EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1094, 2021

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

ZHENYA GAO1, HUANXIA JIA1, FANG YU1, HONGFANG GUO1 and BAOYU LI2

1Department of Clinical Medicine, School of Medicine, Xuchang University, Xuchang, Henan 461000; 2Department of General Surgery, The Secondary Hospital of Tianjin Medical University, Tianjin 300211, P.R. China

Received March 7, 2020; Accepted August 26, 2020

DOI: 10.3892/etm.2021.10528

Abstract. Hepatocellular carcinoma (HCC) is one of the most eter arterial chemoembolization, have to some extent improved common malignancies with high mortality and morbidity rates. the prognosis of patients with HCC (5,6). In recent years, the In recent years, HCC targeted therapy has gained increasing focus has increased on targeted therapy for HCC, which has attention. Due to the heterogeneity and high metastasis of HCC, achieved good therapeutic effects with its strong pertinence (7). more effective therapeutic targets are needed. family However, due to the heterogeneity and high metastasis of HCC, member 2C (KIF2C), also known as mitotic ‑asso‑ novel and promising therapeutic targets are still required (8). ciated kinesin, is a ‑based motor which is Kinesin family member 2C (KIF2C), also known as involved in a variety of important cellular processes, such as mitotic centromere‑associated kinesin, is a microtubule‑based mitosis. The effects of KIF2C on cancer progression and devel‑ with a variety of important cellular regula‑ opment have been widely studied; however, its potential effects tory functions, such as the regulation of mitosis and genome on HCC remains unclear. In the present study, high expression stability (9‑11). KIF2C interacts with microtubule plus‑end of KIF2C in human HCC tissues was demonstrated using The tracking protein TIP150 and APC‑binding protein EB1 at Cancer Genome Atlas database and immunohistochemistry the plus ends of microtubule and therefore mediates micro‑ assays. KIF2C expression was associated with HCC prognosis, tubule dynamics (12,13). In addition, KIF2C contributed to including overall survival and disease‑free survival. KIF2C the progression of by affecting bipolar spindle expression was also associated with clinical pathological formation and segregation (14,15). Moreover, a characteristics including the number of tumor nodes (P=0.015) previous study demonstrated that KIF2C was associated with and tumor size (P=0.009). KIF2C knockdown inhibited the proline/serine‑rich coiled‑coil protein 1 and promoted chro‑ proliferation of HCC cells in vitro, confirmed by MTT and mosome congression (16,17). colony formation assays, and suppressed tumor growth in mice The effects of KIF2C on cancer progression and develop‑ which was confirmed by a xenograft mouse model. Together, ment have been widely studied (18‑21). KIF2C was revealed to the results suggested that KIF2C may serve as a promising be abnormally expressed in multiple types of cancer, such as therapeutic target for the treatment of HCC. lung cancer and glioma, and was also associated with the prog‑ nosis of these cancers (19‑21). Co‑expression network analysis Introduction revealed an association between KIF2C and the prognosis of lung adenocarcinoma (20). Additionally, KIF2C is hypoth‑ Hepatocellular carcinoma (HCC) is one of the most common esized as a novel marker for glioma prognosis (21). KIF2C malignancies, demonstrating high mortality and an increasing was highly expressed and induced frequent T cell responses morbidity worldwide, with ~748,300 new cases each year (1,2). in patients with colorectal cancer (19). Although KIF2C is The last few decades have witnessed advances in HCC diag‑ involved in the development of a variety of tumors (19‑21), its nosis and treatment (3,4). The most widely used treatments, potential impact on HCC is still unclear. such as surgical resection, chemoradiotherapy and transcath‑ 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 clin‑ ical pathological features of patients with HCC. Furthermore, Correspondence to: Professor Zhenya Gao, Department of Clinical Medicine, School of Medicine, Xuchang University, 1389 Xufan KIF2C knockdown suppressed the proliferation of HCC cells Road, Xuchang, Henan 461000, P.R. China in vitro and inhibited tumor growth in mice, thereby providing E‑mail: [email protected] a promising therapeutic target for HCC treatment.

