Effect of KNL1 on the proliferation and apoptosis of colorectal cancer cells

Abstract

Objective: To identify the expression of kinetochore scaffold 1 (KNL1) in colorectal tumor tissues and to clarify the role of this gene in the proliferation capability of colorectal cancer cells.

Methods: A total of 108 paired colorectal tumor and normal tissue samples were collected from patients with colorectal cancer and subjected to quantitative polymerase chain reaction and immunohistochemistry analyses. Expression levels of KNL1 mRNA and protein were compared between tumor and normal tissues, and KNL1 levels were evaluated in relation to the patients' tumor differentiation, sex, lymph node metastasis, TNM stage, infiltration depth, age, and tumor location. Survival curves were also constructed and compared between patients with tumor samples with and without KLN1 protein expression. KNL1 was under-expressed in colorectal cancer cells in vitro using lentiviral transfection with short hairpin RNA, and its function was evaluated by proliferation, colony-formation, and apoptosis assays. Expression levels of BUB1 protein were also compared between tumor and normal tissues, and the correlation between KNL1 expression and BUB1 expression in colorectal cancer tissues was examined.

Results: KNL1 mRNA and protein were both highly expressed in colorectal tumor tissues compared with paired normal tissues. KNL1 downregulation significantly inhibited colorectal cancer cell proliferation and colony formation, and promoted apoptosis. KNL1 protein expression was significantly associated with tumor differentiation, but not with sex, lymph node metastasis, TNM stage, infiltration depth, age, or tumor location. KNL1 protein expression was also significantly associated with poorer survival. Moreover, there was a significant correlation between KNL1 and BUB1 in colorectal cancer tissues.

Conclusions: KNL1 plays an effective role in decreasing apoptosis and promoting the proliferation of colorectal cancer cells, suggesting that its inhibition may represent a promising therapeutic approach in patients with colorectal cancer.

Keywords: KNL1; Kinetochore scaffold 1; cell growth; colorectal tumor; gene expression; prognosis; protein expression.

Figure 1.

Expression of KNL1 in CRC and normal tissues. (A) KNL1 mRNA expression levels in 108 paired CRC and normal adjacent tissues detected by RT-qPCR and normalized against GAPDH. Data analyzed by paired t-test (n=3). (B) KNL1 protein expression levels in 108 paired CRC and normal adjacent tissues detected by western blot analysis. Representative sample of KNL1 western blot band. Data analyzed by paired t-test (n=3). (C-F) Representative samples of negative and positive KNL1 and BUB1 immunostaining. Original magnification, ×400. (G) KNL1 expression data obtained from The Cancer Genome Atlas website. (I and J) Representative immunostaining of KNL1 and BUB1 in adjacent normal tissues. Original magnification, ×400. D and I, and G and J represent paired tissues from the same patients, respectively. *P<0.05.

Figure 2.

KNL1 mRNA and protein levels in colorectal cancer (CRC) and normal colonic epithelial cell lines. (A) KNL1 mRNA expression levels determined by RT-qPCR and (B and C) KNL1 protein expression levels determined by western blotting in CRC (SW480, RKO, HT-29, and HCT116) and normal colonic epithelial (NCM460) cell lines (n = 3) (**P<0.01). (D) Survival curves, correlated with overall survival, in patients with positive and negative expression of KNL1 (P = 0.01).

Figure 3.

Knockdown of KNL1 in RKO and HT-29 cells infected with shKNL1 or shCtrl. (A and B) Representative images of cells examined by fluorescent and light microscopy at day 3 post-infection (×200 magnification). (C and D) KNL1 mRNA levels were analyzed by RT-qPCR at day 5 post-infection. KNL1 mRNA levels decreased significantly after KNL1 knockdown (**P<0.01). (E and F) KNL1 protein expression was analyzed by western blotting in transduced RKO and HT-29 cells at day 5 post-infection. KNL1 protein levels were significantly lower in the experimental compared with the control group.

Figure 4.

KNL1 knockdown inhibited RKO cell growth. (A) Cell counts increased in time-dependent manners in RKO cells infected with shCtrl and shKNL1, respectively, for 5 days, as shown by fluorescence microscopy (green staining). Cell growth was significantly slower in the shKNL1 compared with either the shCtrl or blank control group according to (B) Celigo Cell Counting, (C) count-fold curves, and (D and E) MTT assay. KNL1 knockdown inhibited HT-29 cell growth compared with the control group. (F) The cell count increased in a time-dependent manner in HT-29 cells infected with shCtrl and shKNL1, respectively, for 5 days, as shown by fluorescence microscopy. Cell growth was significantly slower in the shKNL1 compared with either the shCtrl or blank control group according (G) Celigo Cell Counting, (H) count-fold curves, and (I and J) MTT assay. BC, blank control.**P<0.01.

Figure 5.

KNL1 silencing repressed RKO and HT-29 cell colony formation. (A and B) Photomicrographs of crystal violet-stained colonies of RKO and HT-29 cells growing in 6-well plates at 10 days after infection. (C and D) There were significantly fewer colonies in the shKNL1-transfected RKO and HT-29 cell cultures compared with the respective control cultures. RKO cell apoptosis analyzed by flow cytometry. (E) Cells were transfected with shKNL1 and shCtrl and apoptosis was analyzed by flow cytometry. Each group is shown in triplicate. (F) Apoptosis was significantly higher in the shKNL1 compared with the shCtrl group. (G and H) Proportions of cells in various cell cycle stages, determined by flow cytometry.*P<0.05; **P<0.01.