Roles for Rat Hepatocyte Malignant Transforming Factor (HMTF) in Late Stage of Hepatocarcinogenesis

Animals

Details of the animal experiments were previously reported (Suzuki et al. 2009). Six-week-old male F344 rats (Charles River Japan Inc., Atsugi, Japan) continuously received either 0 or 50 ppm diethylnitrosamine (DEN; Tokyo Kasei Kogyo Co. Ltd. Tokyo, Japan) in their drinking water for up to eighteen weeks and were sacrificed six, twelve, or eighteen weeks after starting DEN treatment (six, six, and nine animals, respectively). The livers were immediately excised for analysis. Slices were fixed in 10% buffered formalin for immunohistochemical examination and hematoxylin and eosin staining, and the remaining liver tissue was immediately frozen in liquid nitrogen and stored at –80°C until processed for RNA and protein extraction.

Normal organs of twelve-week-old male rats were excised and frozen in liquid nitrogen and stored at –80°C until processing. The remaining tissues were fixed in 10% buffered formalin for in situ hybridization and hematoxylin and eosin staining. The definition of GST-P foci is GST-P immunohistochemically positive foci exclusive of adenoma and carcinoma.

All animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Nagoya City University School of Medical Sciences.

Diagnosis of Liver Lesions

Liver lesion diagnosis was based on “Proliferative and Nonproliferative Lesions of the Rat and Mouse Hepatobiliary System” (Thoolen et al. 2010).

Cell Lines

Rat HCC cell lines (C1, C2, C5F, C6, N1, and L2 cells) were established in our laboratory from a DEN plus N-nitrosomorpholine–induced hepatocellular carcinoma in a male F344 rat (Ogawa et al. 2001). In our present study, the N1 cell line was employed because it proved to be the most highly metastatic among the six cell lines (Ogawa et al. 2001).

RNA Extraction, Reverse Transcription, and Quantitative PCR

Total RNAs from cultured cells were also isolated with ISOGEN (Wako, Tokyo, Japan) according to the manufacturer’s instructions.

cDNAs were prepared using superscript II (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The resulting cDNAs were used for polymerase chain reaction (PCR; PCR was done in triplicate), and data collection was performed on a LightCycler (Roche Diagnostics, Mannheim, Germany). All quantitative values were normalized to endogenous cyclophilin levels, and expression by nontreated cells was used as the reference. HMTF primers were 5'-GACCCAGGAGCACAATGAAT-3' and 5'-CTGGCAACAAGGCAGTAGTA-3'. Cyclophilin Reverse Transcription (RT)-PCR primers were 5'-TGCTGGACCAAACACAAATG-3' and 5'-GAAGGGGAATGAGGAAAATA-3'.

Northern Blot Analysis for HMTF

Ten µg aliquots of total RNAs from the liver tissues of twenty-four-week-old control rats, rats treated with DEN for eighteen weeks (twenty-four-week-old rats), rat HCC cell lines, and each normal organ were separated by electrophoresis in 1% agarose-formaldehyde gels and transferred to nylon membranes (Hybond-N+; GE Healthcare UK Ltd, Buckinghamshire, England) for autoradiography (Hyperfilm MP; GE Healthcare UK Ltd). The HMTF probe was a cDNA (269 to 542 of HMTF cDNA) constructed with the HMTF primers depicted previously and labeled with [alpha-32P]dCTP (GE Healthcare UK Ltd).

