FBXL20 acts as an invasion inducer and mediates E-cadherin in colorectal adenocarcinoma

Abstract

The mechanisms eliciting colorectal adenocarcinoma are not well understood and the FBXL20 gene is problematic as it exhibits an abnormal expression in colorectal cancer cells. In the present study a recombinant plasmid, pReceiver-M03-FBL20 expression plasmid was constructed, which overexpressed FBXL20; this was transfected into Lovo cells to form Lovo-FBL20 cells. The FBXL20 expression level was examined by quantitative polymerase chain reaction (qPCR) and western blot analysis. The cell viability and invasion capacity were measured using cell counting kit 8, Transwell chamber and wound healing assays, respectively. The associated genes, including E-cadherin, β-catenin, c-Myc, SET nuclear oncogene, protein phosphatase-2A, Axin, p53 and caspase 3, were detected by qPCR and western blotting. It was demonstrated that the FBXL20 expression level was markedly upregulated in the Lovo-FBL20 cells transfected with pReceiver-M03-FBL20 expression plasmid, compared with that of the Lovo cells. In addition, the cell viability and invasion capacity of the Lovo-FBL20 cells were significantly increased. These increases correlated with a significant upregulation in the expression level of β-catenin and c-Myc, and a downregulated expression level of E-cadherin. The results of the present study indicate that FBXL20 may mediate the ubiquitin degradation of E-cadherin resulting in an increased invasive ability of malignant cells.

Keywords: E-cadherin; FBXL20; colorectal adenocarcinoma; invasion.

Figure 1

mRNA and protein expression of FBXL20 as detected by quantitative polymerase chain reaction and western blotting, respectively. (A) The FBXL20 expression level in Lovo cells is significantly lower compared with the SW480, SW620, Ls174T, HCT116 and HT29 cells. (B) The pReceiver-M03-FBL20 expression plasmid specifically upregulated the FBXL20 expression in Lovo-FBL20 cells. (C) The protein expression level of FBXL20 was increased by the pReceiver-M03-FBL20 expression plasmid. (D) The mRNA expression level of FBXL20 was also increased by the pReceiver-M03-FBL20 expression plasmid. mRNA and protein were extracted two days after transient transfection with the proper mixture of pReceiver-M03-FBL20 expression plasmid and Lipofectamine 2000. GAPDH and β-actin were used for normalization of mRNA and protein loading, respectively. The results were expressed as the mean ± standard deviation for three independent experiments. ^*P<0.05 vs. Lovo cels, as determined by analysis of variance test. NC, negative control vector.

Figure 2

Cell proliferation effect of FBXL20 overexpressed in colorectal adenocarcinoma Lovo cells. (A) Cell viability assay. Human colorectal adenocarcinoma cells were incubated for the indicated time-periods. As a measurement of cell viability, the relative absorbances (means ± SD; n=6), obtained from the CCK8 assay, are shown. The mRNA expression level of (B) c-Myc and (C) β-catenin were upregulated by the pReceiver-M03-FBL20 expression plasmid vector in Lovo-FBL20 cells. RNA was extracted two days after transient transfection with the proper mixture of pReceiver-M03-FBL20 expression plasmid and Lipofectamine 2000. GAPDH was used for normalization of the RNA. The results were expressed as means ± SD for three independent experiments. ^*P<0.05 compated with the Lovo cells, as determined by Student’s t-test. NC, negative control vector; CCK-8, cell counting kit 8; SD, standard deviation.

Figure 3

Cell invasion effect of FBXL20 overexpression on colorectal adenocarcinoma Lovo cells. (A) Transwell chamber assay (20 h). Human colorectal adenocarcinoma Lovo cells were incubated in the upper chamber subsequent to transfecting the pReceiver-M03-FBL20 expression plasmid into the cells. As a measurement of cell invasion, the cells in the lower membrane were counted (means ± SD; n=6) as shown. (B) Wound healing assay (24 h). (C) Protein expression using immunoblotting of whole proteins from the Lovo-FBL20 and Lovo cells. E-cadherin expression levels were significantly downregulated in Lovo-FBL20 cells compared with those of the Lovo cells. Representative blots of E-cadherin and β-actin expression levels. The (D) protein and (E) mRNA expression levels of E-cadherin were significantly downregulated by the pReceiver-M03-FBL20 expression plasmid in Lovo-FBL20 cells. mRNA was extracted two days after transient transfection with the proper mixture of pReceiver-M03-FBL20 expression plasmid and Lipofectamine 2000. GAPDH and β-actin were used for normalization of the mRNA and protein. The results were expressed as means ± SD for three independent experiments. ^*P<0.05 indicates a significant increase compared with the control value. SD, standard deviation.

Figure 4

Protein expression using immunobloting of whole proteins from the Lovo-FBL20 and Lovo cells. Caspase 3 and p53 protein expression levels were significantly upregulated in Lovo-FBL20 cells compared with those of the Lovo cells. (A) Representative blots of caspase 3, p53 and β-actin expression levels. (B) Average signal intensity values plotted for caspase 3 protein expression and (C) p53 protein expression. 50 μg protein loaded per lane. All signals were normalized to β-actin. Values are expressed as means ± SD for three independent experiments. ^* P<0.05 indicates a significant increase compared with the control value. SD, standard deviation.

Figure 5

Protein expression using immunobloting of whole proteins from the Lovo-FBL20 and Lovo cells. No statistically significant differences in the expression levels of SET, PP2A and Axin proteins were identified between the Lovo-FBL20 and Lovo cells. (A) Representative blots of SET, PP2A and Axin expression levels. Average signal intensity values are plotted for (B) SET, (C) PP2A and (D) Axin protein expression. 50 μg protein loaded per lane. All signals were normalized to β-actin. Values are expressed as means ± SD for three independent experiments. ^* P<0.05 indicates a significant increase compared with the control value. SET, SET nuclear oncogene; PP2A, protein phosphatase-2A; SD, standard deviation.