Functional analysis of fatty acid binding protein 7 and its effect on fatty acid of renal cell carcinoma cell lines

Reagents and their sources were as follows: RPMI 1640 medium, Oligo(dT)_12–18 Primer, SuperScript® III Reverse Transcriptase, SYBR® Green PCR Master Mix, pENTR™/D-TOPO® vector, Gateway® pT-Rex™-DEST30 vector, pT-Rex/GW-30/lacZ vector, pcDNA™6/TR vector, Lipofectamine® 2000 Transfection Reagent and blasticidin S HCl (Thermo Fisher Scientific, Waltham, MA, USA); docosatetraenoic acid, eicosapentaenoic acid (EPA) (NU-CHEK PREP, Inc.; Elysian, MN, USA); oligopeptides (Hokkaido System Science, Sapporo, Hokkaido, Japan); Tris, dithiothreitol, sodium orthovanadate, phenylmethanesulfonyl fluoride, and doxycycline hyclate (Sigma-Aldrich, St. Louis, MO, USA); sodium chloride (Nacalai Tesque, Kyoto, Japan); EDTA, sodium deoxycholate, sodium fluoride, sodium dodecyl sulfate (SDS), 4% paraformaldehyde and crystal violet (Wako, Osaka, Japan); IGEPAL CA-630 (MP Biomedicals, Santa Ana, CA, USA); protease inhibitor cocktail tablet (Complete, Mini, EDTA-free), geneticin (G418) (Roche Diagnostics GmbH, Mannheim, Germany); and SacI, XhoI (Takara Bio Inc., Otsu, Shiga, Japan).

The 786-O cell line (CRL-1932) was purchased from the American Type Culture Collection (Manassas, VA, USA). The TUHR14TKB cell line (RCB1383) was provided by RIKEN (Tsukuba, Ibaraki, Japan). Short tandem-repeat typing was performed to confirm the identity of high-passage TUHR14TKB cells, and the data were verified using the RIKEN short tandem-repeat database [26]. All cell lines were grown in RPMI 1640 medium supplemented with 10% (v/v) or 1% fetal bovine serum (FBS) (Nichirei Biosciences Inc., Tokyo, Japan). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. Docosatetraenoic acid or EPA (100 mM each) was dissolved in ethanol, and a 1:2000 dilution of each fatty acid was added to the culture medium.

Clones were isolated from low-passage cultures of TUHR14TKB cells by plating the cells at limiting dilution in 96-well plates. The cells were serially diluted to 128 to 4 viable cells/mL, and 50 μL was added per each well of a 96-well plate. After incubation at 37 °C in a humidified atmosphere containing 5% CO₂, single colonies in the wells were expanded.

Real-time PCR assays were performed using a modified version of the method described by Takaoka et al. [27]. Cells were cultured in 10-cm dishes. Total RNA was isolated from cultured cell lines using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Two micrograms of RNA was reverse transcribed using SuperScript® III Reverse Transcriptase primed by 500 ng of Oligo(dT)_12–18 Primer according to the manufacturer’s protocol. Real-time PCR analysis of FABP7 expression was performed using an Applied Biosystems StepOnePlus (Thermo Fisher Scientific). The final PCR reaction mix (20 μL) included 2 μL of each specific primer (5 μM), 1 μL of first-strand cDNA, and 10 μL of SYBR® Green PCR Master Mix. Plasmids that encode FABP7 and TATA box binding protein (TBP) were synthesized as described previously [27], and standard curves for each gene were generated using seven serial dilutions of plasmid templates (0.1 nM to 0.1 fM). TBP was used as an internal control. Takaoka et al. [27] and Jung et al. [28] reported the sequences of the primers used to amplify FABP7 and TBP, respectively.

