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Hepatology Research 2014
doi: 10.1111/hepr.12428
Original Article
TPX2 as a novel prognostic biomarker for hepatocellular carcinoma Qingquan Liu, Kangsheng Tu, Hongyong Zhang, Xin Zheng, Yingmin Yao and Qingguang Liu Department of Hepatobiliary Surgery, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China Aim: Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein that impacts spindle assembly in human cells. Several studies have shown that the overexpression of TPX2 is correlated with multiple tumor types. However, the role of TPX2 in hepatocellular carcinoma (HCC) remains undetermined. Methods: TPX2 expression was detected by quantitative reverse transcription polymerase chain reaction and immunoblotting in six cell lines and 130 pairs of HCC and adjacent non-cancerous liver tissues. Matrix metalloproteinase (MMP)2 and MMP9 expression was detected by immunohistochemistry to evaluate their correlations with TXP2. TPX2 siRNA was used to knock down TPX2 expression, and cell migration and invasion were determined. Moreover, clinical significance of TPX2 in HCC was analyzed. Results: TPX2 expression was found to be obviously higher in HCC tissues than that in non-tumor tissues. Elevated TPX2 expression was observed in HCC cell lines as compared with
INTRODUCTION
H
EPATOCELLULAR CARCINOMA (HCC) is one of the most lethal tumors and is the third leading cause of cancer-related death in the world, especially in Asia and sub-Saharan Africa.1,2 Recently, HCC incidence rates are increasing in several countries including the United States, partly due to the rise in hepatitis C virus infection and obesity epidemic.3,4 More than 600 000 HCC cases were diagnosed per year, and the incidence of HCC-related death exceeded 500 000 patients worldwide in 2008.5,6 At present, liver resection is the main curative therapy for HCC, but the prognosis of the
Correspondence: Drs Kangsheng Tu and Qingguang Liu, Department of Hepatobiliary Surgery, the First Affiliated Hospital of Xi’an Jiaotong University, 277 West Yanta Road, Xi’an 710061, China. Emails: [email protected], [email protected] Received 14 April 2014; revision 2 September 2014; accepted 24 September 2014.
© 2014 The Japan Society of Hepatology
that in a non-transformed hepatic cell line. Clinical analysis indicated that TPX2 expression in the HCC tissues was evidently correlated with the tumor–node–metastasis stage, tumor number and tumor differentiation. TPX2 is a novel prognostic marker for predicting 5-year overall survival and disease-free survival of HCC patients. TPX2 expression was positively correlated with MMP2 and MMP9. In vitro studies found that TPX2 knockdown prominently reduced cell invasion and migration and decreased phosphorylated AKT, MMP2 and MMP9 expression in MHCC-97H cells.
Conclusion: Overexpression of TPX2 was associated with clinicopathological characteristics and poor patient outcomes. TPX2 may serve as a novel prognostic marker for HCC. Key words: hepatocellular carcinoma, matrix metalloproteinase 2, matrix metalloproteinase 9, prognostic factor, TPX2
patients remains poor due to the high recurrence rate and early metastasis.7 Therefore, understanding the recurrence and metastasis mechanisms and identifying novel prognostic biomarkers of HCC will contribute to cancer recurrence prediction and providing potential therapeutic targets for the patients with HCC. Targeting protein for Xenopus kinesin-like protein 2 (TPX2), a microtubule-associated protein that impacts spindle assembly in human cells, was first found by Heidebrecht et al. in 1997.8 Subsequent researches explored that TPX2 was essential for spindle formation and microtubule nucleation surrounding the chromosomes.9 Notably, the TPX2 protein is a nuclear proliferation-related protein that is implicated in the regulation of the cell mitosis and is adjusted by the cell cycle.10 During the period of cell mitosis, TPX2 is evidently associated with the mitotic spindle, targeting Xklp2 to the spindle microtubule for the stability of the spindle pole. The overexpression of TPX2 induces the amplification of centrosome and leads to DNA
1
2
Q. Liu et al.
polyploidy.11 Recently, a variety of studies have paid attention to the relationship between TPX2 and human malignancies.12,13 There is now strong research evidence that the aberrant expression of TPX2 may play an important role in the invasion and progression of multiple tumor types.14 TPX2 has been verified to be overexpressed in various cancers including lung, colon and bladder tumors.15–17 High expression level of TPX2 was correlated with the aggressiveness of ovarian and salivary gland cancers.18,19 Importantly, TPX2 as an oncogenic protein could upregulate the expression of matrix metalloproteases (MMP) through the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in colon cancer.16 Considering the fact that the MMP and the PI3K/Akt signaling pathway play an important role in the development and progression of HCC, we hypothesized that the overexpression of TPX2 in HCC was associated with the high expression of MMP2 and MMP9 and TPX2 may be a novel prognostic biomarker for HCC.20,21 So far, TPX2 has been associated with a great number of malignancies due to its expression correlating with cancer invasion and metastasis.22 However, little is known about the clinical role of TPX2 in HCC. Recently, Satow et al.23 reported that TPX2 was highly expressed in HCC through combined functional genome survey, but the role and clinical implications of TPX2 in HCC were not investigated. To our knowledge, this is the first study assessing the effect of TPX2 expression on the survival of patients with HCC. In this study, we demonstrate that elevated TPX2 expression is observed in the HCC tissues and cells. The high expression of TPX2 is correlated with poor clinicopathological features of HCC. Moreover, high TPX2 expression confers a worse 5-year survival of HCC patients. The inverse correlation between TPX2 and MMP2/9 is observed in HCC cases. TPX2 knockdown reduces HCC cell migration and invasion. Furthermore, TPX2 knockdown decreases phosphorylated (p)-AKT, MMP2 and MMP9 protein expression in HCC cells. Our results demonstrate that TPX2 acts as a potent prognostic marker and contributes to tumor invasion and metastasis in HCC.
METHODS Cell lines
T
HE IMMORTALIZED NORMAL human liver cell line L-02 was obtained from the American Type Culture Collection (Manassas, VA, USA). Five human HCC cell lines, including SMMC-7721, BEL-7402,
© 2014 The Japan Society of Hepatology
Hepatology Research 2014
Huh-7, HepG2 and MHCC-97H were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained under recommended culture conditions. Cells were incubated in a 37°C humidified incubator containing 5% CO2. RNA was extracted from exponentially growing cells for the purpose of detecting the expression of TPX2.
