A total of 186 HCC tissue specimens and matched adjacent normal tissues collected at Gansu Provincial Hospital between 2013 and 2018 were included in the study. In accordance with the protocol used to obtain the tissue samples, informed consent was obtained from all donors and recipients. The study was approved by the Ethics Committee of Gansu Provincial Hospital. The characteristics of patients with HCC are summarized in Table 1. Two independent pathologists diagnosed HCC based on the World Health Organization criteria. None of the patients had received chemotherapy, radiation therapy, or transarterial chemoembolization prior to surgery. HCC tissues and adjacent normal tissues were collected, fixed with 10% formaldehyde solution, and embedded in paraffin.

The KIF21B expression profile and relative clinicopathologic features of patients with HCC were selected from the TCGA database (https://portal.gdc.cancer.gov/legacy-archive/search/f). The database included 50 paired cancer and adjacent normal tissues, which were used to examine the differential expression of KIF21B between HCC and adjacent normal tissues. The data were standardized using the Trimmed Mean of M-values method, and biological coefficient of variation was performed for the quality control. Differentially expressed genes were accessed using the TCGA analyze-DEA function considering a log2 fold change > 1 or < −1. The fold change was the ratio of gene expression in the cancer samples to that in the adjacent normal samples. Differences with P < 0.05 were considered statistically significant.

HCC cell lines Hep-G2, BEL7402, BEL-7404, and SMMC-7721 and the normal liver cell line Chang liver were purchased from Shanghai Genechem Co., Ltd. (Shanghai, China). BEL7402, BEL-7404, and Chang liver cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc.). SMMC-7721 and HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, United States) supplemented with 10% fetal bovine serum. The cell lines were cultured in a 37 °C incubator with 5% CO₂. To further probe the role of KIF21B in HCC cells, KIF21B expression in BEL-7404 cells was silenced using lentivirus-mediated siRNA. In brief, cells were transfected for 24 h with lentiviral constructs expressing short hairpin RNA (shRNA) specific for KIF21B (shKIF21B; Shanghai Genechem Co., Ltd.) or control shRNA (shCtrl; Shanghai Genechem Co., Ltd.). Green fluorescence was used to estimate the efficiency of transfection. Stable knockdown cells were selected using puromycin.

TRIzol reagent (Shanghai Pufei Biotech Co., Ltd, Shanghai, China) was used to extract total RNA from BEL-7404 cells, which was used as a template for synthesis of cDNA using M-MLV Reverse Transcriptase (Promega, Beijing, China). For real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR), cDNA was mixed with SYBR Master Mixture (TAKARA, Kyoto, Japan) and amplified using a real-time PCR thermocycler (Agilent Technologies, Beijing, China). The PCR primers used have the following sequences: KIF21B forward, 5ʹ-GGATGCCACAGATGAGTT-3ʹ and reverse, 5ʹ-TGTCCCGTAACCAAGTTC-3ʹ; GAPDH forward, 5ʹ-TGACTTCAACAGCGACACCCA-3ʹ and reverse, 5ʹ-CACCCTGTTGCTGTAGCCAAA-3ʹ. PCR amplification was quantitated using the 2^-∆∆Ct method. Each sample was amplified in triplicate, and GAPDH was used as an internal control. The KIF21B and GAPDH primers were designed by Shanghai Genechem Co., Ltd.

Concentrations of proteins extracted from cells were measured using a Bicinchoninic acid Protein Assay Kit (Beyotime Biotechnology, China) based on the manufacturer’s instructions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the total proteins, and the proteins were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). The membranes were incubated with the following primary antibodies overnight at 4 °C followed by incubation with the corresponding secondary antibodies: Mouse-human KIF21B antibody (1:400; Sigma-Aldrich) and rabbit-human anti-GAPDH (1:2000; Santa Cruz Biotechnology, CA, United States); goat anti-mouse IgG (1:2000; Santa Cruz Biotechnology CA, United States) and goat anti-rabbit IgG (1:2000; Santa Cruz Biotechnology). GAPDH was used as an internal control.