In Situ Hybridization

A fragment of the HMTF cDNA, corresponding to nucleotides 269 to 542, was cloned into the vector pGEM-T (Promega, Madison, WI, USA), and antisense and sense HMTF riboprobes were prepared using pGEM-T/HMTF as the template with a digoxigenin RNA labeling kit (Roche Diagnostics). After deparaffinization, sections were treated with 3 µg/ml proteinase K for twenty min at 37°C, postfixed in 4% paraformaldehyde for ten min, and then placed in hybridization buffer containing digoxigenin-labeled antisense riboprobes for sixteen to eighteen hr at 42°C. After hybridization, the sections were washed in 2 × SSC/50% formamide for thirty min at 42°C, then TNE buffer (10 mM Tris-HCl [pH 8.0], 500 mM NaCl,1 mM EDTA) for ten min at 37°C. Thereafter, the sections were treated with 10 µg/ml of RNase (Roche Diagnostics) for thirty min at 37°C, rinsed in buffer A (100 mM maleic acid, 150 mM NaCl, pH 7.5) for ten min, and then incubated with buffer C (1% blocking reagent [Roche Diagnostics] dissolved in buffer A). Horse-radish peroxidase conjugated sheep anti-digoxigenin Fab fragments (1:100, Roche Diagnostics) were applied for thirty min. The slides were then washed in buffer A followed by incubation with biotinylated tyramide in amplification diluent (NEN Life Science Products, Inc., Boston, MA, USA) for ten min, washed three times in buffer A for five min, and then incubated with alkaline phosphate conjugated streptoavidin (1:5000, Roche Diagnostics) for thirty min. After sequential washing in buffer A containing 0.2% Tween 20 and buffer B (100 mM Tris-HCl [pH 9.5], 100 mM NaCl, 50 mM MgCl2), hybridization signals were detected with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitro blue tetrazolium chloride (NBT) solution (Roche Diagnostics). The specificity of the in situ hybridization (ISH) was assessed by parallel hybridization of the sections with sense riboprobes.

Determination of the 5’ End Sequence of HMTF mRNA

To determine the 5’ end sequence of HMTF mRNA, a GeneRacer kit (Life Technologies) was used according to the manufacturer’s instructions using the AW434047 specific primer 5'-TTCAGACATCGCCCTTCCTT-3'. The resultant HMTF cDNA was cloned into a pGEM-T vector (Promega), and its sequence was determined with a BigDye^® Terminator v1.1 Cycle Sequencing Kit (Life Technologies) and an ABI PRISM^® 310 Genetic Analyzer (Life Technologies) following the manufacturer’s instructions.

HMTF RNA-Interference

N1 cells were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies) containing 10% fetal bovine serum and 1% Penicillin-Streptomycin (Life Technologies). An HMTF siRNA was designed and obtained from RNAi Co. Ltd. (Tokyo, Japan) with the sequence 5'-ACCACCGUCUGCCUUCUUCCUGUUU-3'. Stealth RNAi^TM siRNA Negative Control Med GC Duplex (GE Healthcare UK Ltd) was used as a control. N1 cells were transfected with the siRNAs using Lipofectamine RNAiMAX (Life Technologies).

Cell Proliferation, Cell Cycle, and Apoptosis Assays

N1 cells were seeded into twenty-four-well plates at a density of 2 × 10⁴ cells per well in 500 µl medium and incubated for twenty-four hr at 37°C. Then the cells were transfected with siRNAs. After twenty-four-hr transfection, the culture supernatant was removed and replaced with fresh complete culture medium (DMEM) and culture was continued. For cell proliferation, live cells were counted using Trypan blue staining (Life Technologies) one and three days after addition of fresh culture medium. For cell cycle analysis, one day after addition of fresh culture medium, cells were harvested and stained with propidium iodide (Guava^® cell cycle reagent, Guava Technologies, Hayward, CA, USA) according to the Guava^® Cell Cycle Assay protocol. Cell cycle phase distributions were determined on a Guava^® PCA Instrument using CytoSoft Software. For analysis of apoptosis, one day after addition of fresh culture medium, cell suspensions were prepared and stained with Guava^® Nexin reagent for twenty min and immediately processed with the Guava^® PCA system (Guava Technologies) according to the manufacturer’s protocol. Cells were gated based on forward scatter (size), and results reported as the percentage of gated cells positive for both annexin V and 7-AAD. Each assay was carried out in triplicate.

