Hepatocellular carcinoma (HCC), a type of liver cancer with a high mortality rate, is a common malignancy worldwide and is the most common cause of mortality in patients with cirrhosis in China (1–3). The incidence of HCC is on the rise in the USA and in developing countries due to the rise in hepatitis c virus infections (4,5). With the clinical measurement of serum α-fetoprotein (AFP) levels and developments in various imaging techniques, particularly ultrasonography, individuals at high risk of liver cancer can be monitored and HCC can be clinically diagnosed even in the presence of minor symptoms such as loss of appetite, nausea and stomach discomfort (6,7). Additionally, advances in a variety of treatment methods, including surgery, radiotherapy and targeted drug therapy, have improved the survival rate of patients with HCC (8,9). However, given the high heterogeneity and metastasis of HCC, targeted therapy remains the best method, and novel therapeutic targets for HCC are urgently required (10).Kinesin family member C1 (KIFC1; also termed HSET) is a member of the kinesin superfamily (11). KIFC1 moves towards the minus-end of microtubules (12) and is involved during mitotic spindle formation and ciliogenesis (13,14). Previous studies have reported that KIFC1 mediates the positioning and architecture of the Golgi apparatus, and therefore promotes cargo transport from the Golgi apparatus (12,15). In addition, KIFC1 has been demonstrated to be involved in acrosome formation and nuclear shaping, which contributes to spermiogenesis (11,16). In the past 20 years, the role of KIFC1 in tumorigenesis has received an increasing amount of attention.KIFC1 has been reported to be highly expressed in several types of tumor, such as non-small lung cancer and renal cell carcinoma, and is associated with the proliferation and survival of patients with these tumors (17,18). Additionally, nuclear KIFC1 has been identified as a biomarker of poor prognosis in patients with triple-negative breast cancer (19). KIFC1 can also induce docetaxel resistance and is associated with the prognosis of patients with prostate cancer (20). Due to the key role of KIFC1 in the clustering of multiple centrosomes in various types of cancer cells, it is considered to be a novel antitumor therapeutic target (21). A previous study has indicated that KIFC1 is involved in the occurrence and development of HCC, and is associated with tumor metastasis, however, to the best of our knowledge, whether there is an effect on HCC cell proliferation remains unclear (22).The present study demonstrated the association between KIFC1 expression level and the prognosis of patients with HCC. Further results indicated that knockdown of KIFC1 inhibited the proliferation of two HCC cell lines. Additionally, depletion of KIFC1 inhibited HCC formation and growth in mice. In summary, these data support a tumor promoting role of KIFC1 in the growth and development of HCC.
Materials and methods
Cell culture
Hep3B and SNU-475humanHCC cell lines were purchased from the American Type Culture Collection and maintained in the Eagle's minimum essential medium and RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), respectively, supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2.
Antibodies and primers
Anti-KIFC1 [1:10,000 for western blot analysis; 1:100 for immunohistochemistry (IHC); cat. no. ab172620], anti-β-actin (1:1,000; cat. no. ab8226), anti-Ki67 (1:500; cat. no. ab6615), anti-proliferating cell nuclear antigen (PCNA) (1:500; cat. no. ab29) were all purchased from Abcam. The primer sequences used for quantitative PCR (qPCR) were as follows: KIFC1 forward, 5′-TGAGCAACAAGGAGTCCCAC-3′ and reverse, 5′-TCACTTCCTGTTGGCCTGAG-3′; and β-actin forward, 5′-CAGCTCACCATGGATGATGATATC-3′ and reverse, 5′-AAGCCGGCCTTGCACAT-3′. The short hairpin (sh)RNA sequences used for transfection were as follows: KIFC1, 5′-AAATTACCACATCCCACCCAAGA-3′; non-targeting control, 5′-ACATTACTACATCCCAGCCACTA-3′.
Transfection
Ready-to-package AAV short hairpin (sh)RNA plasmids targeting KIFC1 were obtained from Addgene, Inc. The KIFC1 shRNA plasmids (100 nM) were transfected into Hep3B and SNU-575 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Additionally, control cells were transfected with 100 nM control shRNA, which did not match any known human coding cDNA. Cells were plated in 6-well plates at a density of 1×104/well and transfected with Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for ~30 min in room temperature according to the manufacturer's protocol. Three groups of cells were generated: i) A sh-KIFC1 group, which was transfected with shRNA targeting KIFC1; ii) a negative control group, which was transfected with control shRNA; and iii) a mock group, which received no transfection (data not shown). Transfection efficiency was assessed by RT-qPCR and western blot 48 h after transfection. The stably transfected cell lines were screened and used for the in vitro and in vivo assays.
