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. 2018 Nov 21:24:8391-8400.
doi: 10.12659/MSM.911124.

Methylenetetrahydrofolate Dehydrogenase 1 (MTHFD1) is Underexpressed in Clear Cell Renal Cell Carcinoma Tissue and Transfection and Overexpression in Caki-1 Cells Inhibits Cell Proliferation and Increases Apoptosis

Donglin He  1 , Zhihai Yu  1 , Sheng Liu  1 , Hong Dai  1 , Qing Xu  1 , Feng Li  1
Affiliations

Methylenetetrahydrofolate Dehydrogenase 1 (MTHFD1) is Underexpressed in Clear Cell Renal Cell Carcinoma Tissue and Transfection and Overexpression in Caki-1 Cells Inhibits Cell Proliferation and Increases Apoptosis

Donglin He et al. Med Sci Monit. .

Abstract

BACKGROUND The aims of this study were to investigate the expression of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) in human tissue containing clear cell renal cell carcinoma (CCRCC) compared with normal renal tissue, and the effects of upregulating the expression of MTHFD1 in the human CCRCC cell line, Caki-1. MATERIAL AND METHODS Tumor and adjacent normal renal tissue were obtained from 44 patients who underwent radical nephrectomy for CCRCC. Caki-1 human CCRCC cells were divided into the control group, the empty vector (EV) group, and the plasmid-treated group that overexpressed MTHFD1. MTHFD1 mRNA and protein levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The cell counting kit-8 (CCK-8) assay measured cell viability. Flow cytometry evaluated apoptosis and the cell cycle. Western blot measured the protein levels of MTHFD1, Bax, Bcl-2, Akt, p53, and cyclin D1, and qRT-PCR determined the gene expression profiles. RESULTS MTHFD1 mRNA and protein levels in CCRCC tumor tissues were significantly lower compared with adjacent normal renal tissue. MTHFD1 over-expression in Caki-1 cells inhibited cell proliferation, arrested cells in the G1 phase, increased cell apoptosis, and upregulated gene and protein expression of Bax/Bcl-2 and p53 and inhibited p-Akt, and cyclin D1. CONCLUSIONS MTHFD1 was underexpressed in CCRCC tissue when compared with normal renal tissue. MTHFD1 transfection of human CCRCC Caki-1 cells in vitro inhibited cell proliferation and promoted apoptosis, associated with reduced expression of cyclin D1, reduced Akt phosphorylation, and increased expression of Bax/Bcl-2 and p53.

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Conflict of interest statement

Conflict of interest

None.

Figures

Figure 1
Figure 1
Expression levels of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) in clear cell renal cell carcinoma (CCRCC) tissues and adjacent normal renal tissues and survival curve analysis. (A) For the 44 patients who underwent radical nephrectomy for clear cell renal cell carcinoma (CCRCC), the relative mRNA levels of MTHFD1 in CCRCC tissue and adjacent normal renal tissue are shown by quantitative real-time polymerase chain reaction (qRT-PCR). (B) Western blot shows that MTHFD1 protein levels are decreased in the four groups of CCRCC tissue when compared with matched adjacent normal renal tissue. (C) The relative protein expression levels are shown as bar diagrams. (D) Kaplan-Meier curve analysis of patient survival rates in the lower expression and higher expression groups MTHFD1 in patients with CCRCC. Data are expressed as the mean ±SD from three independent experiments. * Compared with adjacent tissues. * P<0.05.
Figure 2
Figure 2
Transfection efficiency of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and the effect on cell viability of Caki-1 cells. (A) mRNA levels of MTHFD1 in Caki-1 cells. (B) Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protein levels are elevated in Caki-1 cells. (C) The relative protein expression levels are shown as bar diagrams. (D) Caki-1 cell viability, assessed by cell counting kit-8 (CCK-8) assay shows that MTHFD1 inhibited Caki-1 cell proliferation. Data are expressed as the mean ±SD from three independent experiments. * Compared with control. * P<0.05.
Figure 3
Figure 3
Effect of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) transfection on the regulation of Caki-1 apoptosis and cell cycle distribution in vitro. (A) Apoptosis assay was detected using flow cytometric analysis in Caki-1 cells. (B) The apoptosis rate shown as bar diagrams. (C) Cell cycle analysis was assessed by flow cytometry. (D) Quantified data from C. Data are expressed as the mean ±SD from three independent experiments. * Compared with control. * P<0.05; ** P<0.01.
Figure 4
Figure 4
Effects of the mRNA and proteins levels of Bax and Bcl-2 on Caki-1 cells. (A, B) Quantitative real-time polymerase chain reaction (qRT-PCR) shows the mRNA expression of Bax and Bcl-2. (C–E) Western blot results and relative units of protein levels. Expression of each protein in the control, empty vector (EV) or MTHFD1 transfected Caki-1 cells, following normalization with the loading control GAPDH. Data are expressed as the mean ±SD from three independent experiments. * Compared with control. * P<0.05; ** P<0.01.
Figure 5
Figure 5
Effects of the mRNA and proteins levels of Akt-p53-cyclin D1 signaling on Caki-1 cells, (A) Quantitative real-time polymerase chain reaction (qRT-PCR) shows the mRNA expression level of p53. (B) qRT-PCR shows the mRNA expression level of cyclin D1. (C) The proteins were measured using Western blot for p-Akt, Akt, p53, cyclin D1 and with normalization using a loading control of GAPDH. The relative levels of protein expression are shown as bar diagrams for P53 (D), cyclin D1 (E), and p-Akt/Akt (F). Data are expressed as the mean ±SD from three independent experiments. * Compared with control. * P<0.05, ** P<0.01.
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