Chemoinvasion and Migration Assays

Cell culture inserts coated with Matrigel Matrix (BD Biocoat Matrigel invasion chambers; BD Biosciences, Bedford, MA) for invasion assays and the BD Falcon twenty-four-multiwell insert system (BD Biosciences) for migration assays, were placed in twenty-four-well plates filled with DMEM containing 0.1% bovine serum albumin (BSA) and 10 µg/ml rat HGF as a chemoattractant. Twenty-four hours after transfection with siRNA, cells (1 × 10⁵) were suspended in DMEM containing 0.1% BSA and seeded into the cell culture inserts and incubated at 37 °C for forty-eight hours. Cells moving through the Matrigel Matrix (invasion) and cells migrating through uncoated inserts (migration) were then counted using Trypan blue staining. The number of nontreated, invading/migrating cells was used as the reference. Each assay was carried out in triplicate.

Immunoblot Analysis

After transfection and culture for twenty-four hr, N1 cells were washed twice with ice-cold phosphate buffered saline (PBS) and harvested by scraping into ice-cold PBS. After centrifugation at 1,500 rpm for ten min, the supernatant was removed and cell pellets were lysed with RIPA buffer (Pierce Biotechnology, Rockford, IL, USA) containing protease inhibitors (Pierce Biotechnology) for fifteen min on ice. The insoluble matter was removed by centrifugation at 12,000 rpm, for twenty min at 4°C. Supernatants were transferred to new tubes and the debris pellet was discarded. Protein concentrations were determined with a Coomassie Plus^TM-The Better Bradford Assay Kit (Pierce Biotechnology).

Samples were mixed with 2× sample buffer (Bio-Rad Laboratories, Hercules, CA, USA), heated for five minutes at 95°C, and then subjected to 10% SDS-PAGE. The separated proteins were transferred onto nitrocellulose membranes followed by blocking with 5% nonfat milk powder (w/v) in 1× TBS [10 mmol/L Tris-HCl (pH7.5), 100 mmol/L NaCl, 0.1% Tween-20] for one hr at room temperature. Membranes were probed with antibodies against cyclin D1, cyclin E, p21, p27 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), p53 (Epitomics, Inc, Burlingame, CA, USA), the signal transducers and activators of transcription 3 (STAT3), phospoh-STAT3 (pSTAT3; Y705), nuclear factor-kappa B (NF-kB) (Cell Signaling, Technology Inc., Danvers, MA, USA), and c-MYC (Novocastra Laboratories Ltd., Newcastle upon Tyne, UK) in 5% nonfat dry milk, 1× TBS, 0.1% Tween-20 at 4°C overnight, followed by incubation with peroxidase-conjugated secondary antibodies. Binding was visualized with an enhanced chemiluminescence detection system (GE Healthcare UK Ltd). To confirm equal protein loading, each membrane was stripped and reprobed with anti-beta-actin (b-actin) antibody (Sigma-Aldrich, Co., St. Louis, MO, USA).

Statistical Analysis

All data are presented as mean ± standard deviation values. Statistical comparisons were performed with one-way ANOVA followed by Dunnett’s test. Correlations were assessed by Spearman correlation coefficient analysis. Significance was concluded when p < .05. All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA).

Expression and Sequence of HMTF mRNA in Normal Hepatocytes and Malignant Tumors

We utilized a GeneRacer kit to determine the sequence of the mRNA transcript associated with the EST AW434047. The sequence is registered with GenBank under the name rat HMTF (GenBank ID: GU969272).

In the rat liver treated with DEN for eighteen weeks, HMTF expression, determined by ISH, was readily detected in carcinoma cells (Fig. 1B & D). No hybridization was detected with a sense RNA probe (Fig. 1C) or after RNAse treatment before hybridization with the anti-sense RNA probe (Fig. 1E). In the same liver, there was less staining of HMTF in GST-P positive foci and adenomas (supplemental Fig. 1).

