KIF2A is a member of the kinesin-13 family and is involved in cell mitosis and spindle assembly. The abnormal expression and dysfunction of KIF2A are associated with tumorigenesis and the progression of certain human cancer types; however, little is known about the expression and roles of KIF2A in glioma. In the present study, the expression of KIF2A in 35 freshly isolated human glioma tissue samples was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). The association between the expression of KIF2A and clinicopathological parameters were assessed using the χ2 test. The biological functions of KIF2A on the proliferation, apoptosis, migration and invasion of glioma cells were investigated using an A172 cell line. RT-qPCR and IHC results demonstrated that the mRNA and protein expression levels of KIF2A were significantly higher in grade III–IV glioma tissues compared with those in grade I–II glioma tissues (P<0.05). No significant correlation was observed between the expression of KIF2A, and age, gender, tumor location and size (P>0.05).
Glioma is the most common tumor type of the central nervous system and they occur in 6.42/100,000 individuals (
The kinesin superfamily proteins (KIFs) are a conserved class of microtubule-dependent molecular motor proteins exhibiting adenosine triphosphatase activity. KIFs are important in mitosis, meiosis and macromolecular transport (
In the present study, the expression of KIF2A was examined in 35 freshly isolated human glioma tissue samples, and its prognostic value for glioma patients was evaluated. The biological functions of KIF2A in glioma cells were also analyzed.
Intracranial tissue specimens were collected from 35 patients [23 males (65.7%) and 12 females (34.3%)] and included 15 grade I–II cases (42.9%) and 20 grade III–IV cases (57.1%), ranging in age from 4 to 70 years. The patients underwent primary and curative resection for glioma, and were pathologically confirmed at Qilu Hospital of Shandong University (Jinan, China) in 2014, for reverse transcription quantitative-polymerase chain reaction (RT-qPCR) and immunohistochemical (IHC) analyses. The patients were diagnosed based on the World Health Organization (2007) standard classification (
The total RNA was extracted from human intracranial tissues or cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Single-stranded cDNA was synthesized using oligo dT16 primers and Moloney Murine Leukemia Virus Reverse Transcriptase, according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR was performed using a LightCycler 2.0 Instrument (Roche Diagnostics GmbH, Penzberg, Germany) and the cycling conditions were as follows: 30 sec at 95°C, and 40 cycles at 95°C for 5 sec, 60°C for 10 sec and 72°C for 15 sec for PCR amplification, according to the instructions of the SYBR® Green Realtime PCR Master Mix [Toyobo (Shanghai) Co., Ltd.], which was used as the detection dye. Gene-specific amplifications were confirmed through a melting curve analysis at the end of the RT-qPCR. Relative gene expression levels were determined using the 2−ΔΔCq method with endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as the reference gene. The following primers were used: KIF2A, sense: 5′-GCCTTTGATGACTCAGCTCC-3′ and antisense: 5′-TTCCTGAAAAGTCACCACCC-3′ (154 bp); GAPDH, sense: 5′-GGTGGTCTCCTCTGACTTCAACAG-3′ and antisense: 5′-GTTGCTGTAGCCAAATTCGTTGT (127 bp).
The IHC procedure was performed according to an established protocol, with minor modifications (
The tissue sections were assessed by light microscopy (Olympus IX51; Olympus Corp., Tokyo, Japan; magnification, ×200) by two pathologists in a blinded manner. Initially, a proportion score was assigned, representing the estimated proportion of positive tumor cells (0, none; 1, 1/100; 2, 1/100-1/10; 3, 1/10-1/3; 4, 1/3-2/3; 5, >2/3). Next, an intensity score was assigned, representing the average intensity of the positive tumor cells (0, none; 1, weak; 2, intermediate; 3, strong). The proportion and intensity scores were subsequently added to obtain a total score ranging between 0 and 8, with 0–3 indicating negative and 4–8 indicating positive (
Cultured cells (4×105) in a 6-well cell plate were homogenized for 60 min in 150
The human malignant glioma cell lines, A172 and U251 were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO2 humidified incubator. For RNA interference, the A172 cells were transfected with 50 nM KIF2A small interfering (si)RNA (5′-CACCGGCAAAGAGATTGACCTGGTTCAAGAGACCAGGTCAATCTCTTTGCCTTTTTTG-3′) or scrambled siRNA (a universal negative control; 5′-CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAATTTTTTG-3′) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.