Patients and specimens A total of 130 cases of HCC and corresponding adjacent non-tumorous liver tissues were collected from patients who underwent hepatectomy between January 2005 and December 2008 in the First Affiliated Hospital of Xi’an Jiaotong University, and all tumors were histologically confirmed as HCC. None of the patients had received preoperative chemotherapy or radiotherapy treatment, and the patients with evidence of concomitant extrahepatic disease were excluded from the study. The HCC stages were classified according to the sixth edition tumor–node–metastasis (TNM) classification criteria of the Union for International Cancer Control (UICC), and corresponding adjacent non-tumorous liver tissues were defined as 2.0 cm from the resection margin, which were confirmed negative by histological diagnosis. This study enrolled 104 men and 26 women with a median age of 52 years (range, 33–76). All HCC tissues and matched adjacent non-tumorous liver tissues were obtained immediately after hepatectomy, snapfrozen in liquid nitrogen and stored at −80°C until use. All the patients enrolled in this study provided written informed consent before surgical resection, and all the protocols were approved by the ethics committee of the First Affiliated Hospital of Xi’an Jiaotong University according to the 1975 Declaration of Helsinki.
Quantitative reverse transcription polymerase chain reaction (qRT–PCR) Total RNA was extracted from cell lines and liver specimens using TRIzol reagent (Invitrogen, San Diego, CA, USA), according to the manufacturer’s protocol. To avoid any DNA contamination, isolated RNA was treated with RNase-free DNase I (Invitrogen) and quantified by spectrophotometry. The RNA samples were measured by optical density at 260 nm, and then reverse transcribed into cDNA with a RevertAid Premium First Strand cDNA Synthesis Kit (Fermentas, Burlington, ON, Canada). qRT–PCR was performed using the SYBR Premix Ex Taq II (Tli RNaseH Plus) (TakaRa, Dalian, China) in an ABI 7500 system. The primer sequences to detect mRNA were: TPX2, forward 5′-ACCTTGCCCTAC TAAGATT-3′, and reverse 5′-A ATGTGGCACAGGTTG
Hepatology Research 2014
AGC-3′; and β-actin, forward 5′-GGGAAATCGTGCGT GAC AT-3′, and reverse 5′-CTGGAAGGTGGACAG CGAG-3′. The reaction conditions for the PCR program were as follows: initial melting at 95°C for 30 s followed by 40 cycles at 95°C for 5 s, 60°C for 64 s. Analysis of melting curve for the primers was conducted to confirm the specificity of the PCR product, and the threshold cycle (Ct) value for triplicate reactions was averaged. The relative expression of TPX2 mRNA for each sample was calculated as follows: ΔCt = Ct (TPX2) − Ct (β-actin), ΔΔCt (sample) = ΔCt (sample) − ΔCt (calibrator), and the fold changes in mRNA were calculated through relative quantification (2–ΔΔCt). To evaluate the effect of TPX2 mRNA on survival, the weighed expression of TPX2 mRNA were divided into high and low level using the median expression as the cut-off point for the whole group.
Immunoblotting The primary antibodies TPX2 (sc-32863), AKT (sc-8312, which can be used to detect total proteins of AKT1, AKT2 and AKT3), p-AKT (sc-293095, which can be used to detect p-AKT1, p-AKT2 and p-AKT3), MMP2 (sc10736), MMP9 (sc-10737) and β-actin (sc-130656) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary antibody (sc-2004) was also obtained from Santa Cruz Biotechnology. Briefly, the denatured protein samples were separated by polyacrylamide gel electrophoresis, and then electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were incubated with different primary antibodies and examined with the secondary antibody conjugated with horseradish peroxidase (HRP). Reactions were visualized using the HyGLO HRP detection kit (Denville, Metuchen, NJ, USA).
Immunohistochemical staining The primary mouse monoclonal anti-TPX2 antibody (sc-376812), goat polyclonal anti-MMP2 antibody (sc34014) and goat polyclonal anti-MMP9 antibody (sc-6840) were purchased from Santa Cruz Biotechnology. The biotinylated secondary antibodies of goat antimouse and rabbit antigoat were obtained from Wuhan Boster Bio-engineering (Wuhan, China). Immunohistochemical staining was performed on paraformaldehyde-fixed paraffin sections, and was done according to standard procedures. Sections were cut at 5-μm thickness, and heated in a 60°C oven. Briefly, tissue sections were deparaffinized in xylene, rehydrated through a graded series of alcohol and then incubated with 0.3% hydrogen peroxide for 20 min to block
TPX2 in hepatocellular carcinoma 3
endogenous peroxidase activities. After antigen retrieval in citrate buffer, the sections were blocked for 30 min using 10% normal goat serum, then separately incubated overnight at 4°C with the primary antibodies directed against TPX2, MMP2 and MMP9. This was followed by detecting the primary antibody with a biotinylated secondary antibody according to the manufacturer’s recommendations. The staining of the tissue sections was performed using the streptavidin–biotin– peroxidase complex for TPX2, MMP2 and MMP9 (SABC method). Using previously reported immunohistochemical techniques, the staining was visualized by using diaminobenzidine and counterstained with hematoxylin, then dehydrated in a graded series of alcohol, cleared in xylene and mounted onto glass slides. All tissue sections were scored by light microscopy by two experienced pathologists, blindly and evaluated independently, and then assessed at a conference microscope and a consensus determined. For the evaluation of TPX2, MMP2 and MMP9 immunostaining results, a semiquantitative scoring system based on the product of staining intensity and combined with the percentage of positive liver cells was used in the present study.24 Immunostaining intensity was evaluated as four grades: 0, negative; 1, weak; 2, moderate; and 3, strong. The percentage of positive liver cells was categorized as the following grades: 0, 0%; 1, 1–10%; 2, 11–50%; 3, 51–80%; and 4, more than 80%.24 The immunostaining intensity and average percentage of positive cells were evaluated for 10 independent high-magnification fields. By multiplying the staining intensity and the percentage of positive cells, the final weighed expression score was obtained (0–12). The total expression score for TPX2, MMP2 and MMP9 was used as a continuous variable for the correlation analyses. To evaluate the effect of TPX2 protein on survival, the weighed expression scores of TPX2 protein were divided into high and low scores using the median expression score as the cut-off point for the whole group.
siRNA transfection The specific siRNA against TPX2 (sc-37653) and control siRNA (sc-37007) were both from Santa Cruz Biotechnology. MHCC-97H tumor cells were seeded at a concentration of 2 × 106 per well in six-well plates and cultured overnight. Then, cells were transfected with 100 nM siRNA using Lipofectamine RNAi MAX Reagent (Invitrogen). Further experiments were performed 48 h after transfection.