Key words: hepatocellular carcinoma, kinesin family member 2C, Materials and methods proliferation, clinical pathological features, therapeutic target Biological information. Biological information was obtained to investigate the mRNA levels of KIF2C in 2 GAO et al: KIF2C PROMOTES HCC PROLIFERATION

HCC and normal tissues and investigate the association expression in tumor tissues was analyzed using ImageJ 8.0 between KIF2C and prognosis of patients with HCC. Data software (National Institutes of Health). on survival rates were obtained from the TCGA database. Expression Profiling Interactive Analysis (http://gepia. Cell culture and transfection. Hep3B and SNU475 cells cancer‑pku.cn/detail.php?gene=KIF2C/) was used to collate were used as HCC cell models for in vitro experiments. and analyze TCGA (https://www.cancer.gov/about‑nci/orga‑ The cell lines were purchased from the American Type nization/ccg/research/structural‑genomics/tcga) data with a Culture Collection and examined for mycoplasma contami‑ threshold of P<0.05 and LogFC>1 or <‑1 for differential genes, nation. All cell lines were negative for mycoplasma. Both and the median was used as the basis for dividing patients cell lines were maintained in DMEM (Gibco; Thermo into two groups: i) High KIF2C expression, or ii) low KIF2C Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo expression for Kaplan‑Meier survival analysis. Log rank test Fisher Scientific, Inc.) at 37˚C in a 5% CO2 incubator. The was used to determine any statistical significance. targeting sequence of the KIF2C shRNA plasmid was 5'‑AAATTACCACATCCCACCCAAGA‑3'. A plasmid with Tissue specimens. A total of 66 HCC surgical specimens non‑targeting shRNA was used as the shControl plasmid. The were collected from the Department of General Surgery, The aforementioned shRNA plasmids were transfected into both Secondary Hospital of Tianjin Medical University (Tianjin, Hep3B and SNU475 cells using Lipofectamine® 2000 (cat. China) between August 2017 and July 2019, as well as no. 11668019; Invitrogen; Thermo Fisher Scientific, Inc.). In complete clinicopathological data. All patients were treated 6‑well plates, 5 µl transfection reagent and 1.5 µg of the corre‑ with surgery only, and no chemoradiotherapy was applied. sponding shRNA plasmid were mixed in 300 µl serum‑free All patients enrolled provided written informed consent. The DMEM, left to stand for 5 min and subsequently mixed. patients were followed up according to the items listed in Following incubation at room temperature for 20 min, the mix Table I and then summarized for clinicopathological analysis. was added to serum‑starved cells and incubated at 37˚C for All studies were approved by the Ethics Committee of School 4 h. After transfection, Hep3B cells were further treated with of Medicine, Xuchang University. 1 mg/ml puromycin (Sigma‑Aldrich; Merck KGaA) to screen stable KIF2C knockdown cells for use in the in vivo experi‑ Antibodies. The following antibodies were used for western ments. blotting and IHC assays: KIF2C (cat. no. ab71706; 1:1,000 for western blotting, 1:200 for IHC; Abcam), β‑ (cat. RNA isolation and reverse transcription‑quantitative PCR no. ab8226; 1:1,000; Abcam), Ki67 (cat. no. ab16667; 1:1,000; (RT‑qPCR). KIF2C mRNA levels were detected using Abcam) and proliferating cell nuclear antigen (PCNA; cat. RT‑qPCR 24 h after the transfection. Total RNA was isolated no. ab92552, 1:500; Abcam). from Hep3B and SNU475 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA IHC assays. IHC assays were performed to detect KIF2C synthesis was performed using a Reverse Transcription System expression in HCC and adjacent tissues and tumor tissues in (cat. no. M1701; Promega Corporation) according to the manu‑ animal experiments. Tumor tissues were cut into 5 µm slices facturer's instructions. GAPDH was used as a reference gene. and fixed with 4% paraformaldehyde at room temperature for The following primer pairs were used for the qPCR: GAPDH 30 min. After deparaffinization and rehydration, slides were forward, 5'‑CGACCACTTTGTCAAGCTCA‑3' and reverse, immersed in citrate buffer and microwaved at 750 W for 5'‑GGTTGAGCACAGGGTACTTTATT‑3' and KIF2C 30 min for antigen retrieval. Endogenous peroxidase activity forward, 5'‑ACTATGACTGATCCTATCGAAGAG‑3' and was blocked by adding 3% hydrogen peroxide for 10 min at reverse, 5'‑GCCAATTCTTGCTTATTCAGTG‑3'. qPCR was room temperature and washed by PBS buffer. Sections were conducted using SYBR PrimeScript RT‑PCR Kit II (cat. subsequently blocked with 2% BSA for 1 h at room tempera‑ no. DRR083; Takara Biotechnology Co., Ltd.). The following ture. The sections were then incubated with rabbit KIF2C thermocycling conditions were used for qPCR: Initial denatur‑ antibody (1:200; cat. no. ab71706; Abcam) for 2 h at room ation at 95˚C for 3 min; followed by 30cycles of denaturation temperature followed by polymer conjugated IgG H&L horse‑ at 95˚C for 30 sec, annealing at 58˚C for 30 sec and extension radish peroxidase antibody (rabbit; 1:500; cat. no. ab205718; at 72˚C for 30 sec. The 2‑ΔΔCq method was used to quantify the Abcam) in a humidified chamber at room temperature for results (22). 1 h. Standard 3,3‑diaminobenzidine staining was performed for chromogenic detection for 5 min at room temperature. Western blotting. Western blotting was performed to detect Photographs were taken using an Olympus inverted fluores‑ KIF2C, β‑actin, Ki67 and PCNA protein levels. Hep3B cence microscope (magnification, x100 and x200). and SNU475 cells were washed with PBS and subsequently The scoring method was as follows. The proportion lysed with RIPA Lysis and Extraction Buffer (cat. no. 89900; of positively‑stained cells: 0, <20% positive tumor cells; Thermo Scientific.). Protein determination was performed 1, 20‑60% positive tumor cells; and 2, >60% positive tumor using the BCA method. A total of 10 µg of each protein cells. Staining intensity was assessed on a score of 0 (nega‑ sample was loaded per lane, separated on 8% SDS‑PAGE tive), 1 (modest) and 2 (strong). KIF2C levels were calculated gels and transferred onto PVDF membranes (250 mA, 2 h). according to the staining scores: Staining intensity score + Subsequently, the membranes were blocked with 5% milk in positive tumor cell staining score. Staining scores of 0‑2 were TBS‑Tween‑20 (0.05%; TBS‑T) for 2 h at room temperature, considered low KIF2C expression, and 3‑4 were considered and incubated with antibodies against KIF2C, β‑actin, Ki67 high KIF2C high‑expression. The quantification of KIF2C and PCNA for 2 h at room temperature. Following which, EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1094, 2021 3