A172 cells were transfected with either KIF2A siRNA or scrambled siRNA and seeded at a density of 1.0×104 cells/well in a 96-well plate. Following incubation for 0, 24, 48 and 72 h, a CCK-8 assay (Yiyuan Biotech Inc., Guangzhou, China) was performed to measure cell proliferation, according to the manufacturer's protocol. The optical densities were measured at 450 nm using a TECAN Infinite® M200 microplate reader (Tecan US, Durham, NC, USA).
The effects of KIF2A on the invasion and migration of glioma cells were evaluated using a Matrigel invasion and a transwell migration assay in a 24-well Transwell, containing polycarbonate filters with 8 mm pores (Costar, Corning, NY, USA). For the Matrigel cell invasion assay, the inserts were precoated with 50
Gelatin zymography assays were performed, as previously described (
An Annexin V-fluorescein isothiocyanate (FITC) and Propidium Iodide (PI) Apoptosis Detection kit (BestBio, Shanghai, China) was used to assess the apoptotic rate of the A172 cells pretreated with scrambled siRNA or KIF2A siRNA, according to the manufacturer's protocol. Briefly, A172 cells transfected with scrambled siRNA or KIF2A siRNA were seeded into 12-well plates and cultured under normal conditions for 48 h. The cells were subsequently collected with trypsin (no EDTA), washed twice with cold PBS, resuspended with binding buffer and incubated with Annexin V-FITC and PI staining solution in the dark for 10 min at room temperature, and the apoptotic rate was immediately quantified using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Three independent experiments were performed.
Statistical analysis was performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) for Windows. The χ2 test was used to examine the correlation between the expression of KIF2A and various clinicopathological parameters. Student's t-test or Mann-Whitney U test was used for statistical analyses. P<0.05 was considered to indicate a statistically significant difference.
A summary of the patient clinicopathological features is provided in
The χ2 test results revealed that the expression of KIF2A correlated closely with the World Health Organization grade of glioma (
To identify the functions of KIF2A in malignant glioma cells, the expression of KIF2A in the A172 and U251 glioma cell lines was analyzed. RT-qPCR results revealed that the mRNA expression of KIF2A was significantly higher in A172 cells compared with in U251 cells (
The CCK-8 assay was used to investigate the effect of KIF2A on the proliferation of A172 cells. KIF2A knockdown inhibited A172 cell proliferation in a time-dependent manner (
Matrigel cell invasion and Transwell cell migration models were performed to analyze the effect of KIF2A expression on the invasion and migration capacities of A172 cells. Compared with the cells transfected with scrambled siRNA, the invasion and migration capacities of A172 cells were significantly inhibited by KIF2A knockdown (
Matrix metalloproteinases (MMPs) are a group of peptidases involved in the degradation of the extracellular matrix, and accumulated data have suggested that MMPs contribute to the invasion and migration processes of glioma cells (
The aim of the present study was to evaluate the expression of KIF2A in glioma tissues, assess the association between the expression of KIF2A and clinical parameters, as well as identify the functions of KIF2A in malignant glioma cells.
KIF2A, a member of the kinesin-13 family, was first cloned from the murine central nervous system by degenerate PCR in 1992 (
In the present study, the findings of RT-qPCR and IHC analysis demonstrated that the mRNA and protein expression levels of KIF2A were significantly higher in grade III–IV glioma tissues compared with those in grade I–II glioma tissues, and that the expression of KIF2A was notably correlated with glioma grade (
Previous studies have reported that the knockdown of KIF2A in somatic or cancer cells can lead to a marked increase in monopolar spindles (
Cellular morphology is supported by the cytoskeleton, and cytoskeletal reorganizations have important effects on the migration of neoplastic cells (
In conclusion, the findings of the present study demonstrated that the expression of KIF2A was upregulated in grade III–IV glioma tissues compared with that in grade I–II glioma tissues, and that it was markedly correlated with glioma grade. Furthermore, it was revealed that KIF2A knockdown significantly inhibited proliferation and induced apoptosis in A172 cells, as well as significantly inhibited the invasion and migration capacities of A172 cells by regulating the activity and expression of MMP-2. These results suggested that KIF2A has a central role in glioma development and that the inhibition of KIF2A expression may prove to be a promising target in the control of glioma.