IHC
HumanHCC tissues were obtained from 82 patients receiving surgical resection treatment at the Affiliated Hospital of Weifang Medical University between July 2014 and May 2016 (aged 44–62 years; mean age 53.4 years). For the analysis of KIFC1 expression in surgical samples, IHC was performed as described previously (17,18).Briefly, specimens were fixed with 10% formaldehyde, embedded in paraffin, sectioned (5 µm), deparaffinized at room temperature and rehydrated with xylene and graded ethanol (100, 95, 85 and 75%). Following antigen retrieval in citrate buffer (pH 6.0; 140°C) and inactivation of endogenous peroxidase with 3% H2O2 at room temperature for 10 min avoiding light, the sections were blocked with 5% BSA (cat. no. A8010; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at 4°C and incubated with the anti-KIFC1 antibody for 1.5 h at 37°C. Subsequently, the sections were incubated with a biotinylated secondary antibody (Goat anti-rabbit immunoglobulin G; cat. no. ZB-2301; OriGene Technologies, Inc.) for 1.5 h at 37°C, and 3,3′-diaminobenzidine was used as a chromogen substrate. The sections were observed under a light microscope (magnification, ×100 and ×200).The expression level of KIFC1 was scored according to the percentage of positive tumor cells, using the following cutoffs: <5% scored 0, between 5 and 25% scored 1, between 25 and 75% scored 2, and >75% scored 3. The membrane and plasma staining intensity of tumor cells with positive staining was also evaluated and those with no staining scored 0, weak positive staining scored 1, moderate positive staining scored 2, and strong positive staining scored 3. Cells were classified as having high (2–3) or low (0–1) expression levels based on the positive cell percentage score and the staining intensity score. The sections of each patient were observed in five visual fields and the results were judged using the double-blind method.
Total RNA isolation and reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was isolated from Hep3B and SNU-475 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc). Subsequently, total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega Corporation) with 10 µl template RNA and primers, 4 µl 5X Prime Script buffer (Promega Corporation), 1 µl Prime Script (Promega Corporation) and 20 µl RNase free H2O; the mixture was incubated at 42°C for 60 min and 70°C for 15 min. RT-qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Pre-denaturation at 94°C for 5 min; 33 cycles of 94°C for 30 sec, 64°C for 30 sec and 72°C for 45 sec; and 72°C for 10 min. The obtained PCR products were routinely subjected to agarose gel electrophoresis and scanned by a gel imaging system. The gray-scale ratio of target genes and internal parameters was used to represent the relative mRNA expression levels of each target gene, and the relative expression level (2−DDCq) of KIFC1 was normalized to β-actin (17,18).
Western blot analysis
The whole cell extracts were prepared using CelLytic™ M cell lysis reagent (cat. no. C2978; Sigma-Aldrich; Merck KGaA). Total proteins were quantified using a bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.). Protein samples (50 µg each) were separated using 10% SDS-PAGE and transferred onto PVDF membranes, followed by blocking with goat serum (dilution, 1:1,000; cat. no. ZLI-9022; OriGene Technologies, Inc.) at room temperature for 60 min, incubation with primary antibodies for the detection of KIFC1, β-actin, Ki67 and PCNA for 2 h at 37°C, and subsequently incubated with horseradish peroxidase-conjugated polyclonal goat anti-rabbit/mouse secondary antibody (cat. no. RI2341; dilution, 1:5,000; Rockland Immunochemicals Inc.) for 45 min. The visualization reagent used was Coomassie brilliant blue G-250 (cat. no. C8420; Beijing Solarbio Science & Technology Co., Ltd). The gray values were analyzed using Odyssey v3.0 software (Thermo Fisher Scientific, Inc.).
Colony formation assay
A total of 1×103 Hep3B (KIFC1 shRNA and control) and SNU-475 (KIFC1 shRNA and control) cells/well were plated in 6-well plates and cultured at 37°C with 5% CO2 for 2 weeks. The colonies were then fixed with 4% paraformaldehyde for 25 min and stained with crystal violet for 15 min. Images were obtained and subsequently the crystal violet was extracted and the absorbance value was quantified using a microplate reader at a wavelength of 570 nm.
