultured cells using TRIzol Reagent (Invitrogen) according to the manufacturer's instruction. 2 μg of total RNA was reversed transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kits (Thermo). cDNA were prepared for subsequent quantitative PCR amplification with

SUPPLEMENTARY MATERIALS AND METHODS
www.impactjournals.com/oncotarget/ SYBR Premix Ex Taq (TAKARA) using IQ5 (Bio-Rad). The experimental Ct (cycle threshold) was calibrated against that of beta-actin control product. The used paired primers for each gene were listed in Supporting Table 3.

TUMOR FORMATION ASSAYS
Male BALB/C nude mice (5-6 week old) were obtained from Shanghai Institute of Materia Medica (Chinese Academy of Sciences, Shanghai, China). The in vivo experiments were carried out strictly in accordance with a protocol approved by the Shanghai Medical Experimental Animal Care Committee (Permit Number: 2009-0082). 1×10 7 cells were injected subcutaneously into the upper left flank region of nude mice. When the tumor reached 1 cm in diameter, they were cut into 2×2×2 mm 3 sized pieces, and implanted into livers of nude mice. The mice were sacrificed at the 27 th day after tumor implantation.The tumor size and weight were measured. were 0.02% and 2% in WB7 and WB11 cells, but WB cells could not grow in soft agar (Fig. 1A). The subcutaneous tumor formation in WB11 cells, and WB, WB7 cells could not form tumor (Fig. 1B). CCK8 assay displayed that transformed cells proliferated faster than WB cells (Fig. 1C). Cell migration showed that the migration ability was enhanced in WB7 and WB11, and WB11 cells has more migration ability than WB7 cells (Fig. 1D). These results strongly indicated that MNNG induced WB cells gained the characteristics of transformed cells. It implied that the WB7 cell may be in precancer status and WB11 cell, definitely in real cancer. Characteristics of transformed WB, WB7, and WB11 cells. A. Soft agar clone formation in WB7 and WB11. B. Subcutaneous tumor formation in WB7, WB11. C. The proliferation of WB, WB7, WB11 cells. D. The migration of WB, WB7, WB11 cells, *P < 0.05.