BEL-7404 cells were transfected with shKIF21B or shCtrl. Three days later, the cells were seeded into 96-well plates at a density of 2000 cells/well. The cells were incubated at 37 °C with 5% CO₂ for 5 d. The cells were counted daily using a Celigo Imaging Cytometer (Nexcelom Bioscience, Lawrence, MA, United States). Each experiment was performed in triplicate.

Lentivirus-infected BEL-7404 cells were seeded into 96-well plates at a density of 2000 cells/well, and cell viability was assessed using MTT (Genview, Beijing, China). MTT (5 mg/mL) was added to each well (20 μL) and incubated for 4 h at 37 °C. Dimethyl sulfoxide (Shanghai Shiyi Chemical Technology Co., LTD, Shanghai, China) was added to each well (100 μL). The MTT colorimetric assay was performed to detect cell proliferation after 1, 2, 3, 4, and 5 d of incubation. Absorbance by the resulting formazan crystals (solubilized with DMSO) was read at 490 nm using an enzyme-linked immunosorbent assay plate reader.

To quantify the effects of shKIF21B on cell apoptosis, the transfected cells were fixed with ice-cold 75% ethyl alcohol at 4 °C overnight. The cells were stained using an Annexin V-APC/7-AAD Kit (eBioscience, Shanghai, China) according to the manufacturer’s instructions. The cells were incubated with Annexin V–APC for 15–20 min at room temperature in the dark.

Colony formation assays were used to detect the growth of cells. BEL-7404 cells were transfected with shKIF21B or shCtrl. Three days later, 1000 cells/well were seeded into six-well plates and cultured for 14 d at 37 °C. Cell status was evaluated, and the medium replaced every 3 d. At the end of the culture period, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 30–60 min. The fixed cells were washed with PBS, stained with Crystal Violet Staining Solution (Shanghai Yuanye Bio-Technology Co., Ltd. Shanghai, China) for 10–20 min, and washed several times with double-distilled water. The number of colonies formed was counted using a microscope and photographed.

Sections of each specimen (5 μm thick) were placed on glass slides, deparaffinized, dehydrated, and boiled in 10 mmol/L citrate buffer for antigen retrieval. After inhibition of endogenous peroxidase activity by incubation for 10 min with methanol containing 0.3% H₂O₂, the sections were blocked with 2% bovine serum albumin for 30 min and incubated overnight at 4 °C with a primary polyclonal rabbit-human KIF21B antibody (1:200; Bioss, Beijing, China). After three washes with PBS, the slides were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG for 30 min, reacted with diaminobenzidine, and counterstained with hematoxylin.

Expression of KIF21B was independently evaluated by two pathologists who were blinded to the clinical and pathological stage of the patients. Staining results were assessed by taking into consideration both the percentage of positive cells and the intensity of staining. The positive percentage of KIF21B in the cells was evaluated as follows; 0 (< 10%); 1 (10%-25%); 2 (25%-50%); 3 (50%-70%); and 4 (> 70%). Staining intensity was considered as: 0 (negative); 1 (weak); 2 (moderate); and 3 (strong). According to the overall score of the percentage of positive cells × the intensity of staining, which ranged from 0-12, we graded the staining as follows: 0 (negative); 1-4 (weak); 5-9 (moderate); 10-12 (strong). An overall score > 5 was considered as high expression.

All the data shown are the results of at least three independent experiments and are expressed as the mean ± SD. The differences between groups were compared using Student’s t-test. Differences between groups were analyzed using one-way analysis of variance (ANOVA) or χ² test. Cumulative survival was compared using the Kaplan–Meier method with the log-rank test. A multivariate Cox proportional-hazards regression model was used to identify independent prognostic factors. Statistical significance was set at P < 0.05.

Based on the screening of the 50 pairs of HCC/adjacent normal tissues in TCGA database, KIF21B expression in HCC tissue was upregulated in 31 specimens, unchanged in 14, and downregulated in 5 compared to expression in the corresponding normal tissues (Figure 1A-B). We utilized RT-qPCR to detect the expression of KIF21B in the HCC cell lines (BEL-7404, BEL-7402, HepG2, and SMMC7721) and the normal liver cell line (Chang liver). The results showed that KIF21B mRNA expression was higher in HCC cell lines than in normal liver cells (Figure 1C). The BEL-7404 cell line was selected for use in subsequent investigations due to the KIF21B expression being at the highest level in this cell line among the five HCC cell lines evaluated.