© 2014 The Japan Society of Hepatology
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Cell invasion and migration assays Transwell cell migration assays were performed in 12-well plates with 8-μm BioCoat control inserts (Becton Dickinson Labware, Bedford, MA, USA). TPX2 siRNA or control siRNA transfected MHCC97H cells (1–2 × 105) that were suspended in 500 μL serum free Dulbecco’s modified Eagle’s medium (DMEM) were seeded in the upper well and DMEM medium with 10% fasting blood sugar, as indicated, in the lower well. After completion, membranes were removed, wiped on the side facing the upper well and stained with crystal violet. At least five representative images of each well were taken and cell numbers were counted using ImageJ. BioCoat Matrigel invasion chamber (Becton Dickinson Labware) was used for transwell cell invasion assays and the following protocols were the same as transwell cell migration assays. The experiments were performed in triplicate.
Follow up The follow up of this study finished on 31 December 2013, and the duration was defined as the interval between the date of surgical operation and the date of death or last follow up. The median follow-up time was 55 months (range, 3–79). All patients were monitored every 1–3 months in the first year and every 3–6 months thereafter. The monitor protocol included physical examination, serum α-fetoprotein (AFP) level, chest X-ray and abdominal ultrasonography. For the patients with follow-up results suggestive of tumor recurrence, computed tomography, magnetic resonance imaging and/or positron emission tomography were performed to verify whether tumor recurrence had occurred. Intrahepatic recurrence or distant tumor metastasis detected only by imaging diagnosis after surgical operation was designated as tumor recurrence. During the duration of follow up, 90 of 130 patients (69.2%) developed intrahepatic tumor recurrence and 22 patients (16.9%) developed distant tumor metastases.
Statistical analysis Statistical analysis was carried out using the SPSS version 16.0 statistical software package (SPSS, Chicago, IL, USA). To assess the association between ordinal data, Spearman’s rank correlation coefficient was used, and the χ2-test or Fisher’s exact test were used for categorical data. Group expression level was compared by Mann– Whitney U-test. Overall survival, with deaths due to any cause as event, and disease-free survival, with tumor recurrence and metastases as events, was analyzed using
© 2014 The Japan Society of Hepatology
Hepatology Research 2014
the Kaplan–Meier method, and the difference between curves was evaluated by the log–rank test. Independent prognostic factors were assessed by the Cox proportional hazards stepwise regression model. Data were showed as the Mean 1 standard error of the mean. P-values were two-sided, and P < 0.05 was considered statistically significant.
RESULTS Expression of TPX2 in HCC tissues and cells
W
E FIRST DETECTED the expression of TPX2 in HCC cell lines (SMMC-7721, BEL-7402, HUH-7, HepG2 and MHCC-97H) and a normal hepatocyte cell line (LO2) using qRT–PCR and immunoblotting. Our results showed that the expression of TPX2 in the immortalized liver cell line, L02, was significantly lower than those in HCC cell lines (P < 0.01, respectively; Fig. 1a,b). In 130 pairs of HCC and normal tumoradjacent tissues, qRT–PCR and immunoblotting showed that TPX2 expression in HCC was obviously higher than that in adjacent non-cancerous liver tissues (P < 0.01, respectively; Fig. 1c,d). We also examined the expression of TPX2 protein in 130 cases of HCC and matched tumor-adjacent liver tissues using immunohistochemical staining, and found that the expression score of TPX2 protein was significantly higher in the HCC tissues than that in the noncancerous tissues (4.82 1 0.29 vs 1.37 1 0.14; P < 0.01) (Fig. 2a–c). These results indicated TPX2 to be more highly expressed in HCC tissues than in adjacent nontumorous liver tissues.
Correlation between TPX2 expression and clinicopathological parameters To further characterize the clinical role of TPX2 in HCC, we tried to precisely identify the correlations of the TPX2 expression with clinicopathological parameters, including patient sex, age, hepatitis B surface antigen (HBsAg), AFP level, tumor size, tumor number, vascular invasion, cirrhosis, capsule formation, Edmondson–Steiner grade and TNM stage. The median expression score of TPX2 protein and mRNA was used as the cut-off point to divide into low- and high-expressing groups. In our study, the expression of TPX2 was significantly correlated with the Edmondson–Steiner grade, tumor number and TNM stage (P < 0.05). However, no evident correlation was found between the expression of TPX2 and patient sex, age, HBsAg, AFP level, tumor size, vas-
Hepatology Research 2014
TPX2 in hepatocellular carcinoma 5
(a)
(b) TPX2 mRNA level
TPX2 mRNA level
4
∗
∗
∗
3
∗
∗
2 1
10
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-9 7
21 C
C
-7 7 C M
5 0
0
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P < 0.01
15
5
(c)
(d) TPX2
β-actin
β-actin
N
T
N
T
N
T
N
T
M
SM
M
C H 772 C C 1 -9 7 H H ep G H 2 BE uh L- -7 74 02 L0 2
TPX2
Figure 1 Expression of TPX2 in cell lines and hepatocellular carcinoma (HCC) tissues. (a) TPX2 mRNA expression in the immortalized normal human liver cell line L-02 and five hepatoma cell lines (SMMC-7721, BEL-7402, Huh-7, HepG2 and MHCC-97H) using quantitative reverse transcription polymerase chain reaction (qRT–PCR) (n = 3, *P < 0.01, vs L-02 group, respectively). (b) TPX2 mRNA expression in HCC and corresponding adjacent non-tumorous liver tissues (NT) using qRT–PCR (n = 130, P < 0.01). (c) TPX2 protein expression in the immortalized normal human liver cell line L-02 and five hepatoma cell lines (SMMC-7721, BEL-7402, Huh-7, HepG2 and MHCC-97H) using immunoblotting (n = 3, P < 0.01). (d) TPX2 protein expression in HCC tissues (T) and corresponding adjacent non-tumorous liver tissues (N) using immunoblotting (n = 3, P < 0.01).
cular invasion, cirrhosis and capsule formation (P > 0.05). The results are listed in Table 1.