Western blotting was performed using a modified version of a published method [27]. Cells were cultured in 6-well culture plates or in 10-cm culture dishes. The cells were detached using trypsin-EDTA, collected by centrifugation, and washed once with phosphate-buffered saline (PBS). The pellets were lysed on ice for 30 min in RIPA buffer (50 mM Tris, pH 8.0, 150 mM sodium chloride, 5 mM EDTA, 0.5% sodium deoxycholate, 1% IGEPAL CA-630, and 0.1% SDS) containing 2 mg/L sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethanesulfonyl fluoride, 2 mM dithiothreitol, and a protease inhibitor cocktail tablet. Lysates were centrifuged for 10 min at 4 °C at 18,000×g. The supernatants were transferred to sterile microcentrifuge tubes. Protein concentrations were determined using the Bio-Rad Protein Assay Kit II (Bio-Rad, Hercules, CA, USA). Cell extracts (20 μg) were electrophoresed through an 18% (w/v) polyacrylamide-SDS gel. The proteins were transferred electrophoretically onto a PVDF membrane (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire HP7 9NA, England), and the membrane was incubated with 1 g/L of an FABP7 antibody (AF3166; R&D Systems, Minneapolis, MN, USA) diluted 1:5000. Antibody-antigen complexes were visualized using peroxidase-conjugated anti-goat IgG (86,285; Jackson ImmunoResearch Laboratories, West Grove, PA) and Immobilon Western HRP Substrate (Millipore, Billerica, MA, USA). A mouse monoclonal anti-α-tubulin antibody (T6074; Sigma-Aldrich, St. Louis, MO, USA) served as an internal control.

Cells were plated in 10-cm culture dishes at a density of 2 × 10⁶ cells per plate and incubated for two days at 37 °C in an atmosphere containing 5% CO₂. After incubation, the cells were harvested with trypsin/EDTA, washed once with PBS, and then resuspended to 1 × 10⁶ cells/0.2 mL in PBS containing 0.25% Triton X-100 for 5 min at room temperature. Cellular DNA in each cell suspension was stained using 0.6 mL of 50 mg/L propidium iodide for 10 min at room temperature. Cell-cycle analysis was performed using an EPICS-XL flow cytometer (Beckman-Coulter, Brea, CA, USA).

To generate FABP7 expression constructs, the FABP7 cDNA sequence was amplified using PCR with the primers Full B-FABP F2 (5′-CACCATGGTGGAGGCTTTCTGT) and Full B-FABP R3 (5′-TTATGCCTTCTCATAGTGGCG). The PCR product was inserted into pENTR™/D-TOPO® via TOPO cloning (Invitrogen, CA, USA). The cloning vector (pENTR-FABP7) was transferred to the Gateway® pT-Rex™-DEST30 vector via gateway recombination (Invitrogen, CA, USA). The plasmid generated (DEST30-FABP7) was verified by direct DNA sequencing. The pT-Rex/GW-30/lacZ vector that expresses β-galactosidase (lacZ) served as a control.

TUHR14TKB and 786-O cells were transfected with 2 μg of XhoI-digested pcDNA6/TR using FuGene® HD transfection reagent (Promega, Madison, WI, USA). The transfectants were cultured in RPMI 1640 medium containing 10% FBS and 5 mg/L blasticidin S HCl. The pcDNA6™/TR transfectants (TUHR-TR, TUHR14TKB pcDNA6™/TR transfectant; 786-O TR, 786-O pcDNA6™/TR transfectant) were expanded and transfected in the presence of Lipofectamine® 2000 transfection reagent with 4 μg of DEST30-FABP7 or the empty vector pT-Rex/GW-30/lacZ digested with SacI. The transfectants were cultured in RPMI 1640 medium containing 10% FBS, 5 mg/L blasticidin S HCl, and 0.3 g/L G418, and blasticidin- and G418-resistant cells were expanded. The FABP7 or control-vector transfectants of TUHR-TR or 786-O TR were cultured in RPMI 1640 medium containing 10% FBS or 1% FBS with 5 mg/L blasticidin S HCl, 0.3 g/L G418, and 1 mg/L doxycycline hyclate for one to three days and then subjected to western blotting or the following assays: MTS, cell counting, or wound-healing. SRL Inc. (Tokyo, Japan) performed the analyses of the fatty acid composition of the TUHR-TR transfectants.