Figure 1. KIF2C is highly expression in HCC tissues and associated with the prognosis of patients with HCC. (A) Expression levels of KIF2C in tumor tissues and corresponding normal tissues were presented according to TCGA database analysis. *P<0.05. (B) TCGA database analysis demonstrated the association between overall survival, disease‑free survival and KIF2C expression in patients with HCC. The 95% confidence interval is marked with a dotted line. LIHC, liver hepatocellular carcinoma; num (T), tumor tissue number; num (N), normal tissue number; KIF2C, kinesin family member 2C; HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; TPM, transcript per million; HR, hazard ratio. membranes were washed with TBS‑T buffer. β‑actin was used food and water, and clean and hygienic feeding conditions as the internal reference gene. Membranes were subsequently were maintained. Mice were randomized into two groups and incubated with polymer conjugated IgG H&L horseradish treated with control or KIF2C stably depleted Hep3B cells. peroxidase antibodies (1:3,000; cat. no. ab205718; Abcam) Cells in PBS buffer (~5x106) were injected subcutaneously and (1:3,000; cat. no. ab6728; Abcam) at room temperature into nude mice. After injection, the animals were housed. The for 1 h. Protein signals were developed using ECL (Novex™ weight and growth status of the mice were monitored every ECL Chemiluminescent Substrate Reagent kit; Thermo Fisher day. After 15 days, the volumes of the tumors were measured Scientific, Inc.) and visualized by ImageJ version 8.0 (National every 4 days using a vernier caliper until the experiment had Institutes of Health). reached 35 days. Tumor volume was calculated as follows: Tumor volume (mm3) = Tumor length (mm) x Tumor width Colony formation assays. Colony formation assays were (mm)2/2. Mice were euthanized with intraperitoneal injection performed to detect the effects of KIF2C in Hep3B and of 120 mg/kg sodium pentobarbital before the tumor was SNU475 cells. Approximately 1x103 HCC cells were seeded removed. The hearts of the mice were then monitored, and in 6‑well plates with three replicates. After 4 weeks, colonies death was confirmed by cardiac arrest. There were eight mice were fixed with 4% paraformaldehyde for 30 min at room in each group. temperature and stained with 0.2% crystal violet for 30 min at room temperature. Colonies were imaged by a camera, and the Statistical analysis. GraphPad 5.0 (GraphPad Software, colony number was manually counted. Inc.) was used for statistical analysis. Three repeats were performed for each experiment. Data were presented as the MTT assays. MTT assays were performed to detect the effects mean ± standard deviation. The association between clinical of KIF2C on the proliferation of HCC cells. 1,000 Hep3B characteristics and KIF2C expression was calculated using and SNU475 cells transfected with control or KIF2C shRNA Fisher's exact test and χ2 analysis. Student's t‑test was used plasmids were seeded into 96‑well plates and incubated for for statistical comparisons between two groups. P<0.05 was 48 h at 37˚C. HCC cells were treated with MTT for 4 h at 37˚C, considered to indicate a statistically significant difference. before dissolving the purple formazan crystals in DMSO. The absorbance of each well was measured using a microplate Results reader at a wavelength of 570 nm. KIF2C expression is increased in human HCC tissues and In vivo xenograft assays. This experiment was approved by the is associated with the prognosis and clinical pathological Experimental Animal Ethics Committee of The Secondary characteristics of patients with HCC. To investigate the Hospital of Tianjin Medical University (no. SYXK 2019‑0311). possible involvement of KIF2C in the progression and In vivo xenograft assays were performed to detect the effects development of HCC, KIF2C mRNA levels were analyzed in of KIF2C on the tumor growth of mice. A total of 16 sterilized 369 human liver hepatocellular carcinoma (LIHC) tissues and BALB/c nude mice (female, 5 weeks old and 18‑22 g) 160 normal tissues from the TCGA database. KIF2C mRNA were provided by Beijing Vital River Laboratory Animal levels of tumor tissues were significantly higher compared Technology Co., Ltd. Mice were randomized and housed in with normal tissues (Fig. 1A). The effects of KIF2C on the pathogen‑free animal facilities (n=8 mice per group). The prognosis of patients with HCC from TCGA database was mice were kept in a 20˚C environment with 40‑60% humidity also analyzed. Patients with HCC were divided into low and a 12/12 h light/dark cycle. Animals had free access to KIF2C transcript per million (TPM) and high TPM groups 4 GAO et al: KIF2C PROMOTES HCC PROLIFERATION