Correlation between TPX2 and MMP2 protein expression To address whether the TPX2 expression level was correlated with MMP2 expression, immunohistochemical staining was applied to examine the expression score of MMP2 in HCC and corresponding adjacent nontumorous liver specimens. MMP2 protein level was significantly higher in the HCC tissues than that in the corresponding adjacent non-tumorous liver tissues (5.01 1 0.29 vs 1.32 1 0.13; P < 0.01) (Fig. 2d–f). We next analyzed the correlation between the TPX2 expression and the MMP2 expression. There was indeed an evident positive correlation between the protein expression level of TPX2 and MMP2 (r = 0.827, P < 0.01, Spearman’s rank correlation coefficient) (Fig. 3a).
Correlation between TPX2 and MMP9 protein expression Additionally, to explore the correlation between TPX2 and MMP9 expression, the protein expression of MMP9
was examined in 130 cases of HCC and corresponding adjacent non-tumorous liver tissues using immunohistochemical staining. The expression score of MMP9 protein was significantly higher in the HCC tissues than in the corresponding adjacent non-tumorous liver tissues (5.32 1 0.33 vs 1.56 1 0.14; P < 0.01) (Fig. 2g–i). Notably, there was also a significant positive correlation between the protein expression level of TPX2 and MMP9 (r = −0.772, P < 0.01, Spearman’s rank correlation coefficient) (Fig. 3b).
Prognostic of HCC subtypes defined by TPX2 level As described in the Methods section, the median expression score of TPX2 protein and mRNA was used as the cut-off point to divide into low- and high-expressing groups for survival. To assess the effect of TPX2 on overall survival and disease-free survival, univariate prognostic analysis and multivariate Cox regression models were used to calculate. The patients with high expression level of TPX2 had significantly reduced overall survival and disease-free survival. The 5-year overall survival rate of the low-expressing group was
© 2014 The Japan Society of Hepatology
Q. Liu et al.
Hepatology Research 2014
(a)
(b)
(c)
(d)
(e)
(f)
Tumor-adjacent tissue
HCC tissue
HCC tissue
6
(g)
(h)
(i)
100 µm TPX2
MMP2
69.6%, which was evidently higher than that of the high-expressing group (31.5%, P < 0.001). The 5-year disease-free survival rate of the low-expressing group was 55.5%, which was evidently higher than that of the high-expressing group (16.1%, P < 0.001) (Fig. 4). Subsequently, the associations of overall survival and disease-free survival with clinicopathological parameters in the HCC tissues were calculated by univariate analysis. The univariate analysis showed that HBsAg status, tumor number, Edmonson–Steiner classification, TNM stage and the expression level of TPX2 were the prognosis factors for HCC. The results are listed in Table 2.
Stratified univariate and multivariate analysis In a multivariate analysis model, the expression of TPX2 was significantly associated with overall survival (hazard ratio [HR], 2.580; 95% confidence interval [CI], 1.520– 4.378; P < 0.001) and disease-free survival (HR, 2.671; 95% CI, 1.683–4.237; P < 0.001) (Table 3). Multivariate analysis established that the TPX2 expression level, HBsAg status and tumor number were the independent prognostic factors of HCC patients (Table 3). Considering the fact that patient survival may be associated with the TNM stage, we further stratified the data according to
© 2014 The Japan Society of Hepatology
MMP9
Figure 2 Immunohistochemical staining of TPX2, matrix metalloproteinase (MMP)2 and MMP9 in hepatocellular carcinoma tissues (HCC) and matched tumor-adjacent tissues. (a) In cases of high TPX2 protein expression, (d) there was strong MMP2 and (g) MMP9 protein expression in the same tissue section. (b,c) Similarly, in the case of low TPX2 protein expression, (e,f) there was no detectable MMP2 or (h,i) MMP9 protein expression (scale bar = 100 μm).
TNM stage and evaluated the prognostic value of TPX2 expression in TNM stage I. For the 63 TNM stage I HCC patients, evident correlations were found between the expression level of TPX2 and overall survival and disease-free survival (Fig. 5). For the TNM stage II or III HCC patients, TPX2 expression level had no prognostic value regarding overall survival and disease-free survival (P > 0.05, respectively). The associations of overall survival and disease-free survival with clinicopathological parameters in TNM stage I patients were calculated by univariate analysis (Table 4). Furthermore, multivariate analysis showed that the expression level of TPX2 was an independent negative prognostic factor for the survival of TNM stage I patients (Table 5). The patients with high expression of TPX2 had poorer overall survival (HR, 3.440; 95% CI, 1.430–8.273; P = 0.006) and disease-free survival (HR, 2.533; 95% CI, 1.122–5.722; P = 0.025) than those with low expression level of TPX2 in TNM stage I.
TPX2 knockdown suppressed cell migration and invasion To further explore the mechanism of TPX2 in HCC cells, we investigated the effect of TPX2 knockdown on HCC cell migration and invasion in TPX2 siRNA transfected MHCC-97H cells. As compared with the control siRNA
Hepatology Research 2014
TPX2 in hepatocellular carcinoma 7
Table 1 Correlations between TPX2 expression and clinicopathological features in HCC Characteristics
Sex Female Male Age (years) 245 >45 HBsAg status Negative Positive Cirrhosis No Yes AFP (μg/L) 2400 >400 Tumor size 25 cm >5 cm Tumor number Single Multiple Tumor capsule Complete Incomplete Vascular invasion No Yes Edmondson grade I/II III/IV TNM stage I II III
n
TPX2 mRNA High
Low
26 104
15 50
11 54
39 91
15 50
28 102
P
TPX2 protein
P
High
Low
0.511
15 47
11 57
0.279
24 41
0.125
15 47
24 44
0.185
10 55
18 47
0.134
10 52
18 50
0.200
42 88
20 45
22 43
0.851
19 43
23 45
0.712
58 72
28 37
30 35
0.860
26 36
32 36
0.599
49 81
22 43
27 38
0.469
22 40
27 41
0.718
103 27
46 19
57 8
0.029*
43 19
60 8
0.010*
32 98
17 48
15 50
0.839
17 45
15 53
0.543
110 20
53 12
57 8
0.467
51 11
59 9
0.627
78 52
31 34
47 18
0.007*
27 35
51 17
45 HBsAg status Negative Positive Cirrhosis No Yes AFP (μg/L) 2400 >400 Tumor size 25 cm >5 cm Tumor number Single Multiple Tumor capsule Complete Incomplete Vascular invasion No Yes Edmondson grade I/II III/IV TNM stage I II III TPX2 protein level Low High
n
Overall survival rate, years (%) 3
5
26 104
80.4 64.2
63.9 48.8
39 91
79.3 62.3
28 102
P
Disease-free survival rate, years (%) 3
5
0.289
57.7 51.0
46.2 34.3
0.214
55.5 43.9
0.561
64.1 47.3
41.0 34.9
0.565
89.3 66.4
85.6 42.1
0.001*
82.1 44.1
64.3 29.1
Hepatology Research 2014
doi: 10.1111/hepr.12428
Original Article