Northern blotting demonstrated HMTF RNA expression in the livers of DEN treated rats and in the rat HCC cell lines (Fig. 1F) with bands localized around 1 kb. HMTF expression was higher in the livers of twenty-four-week-old rats treated with DEN for eighteen weeks (these livers contained HCC) than liver tissue from untreated twenty-four-week-old rats. HMTF expression was particularly high in all rat HCC cell lines tested (C1, C2, C5F, C6, N1, and L2). These data confirm the high expression of HMTF in HCC originally indicated by microarray analysis.

Knock-Down of HMTF Expression in N1 Cells

To investigate HMTF function in HCC, HMTF siRNA was used in N1 cells. HMTF expression at the mRNA level was significantly suppressed after transfection of HMTF siRNA (1.00 ± 0.06, 0.92 ± 0.11, and 0.47 ± 0.05, nontreated, control siRNA-treated, and HMTF siRNA-treated cells, respectively, at day 1) (Fig. 2A). At day 4 no statistically significant effects were evident (1.00 ± 0.14, 1.14 ± 0.11, and 0.80 ± 0.11, nontreated, control siRNA-treated, and HMTF siRNA-treated cells, respectively, data not shown).

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Figure 2.

Functions of hepatocyte malignant transforming factor (HMTF) as assessed by RNA interference. (A) Quantitative RT-PCR. (B) Cell growth assay. (C) Invasion assay. (D) Migration assay. (E) Percentages of cells in the different cell cycle phases. (F) Percentages of living cells and cells undergoing early and late apoptosis. Control: nontreated. Control siRNA: treated with negative control siRNA. HMTF siRNA: treated with HMTF siRNA. For the data in panels A, C, and D, the control was set to 1.0. *, ***, significantly different from control and control siRNA at p < .05 and p < .001, respectively. #, significantly different from control at p < .05 and from control siRNA at p < .001. All values are means ± standard deviations.

RNAi-mediated HMTF knock-down led to significant suppression of cell growth (79%; Fig. 2B), invasion (62%; Fig. 2C), and migration potential (52%; Fig. 2D) in N1 cells. The growth suppression was associated with G1 arrest of cell cycle (Fig. 2E) and not with the induction of early (annexin V-positive cells) or late apoptosis (annexin V- and 7-AAD-positive cells) (Fig. 2F).

HMTF-Associated Signaling Pathways

For determination of HMTF-regulated genes, analysis by immunoblot revealed that cyclin D1 and PCNA were down-regulated and p27 up-regulated in HMTF siRNA-treated cells (Fig. 3 ). There were no differences in expression of p53, p21, or cyclin E among the groups (Fig. 3). Total and activated STAT3 levels were also reduced in the HTMF siRNA-treated cells. No differences in the expression of c-myc or NF-kB were found among the groups. The protein blots were quantified using ImageJ (National Institute of Mental Health, Maryland, USA), and the data are presented in supplemental Fig. 2.

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Figure 3.

Changes in protein expression in N1 cells treated with siRNAs. Control: nontreated. Control siRNA: treated with negative control siRNA. Hepatocyte malignant transforming factor (HMTF) siRNA: treated with HMTF siRNA.

mRNA Expression and Localization of HMTF in Rat Normal Organs

HMTF RNA expression was detected in the spleen, colon, brain, and especially the testis of nontreated control rats (Fig. 4A). ISH performed for these four organs demonstrated HMTF expression in the lymphocytes in the spleen, mucosal epithelia of the colon, granule cells of hippocampus, and spermatocytes of the testis (Fig. 4B).

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Figure 4.

Expression and localization of hepatocyte malignant transforming factor (HMTF) in normal organs by Northern blotting (A), histology with H&E (left) and in situ hybridization with antisense HMTF (right; B). rRNA: ribosomal RNA.