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01.12.2017 | Research article | Ausgabe 1/2017 Open Access

BMC Cancer 1/2017

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

Zeitschrift:
BMC Cancer > Ausgabe 1/2017
Autoren:
Naohisa Takaoka, Tatsuya Takayama, Seiichiro Ozono
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12885-017-3184-x) contains supplementary material, which is available to authorized users.

Abstract

Background

Renal cell carcinomas (RCCs) overexpress fatty acid binding protein 7 (FABP7). We chose to study the TUHR14TKB cell line, because it expresses higher levels of FABP7 than other cell lines derived from renal carcinomas (OS-RC-2, 786-O, 769-P, Caki-1, and ACHN).

Methods

FABP7 expression was detected using western blotting and real-time PCR. Cell proliferation was determined using an MTS assay and by directly by counting cells. The cell cycle was assayed using flow cytometry. Cell migration was assayed using wound-healing assays. An FABP7 expression vector was used to transfect RCC cell lines.

Results

The levels of FABP7 expressed by TUHR14TKB cells and their doubling times decreased during passage. High-passage TUHR14TKB cells comprised fewer G0/G1-phase and more S-phase cells than low-passage cells. Cell proliferation differed among subclones isolated from cultures of low-passage TUHR14TKB cells. The proliferation of TUHR14TKB cells decreased when FABP7 was overexpressed, and the cell migration property of TUHR14TKB cells were decreased when FABP7 was overexpressed. High concentrations of docosatetraenoic acid and eicosapentaenoic acid accumulated in TUHR14TKB cells that overexpressed FABP7, and docosatetraenoic acid enhanced cell proliferation.

Conclusions

The TUHR14TKB cell line represents a heterogeneous population that does not express FABP7 when it rapidly proliferates. The differences in FABP7 function between RCC cell lines suggests that FABP7 affects cell proliferation depending on cell phenotype.
Zusatzmaterial
Additional file 1: Figure S1. Effect of FABP7 overexpression on the 786-O cell line. 786-O cells were cultured for two days in RPMI 1640 medium containing 10% FBS, 5 mg/L blasticidin S HCl, 0.3 g/L G418, and 1 mg/L doxycycline hyclate. a, Western blot analysis of FABP7 expression by cells transfected with the FABP7 vector or control (lacZ) vector. b, Real-time PCR analysis of FABP7 expression of cells transfected with the FABP7 vector or lacZ vector. c-d, The 786-O transfectants 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 and subjected to cell proliferation and migration assays. c, The doubling times of cell transfected with the FABP7- or the lacZ-expression vector were determined using an MTS assay. The data represent the average and standard deviation (error bars) of five experiments. d, The migration of 786-O cells transfected with the FABP7- or lacZ-expression vector was determined using a wound-healing assay. The data represent the average and standard deviation (error bars) of four experiments. (TIFF 2702 kb)
12885_2017_3184_MOESM1_ESM.tiff
Additional file 2: Figure S2. Proliferation of TUHR14TKB cells transfected with an FABP7 expression vector. The proliferation of cells transfected with the FABP7 expression vector or lacZ expression vector was determined using an MTS assay. The data represent of five experiments. Transfectants were cultured in RPMI 1640 medium containing 5 mg/L blasticidin S HCl, 0.3 g/L G418, and 1 mg/L doxycycline hyclate with 1% FBS (a-b) or 10% FBS (c-d). a, c, TUHR14TKB lacZ. b, d, TUHR14TKB FABP7. (TIFF 1521 kb)
12885_2017_3184_MOESM2_ESM.tiff
Additional file 3: Table S1. Fatty acid concentrations of TUHR14TKB transfectants. The concentrations of fatty acids accumulated by TUHR14TKB cells transfected with vectors expressing FABP7 or lacZ were assayed in four independent cultures. (XLSX 46 kb)
12885_2017_3184_MOESM3_ESM.xlsx
Literatur
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