TPX2 as a novel prognostic biomarker for hepatocellular carcinoma Qingquan Liu, Kangsheng Tu, Hongyong Zhang, Xin Zheng, Yingmin Yao and Qingguang Liu Department of Hepatobiliary Surgery, the First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China Aim: Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a microtubule-associated protein that impacts spindle assembly in human cells. Several studies have shown that the overexpression of TPX2 is correlated with multiple tumor types. However, the role of TPX2 in hepatocellular carcinoma (HCC) remains undetermined. Methods: TPX2 expression was detected by quantitative reverse transcription polymerase chain reaction and immunoblotting in six cell lines and 130 pairs of HCC and adjacent non-cancerous liver tissues. Matrix metalloproteinase (MMP)2 and MMP9 expression was detected by immunohistochemistry to evaluate their correlations with TXP2. TPX2 siRNA was used to knock down TPX2 expression, and cell migration and invasion were determined. Moreover, clinical significance of TPX2 in HCC was analyzed. Results: TPX2 expression was found to be obviously higher in HCC tissues than that in non-tumor tissues. Elevated TPX2 expression was observed in HCC cell lines as compared with
INTRODUCTION
H
EPATOCELLULAR CARCINOMA (HCC) is one of the most lethal tumors and is the third leading cause of cancer-related death in the world, especially in Asia and sub-Saharan Africa.1,2 Recently, HCC incidence rates are increasing in several countries including the United States, partly due to the rise in hepatitis C virus infection and obesity epidemic.3,4 More than 600 000 HCC cases were diagnosed per year, and the incidence of HCC-related death exceeded 500 000 patients worldwide in 2008.5,6 At present, liver resection is the main curative therapy for HCC, but the prognosis of the
Correspondence: Drs Kangsheng Tu and Qingguang Liu, Department of Hepatobiliary Surgery, the First Affiliated Hospital of Xi’an Jiaotong University, 277 West Yanta Road, Xi’an 710061, China. Emails: [email protected], [email protected] Received 14 April 2014; revision 2 September 2014; accepted 24 September 2014.
© 2014 The Japan Society of Hepatology
that in a non-transformed hepatic cell line. Clinical analysis indicated that TPX2 expression in the HCC tissues was evidently correlated with the tumor–node–metastasis stage, tumor number and tumor differentiation. TPX2 is a novel prognostic marker for predicting 5-year overall survival and disease-free survival of HCC patients. TPX2 expression was positively correlated with MMP2 and MMP9. In vitro studies found that TPX2 knockdown prominently reduced cell invasion and migration and decreased phosphorylated AKT, MMP2 and MMP9 expression in MHCC-97H cells.
Conclusion: Overexpression of TPX2 was associated with clinicopathological characteristics and poor patient outcomes. TPX2 may serve as a novel prognostic marker for HCC. Key words: hepatocellular carcinoma, matrix metalloproteinase 2, matrix metalloproteinase 9, prognostic factor, TPX2
patients remains poor due to the high recurrence rate and early metastasis.7 Therefore, understanding the recurrence and metastasis mechanisms and identifying novel prognostic biomarkers of HCC will contribute to cancer recurrence prediction and providing potential therapeutic targets for the patients with HCC. Targeting protein for Xenopus kinesin-like protein 2 (TPX2), a microtubule-associated protein that impacts spindle assembly in human cells, was first found by Heidebrecht et al. in 1997.8 Subsequent researches explored that TPX2 was essential for spindle formation and microtubule nucleation surrounding the chromosomes.9 Notably, the TPX2 protein is a nuclear proliferation-related protein that is implicated in the regulation of the cell mitosis and is adjusted by the cell cycle.10 During the period of cell mitosis, TPX2 is evidently associated with the mitotic spindle, targeting Xklp2 to the spindle microtubule for the stability of the spindle pole. The overexpression of TPX2 induces the amplification of centrosome and leads to DNA
1
2
Q. Liu et al.
polyploidy.11 Recently, a variety of studies have paid attention to the relationship between TPX2 and human malignancies.12,13 There is now strong research evidence that the aberrant expression of TPX2 may play an important role in the invasion and progression of multiple tumor types.14 TPX2 has been verified to be overexpressed in various cancers including lung, colon and bladder tumors.15–17 High expression level of TPX2 was correlated with the aggressiveness of ovarian and salivary gland cancers.18,19 Importantly, TPX2 as an oncogenic protein could upregulate the expression of matrix metalloproteases (MMP) through the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in colon cancer.16 Considering the fact that the MMP and the PI3K/Akt signaling pathway play an important role in the development and progression of HCC, we hypothesized that the overexpression of TPX2 in HCC was associated with the high expression of MMP2 and MMP9 and TPX2 may be a novel prognostic biomarker for HCC.20,21 So far, TPX2 has been associated with a great number of malignancies due to its expression correlating with cancer invasion and metastasis.22 However, little is known about the clinical role of TPX2 in HCC. Recently, Satow et al.23 reported that TPX2 was highly expressed in HCC through combined functional genome survey, but the role and clinical implications of TPX2 in HCC were not investigated. To our knowledge, this is the first study assessing the effect of TPX2 expression on the survival of patients with HCC. In this study, we demonstrate that elevated TPX2 expression is observed in the HCC tissues and cells. The high expression of TPX2 is correlated with poor clinicopathological features of HCC. Moreover, high TPX2 expression confers a worse 5-year survival of HCC patients. The inverse correlation between TPX2 and MMP2/9 is observed in HCC cases. TPX2 knockdown reduces HCC cell migration and invasion. Furthermore, TPX2 knockdown decreases phosphorylated (p)-AKT, MMP2 and MMP9 protein expression in HCC cells. Our results demonstrate that TPX2 acts as a potent prognostic marker and contributes to tumor invasion and metastasis in HCC.
METHODS Cell lines
T
HE IMMORTALIZED NORMAL human liver cell line L-02 was obtained from the American Type Culture Collection (Manassas, VA, USA). Five human HCC cell lines, including SMMC-7721, BEL-7402,
© 2014 The Japan Society of Hepatology
Hepatology Research 2014
Huh-7, HepG2 and MHCC-97H were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained under recommended culture conditions. Cells were incubated in a 37°C humidified incubator containing 5% CO2. RNA was extracted from exponentially growing cells for the purpose of detecting the expression of TPX2.