Table I. Association between KIF2C and clinicopathological characteristics in 66 patients with hepatocellular carcinoma.

KIF2C expression ------No. of patients Low, High, Feature (total, n=66) n=36 n=30 χ2 P‑value

Age, years 0.344 0.557 <55 46 24 22 ≥55 20 12 8 Sex 1.861 0.173 Male 38 18 20 Female 28 18 10 Number of tumor nodes 5.942 0.015a Single 26 19 7 Multiple ≥2 40 17 23 Tumor grade 0.405 0.524 Low 28 14 14 High 38 22 16 Tumor size, cm 6.875 0.009a <5 22 17 5 ≥5 44 19 25 Lymph node metastasis 1.306 0.253 No 39 19 20 Yes 27 17 10 AFP, ng/ml 2.475 0.116 <50 16 6 10 ≥50 50 30 20 aP<0.05. KIF2C, kinesin family member 2C; AFP, α fetoprotein.

Figure 2. High expression levels of KIF2C in human HCC tissues. Representative images of IHC assays, which detected KIF2C expression in (A) human HCC tissues (x100 and x200 magnification for the upper and lower images, respectively) and (B) normal tissues (x100 and x200 magnification for the upper and lower images, respectively). KIF2C, kinesin family member 2C; HCC, hepatocellular carcinoma; IHC, immunohistochemistry.

based on their KIF2C mRNA levels. As hypothesized, KIF2C disease‑free survival rates (Fig. 1B). High KIF2C expression mRNA expression was associated with overall survival and in human HCC tissues and an association between KIF2C EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1094, 2021 5

Figure 3. KIF2C expression is effectively reduced in Hep3B and SNU‑475 cells following the transfection of KIF2C shRNA plasmids. (A) Reverse tran‑ scription‑quantitative PCR assays demonstrated a decrease in KIF2C expression after the transfection of its shRNA plasmids in Hep3B and SNU‑475 cells (B) Western blot assays revealed KIF2C expression following the transfection of the indicated shRNA plasmids in Hep3B and SNU‑475 cells. Results are presented as the mean ± standard deviation. *P<0.05. KIF2C, kinesin family member 2C; sh, short hairpin; Con, control.