MTT assay
Hep3B (KIFC1 shRNA and control) and SNU-475 (KIFC1 shRNA and control) cells were plated in 96-well plates at a density of 1×103 cells/well and cultured in Eagle's minimum essential medium or RPMI-1640 medium, respectively, at 37°C with 5% CO2 for 48 h. Cells were then incubated with MTT (Roche Diagnostics GmbH) for 3 h and the medium was removed. Subsequently, MTT was dissolved using DMSO and the absorbance value was quantified using a microplate reader at 570 nm.
Examination of tumor growth in mice
The animal study was approved by the Institutional Animal Care Committee of the Affiliated Hospital of Weifang Medical University. Nude BalB/c female mice (6–8 weeks; 18–22g; n=8) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in a pathogen-free animal facility at 25°C with a 12-h light/dark cycle and free access to food and water, and randomly assigned to the control or experimental group. To detect tumor formation and growth in vivo, Hep3B cells, transfected with KIFC1 shRNA or control shRNA were injected subcutaneously into the right flank of the mice. The tumor volume was measured using Vernier calipers every four days after the tumors were established for two weeks. The maximum tumor length was 22 mm. On day 35, the mice were sacrificed by cervical dislocation, the tumors were isolated and images were obtained. The following formula was used to calculate tumor volume: Tumor volume (mm3)=tumor length (mm) × tumor width (mm2)/2. For the IHC analysis of KIFC1 expression in the tumors, the fresh tissues were frozen and then lapped in liquid nitrogen, the protocol described above was used, and stained cells were counted under a light microscope in five fields of view.
Statistical analysis
All in vitro experiments were performed independently three times. Data were analyzed using SPSS v22.0 software (IBM Corp.). For the IHC experiments, associations between KIFC1 expression and the clinicopathological features were evaluated using χ2 tests. Associations between survival, tumor progression and KIFC1 expression were estimated using the Kaplan-Meier method and log-rank tests. The quantitative data are reported as the mean ± standard deviation, and Student's t-test was used to analyze significant differences between two sample means. P<0.05 was considered to indicate a statistically significant difference.
Results
KIFC1 is associated with poor prognosis of patients with HCC
To investigate the potential role of KIFC1 in HCC, IHC assays were performed with tissue samples from patients with HCC, and the staining intensity of KIFC1 was then detected. The results demonstrated that KIFC1 was predominantly expressed in the cytoplasm and highly expressed in the tissue of HCC (Fig. 1A). Tissue samples were divided into low and high KIFC1 expression groups (Fig. 1A). Furthermore, no high KIFC1 expression was detected in adjacent tissues, further suggesting the possible involvement of KIFC1 in the development of HCC (Fig. 1B).
Figure 1.
KIFC1 expression levels in HCC tissues are associated with poor prognosis (A) Immunohistochemical staining demonstrated the low and high expression levels of KIFC1 in HCC tissues. Magnification, ×100 and ×200. (B) Immunohistochemical staining revealed the low expression level of KIFC1 in the normal liver tissues. Magnification, ×100 and ×200. (C) Kaplan-Meier method and log-rank analysis of overall survival and relapse-free survival rates between low and high-expression groups of KIFC1. HCC, hepatocellular carcinoma; KIFC1, kinesin family member C1.
The high and low expression groups of KIFC1 were analyzed with clinicopathological characteristic data. The results showed that the expression of KIFC1 in the HCC tissues was associated with clinical features, including the number of tumor nodes and tumor size, suggesting a potential association between KIFC1 and HCC (Table I). However, there was no significant difference between high and low KIFC1 expression groups in other clinicopathological characteristics, such as patient age, sex, tumor differentiation, lymphatic metastasis and AFP level (Table I).
Table I.
Associations between KIFC1 and clinicopathological characteristics of 82 patients with hepatocellular carcinoma.
KIFC1 expression
Characteristic
Total (n=82)
Low (n=42)
High (n=40)
χ2
P-value
Age, years
2.905
0.088
<55
54
24
30
≥55
28
18
10
Sex
1.179
0.278
Male
46
26
20
Female
36
16
20
Number of tumor nodes
4.228
0.040[a]
Single
34
22
12
Multiple ≥2
48
20
28
Tumor differentiation
2.513
0.113
Low
36
22
14
High
46
20
26
Tumor size, cm
4.518
0.034[a]
<5
30
20
10
≥5
52
22
30
Lymph node metastasis
0.230
0.632
No
47
23
24
Yes
35
19
16
AFP, ng/ml
3.241
0.072
<50
24
16
8
≥50
58
26
32
P<0.05. AFP, α-fetoprotein; KIFC1, kinesin family member C1.
