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KIF2C promotes the proliferation of hepatocellular carcinoma cells in vitro and in vivo

Hepatocellular carcinoma (HCC) is one of the most common malignancies with high mortality and morbidity rates. In recent years, HCC targeted therapy has gained increasing attention. Due to the heterogeneity and high metastasis of HCC, more effective therapeutic targets are needed. Kinesin family member 2C (KIF2C), also known as mitotic centromere-associated kinesin, is a microtubule-based motor protein which is involved in a variety of important cellular processes, such as mitosis. The effects of KIF2C on cancer progression and development have been widely studied; however, its potential effects on HCC remains unclear. In the present study, high expression of KIF2C in human HCC tissues was demonstrated using The Cancer Genome Atlas database and immunohistochemistry assays. KIF2C expression was associated with HCC prognosis, including overall survival and disease-free survival. KIF2C expression was also associated with clinical pathological characteristics including the number of tumor nodes (P=0.015) and tumor size (P=0.009). KIF2C knockdown inhibited the proliferation of HCC cells in vitro, confirmed by MTT and colony formation assays, and suppressed tumor growth in mice which was confirmed by a xenograft mouse model. Together, the results suggested that KIF2C may serve as a promising therapeutic target for the treatment of HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies, demonstrating high mortality and an increasing morbidity worldwide, with ~748,300 new cases each year (1,2). The last few decades have witnessed advances in HCC diagnosis and treatment (3,4). The most widely used treatments, such as surgical resection, chemoradiotherapy and transcath-eter arterial chemoembolization, have to some extent improved the prognosis of patients with HCC (5,6). In recent years, the focus has increased on targeted therapy for HCC, which has achieved good therapeutic effects with its strong pertinence (7). However, due to the heterogeneity and high metastasis of HCC, novel and promising therapeutic targets are still required (8).
Kinesin family member 2C (KIF2C), also known as mitotic centromere-associated kinesin, is a microtubule-based motor protein with a variety of important cellular regulatory functions, such as the regulation of mitosis and genome stability (9)(10)(11). KIF2C interacts with microtubule plus-end tracking protein TIP150 and APC-binding protein EB1 at the plus ends of microtubule and therefore mediates microtubule dynamics (12,13). In addition, KIF2C contributed to the progression of cell division by affecting bipolar spindle formation and chromosome segregation (14,15). Moreover, a previous study demonstrated that KIF2C was associated with proline/serine-rich coiled-coil protein 1 and promoted chromosome congression (16,17).
The effects of KIF2C on cancer progression and development have been widely studied (18)(19)(20)(21). KIF2C was revealed to be abnormally expressed in multiple types of cancer, such as lung cancer and glioma, and was also associated with the prognosis of these cancers (19)(20)(21). Co-expression network analysis revealed an association between KIF2C and the prognosis of lung adenocarcinoma (20). Additionally, KIF2C is hypothesized as a novel marker for glioma prognosis (21). KIF2C was highly expressed and induced frequent T cell responses in patients with colorectal cancer (19). Although KIF2C is involved in the development of a variety of tumors (19)(20)(21), its potential impact on HCC is still unclear.
In the present study, high KIF2C expression in human HCC tissues was demonstrated according to The Cancer Genome Atlas (TCGA) database and immunohistochemistry (IHC) assays. KIF2C was also associated with the prognosis and clinical pathological features of patients with HCC. Furthermore, KIF2C knockdown suppressed the proliferation of HCC cells in vitro and inhibited tumor growth in mice, thereby providing a promising therapeutic target for HCC treatment.

Materials and methods
Biological information. Biological information was obtained to investigate the mRNA levels of KIF2C in Photographs were taken using an Olympus inverted fluorescence microscope (magnification, x100 and x200). The scoring method was as follows. The proportion of positively-stained cells: 0, <20% positive tumor cells; 1, 20-60% positive tumor cells; and 2, >60% positive tumor cells. Staining intensity was assessed on a score of 0 (negative), 1 (modest) and 2 (strong). KIF2C levels were calculated according to the staining scores: Staining intensity score + positive tumor cell staining score. Staining scores of 0-2 were considered low KIF2C expression, and 3-4 were considered high KIF2C high-expression. The quantification of KIF2C expression in tumor tissues was analyzed using ImageJ 8.0 software (National Institutes of Health).