expression and the prognosis of patients with HCC, was RT‑qPCR, the effective knockdown of KIF2C mRNA identified. expression was demonstrated in both Hep3B and SNU‑475 KIF2C expression was analyzed in tumor tissues and cells (Fig. 3A). Similarly, the results of western blot analysis adjacent normal tissues from 66 patients with HCC. IHC assay further confirmed a significant decrease in KIF2C expression results revealed a markedly higher expression of KIF2C in following KIF2C shRNA plasmid transfection in Hep3B and HCC tissues compared with adjacent normal tissues, which SNU‑475 cells (Fig. 3B). was consistent with the bioinformatics analysis results (Fig. 2). The effects of KIF2C on the proliferation of HCC cells Subsequently, the 66 patients were divided into high was detected in vitro. Colony formation assays demonstrated KIF2C expression and low KIF2C expression groups based on a significant decrease in colony numbers induced by the KIF2CC staining results. A total of 36 patients (54.5%) exhib‑ depletion of KIF2C in Hep3B and SNU‑475 cells compared ited low KIF2C expression whereas the remaining 30 patients with control groups (Fig. 4A). Similarly, using MTT assays, (45.5%) exhibited high KIF2C expression (Table I). Through it was revealed that KIF2C knockdown significantly clinicopathological analysis, no significant association was decreased the optical density value in Hep3B and SNU‑475 identified between KIF2C expression and clinicopathological cells compared with controls (Fig. 4B). Taken together, the characteristics such as age, sex, tumor grade and lymph node results indicated that KIF2C affected the cytotoxicity of metastasis. However, KIF2C expression in human HCC tissues HCC cells in vitro. was significantly associated with the number of tumor nodes Subsequently, the expression of two cell proliferation and tumor size (Table I). Collectively, high KIF2C expression markers, Ki67 and PCNA, were analyzed in Hep3B and in human HCC tissues was demonstrated and an association SNU‑475 cells. Western blot analysis demonstrated a decrease was identified between KIF2C expression and clinicopatho‑ in Ki67 and PCNA expression in KIF2C‑depleted Hep3B and logical features such as age, sex, tumor grade, lymph node SNU‑475 cells, further confirming the aforementioned results metastasis and α fetoprotein (AFP). (Fig. 4C and D). In conclusion, these data demonstrated the involvement of KIF2C in the regulation of HCC cell prolifera‑ KIF2C depletion impairs HCC cell proliferation in vitro. To tion in vitro. further assess the involvement of KIF2C in HCC progression, KIF2C shRNA plasmids were used to decrease KIF2C expres‑ KIF2C contributes to tumor growth of HCC cells in mice. As sion in two HCC cell lines, Hep3B and SNU‑475. Through demonstrated by the aforementioned data, KIF2C depletion 6 GAO et al: KIF2C PROMOTES HCC PROLIFERATION

Figure 4. KIF2C promotes the proliferation of hepatocellular carcinoma cells in vitro. (A) Colony formation assays were performed in Hep3B and SNU‑475 cells transfected with the indicated shRNA plasmids, and the number of colonies were manually counted. Scale bar, 1 cm. (B) MTT assays were performed to assess the proliferative ability of Hep3B and SNU‑475 cells transfected with control or KIF2C shRNA plasmids. Western blot assays were performed to assess the expression of (C) Ki67 and (D) PCNA after transfection with control or KIF2C shRNA plasmids in Hep3B and SNU‑475 cells. *P<0.05. KIF2C, kinesin family member 2C; sh, short hairpin; Con, control; PCNA, proliferating cell nuclear antigen; OD, optical density. led to the impairment of HCC cell proliferation. To further KIF2C shRNA plasmids were used to stably knockdown confirm the potential effects of KIF2C on tumor growth its expression in Hep3B cells. Subsequently, control or in vivo, xenograft animal assays were performed. KIF2C‑depleted cells were injected into nude mice. After EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1094, 2021 7

Figure 5. KIF2C contributes to tumor growth of hepatocellular carcinoma cells in mice. (A) Hep3B cells infected with control or KIF2C shRNA lentivirus were subcutaneously implanted into nude mice. After 15 days, tumors were imaged and tumor volume was measured every 4 days. Tumor growth curves were compared between KIF2C‑depleted and control groups. Scale bar, 5 mm. (B) Western blot assays were used to detect the expression levels of KIF2C in tumors from control or KIF2C‑depletion groups. (C) Immunohistochemistry assays showed KIF2C expression levels in tumors from control or KIF2C‑depletion groups. Results are presented as the mean ± standard deviation. Scale bar, 5 mm. *P<0.05, **P<0.01. KIF2C, kinesin family member 2C; sh, short hairpin; Con, control.