Cells were plated in 96-well cell culture plates at 400 (786-O transfectant) or 2000 cells per well (low-passage or high-passage TUHR14TKB or TUHR14TKB transfectants, respectively) in 100 μL of culture medium. The plates were incubated at 37 °C in an atmosphere containing 5% CO₂. The cells were analyzed using a CellTiter 96® AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Absorbance (490 nm) was measured one, two, and three days after cell plating. Doubling times were determined from four replicate samples per point.

TUHR14TKB transfectants were plated in 24-well cell culture plates (10,000 cells per well) in 500 μL of RPMI 1640 medium containing 10% FBS with 5 mg/L blasticidin S HCl, 0.3 g/L G418, and 1 mg/L doxycycline hyclate. The plates were incubated at 37 °C in an atmosphere containing 5% CO₂. Cells on the plate were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet one, two, and three days after plating. The numbers of cells were counted in five random fields using a light microscope (×100).

Cells (1 × 10⁶) were seeded in 24-well plates. After incubation overnight (786-O TR transfectant) or for one day (TUHR-TR transfectant), an artificial wound was created (0 h) using a 200-μL tip to introduce a gap in the confluent cell monolayer, and the culture medium was changed. Images were acquired at 0 h and 6 h (786-O TR transfectant) or 16 h (TUHR-TR transfectant). The wounded areas were measured before and after healing.

Cell proliferation and migration data were analyzed using the Student t test. Statistical significance was defined as p < 0.05.

High levels of FABP7 were detected during passages 6–8 of TUHR14TKB cells, but not during passages 16–18 (Fig. 1a). The levels of FABP7 expressed by TUHR14TKB cells decreased by approximately four-fold between two cell passages (Fig. 1b). In contrast, the doubling time of low-passage cells was approximately twice that of high-passage cells (Fig. 2a). The doubling times differed among cells that were isolated from individual colonies of low-passage TUHR14TKB cells (Fig. 2a). Further, the percentage of S-phase cells in high-passage TUHR14TKB cells increased and was accompanied by a decrease in the percentage of G0/G1-phase cells compared with low-passage TUHR14TKB cells (Fig. 2b).

We transfected FABP7 low-expressing TUHR14TKB and 786-O cells with an FABP7 expression vector (Fig. 3a and b and Additional file 1: Figure S1a and S1b). In the presence of 10% FBS, the doubling time of TUHR14TKB cells that overexpressed FABP7 was significantly longer than that of cells transfected with the control vector (Fig. 4a and b). Although TUHR14TKB cells transfected with the control vector were able to proliferate, the cells that overexpressed FABP7 were unable to proliferate in the presence of 1% FBS (Additional file 2: Figure S2). Further, the percentage of TUHR14TKB FABP7 in G2/M increased compared with that of TUHR14TKB lacZ cells (Fig. 4c), indicating that FABP7 induced the arrest of TUHR14TKB in G2. In contrast, overexpression of FABP7 stimulated the proliferation of the 786-O cell line cultured in medium containing 1% FBS (Additional file 1: Figure S1c).

Wound-healing assays revealed that TUHR14TKB cells that overexpressed FABP7 migrated significantly slower than TUHR14TKB cells transfected with the control vector (Fig. 3c), although overexpression of FABP7 did not affect the migration of 786-O cells (Additional file 1: Figure S1d).

Although FABP7 binds to fatty acids, it does not catalyze de novo fatty acid synthesis, suggesting that FABP7 expression leads to the accumulation of fatty acid in cells. Docosatetraenoic acid and EPA accumulated in TUHR14TKB cells that expressed FABP7 (Fig. 5a). In contrast, other fatty acids did not accumulate in TUHR14TKB cells that expressed FABP7 (Additional file 3: Table S1). Therefore, we tested the effects of docosatetraenoic acid or EPA on the proliferation of TUHR14TKB cells. The addition of docosatetraenoic acid significantly stimulated the proliferation of TUHR14TKB cells that expressed β-galactosidase (Fig. 5b).