Hepatoma upregulated protein expression is involved in the pathogenesis of human breast carcinogenesis

Abstract

In the last decade, the overexpression of hepatoma upregulated protein (HURP) has been reported in hepatocellular carcinoma, adrenocortical tumors and urogenital carcinoma. However, the role of HURP in breast cancer remains unknown. In the present study, a comprehensive analysis was performed to examine the HURP expression level in 43 breast cancer tumor samples and paired adjacent normal tissues. The correlation between the HURP expression level and the clinicopathological characteristics was evaluated. The role of HURP in breast cancer was investigated by quantitative polymerase chain reaction, western blot analysis and cell proliferation assays. HURP expression was found to be significantly increased in the breast cancer samples. The HURP expression level was higher in the tumors with advanced-grade metastasis and was strongly associated with tumor-node-metastasis staging (P=0.003). Transfection and cell proliferation assays suggested that the suppression of HURP expression or the interference in HURP activity in the breast cancer cells inhibited cell proliferation significantly. These data suggest that HURP is associated with the degree of malignancy and the proliferation of breast cancer. HURP could be a tumor biomarker for prognosis and a potential therapeutic drug target for human breast cancer.

Keywords: breast carcinogenesis; cell proliferation; hepatoma upregulated protein expression.

Figure 1

Hepatoma upregulated protein (HURP) mRNA expression in breast tumors and adjacent normal tissues. (A) Mean HURP expression in primary breast tumors (5.38±3.71; mean ± standard deviation) compared with adjacent normal tissues (1.37±0.87; P<0.0001, Student’s t-test). (B) Representative images of the HURP protein level from breast tumors (T) and adjacent normal tissues (N) were calculated by western blot analysis. The protein level of HURP was normalized to the level of glyceraldehyde-3 phosphate dehydrogenase (Gapdh) protein. (C) HURP mRNA expression in breast cancer cell lines. The values are expressed as the mean ± standard error of the mean for three independent sets of data. ^*P<0.05 and ^**P<0.01 vs. control group

Figure 2

Suppression of hepatoma upregulated protein (HURP) inhibits breast cancer cell proliferation in vitro. (A) The HURP mRNA normalized expression levels of MDA-MB-231 cells transfected with: A, gene-specific 27mer small interfering (si)RNA duplex 1 (DsiRNA1); B, DsiRNA2; C, DsiRNA3; negative control siRNA duplex and non-transfected cells. The expression levels of the HURP mRNA were markedly suppressed in HURP DsiRNA3-transfected cells when compared with the others. (B) MDA-MB-231 cell viability was measured using a Cell Counting Kit-8 assay for 5 days. The viability of the HURP DsiRNA3-transfected cells was lower compared with the parent cells and cells transfected with the negative control siRNA duplex (^*P<0.05 and ^**P<0.01 vs. control group).

Figure 3

Interference in hepatoma upregulated protein (HURP) expression inhibits breast cancer cell proliferation in vitro. (A) MDA-MB-231 cells transfected with 2μg β-galactosidase (β-gal) using Xfect Protein Transfection Reagent. At 1 h post-transfection, the cells were assayed for β-gal activity using a β-Galactosidase Staining kit. The image was captured using an inverted microscope with ×100 magnification. The Xfect Protein Transfection Reagent displayed a markedly higher signal for β-gal. (B) MDA-MB-231 cell viability was measured using the Cell Counting Kit-8 assay for four days. The viability of the anti-HURP Ab-transfected cells was lower than that of the parent cells. The cells transfected with 5μg anti-HURP Ab grew slower than the other transfected cells (^*P<0.05 and ^**P<0.01).