KIF2C promotes the proliferation of hepatocellular carcinoma cells in vitro and in vivo
Cell culture and transfection. Hep3B and SNU475 cells were used as HCC cell models for in vitro experiments. The cell lines were purchased from the American Type Culture Collection and examined for mycoplasma contamination. All cell lines were negative for mycoplasma. Both cell lines were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in a 5% CO 2 incubator. The targeting sequence of the KIF2C shRNA plasmid was 5'-AAATTACCACATCCCACCCAAGA-3'. A plasmid with non-targeting shRNA was used as the shControl plasmid. The aforementioned shRNA plasmids were transfected into both Hep3B and SNU475 cells using Lipofectamine ® 2000 (cat. no. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.). In 6-well plates, 5 µl transfection reagent and 1.5 µg of the corresponding shRNA plasmid were mixed in 300 µl serum-free DMEM, left to stand for 5 min and subsequently mixed. Following incubation at room temperature for 20 min, the mix was added to serum-starved cells and incubated at 37˚C for 4 h. After transfection, Hep3B cells were further treated with 1 mg/ml puromycin (Sigma-Aldrich; Merck KGaA) to screen stable KIF2C knockdown cells for use in the in vivo experiments.

RNA isolation and reverse transcription-quantitative PCR (RT-qPCR).
KIF2C mRNA levels were detected using RT-qPCR 24 h after the transfection. Total RNA was isolated from Hep3B and SNU475 cells using TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA synthesis was performed using a Reverse Transcription System (cat. no. M1701; Promega Corporation) according to the manufacturer's instructions. GAPDH was used as a reference gene. The following primer pairs were used for the qPCR: GAPDH forward, 5'-CGACCACTTTGTCAAGCTCA-3' and reverse, 5'-GGTTGAGCACAGGGTACTTTATT-3' and KIF2C forward, 5'-ACTATGACTGATCCTATCGAAGAG-3' and reverse, 5'-GCCAATTCTTGCTTATTCAGTG-3'. qPCR was conducted using SYBR PrimeScript RT-PCR Kit II (cat. no. DRR083; Takara Biotechnology Co., Ltd.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95˚C for 3 min; followed by 30cycles of denaturation at 95˚C for 30 sec, annealing at 58˚C for 30 sec and extension at 72˚C for 30 sec. The 2 -ΔΔCq method was used to quantify the results (22).
Western blotting. Western blotting was performed to detect KIF2C, β-actin, Ki67 and PCNA protein levels. Hep3B and SNU475 cells were washed with PBS and subsequently lysed with RIPA Lysis and Extraction Buffer (cat. no. 89900; Thermo Scientific.). Protein determination was performed using the BCA method. A total of 10 µg of each protein sample was loaded per lane, separated on 8% SDS-PAGE gels and transferred onto PVDF membranes (250 mA, 2 h). Subsequently, the membranes were blocked with 5% milk in TBS-Tween-20 (0.05%; TBS-T) for 2 h at room temperature, and incubated with antibodies against KIF2C, β-actin, Ki67 and PCNA for 2 h at room temperature. Following which, membranes were washed with TBS-T buffer. β-actin was used as the internal reference gene. Membranes were subsequently incubated with polymer conjugated IgG H&L horseradish peroxidase antibodies (1:3,000; cat. no. ab205718; Abcam) and (1:3,000; cat. no. ab6728; Abcam) at room temperature for 1 h. Protein signals were developed using ECL (Novex™ ECL Chemiluminescent Substrate Reagent kit; Thermo Fisher Scientific, Inc.) and visualized by ImageJ version 8.0 (National Institutes of Health).
Colony formation assays. Colony formation assays were performed to detect the effects of KIF2C in Hep3B and SNU475 cells. Approximately 1x10 3 HCC cells were seeded in 6-well plates with three replicates. After 4 weeks, colonies were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 0.2% crystal violet for 30 min at room temperature. Colonies were imaged by a camera, and the colony number was manually counted.
MTT assays. MTT assays were performed to detect the effects of KIF2C on the proliferation of HCC cells. 1,000 Hep3B and SNU475 cells transfected with control or KIF2C shRNA plasmids were seeded into 96-well plates and incubated for 48 h at 37˚C. HCC cells were treated with MTT for 4 h at 37˚C, before dissolving the purple formazan crystals in DMSO. The absorbance of each well was measured using a microplate reader at a wavelength of 570 nm.
In vivo xenograft assays. This experiment was approved by the Experimental Animal Ethics Committee of The Secondary Hospital of Tianjin Medical University (no. SYXK 2019-0311). In vivo xenograft assays were performed to detect the effects of KIF2C on the tumor growth of mice. A total of 16 sterilized BALB/c nude mice (female, 5 weeks old and 18-22 g) were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were randomized and housed in pathogen-free animal facilities (n=8 mice per group). The mice were kept in a 20˚C environment with 40-60% humidity and a 12/12 h light/dark cycle. Animals had free access to food and water, and clean and hygienic feeding conditions were maintained. Mice were randomized into two groups and treated with control or KIF2C stably depleted Hep3B cells. Cells in PBS buffer (~5x10 6 ) were injected subcutaneously into nude mice. After injection, the animals were housed. The weight and growth status of the mice were monitored every day. After 15 days, the volumes of the tumors were measured every 4 days using a vernier caliper until the experiment had reached 35 days. Tumor volume was calculated as follows: Tumor volume (mm3) = Tumor length (mm) x Tumor width (mm) 2 /2. Mice were euthanized with intraperitoneal injection of 120 mg/kg sodium pentobarbital before the tumor was removed. The hearts of the mice were then monitored, and death was confirmed by cardiac arrest. There were eight mice in each group.
Statistical analysis. GraphPad 5.0 (GraphPad Software, Inc.) was used for statistical analysis. Three repeats were performed for each experiment. Data were presented as the mean ± standard deviation. The association between clinical characteristics and KIF2C expression was calculated using Fisher's exact test and χ 2 analysis. Student's t-test was used for statistical comparisons between two groups. P<0.05 was considered to indicate a statistically significant difference.