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Figure 1 Differential expression of KIF21B in The Cancer Genome Atlas database and hepatocellular carcinoma cell lines. A and B: Differential expression levels of KIF21B in 50 pairs of matched hepatocellular carcinoma and adjacent normal tissues from the The Cancer Genome Atlas database (P < 0.01); C: KIF21B expression was examined in BEL-7404, BEL-7402, Hep-G2, SMMC-7721, and Chang liver cells. All the data were normalized to mRNA expression levels of human GAPDH using the 2^-ΔΔCT method. All the experiments were conducted in triplicate. Data were analyzed by ANOVA or t-test. The data are reported as the mean ± SD. P < 0.05 was considered significant, ^aP < 0.05, ^bP < 0.01.

To investigate the role of KIF21B, we knocked down KIF21B expression in BEL-7404 cells using shRNA-mediated interference. As shown in Figure 2A, at 72 h after transfection, the proportion of infected cells in both the shCtrl and shKIF21B groups had reached 80%. Based on the RT-qPCR results (Figure 2B) and Western blot analysis (Figure 2C), the expression of KIF21B mRNA and KIF21B protein in the shCtrl group was significantly higher than that of the shKIF21B group.

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Figure 2 Knockdown of KIF21B using lentivirus-mediated small interfering RNA in hepatocellular carcinoma BEL-7404 cells. A: The proportion of infected cells in both the shCtrl and shKIF21B groups reached 80%; B: Expression level of KIF21B mRNA in BEL-7404 cells was significantly decreased in the shKIF21B-treated group compared to the shCtrl-treated group; C: Expression level of KIF21B protein significantly increased in the shCtrl-treated group compared to the shKIF21B-treated group (P < 0.01). All the experiments were conducted in triplicate. Data were analyzed by ANOVA or t-test. The data are reported as the mean ± SD. P < 0.05 was considered significant, ^bP < 0.01.

BEL-7404 cells transfected with shKIF21B or shCtrl were quantitated for 5 d using Celigo Imaging Cytometer analysis to evaluate cell growth. Silencing KIF21B significantly decreased the total number of cells and the rate of cell growth (Figure 3A-B).

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Figure 3 Growth of BEL-7404 cells detected using a Celigo Imaging Cytometer after transfection with short hairpin RNA shCtrl or shKIF21B. A and B: Fluorescence images of cells for each day post-transfection. Data were analyzed by ANOVA or t-test. The data are reported as the mean ± SD. P < 0.05 was considered significant, ^bP < 0.01.

MTT analysis was used to evaluate the effect of KIF21B on cell proliferation. As shown in Figure 4, the shKIF21B group had lower cell viability compared to the shCtrl group with the difference being statistically significant at 4 d and 5 d post-transfection. Thus, silencing the expression of KIF21B inhibited cell proliferation.

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Figure 4 Growth of BEL-7404 cells detected by MTT assay after transfection with short hairpin RNA shCtrl or shKIF21B. Cell proliferation was significantly inhibited in the shKIF21B-transfected group cells. All the experiments were conducted in triplicate. Data were analyzed by ANOVA or t-test. The data are reported as the mean ± SD. P < 0.05 was considered significant, ^bP < 0.01.

Fluorescence-activated cell sorting analysis was used to determine the effect of KIF21B knockdown in inducing apoptosis. The results showed that the level of apoptosis was significantly increased in shKIF21B-transfected cells compared with that of the shCtrl group. This result suggested that knockdown of KIF21B induced cell apoptosis (Figure 5).

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Figure 5 Apoptosis of BEL-7404 cells detected by fluorescence-activated cell sorting after transfection with short hairpin RNA shCtrl or shKIF21B. Cell apoptosis was significantly inhibited in the shCtrl-transfected group compared with the shKIF21B-transfected group. All the experiments were conducted in triplicate. Data were analyzed by ANOVA or t-test. The data are reported as the mean ± SD. P < 0.05 was considered significant, ^bP < 0.01.

