Prognostic Relevance of HJURP Expression in Patients with Surgically Resected Colorectal Cancer

Abstract

HJURP is a key factor for CENP-A deposition and maintenance in centromeres. The role of mis-regulation of histone chaperones in cancer initiation and progression has been studied. However, its role in colorectal cancer is still unclear. In this study, we aimed to evaluate the expression of HJURP in 162 colorectal cancer tissue. To investigate the function of HJURP in the colorectal cancer cell, we suppressed HJURP expression by siRNA and confirmed proliferation, migration, invasion, and anchorage independent of colony forming ability. The association between HJURP expression levels and clinicopathological factors was evaluated in 162 CRC tissues using immunohistochemistry. The overall survival rate in patients of HJURP high expression was higher than those in HJURP low expression in CRC. Suppressing HJURP expression decreased cellular proliferation, invasion, and migration in four CRC cell lines: HT29, HCT116, SW480, SW620 in vitro study. Our findings revealed that the knockdown of HJURP suppressed the proliferation, migration, invasion, and tumorigenicity in CRC cells. Due to its strong association with CRC, HJURP could be a potential prognostic biomarker and a novel target for drug discovery.

Keywords: Colorectal cancer (CRC); HJURP; prognostic biomarker.

Figure 1

Immunohistochemistry for HJURP expression in colorectal cancer tissues. (A–D). One hundred and sixty-two CRC samples were stained with HJURP antibody and scored based on both staining intensity and staining frequency. Representative images of tissue slides and immunohistochemistry are shown ((A), Normal negative HJURP expression; (B), Tumor negative; (C), Tumor low expression; (D), Tumor high expression). Original magnification × 200.

Figure 2

HJURP expression is related to overall survival in colorectal cancer patients. Patients were divided into two groups based on the expression of HJURP and survival rate was determined using Kaplan-Meier analysis (p = 0.016).

Figure 3

siRNA-mediated HJURP depletion in colorectal cancer cell lines. Colorectal cancer cell lines including HT29, HCT116, SW480, and SW620 were transduced with either control or HJURP siRNA. (A) RT-PCR was performed to determine HJURP mRNA expression cancer cell lines. (B) Immunoblot with anti-HJURP antibody was conducted to analyzed HJRUP protein level. GAPDH and β-actin was used as the loading control for RT-PCR and immunoblot.

Figure 4

HJURP depletion impairs cell proliferation in colorectal cancer cell lines. Control or HJURP-siRNA colorectal cancer cell lines such as HT29, HCT116, SW480, and SW620 were analyzed every 24 h for 3 days to determine the proliferation rate by MTT assay. (A) HT29 cells. 24 h; *** p = 0.001, 48 h; ** p = 0.007, 72 h; ** p = 0.01, (B) HCT116. 48 h; * p = 0.021, 72h; *** p = 0.001, (C) SW480. 24 h; *** p = 0.001, 48 h; * p = 0.011, 72 h; * p = 0.021. (D) SW620. 24 h; *** p = 0.001, 48 h; ** p = 0.003, 72 h; ** p = 0.007.

Figure 5

HJURP depletion impairs migration and invasion of colorectal cancer cell lines in vitro. Control or HJURP-siRNA colorectal cancer cell lines were seeded on a matrigel uncoated or coated Transwell, followed by incubation for 48 h for assessing migration and invasion, respectively. (A) Imaging was done using an inverted microscope and representative images are shown (× 40). (B) HJURP-siRNA cells displayed significantly reduced ability of migration (HT29; * p = 0.027, HCT116; * p = 0.023, SW480; ** p = 0.007, SW620; * p = 0.031). (C) siRNA mediated depletion of HJURP expression was suppressed the invasion ability of colorectal cancer cells (HT29; * p = 0.021, HCT116; * p = 0.028, SW480; * p = 0.011, SW620; * p = 0.026).

Figure 6

HJURP depletion impairs anchorage-independent colony forming ability of colorectal cancer cell lines in vitro. Control or HJURP-siRNA colorectal cancer cell lines were seeded on soft agarose and incubation was done for 14 days to evaluate colony forming ability. (A) Representative images of colonies were shown (× 40). (B) Number of colonies from each colorectal cancer cell lines were quantified (HT29; * p = 0.038, HCT116; * p = 0.05, SW480; ** p = 0.008, SW620; *** p = 0.001).