Clinical significance of Hiwi gene expression in gliomas

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Research Report

Clinical significance of Hiwi gene expression in gliomas Guan Sun 1 , Yingyi Wang 1 , Lihua Sun 1 , Hui Luo, Ning Liu, Zhen Fu, Yongping You⁎ Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China

A R T I C LE I N FO

AB S T R A C T

Article history:

Piwi, highly conserved during evolution, has been reported to play a key role in stem cell

Accepted 30 November 2010

self-renewal in several different organisms. As one of the four human homologues of the

Available online 4 December 2010

Piwi family, Hiwi has been demonstrated to be significantly overexpressed in some human cancer. However, the relationship between Hiwi and human gliomas is unclear. In this

Keywords:

study, we investigated the clinical significance of Hiwi in human gliomas. Hiwi was found to

Hiwi

be specifically expressed in the majority of glioma tissues, and the expression was greatly

Glioma

increased with the ascending of tumor grades. In addition, it was statistically significant

Prognosis

that the patients with high Hiwi positive had poorer outcome than the patients with low Hiwi positive. Our results suggest that Hiwi may be a critical factor in glioma progression and could be used as a potential molecular marker for pathological diagnosis and prognosis evaluation for malignant gliomas. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Glioma, one of the leading common primary tumors in brain, has poor clinical outcome, and the incidence and mortality of gliomas have been rising in recent years. To multiforme glioblastoma (GMB), after surgery and radiotherapy and chemotherapy comprehensive treatment, the average survival time is still only 14.6 months (Stupp et al., 2005). Thus, to understand more deeply the pathogenesis of gliomas and to explore a more effective treatment become the focus of basic and clinical research. Piwi was found in the drosophila germline stem cells firstly by Lin and Spradling in 1970 (Lin and Spradling, 1997). As a subfamily of Argonaute proteins, Piwi is highly conserved during evolution (Cox et al., 1998). It has been reported that Piwi plays a key role in stem cell self-renewal, spermiogenesis, RNA silencing and translational regulation in several different organisms (Cox et al., 1998; Hutvagner and Simard, 2008; Lingel and Sattler, 2005; Seto et al., 2007). Hiwi is one of the four human

homologues of the Piwi family, which maps to the long arm of chromosome 12, band 12q24.33, and originally isolates from a human testis cDNA library (Cox et al., 1998). Further analysis demonstrated that Hiwi is also expressed in a wide range of tissues including the prostate, heart, liver, kidney, and so on (Sharma et al., 2001). As a human member of Piwi gene family, Hiwi contains a conserved architecture with a PAZ motif in the middle and Piwi motif in the C-terminal region (Cerutti et al., 2000). Recent studies have reported that Hiwi is present in human CD34+ hematopoietic progenitor cells but not in more differentiated cell populations. The studies suggested that Hiwi may be an important negative developmental regulator underlying the unique biologic properties associated with hematopoietic stem cells (Sharma et al., 2001). Recently, accumulating data have revealed that Hiwi not only plays an important role in the proliferation of stem cells, but also exerts critical effects on tumorigenesis, such as adenocarcinoma of the pancreas, and gastric cancer (Grochola et al., 2008; Liu et al., 2006). However, it has been unknown that whether Hiwi

⁎ Corresponding author. Fax: +86 025 83716602. E-mail address: [email protected] (Y. You). 1 Contributed equally. 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.11.097

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expressed in gliomas, and that the relationship between Hiwi expression and overall survival of the patients. In the present study, both Hiwi protein and mRNA level were examined in different grades of primary human gliomas and normal brain tissues as well as glioblastoma cell lines. Moreover, we analyzed the association of patient prognosis with Hiwi expression. To our knowledge, it is the first report to show the expression of stem cell-associated gene Hiwi in human gliomas and a correlation between its expression level and clinical outcome.

2.

Results

2.1.

