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

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.

Introduction

Colorectal carcinoma (CRC) has been reported to be the second deadliest cancer.[1-4] Although the overall morbidity and mortality of CRC have decreased in recent decades, there has been a considerable increase in patients <50 years of age.[4-6] Additionally, the tumor is often not completely resectable with a 5-year survival rate of only 14%,[5,6] and despite advances in therapeutic strategies and diagnostic approaches, the prognoses for patients with CRC typically remain poor.[7] There is thus an urgent need to develop new treatment approaches for CRC.

We previously reported the effects of cloning D40 on chromosome 15.[8] D40 and BUB1 (budding uninhibited by benzimidazoles 1) were characterized as cancer-related genes primarily expressed in the testis under normal conditions, but which were also widely expressed in primary tumors of various origins and in assorted cancer cell lines.[9-16] AF15q14, a human gene on chromosome 15, was shown to be identical to the D40 gene,[8] and a previous study reported the mutual translocation of AF15q14 with the mixed lineage leukemia gene MLL in acute myeloid leukemia.[17] Furthermore, D40 expression has been reported to be associated with pathological features of primary lung tumors, such as the degree of differentiation and the patient’s smoking history.[10] In a study of human testes, D40 was found to be expressed in pre-acrosomes and spermatocytes.[18] A follow-up study revealed that blinkin, a type of kinetochore protein in the mitotic machinery, was identical to kinetochore scaffold 1 (KNL1).[19] In addition to binding with Ndc80 and Mis12 complexes to create the KNL1, MIS12, NDC80 (KMN) network, KNL1/blinkin also bound to other proteins, including tubulin, the spindle assembly checkpoint (SAC) proteins BUBR1 and BUB1, and protein phosphatase 1.[19-23] Such binding suggests that KNL1/blinkin and BUB1 may have crucial impacts on kinetochore formation in terms of the connection between spindles and chromosomes, as well as regulation of the SAC.[19-28]

Primary microcephaly (MCPH) is a rare congenital neurodevelopmental disorder with autosomal recessive features.[29,30] Patients with MCPH have a smaller head circumference and smaller brains, leading to varying levels of mental retardation. However, patients with MCPH generally do not have any additional somatic or neurological disorders. MCPH is a genetically heterogeneous condition controlled by several chromosomal loci.[29] Previous studies identified an MCPH locus on chromosome 15,[30] and the gene responsible was identified as CASC5, which is identical to KNL1.[31] Brain development requires rapid cell division, thus suggesting that KNL1 may play a significant role in in vivo cell division.

As noted above,[8,10,17,19,22] there are several different names for the same gene/protein (KNL1, AF15q14, KIAA1570, CASC5, blinkin, D40) and we have therefore used the term KNL1 throughout the present study.

In the present study, we analyzed KNL1 gene and protein expression levels in CRC and matched adjacent noncancerous tissues and in CRC cell lines. We also examined the effect of KNL1 knockdown on CRC cell growth in vitro by transfecting RKO and HT-29 CRC cell lines with short hairpin KNL1 RNA (shKNL1).

Materials and Methods

Bioinformatics analysis

Gene expression data were obtained from The Cancer Genome Atlas website (TCGA, https://tcga-data.nci.nih.gov/tcga/) in relation to the colon adenocarcinoma (COAD) project. In the COAD project, gene set enrichment analysis (GSEA) was carried out to gain further insights into the biological pathways involved in the pathogenesis of CRC. GSEA provides a method for analyzing and interpreting microarray data in relation to biological states.[32]

Patients and samples

We collected CRC and paired adjacent noncancerous tissue samples from 108 patients with colorectal cancer at the Department of General Surgery of the Fourth Hospital of Hebei Medical University (Shijiazhuang, Hebei, China). Intraoperative colorectal cancer tissues and adjacent normal tissues >10 cm away from colorectal cancer tissues were studied. All patients were diagnosed for the first time and had undergone no other treatments such as radiotherapy, chemotherapy, or immunotherapy before surgery. All experiments were approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University, and all clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. All patients provided written informed consent for participation in this study. All patients underwent outpatient review and telephone follow-up to obtain information on postoperative survival status, quality of life, tumor differentiation, sex, lymph node metastasis, TNM stage, infiltration depth, age, tumor location, and imaging findings. Survival time was determined from the date of initial diagnosis to the date of last postoperative follow-up or death. Postoperative staging was performed according to the American Joint Committee on Cancer Seventh Edition staging method.