The prognosis of patients with HCC and KIFC1 expression was also investigated, and patients with high KIFC1 expression had lower overall survival and relapse-free survival rates compared with patients with low expression (P=0.011 and P=0.016, respectively; Fig. 1C). All these data revealed that KIFC1 was associated with poor prognosis of patients with HCC.
Knockdown of KIFC1 blocks HCC proliferation in vitro
To further investigate the effects of KIFC1 in the progression of HCC, shRNA targeted against KIFC1 was used to specifically knockdown the expression of KIFC1 in Hep3B and SNU-475 HCC cell lines. The results of RT-qPCR and western blot revealed that the transfection of KIFC1 shRNA plasmids significantly reduced the expression of KIFC1 in the two cell lines at the mRNA and protein levels, respectively (Fig. 2).
Figure 2.
KIFC1 is significantly downregulated by its targeted shRNA in Hep3B and SNU-475 hepatocellular carcinoma cells. (A) Reverse transcription- quantitative PCR assays and (B) western blot analysis revealed the expression level of KIFC1 was significantly reduced in Hep3B and SNU-475 cells following transfection. Data are presented as mean ± standard deviation. *P<0.05. KIFC1, kinesin family member C1; shRNA, short hairpin RNA.
To investigate the potential role of KIFC1 in the development of HCC, colony formation assays were performed. Results indicated that the proliferation capacity of Hep3B and SNU-475 cells was significantly inhibited by KIFC1 shRNA (Fig. 3A). MTT assays were also performed to detect the effect of KIFC1 on the survival of HCC cells in vitro. The transfection of KIFC1 shRNA significantly reduced the proliferation of Hep3B and SNU-475 cells, which is consistent with the lower optical density value as compared with that in the control group (Fig. 3B). To further confirm the effects of KIFC1 in HCC proliferation, the expression of Ki67 and PCNA, two biomarkers of proliferation was examined. The expression of both Ki67 and PCNA was significantly reduced in Hep3B and SNU-475 cells following transfection (Fig. 3C and D). In conclusion, these results demonstrate that KIFC1 promotes HCC proliferation in vitro.
Figure 3.
Knockdown of KIFC1 reduces proliferation of Hep3B and SNU-475 cells. (A) Colony formation and (B) MTT assays examined Hep3B and SNU-475 cell lines transfected with control or KIFC1 shRNA plasmids. (C and D) Western blot analysis demonstrated that (C) Ki67 and (D) PCNA expression was downregulated in Hep3B and SNU-475 cells. Data are presented as mean ± standard deviation. *P<0.05. PCNA, proliferating cell nuclear antigen; KIFC1, kinesin family member C1; shRNA, short hairpin RNA; OD, optical density.
KIFC1 promotes HCC proliferation in mice
Since KIFC-knockdown inhibited the proliferation of HCC in vitro, the effects of KIFC1 on the growth of HCC in mice was examined. Hep3B cells transfected with control or KIFC1 shRNA lentivirus were injected subcutaneously into nude mice, and tumor volume was measured after 15 days following injection. The tumor volume in the KIFC1-knockdown group was significantly smaller compared with that in the control (Fig. 4A), and representative images from each group are also presented (Fig. 4A). In addition, the expression of KIFC1 in the tumors of the control and KIFC1-knockdown groups was also investigated, and the results of both western blot and IHC assays confirmed that the expression of KIFC1 in KIFC1-knockdown group was significantly reduced compared with that in the control group (Fig. 4B and C).
Figure 4.
KIFC1 promotes tumor formation and growth of hepatocellular carcinoma cells in nude mice. (A) Subcutaneous tumors formed by Hep3B cells transfected with control and KIF5A shRNA lentivirus in nude mice were measured after two weeks, every 4 days. n=6 per group. Representative images of tumors from each group were obtained. (B) Western blot analysis and quantification of the expression of KIFC1 and β-actin in control and KIFC1 shRNA groups. (C) Immunohistochemical analysis of KIFC1 expression levels in control and KIFC1 shRNA groups. Magnification, ×200. Data are presented as mean ± standard deviation. *P<0.05. shRNA, short hairpin RNA; KIFC1, kinesin family member C1.