Patients and specimens A total of 130 cases of HCC and corresponding adjacent non-tumorous liver tissues were collected from patients who underwent hepatectomy between January 2005 and December 2008 in the First Affiliated Hospital of Xi’an Jiaotong University, and all tumors were histologically confirmed as HCC. None of the patients had received preoperative chemotherapy or radiotherapy treatment, and the patients with evidence of concomitant extrahepatic disease were excluded from the study. The HCC stages were classified according to the sixth edition tumor–node–metastasis (TNM) classification criteria of the Union for International Cancer Control (UICC), and corresponding adjacent non-tumorous liver tissues were defined as 2.0 cm from the resection margin, which were confirmed negative by histological diagnosis. This study enrolled 104 men and 26 women with a median age of 52 years (range, 33–76). All HCC tissues and matched adjacent non-tumorous liver tissues were obtained immediately after hepatectomy, snapfrozen in liquid nitrogen and stored at −80°C until use. All the patients enrolled in this study provided written informed consent before surgical resection, and all the protocols were approved by the ethics committee of the First Affiliated Hospital of Xi’an Jiaotong University according to the 1975 Declaration of Helsinki.
Quantitative reverse transcription polymerase chain reaction (qRT–PCR) Total RNA was extracted from cell lines and liver specimens using TRIzol reagent (Invitrogen, San Diego, CA, USA), according to the manufacturer’s protocol. To avoid any DNA contamination, isolated RNA was treated with RNase-free DNase I (Invitrogen) and quantified by spectrophotometry. The RNA samples were measured by optical density at 260 nm, and then reverse transcribed into cDNA with a RevertAid Premium First Strand cDNA Synthesis Kit (Fermentas, Burlington, ON, Canada). qRT–PCR was performed using the SYBR Premix Ex Taq II (Tli RNaseH Plus) (TakaRa, Dalian, China) in an ABI 7500 system. The primer sequences to detect mRNA were: TPX2, forward 5′-ACCTTGCCCTAC TAAGATT-3′, and reverse 5′-A ATGTGGCACAGGTTG
Hepatology Research 2014
AGC-3′; and β-actin, forward 5′-GGGAAATCGTGCGT GAC AT-3′, and reverse 5′-CTGGAAGGTGGACAG CGAG-3′. The reaction conditions for the PCR program were as follows: initial melting at 95°C for 30 s followed by 40 cycles at 95°C for 5 s, 60°C for 64 s. Analysis of melting curve for the primers was conducted to confirm the specificity of the PCR product, and the threshold cycle (Ct) value for triplicate reactions was averaged. The relative expression of TPX2 mRNA for each sample was calculated as follows: ΔCt = Ct (TPX2) − Ct (β-actin), ΔΔCt (sample) = ΔCt (sample) − ΔCt (calibrator), and the fold changes in mRNA were calculated through relative quantification (2–ΔΔCt). To evaluate the effect of TPX2 mRNA on survival, the weighed expression of TPX2 mRNA were divided into high and low level using the median expression as the cut-off point for the whole group.
Immunoblotting The primary antibodies TPX2 (sc-32863), AKT (sc-8312, which can be used to detect total proteins of AKT1, AKT2 and AKT3), p-AKT (sc-293095, which can be used to detect p-AKT1, p-AKT2 and p-AKT3), MMP2 (sc10736), MMP9 (sc-10737) and β-actin (sc-130656) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary antibody (sc-2004) was also obtained from Santa Cruz Biotechnology. Briefly, the denatured protein samples were separated by polyacrylamide gel electrophoresis, and then electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were incubated with different primary antibodies and examined with the secondary antibody conjugated with horseradish peroxidase (HRP). Reactions were visualized using the HyGLO HRP detection kit (Denville, Metuchen, NJ, USA).
Immunohistochemical staining The primary mouse monoclonal anti-TPX2 antibody (sc-376812), goat polyclonal anti-MMP2 antibody (sc34014) and goat polyclonal anti-MMP9 antibody (sc-6840) were purchased from Santa Cruz Biotechnology. The biotinylated secondary antibodies of goat antimouse and rabbit antigoat were obtained from Wuhan Boster Bio-engineering (Wuhan, China). Immunohistochemical staining was performed on paraformaldehyde-fixed paraffin sections, and was done according to standard procedures. Sections were cut at 5-μm thickness, and heated in a 60°C oven. Briefly, tissue sections were deparaffinized in xylene, rehydrated through a graded series of alcohol and then incubated with 0.3% hydrogen peroxide for 20 min to block
TPX2 in hepatocellular carcinoma 3
endogenous peroxidase activities. After antigen retrieval in citrate buffer, the sections were blocked for 30 min using 10% normal goat serum, then separately incubated overnight at 4°C with the primary antibodies directed against TPX2, MMP2 and MMP9. This was followed by detecting the primary antibody with a biotinylated secondary antibody according to the manufacturer’s recommendations. The staining of the tissue sections was performed using the streptavidin–biotin– peroxidase complex for TPX2, MMP2 and MMP9 (SABC method). Using previously reported immunohistochemical techniques, the staining was visualized by using diaminobenzidine and counterstained with hematoxylin, then dehydrated in a graded series of alcohol, cleared in xylene and mounted onto glass slides. All tissue sections were scored by light microscopy by two experienced pathologists, blindly and evaluated independently, and then assessed at a conference microscope and a consensus determined. For the evaluation of TPX2, MMP2 and MMP9 immunostaining results, a semiquantitative scoring system based on the product of staining intensity and combined with the percentage of positive liver cells was used in the present study.24 Immunostaining intensity was evaluated as four grades: 0, negative; 1, weak; 2, moderate; and 3, strong. The percentage of positive liver cells was categorized as the following grades: 0, 0%; 1, 1–10%; 2, 11–50%; 3, 51–80%; and 4, more than 80%.24 The immunostaining intensity and average percentage of positive cells were evaluated for 10 independent high-magnification fields. By multiplying the staining intensity and the percentage of positive cells, the final weighed expression score was obtained (0–12). The total expression score for TPX2, MMP2 and MMP9 was used as a continuous variable for the correlation analyses. To evaluate the effect of TPX2 protein on survival, the weighed expression scores of TPX2 protein were divided into high and low scores using the median expression score as the cut-off point for the whole group.
siRNA transfection The specific siRNA against TPX2 (sc-37653) and control siRNA (sc-37007) were both from Santa Cruz Biotechnology. MHCC-97H tumor cells were seeded at a concentration of 2 × 106 per well in six-well plates and cultured overnight. Then, cells were transfected with 100 nM siRNA using Lipofectamine RNAi MAX Reagent (Invitrogen). Further experiments were performed 48 h after transfection.