Cell lines and cell culture

Human CRC (SW480, HT-29, HCT-116, and RKO) and normal colonic epithelial cell lines (NCM460) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere containing 5% CO2 at 37°C.

Immunohistochemistry

Immunohistochemistry was carried out using the streptavidin–biotin–peroxidase method according to the manufacturer’s protocol (LSAB Universal Kit; DAKO Corporation, Carpinteria, CA, USA). The sections were deparaffinized by xylene and dehydrated in graded ethanol, followed by antigen retrieval in a steam pressure cooker. The sections were treated in 3% hydrogen peroxide in phosphate-buffered saline (PBS) for 15 minutes and then rinsed in PBS. The sections were placed in 500 mL of 0.01 M citric acid-buffered solution (pH 7.0) and microwaved at 500 W for 5 minutes. After thorough washing, the sections were incubated with normal rabbit serum for 20 minutes at room temperature to avoid nonspecific binding of the antibodies. Endogenous peroxidase activity was inhibited by incubation with 0.3% hydrogen peroxide in methanol. After blocking nonspecific binding, the sections were incubated overnight with antibodies against BUB1 and KNL1 (dilution, 1:300; Biorbyt Ltd., Cambridge, UK). Sections were then incubated with streptavidin peroxidase complex, developed with 3,3′-diaminobenzidine solution, counterstained with Mayer’s/Lillie Mayer’s hematoxylin, and mounted.

Evaluation of KNL1 and BUB1 protein expression

The immunostained specimens were evaluated independently by two observers (authors Y.B. Liu and B.H. Li) with no knowledge of the clinicopathological data. In the event of a discrepancy, a consensus was reached following additional assessment. The staining intensity of cancer cells was graded on a 4-point scale: 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The percentage of stained cancer cells was also graded on a 4-point scale: 0 = no cells, 1 = <10%, 2 = 10% to 50%, and 3 = >50%. The intensity score was multiplied by the percentage score to obtain an overall score.

RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Whole RNA was extracted from the sampled cells and tissues using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was generated by RT using M-MLV Reverse Transcriptase (Promega Corp., Madison, WI, USA) according to the manufacturer’s protocol. A total of 1 µL cDNA was used as the template for qPCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primer sequences were as follows: KNL1, forward 5′-ACA TTG GAA AAA GCG CAA GTTG-3′ and reverse 5′-TTG CAC TGG GCA ATA ATT GGC-3′; GAPDH, forward 5′-TGA CTT CAA CAG CGA CAC CCA-3′ and reverse 5′-CAC CCT GTT GCT GTA GCC AAA-3′. Thermocycling comprised initial denaturation at 95°C for 30 s followed by 45 cycles of denaturation at 95°C for 5 s and extension at 60°C for 30 s. Each experiment was performed in triplicate and relative quantitative gene expression was calculated as described previously.[33]

Western blotting

Cells and tissues were lysed using RIPA buffer (Thermo Fisher Scientific, Inc.). Lysates were electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific, Inc.) and transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Inc.). After blocking for 1 hour in 5% skimmed milk, the membranes were incubated overnight at 4°C with rabbit antibodies against KNL1 (dilution, 1:500; Biorbyt Ltd.) and GAPDH (dilution, 1:800; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), washed four times in TBST, and then incubated at room temperature for 1 hour with a horseradish peroxidase-conjugated secondary antibody (dilution, 1:10,000; catalog no., 323-065-021; Jackson ImmunoResearch, Inc., West Grove, PA, USA). Protein bands were visualized using a chemiluminescence system (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. The band intensities were quantified using Image-Quant Software v3.0 (LI-COR Biosciences, Lincoln, NE, USA). This experiment was repeated in triplicate.