15 days, tumors were collected and the volume of tumors was tumor nodes and tumor size. Taken together, this indicated detected every 4 days. Tumor growth curves and representa‑ KIF2C as a potential molecular target for HCC treatment. tive tumor images are presented in Fig. 5A. As hypothesized, As a potential oncogene, the function of KIF2C in tumor significantly smaller tumors were observed following KIF2C development has been widely studied (18‑21). Previous studies depletion compared with control mice (Fig. 5A). Western demonstrated that the proliferation and metastasis of NSCLC blotting and IHC assays further confirmed KIF2C expression cells were inhibited after KIF2C depletion (18‑21). KIF2C levels in tumors from the KIF2C‑depleted group were signifi‑ also served as a potential prognostic biomarker for breast cantly lower compared with the control group (Fig. 5B and C). cancer and was a target of miR‑485‑5p (29). In addition, KIF2C was aberrantly regulated in breast and lung cancer Discussion cells, and further affected cancer proliferation, metastasis and drug resistance (30). In the present study, it was determined Liver cancer is the sixth most diagnosed malignancy and that KIF2C affected the proliferation of HCC cells in vitro, the third leading cause of cancer‑related deaths worldwide, which was confirmed by colony formation and MTT assays. accounting for 8.2% of annual deaths (23). HCC is the most Concordantly, two cell proliferation markers, Ki67 and PCNA, predominant type of liver cancer, accounting for nearly 80% were decreased in KIF2C‑depleted HCC cells. Furthermore, cases (24); however, most patients with HCC are diagnosed at KIF2C knockdown also suppressed tumor growth in mice an advanced stage due to its high level of metastasis (25). HCC injected with HCC cells. Both the in vitro and in vivo data is prone to metastasis and targeted therapy with liposomes has suggested the involvement of KIF2C in the progression of been shown to be effective (26,27). In recent years, targeted HCC. However, the precise regulatory mechanism underlying therapy has demonstrated the most effective results for HCC, KIF2C promotion of HCC cell proliferation requires further and several targeted therapy drugs remain in clinical trials (28). study. In the present study, using TCGA database analysis and IHC A major limitation of the present study was the small staining assays, high KIF2C expression levels were identi‑ clinical sample size of 66 patients. In future studies, the fied in human HCC tissues. Clinicopathological and survival clinical sample size should be increased to further confirm the analysis revealed that KIF2C expression was associated with difference in KIF2C expression between tumor and adjacent HCC prognosis and clinical features such as the number of tissues. As a member of the kinesin family, KIF2C is involved 8 GAO et al: KIF2C PROMOTES HCC PROLIFERATION in the regulation of various cell functions, such as the regula‑ conceived the study, participated in its design and coordina‑ tion of mitosis (9). In the present study, only the effect on cell tion and helped to draft the manuscript. All authors read and proliferation was demonstrated, but the effect on cell migra‑ approved the final manuscript. tion and invasion was not studied. Therefore, future studies should examine the influence of KIF2C on the migration and Ethics approval and consent to participate invasion of HCC cells to further understand the relationship between KIF2C and HCC. All procedures performed in the present study were approved KIF2C is critical for the regulation of microtubule by the Ethics Committee of School of Medicine Xuchang dynamics and stabilization (31). KIF2C promotes microtubule University. Written informed consent was obtained from all depolymerization, which was negatively mediated by aurora patients or their families. kinases (32). Additionally, the KIF2C C‑terminal region could regulate its activity through a conformational switch, and Patient consent for publication further affect microtubule dynamics and cellular processes including migration and mitosis (33). The potential role of Not applicable. KIF2C in HCC progression has been previously reported, and it was found that KIF2C promoted the progression of HCC Competing interests by interacting with competing endogenous RNA (34). 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