© 2014 The Japan Society of Hepatology
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Cell invasion and migration assays Transwell cell migration assays were performed in 12-well plates with 8-μm BioCoat control inserts (Becton Dickinson Labware, Bedford, MA, USA). TPX2 siRNA or control siRNA transfected MHCC97H cells (1–2 × 105) that were suspended in 500 μL serum free Dulbecco’s modified Eagle’s medium (DMEM) were seeded in the upper well and DMEM medium with 10% fasting blood sugar, as indicated, in the lower well. After completion, membranes were removed, wiped on the side facing the upper well and stained with crystal violet. At least five representative images of each well were taken and cell numbers were counted using ImageJ. BioCoat Matrigel invasion chamber (Becton Dickinson Labware) was used for transwell cell invasion assays and the following protocols were the same as transwell cell migration assays. The experiments were performed in triplicate.
Follow up The follow up of this study finished on 31 December 2013, and the duration was defined as the interval between the date of surgical operation and the date of death or last follow up. The median follow-up time was 55 months (range, 3–79). All patients were monitored every 1–3 months in the first year and every 3–6 months thereafter. The monitor protocol included physical examination, serum α-fetoprotein (AFP) level, chest X-ray and abdominal ultrasonography. For the patients with follow-up results suggestive of tumor recurrence, computed tomography, magnetic resonance imaging and/or positron emission tomography were performed to verify whether tumor recurrence had occurred. Intrahepatic recurrence or distant tumor metastasis detected only by imaging diagnosis after surgical operation was designated as tumor recurrence. During the duration of follow up, 90 of 130 patients (69.2%) developed intrahepatic tumor recurrence and 22 patients (16.9%) developed distant tumor metastases.
Statistical analysis Statistical analysis was carried out using the SPSS version 16.0 statistical software package (SPSS, Chicago, IL, USA). To assess the association between ordinal data, Spearman’s rank correlation coefficient was used, and the χ2-test or Fisher’s exact test were used for categorical data. Group expression level was compared by Mann– Whitney U-test. Overall survival, with deaths due to any cause as event, and disease-free survival, with tumor recurrence and metastases as events, was analyzed using
© 2014 The Japan Society of Hepatology
Hepatology Research 2014
the Kaplan–Meier method, and the difference between curves was evaluated by the log–rank test. Independent prognostic factors were assessed by the Cox proportional hazards stepwise regression model. Data were showed as the Mean 1 standard error of the mean. P-values were two-sided, and P < 0.05 was considered statistically significant.
RESULTS Expression of TPX2 in HCC tissues and cells
W
E FIRST DETECTED the expression of TPX2 in HCC cell lines (SMMC-7721, BEL-7402, HUH-7, HepG2 and MHCC-97H) and a normal hepatocyte cell line (LO2) using qRT–PCR and immunoblotting. Our results showed that the expression of TPX2 in the immortalized liver cell line, L02, was significantly lower than those in HCC cell lines (P < 0.01, respectively; Fig. 1a,b). In 130 pairs of HCC and normal tumoradjacent tissues, qRT–PCR and immunoblotting showed that TPX2 expression in HCC was obviously higher than that in adjacent non-cancerous liver tissues (P < 0.01, respectively; Fig. 1c,d). We also examined the expression of TPX2 protein in 130 cases of HCC and matched tumor-adjacent liver tissues using immunohistochemical staining, and found that the expression score of TPX2 protein was significantly higher in the HCC tissues than that in the noncancerous tissues (4.82 1 0.29 vs 1.37 1 0.14; P < 0.01) (Fig. 2a–c). These results indicated TPX2 to be more highly expressed in HCC tissues than in adjacent nontumorous liver tissues.
Correlation between TPX2 expression and clinicopathological parameters To further characterize the clinical role of TPX2 in HCC, we tried to precisely identify the correlations of the TPX2 expression with clinicopathological parameters, including patient sex, age, hepatitis B surface antigen (HBsAg), AFP level, tumor size, tumor number, vascular invasion, cirrhosis, capsule formation, Edmondson–Steiner grade and TNM stage. The median expression score of TPX2 protein and mRNA was used as the cut-off point to divide into low- and high-expressing groups. In our study, the expression of TPX2 was significantly correlated with the Edmondson–Steiner grade, tumor number and TNM stage (P < 0.05). However, no evident correlation was found between the expression of TPX2 and patient sex, age, HBsAg, AFP level, tumor size, vas-
Hepatology Research 2014
TPX2 in hepatocellular carcinoma 5
(a)
(b) TPX2 mRNA level
TPX2 mRNA level
4
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β-actin
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TPX2
Figure 1 Expression of TPX2 in cell lines and hepatocellular carcinoma (HCC) tissues. (a) TPX2 mRNA expression in the immortalized normal human liver cell line L-02 and five hepatoma cell lines (SMMC-7721, BEL-7402, Huh-7, HepG2 and MHCC-97H) using quantitative reverse transcription polymerase chain reaction (qRT–PCR) (n = 3, *P < 0.01, vs L-02 group, respectively). (b) TPX2 mRNA expression in HCC and corresponding adjacent non-tumorous liver tissues (NT) using qRT–PCR (n = 130, P < 0.01). (c) TPX2 protein expression in the immortalized normal human liver cell line L-02 and five hepatoma cell lines (SMMC-7721, BEL-7402, Huh-7, HepG2 and MHCC-97H) using immunoblotting (n = 3, P < 0.01). (d) TPX2 protein expression in HCC tissues (T) and corresponding adjacent non-tumorous liver tissues (N) using immunoblotting (n = 3, P < 0.01).
cular invasion, cirrhosis and capsule formation (P > 0.05). The results are listed in Table 1.