Construction of recombinant lentiviral vector and cell transduction

KNL1 shRNA (shKNL1) and control shRNA (shCtrl) were designed by GeneChem Co., Ltd. (Shanghai, China) from the unabridged sequence of D40/KNL1, which is targeted at the human KNL1 gene (GenBank no. NM_144508). The targeting sequence of KNL1 was 5′-CAG AGT TGT ATG GTG GAAA-3′. The efficiency of KNL1 knockdown was tested following synthesis and insertion of stem-loop oligonucleotides into a lentivirus-based pGV115-GFP vector (GeneChem Co. Ltd.) with AgeI/EcoRI sites. Lentivirus particles were prepared as described previously.[34] RKO and HT-29 cells (2×105 cells/well) were cultured in 6-well plates and transfected with either negative control (shCtrl) lentivirus or KNL1 (shKNL1) lentivirus at a multiplicity of infection of 5. A blank control group (uninfected) was set up simultaneously. Cells were incubated for 5 days at 37°C in an atmosphere containing 5% CO2. After 72 hours of transfection, cells were observed using a fluorescence microscope (IX71; Olympus Corp., Tokyo, Japan). The knockdown efficiency was measured after 5 days using western blotting and RT-qPCR.

Cell growth assay

ShKNL1-transfected, shCtrl-transfected, and blank control RKO and HT-29 cells in logarithmic growth phase were cultured in 96-well plates at a density of 2,000 cells/well for 5 days at 37°C in an atmosphere containing 5% CO2. Cell numbers were recorded every day using a Celigo Imaging Cytometer (Nexcelom Bioscience, Lawrence, MA, USA). All assays were performed in triplicate.

Methyl-thiazol-tetrazolium (MTT) assay

ShKNL1-transfected, shCtrl-transfected, and blank control RKO and HT-29 cells were seeded and cultured in 96-well plates at a density of 2,000 cells/well at 37°C and their proliferation was assessed each day for 5 days. Briefly, 20 µL MTT (5 mg/mL; Genview, Craigie Burn, Australia) was added to each well and incubated at 37°C for 4 hours. The culture medium was removed and 100 µL of dimethyl sulfoxide was added to each well to dissolve the crystals. Plates were agitated for 2 to 5 minutes and the absorbance at 490 nm was read using a microplate reader (Tecan Infinite, Tecan GmbH, Austria). All experiments were performed in triplicate.

Colony-formation assay

Cells transfected with shKNL1 or shCtrl vectors in logarithmic growth phase were re-suspended in RPMI-1640 and seeded in 6-well plates at a density of 800 cells/well and then incubated for 14 days. Cell colonies were then observed by fluorescence microscopy (IX71; Olympus Corp.). Cells were fixed with paraformaldehyde (1 mL/well; Sangon Biotech Co. Ltd., Shanghai, China) for 30 minutes, washed with phosphate-buffered saline (PBS), and then stained with 500 µL 0.5% crystal violet (Sangon Biotech Co. Ltd.) for 5 minutes. The stained cells were then washed with ddH2O and dried at room temperature. Finally, cell colonies were observed under a microscope (Cai Kang Optical Instrument Co., Ltd, Shanghai, China). All experiments were performed in triplicate.

Fluorescence-activated cell sorting (FACS)

FACS was used to analyze cell apoptosis.[35] In brief, RKO cells transfected with shCtrl or shKNL1 were seeded in 6-well plates for 120 hours and grown to 70% confluence, harvested, and washed with 1× binding buffer, followed by the addition of 10 µL of Annexin V-allophycocyanin Detection kit (eBioscience; Thermo Fisher Scientific, Inc.) per 200 µL of the adjusted suspension for 15 minutes in the dark at room temperature. Flow cytometry analysis was carried out after cell staining. All experiments were performed in triplicate.

Cell cycle detection

Cells collected from each group were washed in cold PBS and then adjusted to a concentration of 1×106 cells/mL. The cells were then fixed with 70% ethyl alcohol overnight, incubated with RNaseA at 37°C in a water bath for 30 minutes, followed by staining with propidium iodide at 4°C for 30 minutes. The DNA content was measured by flow cytometry.