KIF2C expression is increased in human HCC tissues and is associated with the prognosis and clinical pathological characteristics of patients with HCC.
To investigate the possible involvement of KIF2C in the progression and development of HCC, KIF2C mRNA levels were analyzed in 369 human liver hepatocellular carcinoma (LIHC) tissues and 160 normal tissues from the TCGA database. KIF2C mRNA levels of tumor tissues were significantly higher compared with normal tissues (Fig. 1A). The effects of KIF2C on the prognosis of patients with HCC from TCGA database was also analyzed. Patients with HCC were divided into low KIF2C transcript per million (TPM) and high TPM groups based on their KIF2C mRNA levels. As hypothesized, KIF2C mRNA expression was associated with overall survival and disease-free survival rates (Fig. 1B). High KIF2C expression in human HCC tissues and an association between KIF2C Table I. Association between KIF2C and clinicopathological characteristics in 66 patients with hepatocellular carcinoma.  expression and the prognosis of patients with HCC, was identified. KIF2C expression was analyzed in tumor tissues and adjacent normal tissues from 66 patients with HCC. IHC assay results revealed a markedly higher expression of KIF2C in HCC tissues compared with adjacent normal tissues, which was consistent with the bioinformatics analysis results (Fig. 2).

KIF2C depletion impairs HCC cell proliferation in vitro.
To further assess the involvement of KIF2C in HCC progression, KIF2C shRNA plasmids were used to decrease KIF2C expression in two HCC cell lines, Hep3B and SNU-475. Through RT-qPCR, the effective knockdown of KIF2C mRNA expression was demonstrated in both Hep3B and SNU-475 cells (Fig. 3A). Similarly, the results of western blot analysis further confirmed a significant decrease in KIF2C expression following KIF2C shRNA plasmid transfection in Hep3B and SNU-475 cells (Fig. 3B).
The effects of KIF2C on the proliferation of HCC cells was detected in vitro. Colony formation assays demonstrated a significant decrease in colony numbers induced by the depletion of KIF2C in Hep3B and SNU-475 cells compared with control groups (Fig. 4A). Similarly, using MTT assays, it was revealed that KIF2C knockdown significantly decreased the optical density value in Hep3B and SNU-475 cells compared with controls (Fig. 4B). Taken together, the results indicated that KIF2C affected the cytotoxicity of HCC cells in vitro.
Subsequently, the expression of two cell proliferation markers, Ki67 and PCNA, were analyzed in Hep3B and SNU-475 cells. Western blot analysis demonstrated a decrease in Ki67 and PCNA expression in KIF2C-depleted Hep3B and SNU-475 cells, further confirming the aforementioned results ( Fig. 4C and D). In conclusion, these data demonstrated the involvement of KIF2C in the regulation of HCC cell proliferation in vitro.
KIF2C contributes to tumor growth of HCC cells in mice. As demonstrated by the aforementioned data, KIF2C depletion led to the impairment of HCC cell proliferation. To further confirm the potential effects of KIF2C on tumor growth in vivo, xenograft animal assays were performed.
KIF2C shRNA plasmids were used to stably knockdown its expression in Hep3B cells. Subsequently, control or KIF2C-depleted cells were injected into nude mice. After 15 days, tumors were collected and the volume of tumors was detected every 4 days. Tumor growth curves and representative tumor images are presented in Fig. 5A. As hypothesized, significantly smaller tumors were observed following KIF2C depletion compared with control mice (Fig. 5A). Western blotting and IHC assays further confirmed KIF2C expression levels in tumors from the KIF2C-depleted group were significantly lower compared with the control group ( Fig. 5B and C).

