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. 2018 May;15(5):6967-6974.
doi: 10.3892/ol.2018.8211. Epub 2018 Mar 8.

Overexpression of ATP citrate lyase in renal cell carcinoma tissues and its effect on the human renal carcinoma cells in vitro

Lichen Teng  1 , Yongsheng Chen  1 , Yan Cao  1 , Wentao Wang  1 , Yongpeng Xu  1 , Yanjie Wang  1 , Jiayin Lv  1 , Changfu Li  1 , Yajuan Su  2
Affiliations

Overexpression of ATP citrate lyase in renal cell carcinoma tissues and its effect on the human renal carcinoma cells in vitro

Lichen Teng et al. Oncol Lett. 2018 May.

Abstract

ATP citrate lyase (ACLY) is a key enzyme of lipogenesis in cells. However, ACLY expression in renal cell carcinoma (RCC) and its association with clinicopathological parameters remain unclear. The present study aimed to evaluate ACLY expression levels in RCC and adjacent normal tissues. This study included 33 patients with clear cell RCC (ccRCC). ACLY protein was assayed using immunohistochemistry and western blotting methods. ACLY mRNA expression was determined by reverse transcription-quantitative polymerase chain reaction. Serum ACLY concentrations were measured using the ELISA. Compared with adjacent normal tissues, significantly higher levels of ACLY protein expression were observed in all of the ccRCC tissues (P<0.05). ACLY protein levels were positively associated with the T stage and nuclear grade of RCC. ACLY immunostaining was located in the cytoplasm and nucleus. ACLY protein levels and ACC1 mRNA expression in RCC tissues were significantly higher compared with that in adjacent normal tissues (P<0.05). There were no significant differences in serum ACLY concentrations between patients with RCC and health controls (P>0.05). Preliminary evaluation of ACLY function showed that ACLY small interfering RNA downregulation inhibited RCC cell proliferation and migration, but promoted RCC cell apoptosis. ACLY may be a novel biomarker for the evaluation of biological aggressiveness and may be a potential _target for RCC treatment.

Keywords: ATP citrate lyase; lipogenesis; metabolic; renal cell carcinoma.

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Figures

Figure 1.
Figure 1.
ACLY is higher expression in primary RCC tissues than that in adjacent normal tissues. (A) ACLY mRNA expression is increased when compare to adjacent normal tissue by RT-PCR (**P<0.01). (B) Representative bands (N, adjacent normal tissue; T, tumor) show increasing ACLY expression in frozen primary ccRCC, compare with adjacent normal tissues by immunoblotting. (C) Immunoblotting analysis showed higher ACLY expression in tumor (**P<0.01). (D) H&E staining of RCC tissues and adjacent normal kidney tissues, and increased expressions of ACLY in ccRCC with different stage and grade were showed by immunohistochemical staining. Representive images show an increasing tendency for staining intensity of ACLY in primary RCC tissues with increasing TNM stage and Fuhrman grade. Subcellular location of ACLY mainly was in both the cytoplasm (magnification, ×400). ACLY, ATP citrate lyase; RCC, renal cell carcinoma; ccRCC, clear cell RCC; H&E, hematoxylin and eosin.
Figure 2.
Figure 2.
ELISA assay showing increased ACLY level in peripheral blood of patients with RCC than health control. (**P<0.01). ACLY, ATP citrate lyase; RCC, renal cell carcinoma.
Figure 3.
Figure 3.
Transfected siRNA into 786-0 and GRC-1 renal cancer cells _targets the expression of ACLY at both mRNA and protein level. (A) Relative expression of ACLY mRNA in quantified by real-time PCR in 786-0, GRC-1, 786-0 cells transfected with negative control, 786-0 cells transfected with siRNA and HK-2 cells (**P<0.01 compared with the HK-2. #P<0.05 compared with 786-0 and GRC-1). (B) Differential expression ACLY protein levels illustrated by western blotting in above five cell lines. (C) Analysis of ACLY protein expression shows lower expression in transfected with siRNA than non-treated cells or treated cells with negative control (**P<0.01 compared with the HK-2. #P<0.05 compared with 786-0 and GRC-1). ACLY, ATP citrate lyase; RCC, renal cell carcinoma; siRNA, small interferring RNA.
Figure 4.
Figure 4.
ACLY siRNA inhibits RCC cells proliferation and migration. (A) Cell proliferation analysis shows a decreased OD in RCC cells after transfection of ACLY siRNA by CCK-8 assay (**P<0.01 compared with the HK-2. #P<0.05 compared with 786-0 and GRC-1). (B) Inhibition of ACLY by transfected siRNA reduced the migration of 786-0 and HK-2 cells Migration of RCC cells transfected with siRNA was suppressed by transwell assay. The upper panel is the representive microscopic images of crystal violate staining; the lower panel is the statistical results (**P<0.01 compared with the HK-2. #P<0.05 compared with 786-0 and GRC-1). Magnification, ×200. ACLY, ATP citrate lyase; RCC, renal cell carcinoma; siRNA, small interferring RNA; OD, optical density.
Figure 5.
Figure 5.
ACLY siRNA promotes apoptosis of RCC cells. Inhibition of ACLY by transfected siRNA promoted apoptosis of 786-0 cells and HK-2 cells. The upper panel is the typical flow cytometric images, and lower panel is the result of analysis (**P<0.01 compared with the HK-2. #P<0.05 compared with 786-0 and GRC-1). Typical images of apoptosis in 786-0, GRC-1, 786-0 cells transfected with negative control, 786-0 cells transfected with siRNA and HK-2 cells are displayed by flow cytometry analysis. The percentage of apoptotic RCC cells shows a significantly increase after transfection of siRNA (**P<0.01 compared with the HK-2. #P<0.05 compared with 786-0 and GRC-1). ACLY, ATP citrate lyase; RCC, renal cell carcinoma; siRNA, small interferring RNA.
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