Statistical analysis

Statistical analyses were carried out using IBM SPSS Statistics for Windows, Version 19.0 (IBM Corp., Armonk, NY, USA). All data are presented as the mean ± standard deviation. Comparisons between two groups were made using Student’s t-test, and correlations between KNL1 expression and clinicopathological parameters were analyzed by the χ2 test. Survival curves were plotted using the log-rank (Mantel–Cox) test and the Gehan–Breslow–Wilcoxon test. A value of P<0.05 was considered to indicate a significant difference.

Results

Demographics

The patients’ demographic information is presented in Table 1.

Table 1.

Patients’ demographic information.

Clinical parameters	Patients, n
Age (years)
<60	42
≥60	66
Sex
Male	63
Female	45
Differentiation
Level I–II	48
Level III	60
TNM stage
I–II	69
III	39
Lymphatic metastasis
No	66
Yes	42
Infiltration depth
T1–T2	30
T3–T4	78
Tumor location
Colon	42
Rectum	66

Expression of KNL1 and BUB1 in normal and CRC tissues

KNL1 gene and protein expression levels in CRC and para-cancerous tissues were measured by RT-qPCR and western blot, respectively. The PCR products for GAPDH and KNL1 were 121 and 213 base pairs in length, respectively. KNL1 mRNA and protein levels were both significantly higher in CRC compared with normal tissues (P<0.05) (Figure 1A and B).

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.

Immunohistochemical analysis of serial sections of 108 CRC samples showed that KNL1 and BUB1 proteins were located mainly in the nucleus of the same cancer cells (Figure 1C–F). KNL1 mRNA expression levels were also significantly higher in CRC compared with normal tissues in COAD gene expression data collected from the TCGA website (https://tcga-data.nci.nih.gov/tcga/) (P<0.001) (Figure 1G). The calculated regression coefficient between the expression scores of KNL1 and BUB1 showed a significant correlation between KNL1 and BUB1 expression in CRC (P<0.0001) (Figure 1 H). However, KNL1 and BUB1 were hardly expressed in adjacent normal tissues (Figure 1I and J).

The mean overall scores for KNL1 and BUB1 in the 108 tumors were 4.9 and 5.4, respectively, and these were therefore used as the mean overall cut-off scores for discriminating KNL1 and BUB1 protein expression statuses, respectively. Samples with a KNL1 score >4.9 were considered as KNL1-positive and samples with a score ≤4.9 were considered KNL1-negative. Samples with a BUB1 score >5.4 were considered BUB1-positive and samples with a score ≤5.4 were considered BUB1-negative.

To explore the medical significance of KNL1 expression in CRC, we analyzed its expression in relation to various clinicopathological characteristics. Among the 108 patients, KNL1 expression in tumor specimens was substantially associated with tumor differentiation, but not with sex, lymph node metastasis, TNM stage, infiltration depth, age, or tumor location (Supplementary Table 1).

KNL1 expression in colorectal cancer cell lines

KNL1 mRNA and protein expression levels in SW480, RKO, HT-29, and HCT-116 CRC cells and in NCM460 normal colonic epithelial cells were determined by RT-qPCR and western blotting, respectively. Both KNL1 mRNA and protein were significantly more highly expressed in CRC compared with normal cells (all P<0.01) (Figure 2A–C).

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).

Association between KNL1 expression and survival

We created survival curves to analyze the association between KNL1 protein expression and prognosis of CRC patients. Positive expression of KNL1 was significantly associated with poorer survival (P=0.01) (Figure 2D).

Lentivirus-mediated knockdown of KNL1 in CRC cells

To assess the mechanism of KNL1 in CRC, we carried out KNL1 knockdown in RKO and HT-29 cells by transfection with a shKNL1 lentivirus. After 3 days, >80% of the cells were successfully transfected with either shCtrl or shKNL1 lentivirus (Figure 3A and B). KNL1 mRNA expression was significantly lower in the shKNL1 compared with the shCtrl-infected cells after 5 days of transfection, as determined by RT-qPCR (P<0.01) (Figure 3C and D). These results were confirmed by western blotting, indicating that the target gene was successfully knocked down (Figure 3E and F).

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.