Discussion
Liver cancer is the sixth most diagnosed malignancy and the third leading cause of cancer-related deaths worldwide, accounting for 8.2% of annual deaths (23). HCC is the most predominant type of liver cancer, accounting for nearly 80% cases (24); however, most patients with HCC are diagnosed at an advanced stage due to its high level of metastasis (25). HCC is prone to metastasis and targeted therapy with liposomes has been shown to be effective (26,27). In recent years, targeted therapy has demonstrated the most effective results for HCC, and several targeted therapy drugs remain in clinical trials (28). In the present study, using TCGA database analysis and IHC staining assays, high KIF2C expression levels were identified in human HCC tissues. Clinicopathological and survival analysis revealed that KIF2C expression was associated with HCC prognosis and clinical features such as the number of tumor nodes and tumor size. Taken together, this indicated KIF2C as a potential molecular target for HCC treatment.
As a potential oncogene, the function of KIF2C in tumor development has been widely studied (18)(19)(20)(21). Previous studies demonstrated that the proliferation and metastasis of NSCLC cells were inhibited after KIF2C depletion (18)(19)(20)(21). KIF2C also served as a potential prognostic biomarker for breast cancer and was a target of miR-485-5p (29). In addition, KIF2C was aberrantly regulated in breast and lung cancer cells, and further affected cancer proliferation, metastasis and drug resistance (30). In the present study, it was determined that KIF2C affected the proliferation of HCC cells in vitro, which was confirmed by colony formation and MTT assays. Concordantly, two cell proliferation markers, Ki67 and PCNA, were decreased in KIF2C-depleted HCC cells. Furthermore, KIF2C knockdown also suppressed tumor growth in mice injected with HCC cells. Both the in vitro and in vivo data suggested the involvement of KIF2C in the progression of HCC. However, the precise regulatory mechanism underlying KIF2C promotion of HCC cell proliferation requires further study.
A major limitation of the present study was the small clinical sample size of 66 patients. In future studies, the clinical sample size should be increased to further confirm the difference in KIF2C expression between tumor and adjacent tissues. As a member of the kinesin family, KIF2C is involved in the regulation of various cell functions, such as the regulation of mitosis (9). In the present study, only the effect on cell proliferation was demonstrated, but the effect on cell migration and invasion was not studied. Therefore, future studies should examine the influence of KIF2C on the migration and invasion of HCC cells to further understand the relationship between KIF2C and HCC.
KIF2C is critical for the regulation of microtubule dynamics and stabilization (31). KIF2C promotes microtubule depolymerization, which was negatively mediated by aurora kinases (32). Additionally, the KIF2C C-terminal region could regulate its activity through a conformational switch, and further affect microtubule dynamics and cellular processes including migration and mitosis (33). The potential role of KIF2C in HCC progression has been previously reported, and it was found that KIF2C promoted the progression of HCC by interacting with competing endogenous RNA (34). As a comparison, the data of the present study provided further evidence of the involvement of KIF2C in the regulation of HCC cell proliferation. The authors hypothesize that KIF2C promoted this process due to increased chromosomal instability and abnormal cell division in HCC cells.
The association between kinesins and cancers has also been widely demonstrated. Kinesin family members such as KIF3A, KIF18B and KIFC1 have been involved in the growth and metastasis of multiple types of cancers, including breast cancer, gastric cancer and lung cancer (35). Several studies have confirmed that kinesins were associated with the prognosis of cancer and could therefore act as molecular targets (35,36). These studies, together with the findings of the present study, suggested that kinesins may serve as promising cancer therapeutic targets. Future studies should focus on the molecular mechanisms underlying kinesin involvement in cancer and develop novel inhibitors of kinesins.
In the present study, high KIF2C expression was found in human HCC tissues. KIF2C expression was associated with the prognosis and clinicopathological characteristics including the number of tumor nodes and tumor size. KIF2C knockdown inhibited the proliferation of HCC cells in vitro and in vivo.
In conclusion, KIF2C may serve as a promising therapeutic target for HCC treatment.