The present study was supported by grants from the National Natural Science Foundation of China (grant nos. 31470885 and 31270971) and the Natural Science Foundation of Science and Technology Department of Liaoning Province (grant no. 2014022020).
Immunohistochemical staining of KIF2A in glioma tissues. Positive staining for KIF2A was observed in the cytoplasm and/or nuclei of tumor cells, exhibiting a higher expression of KIF2A in (A) high-grade compared with (B) low-grade glioma tissues. (C) Negative staining for KIF2A in low-grade glioma tissues. (D) Negative staining for KIF2A in high-grade glioma tissues. 3,3′-Diaminobenzidine (brown) was used as a chromogen (scale bars=50 mm).
Evaluation of the expression of KIF2A and confirmation of RNA interference targeting KIF2A in glioma cells. (A) RT-qPCR was performed to determine the mRNA expression of KIF2A in glioma cell lines. The expression of KIF2A in A172 cells was demonstrated to be significantly higher compared with that in U251 cells (P<0.001; Student's t-test). (B) Confirmation of RNA interference against KIF2A in A172 cells by RT-qPCR (P>0.05 and P=0.003; Student's t-test). (C) Confirmation of RNA interference against KIF2A in A172 cells by western blot analysis. RT-qPCR, reverse transcription-quantitative polymerase chain reaction.
KIF2A knockdown significantly inhibited proliferation and induced apoptosis in A172 cells. (A) A CCK-8 assay was performed to determine the proliferation of A172 cells in response to the indicated treatments (n=5; data are presented as the mean). (B) Representative image of flow cytometric analysis to show the proportions of apoptotic cells in A172 cells transfected with the indicated siRNAs. (C) Bar graphs showing the ratios of apoptotic cells in A172 cells transfected with the indicated siRNAs (P<0.0001; Student's t-test). OD, optical density; siRNA, small-interfering RNA.
KIF2A siRNA inhibits the ability of A172 cells to invade and migrate. (A) Representative images of invasion and migration by A172 cells treated with the indicated siRNAs, as determined by Matrigel invasion and Transwell migration assays (scale bars, 100
mRNA and protein expression levels of KIF2A and its association with patient clinicopathological features.
Variable | Patients, n | mRNA expression |
Protein expression, n (%)
| ||||
---|---|---|---|---|---|---|---|
Median | IQR | P-value | Positive | Negative | P-value | ||
Age, years | 0.056 |
0.317 | |||||
≤45 | 14 | 0.009 | 0.005–0.020 | 6 (17.14) | 8 (22.88) | ||
>45 | 21 | 0.020 | 0.010–0.050 | 13 (37.14) | 8 (22.88) | ||
Gender | 0.263 |
1.000 | |||||
Male | 23 | 0.020 | 0.008–0.050 | 12 (34.29) | 11 (31.43) | ||
Female | 12 | 0.020 | 0.004–0.028 | 7 (20.00) | 5 (14.29) | ||
Tumor location | 0.143 |
0.541 | |||||
Prefrontal | 14 | 0.020 | 0.005–0.050 | 6 (17.14) | 8 (22.88) | ||
Temporal lobe | 13 | 0.020 | 0.009–0.055 | 8 (22.88) | 5 (14.29) | ||
Other | 8 | 0.090 | 0.039–0.175 | 5 (14.29) | 3 (8.57) | ||
Grade | 0.033 |
0.044 | |||||
I–II | 15 | 0.009 | 0.005–0.020 | 5 (14.29) | 10 (28.57) | ||
III–IV | 20 | 0.025 | 0.010–0.073 | 14 (40.00) | 6 (17.14) | ||
Tumor size, cm | 0.601 |
0.503 | |||||
≥3 | 22 | 0.020 | 0.008–0.050 | 13 (37.14) | 9 (25.71) | ||
<3 | 13 | 0.010 | 0.007–0.030 | 6 (17.14) | 7 (20.00) |
Continuous value calculated from the ratio of KIF2A and GAPDH;
Mann-Whitney U test;
Kruskal-Wallis test;
χ2 test. P<0.05 was considered to indicate a statistically significant difference. The median represents the 50th percentile of the mRNA expression level. The median and IQR were used to describe the mRNA expression level, as the mRNA expression level distribution is non-normal. IQR, inter-quartile range.