Inhibition of CRC cell proliferation by KNL1 knockdown

RKO and HT-29 cells transfected with shCtrl lentivirus or shKNL1 lentivirus and blank control cells were seeded into 96-well plates and analyzed using Celigo for 5 consecutive days. The numbers of cells in the shCtrl groups increased over the 5 days, whereas the numbers of cells in the shKNL1-transduced groups increased only slightly according to cell counting and MTT assays in both RKO (Figure 4A–E) and HT-29 cells (Figure 4F–J) (P<0.01 at 4 and 5 days). These results suggest that KNL1 knockdown significantly inhibited CRC cell proliferation. KNL1 expression thus appeared determine the growth of RKO and HT-29 cells.

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.

Inhibition of CRC colony formation by KNL1 knockdown

RKO and HT-29 cells were stained with crystal violet to assess colony formation (Figure 5A and B). The number of the cells per colony was significantly higher in the shCtrl compared with the shKNL1 group (shCtrl: 229±9 vs. shKNL1: 40±5; shCtrl: 259±2 vs. shKNL1: 55±3; P<0.01) (Figure 5C and D), indicating that the endogenous reduction in KNL1 expression significantly inhibited CRC cell growth.

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.

Effect of KNL1 knockdown on apoptosis of RKO cells

The apoptotic rate of RKO cells was significantly higher in the shKNL1 compared with the shCtrl group (14.86±0.30% vs 4.32±0.13%) (P<0.01) (Figure 5E and F).

Impact of KNL1 knockdown on cell cycle of RKO cells

DNA ploidy assay demonstrated that the proportion of G1-phase RKO cells was significantly higher in the shKNL1 compared with the negative control group (53.05 ± 6.71% vs 35.58 ±5.42%) (P<0.05) (Figure 5G and H). There were also reductions in the percentages of both S phase and G2 phase cells.

Discussion

The morbidity and mortality rates of CRC have risen rapidly, and it has therefore attracted worldwide attention.[36,37] Despite improvements in clinical practice and treatment, outcomes of CRC patients remain poor owing to metastasis or drug resistance.[38,39] It is therefore necessary to identify new therapeutic targets and to improve our understanding of the underlying molecular mechanisms.

The occurrence and development of tumors are associated with both behavioral and environmental factors, and tumorigenesis is associated with the accumulation of genetic mutations over time. Identifying the affected genes may thus aid the development of advanced diagnostic and treatment methods for cancer.

The spindle checkpoint controls mitotic progression. The human kinetochore oncoprotein AF15q14/KNL1, which belongs to the Spc105/Spc7/KNL-1 family, has been reported to provide direct links between the spindle checkpoint proteins BUB1 and BubR1 and the kinetochores, and also to play a crucial role in the alignment of chromosomes and spindle checkpoints. Previous studies[22,27] showed that KNL1 RNA interference resulted in accelerated mitosis due to chromosome misalignment and checkpoint failure, caused by the lack of microtubule attachment and kinetochores. Another study reported that transfection with KNL1 small interfering RNA resulted in cancer cell apoptosis and inhibited the growth of these cells both in vitro and in vivo, regardless of p53 status.[40] However, to the best of our knowledge, no previous specific studies have reported on the role of KNL1 expression in human cancers, particularly CRC.

The results of the present study suggest an obvious relationship between KNL1 and CRC cell apoptosis and proliferation. Although many studies have described abnormal mitosis in cancer cells treated with KNL1 siRNA,[22,41] the current results provide the first evidence to demonstrate that KNL1 downregulation induced apoptosis and inhibited the growth of CRC cell lines.

However, the current study had several limitations. The number of clinical samples was relatively small and a larger sample size is required to verify the expression of KNL1 in colorectal tumors. Furthermore, there was a lack of relevant clinical information regarding patients’ cause of death.

In conclusion, the results of the present study indicated that KNL1 expression was increased in CRC tissues. Furthermore, KNL1 gene knockdown in CRC cells reduced its gene and protein expression levels, which in turn induced apoptosis and inhibited proliferation. KNL1 knockdown may therefore be an effective therapeutic approach for the treatment of CRCs with KNL1 overexpression.

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supplementary_material-_table for Effect of KNL1 on the proliferation and apoptosis of colorectal cancer cells by Tianliang Bai, Yalei Zhao, Yabin Liu, Bindan Cai, Ning Dong and Binghui Li in Technology in Cancer Research & Treatment