Discussion
The mortality rate of HCC is high due to heterogeneity and metastasis (23). Surgical treatment, radiotherapy and chemotherapy have limited effects to combat this disease (24). A recent study has confirmed that targeted therapy was effective in the treatment of HCC and has a promising future (25). However, the existing therapeutic targets, such as protein Mdm4, hepatocyte growth factor receptor and vascular endothelial growth factor A, still do not meet the clinical requirements (26). Novel therapeutic targets, such as monocarboxylate transporter 4, require further validation (27). In the present study, KIFC1 was associated with poor HCC prognosis and regulates the proliferation of HCC. Combined with the previous findings that KIFC1 affected the progression of multiple tumors (23–27), KIFC1 may be a promising novel therapeutic target for HCC.As a member of the kinesin superfamily, the most classic function of KIFC1 is to act as a motor protein and move towards the minus-end of the microtubule to further affect its dynamics and functions, such as spindle formation and minus-end aster organization (28). Additionally, KIFC1 is involved in the regulation of centrosome amplification, and it has been reported that overexpression of KIFC1 leads to the formation of monopolar spindles in tumor cells (29). Based on the results of the above studies, it may be concluded that KIFC1 is likely to further affect cell proliferation through its effect on microtubules, thus participating in the formation and development of tumors. Multiple studies have confirmed that the expression of KIFC1 is associated with the poor prognosis of several tumors, including non-small cell lung cancer, renal cell carcinoma and breast cancer, and predominantly affects the proliferation of tumor cells (17–19). Therefore, it is reasonable to suspect that KIFC1 may participate in the regulation of tumor cell proliferation through the cellular function mediated by microtubules. Notably, in the present study depletion of KIFC1 inhibited the proliferation of HCC in vitro and in vivo, suggesting that KIFC1 may also regulate cell proliferation of HCC in a microtubule-dependent manner.In the present study, the expression of KIFC1 was associated with the prognosis of patients with HCC, and proliferation was a key mechanism regulated by KIFC1 in the development of HCC. In addition to proliferation, several potential mechanisms may affect the formation and growth of HCC. A previous study indicated that KIFC1 could act as a link between the Golgi apparatus and microtubules, promote the central positioning and maintain the structure of the Golgi apparatus (12). The export process of membrane vesicles from the Golgi complex is also regulated by KIFC1 (15). Vesicle transport is also essential for cancer development (30), therefore it is important to investigate whether KIFC1 may affect HCC by regulating the vesicle transport process in future studies. A recent study investigated the effects of KIFC1 on HCC metastasis and indicated that micoRNA-532-3p decreased the expression of KIFC1 and promoted epithelial-mesenchymal transition and metastasis of HCC (22). The present study further confirmed the promotion of KIFC1 on the proliferation of HCC cells. Combined with the present findings, it's clear that the effects of KIFC1 on HCC are complex, and the progress of HCC is comprehensively influenced by both proliferation and metastasis.In addition to KIFC1, a variety of other kinesin family members could regulate tumor development. KIF3B and KIF14 were reported to be associated with the prognosis of patients with HCC, which is similar to our previous research on KIFC1 (31,32). Additionally, KIFC1 is overexpressed and affects the progression of breast cancer (19). Furthermore, KIF2A contributes to the proliferation and migration of breast cancer cells (33). KIFC1, together with other kinesin family members, may serve as novel and promising therapeutic targets for cancer treatment.
Authors: Phuong H D Nguyen; Siming Ma; Salvatore Albani; Pierce K H Chow; Weiwei Zhai; Valerie Chew; Cheryl Z J Phua; Neslihan A Kaya; Hannah L H Lai; Chun Jye Lim; Jia Qi Lim; Martin Wasser; Liyun Lai; Wai Leong Tam; Tony K H Lim; Wei Keat Wan; Tracy Loh; Wei Qiang Leow; Yin Huei Pang; Chung Yip Chan; Ser Yee Lee; Peng Chung Cheow; Han Chong Toh; Florent Ginhoux; Shridhar Iyer; Alfred W C Kow; Yock Young Dan; Alexander Chung; Glen K Bonney; Brian K P Goh Journal: Nat Commun Date: 2021-01-11 Impact factor: 14.919
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