Colony formation assay was used to confirm the effect of KIF21B knockdown on the self-renewal capacity of HCC cells. As shown in Figure 6, the results after transfection of BEL-7404 cells with shKIF21B or shCtrl demonstrated that knockdown of KIF21B significantly reduced the level of apoptosis in the shKIF21B group compared to the shCtrl group.

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Figure 6 Self-renewal capacity of BEL-7404 cells detected using colony-forming assays after transfection with short hairpin RNA shCtrl or shKIF21B. Self-renewal capacity of cells was significantly inhibited in the shKIF21B-transfected group cells compared with the shCtrl-transfected group. All the experiments were conducted in triplicate. Data were analyzed by ANOVA or t-test. The data are reported as the mean ± SD. P < 0.05 was considered significant, ^bP < 0.01.

Immunohistochemistry was used to analyze the relationship between KIF21B expression and clinicopathological characteristics for the 186 patients with HCC. The results revealed that KIF21B was significantly higher in tumor tissues compared to adjacent normal tissue, and it exhibited cytoplasmic expression (Figure 7A-D). Moreover, the results validated the TGCA data regarding KIF21B expression in HCC tissues and adjacent normal tissues. As shown in Table 1, high expression of KIF21B correlated with vascular invasion (P < 0.05), TNM stage (P < 0.05), and HBsAg (P < 0.01). However, KIF21B expression did not correlate with gender, age, tumor size, or alpha fetoprotein (AFP) level (P > 0. 05).

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Figure 7 KIF21B expression in hepatocellular carcinoma tissues and normal adjacent tissues. A and B: Low expression of KIF21B in normal adjacent tissues (magnification: ×100 for A and ×400 for B); C and D: High expression of KIF21B in hepatocellular carcinoma tissues (magnification: ×100 for C and ×400 for D); E and F: Kaplan-Meier’s survival curves using a log-rank test demonstrating the correlation between KIF21B expression and overall survival or disease-free survival. Dashed lines indicate high KIF21B expression, and solid lines indicate low KIF21B expression.

Kaplan-Meier analysis of the 186 patients with HCC was performed to evaluate the relationship between KIF21B expression and prognosis. The Kaplan-Meier survival curves suggested that HCC patients with higher KIF21B expression had significantly lower OS (P = 0.0002) and DFS (P = 0.0005) after hepatectomy (Figure 7E and F). Univariate analysis showed that liver cirrhosis (P < 0.05) and KIF21B expression (P < 0.01) significantly correlated with OS while age (P < 0.05) and KIF21B expression (P < 0.01) significantly correlated with DFS (Tables 2 and 3, respectively). Multivariate analysis indicated that the level of KIF21B expression (P < 0.01) was an independent risk factor for OS (Table 2). KIF21B expression level (P < 0.01) and age were independent risk factors for DFS (Table 3). Overall, the univariate and multivariate analyses confirmed the potential of KIF21B as an independent risk factor for poor prognosis in HCC.

As one of the most frequent cancers, the morbidity and mortality of hepatocellular carcinoma (HCC) is increasing year by year. The kinesin superfamily protein member KIF21B plays an important role in regulating mitotic progression; however, the function and mechanisms of KIF21B in cancer, particularly in HCC, are unknown.

To explore the role of KIF21B in hepatocellular carcinoma and its clinical significance.

The study aimed to investigate the function of KIF21B in HCC and its effect on prognosis after hepatectomy.

First, we analyzed the differential expression of KIF21B in The Cancer Genome Atlas, and used immunohistochemical staining to validate it. Subsequently, after silencing KIF21B expression, the function of KIF21B in HCC lines was investigated by cell growth assay, MTT assay, fluorescence-activated cell sorting assay, and colony formation assay. The Kaplan-Meier method was used to assess its prognostic significance.

KIF21B expression levels were significantly higher in HCC tissues. Functional experiments showed that KIF21B knockdown remarkably suppressed cell proliferation and induced apoptosis. Statistical analyses revealed KIF21B as an independent risk factor in patients with HCC after hepatectomy.

KIF21B plays an important role in HCC progression and may be a potential diagnostic and prognostic marker for HCC.

In the future, more studies are needed to determine the effect that KIF21B may have on other tumor cell lines and to investigate the underlying mechanism.