Correlation between TPX2 and MMP2 protein expression To address whether the TPX2 expression level was correlated with MMP2 expression, immunohistochemical staining was applied to examine the expression score of MMP2 in HCC and corresponding adjacent nontumorous liver specimens. MMP2 protein level was significantly higher in the HCC tissues than that in the corresponding adjacent non-tumorous liver tissues (5.01 1 0.29 vs 1.32 1 0.13; P < 0.01) (Fig. 2d–f). We next analyzed the correlation between the TPX2 expression and the MMP2 expression. There was indeed an evident positive correlation between the protein expression level of TPX2 and MMP2 (r = 0.827, P < 0.01, Spearman’s rank correlation coefficient) (Fig. 3a).
Correlation between TPX2 and MMP9 protein expression Additionally, to explore the correlation between TPX2 and MMP9 expression, the protein expression of MMP9
was examined in 130 cases of HCC and corresponding adjacent non-tumorous liver tissues using immunohistochemical staining. The expression score of MMP9 protein was significantly higher in the HCC tissues than in the corresponding adjacent non-tumorous liver tissues (5.32 1 0.33 vs 1.56 1 0.14; P < 0.01) (Fig. 2g–i). Notably, there was also a significant positive correlation between the protein expression level of TPX2 and MMP9 (r = −0.772, P < 0.01, Spearman’s rank correlation coefficient) (Fig. 3b).
Prognostic of HCC subtypes defined by TPX2 level As described in the Methods section, the median expression score of TPX2 protein and mRNA was used as the cut-off point to divide into low- and high-expressing groups for survival. To assess the effect of TPX2 on overall survival and disease-free survival, univariate prognostic analysis and multivariate Cox regression models were used to calculate. The patients with high expression level of TPX2 had significantly reduced overall survival and disease-free survival. The 5-year overall survival rate of the low-expressing group was
© 2014 The Japan Society of Hepatology
Q. Liu et al.
Hepatology Research 2014
(a)
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69.6%, which was evidently higher than that of the high-expressing group (31.5%, P < 0.001). The 5-year disease-free survival rate of the low-expressing group was 55.5%, which was evidently higher than that of the high-expressing group (16.1%, P < 0.001) (Fig. 4). Subsequently, the associations of overall survival and disease-free survival with clinicopathological parameters in the HCC tissues were calculated by univariate analysis. The univariate analysis showed that HBsAg status, tumor number, Edmonson–Steiner classification, TNM stage and the expression level of TPX2 were the prognosis factors for HCC. The results are listed in Table 2.
Stratified univariate and multivariate analysis In a multivariate analysis model, the expression of TPX2 was significantly associated with overall survival (hazard ratio [HR], 2.580; 95% confidence interval [CI], 1.520– 4.378; P < 0.001) and disease-free survival (HR, 2.671; 95% CI, 1.683–4.237; P < 0.001) (Table 3). Multivariate analysis established that the TPX2 expression level, HBsAg status and tumor number were the independent prognostic factors of HCC patients (Table 3). Considering the fact that patient survival may be associated with the TNM stage, we further stratified the data according to
© 2014 The Japan Society of Hepatology
MMP9
Figure 2 Immunohistochemical staining of TPX2, matrix metalloproteinase (MMP)2 and MMP9 in hepatocellular carcinoma tissues (HCC) and matched tumor-adjacent tissues. (a) In cases of high TPX2 protein expression, (d) there was strong MMP2 and (g) MMP9 protein expression in the same tissue section. (b,c) Similarly, in the case of low TPX2 protein expression, (e,f) there was no detectable MMP2 or (h,i) MMP9 protein expression (scale bar = 100 μm).
TNM stage and evaluated the prognostic value of TPX2 expression in TNM stage I. For the 63 TNM stage I HCC patients, evident correlations were found between the expression level of TPX2 and overall survival and disease-free survival (Fig. 5). For the TNM stage II or III HCC patients, TPX2 expression level had no prognostic value regarding overall survival and disease-free survival (P > 0.05, respectively). The associations of overall survival and disease-free survival with clinicopathological parameters in TNM stage I patients were calculated by univariate analysis (Table 4). Furthermore, multivariate analysis showed that the expression level of TPX2 was an independent negative prognostic factor for the survival of TNM stage I patients (Table 5). The patients with high expression of TPX2 had poorer overall survival (HR, 3.440; 95% CI, 1.430–8.273; P = 0.006) and disease-free survival (HR, 2.533; 95% CI, 1.122–5.722; P = 0.025) than those with low expression level of TPX2 in TNM stage I.
TPX2 knockdown suppressed cell migration and invasion To further explore the mechanism of TPX2 in HCC cells, we investigated the effect of TPX2 knockdown on HCC cell migration and invasion in TPX2 siRNA transfected MHCC-97H cells. As compared with the control siRNA
Hepatology Research 2014
TPX2 in hepatocellular carcinoma 7
Table 1 Correlations between TPX2 expression and clinicopathological features in HCC Characteristics
Sex Female Male Age (years) 245 >45 HBsAg status Negative Positive Cirrhosis No Yes AFP (μg/L) 2400 >400 Tumor size 25 cm >5 cm Tumor number Single Multiple Tumor capsule Complete Incomplete Vascular invasion No Yes Edmondson grade I/II III/IV TNM stage I II III
n
TPX2 mRNA High
Low
26 104
15 50
11 54
39 91
15 50
28 102
P
TPX2 protein
P
High
Low
0.511
15 47
11 57
0.279
24 41
0.125
15 47
24 44
0.185
10 55
18 47
0.134
10 52
18 50
0.200
42 88
20 45
22 43
0.851
19 43
23 45
0.712
58 72
28 37
30 35
0.860
26 36
32 36
0.599
49 81
22 43
27 38
0.469
22 40
27 41
0.718
103 27
46 19
57 8
0.029*
43 19
60 8
0.010*
32 98
17 48
15 50
0.839
17 45
15 53
0.543
110 20
53 12
57 8
0.467
51 11
59 9
0.627
78 52
31 34
47 18
0.007*
27 35
51 17
45 HBsAg status Negative Positive Cirrhosis No Yes AFP (μg/L) 2400 >400 Tumor size 25 cm >5 cm Tumor number Single Multiple Tumor capsule Complete Incomplete Vascular invasion No Yes Edmondson grade I/II III/IV TNM stage I II III TPX2 protein level Low High
n
Overall survival rate, years (%) 3
5
26 104
80.4 64.2
63.9 48.8
39 91
79.3 62.3
28 102
P
Disease-free survival rate, years (%) 3
5
0.289
57.7 51.0
46.2 34.3
0.214
55.5 43.9
0.561
64.1 47.3
41.0 34.9
0.565
89.3 66.4
85.6 42.1
0.001*
82.1 44.1
64.3 29.1