Hiwi expression was associated with glioma grades

To explore the expression of Hiwi in gliomas, glioma tissue specimens and cells were examined by Western blot and RT-PCR analysis. The protein level of Hiwi expression in gliomas was displayed in (Fig. 1A). Hiwi was overexpressed in glioma tissue

specimens, especially in high grade glioma specimens normalized to GAPDH protein control. The expression of Hiwi in grade IV gliomas tissues (2.96 ± 0.21) and grade III gliomas tissues (1.65±0.09) was significantly stronger than low grade gliomas (0.69±0.14) (P<0.01). Moreover, there was also a notable difference between grade III and grade IV glioma tissues (P<0.05). Additionally, as shown in Fig. 1B, the mRNA expression of Hiwi was 0.85±0.42, 1.5±0.28, and 2.25±0.26, respectively for I–II, III, IV grade glioma tissues normalized to β-actin control (P<0.05). Further, both protein and mRNA expressions of Hiwi were also higher in glioblastoma cells, in line with that the expression of Hiwi increased with the ascending of tumor grades.

2.2. Significant prognostic value of Hiwi expression for gliomas In order to determine the relationship between Hiwi expression and clinical outcome in gliomas, we chose 66 glioma patients with complete clinical data. Based on the survival profile of patients through the Log-rank test method, the

Fig. 1 – The Hiwi expression significantly increases with the ascending of glioma grades. A: Expression of Hiwi protein level in different grades of gliomas (1–3: WHO I–II gliomas, 4–6: WHO III gliomas, and 7–9: WHO IV gliomas) and human glioma cell lines (10: U251, 11: U87, and 12: LN229) by Western blot. B: Expression of Hiwi mRNA level in different grades of gliomas (1–3: WHO I–II gliomas, 4–6: WHO III gliomas, and 7–9: WHO IV gliomas) and human glioma cell lines (10: U251, 11: U87, and 12: LN229). C: Graphical representation of the Hiwi protein level expression profiles in (A). * P < 0.05. D: Graphical representation of the Hiwi mRNA level expression profiles in (B). * P < 0.05.

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Fig. 2 – Representative sections for Hiwi immunoreactivity in normal brain and glioma tissues (400×), and gliomas with high grades exhibit stronger Hiwi expression than gliomas with lower grades. A: normal brain tissues; B: WHO I–II gliomas; C: WHO III gliomas; and D: WHO IV gliomas.

overall survival of patients with age ≥50, KPS score <0, and WHO III–IV gliomas was significantly shorter than their corresponding groups respectively (P < 0.05). Additionally, the overall survival of men and women observed has no statistical differences (P = 0.128) (Table 1). Immunostaining analysis showed that Hiwi protein was localized to both cytoplasm and nucleus, and highly expressed

Table 1 – The relationship between clinical characteristics and survival in glioma patients. Parameters

Gender Male Female Age < 50 ≥ 50 KPS < 80 ≥ 80 WHO grade WHO I–II WHOIII–IV

No. of patients

in gliomas. The total positive rate was 72.5%. In addition, gliomas with different grades exhibited mild to strong Hiwi expression. Indeed, 29/42 grade II–-IV gliomas exhibited detectable levels of Hiwi while 19/24 low grade gliomas exhibited undetectable levels of the protein (P<0.05) (Fig. 2, Table 2). Furthermore, only 2/12 was detected with weakly Hiwi positive immunoreactivity in normal brain tissues. Taken together, these results confirm the data from Western blot and RT-PCR analysis. According to the expression profile of Hiwi immunoreactivity score, the tissue samples were categorized as low positive (≤3) and high positive (>3). We analyzed the overall survival of patients

Average 95%confidence P value survival interval (month) (month)

45 21

29.171 33.667

24.977–33.364 22.619–43.715

P = 0.128

44 22

36.075 20.909

32.605–39.544 15.881–25.936

P < 0.05

26 30

23.346 35.998

18.341–28.350 32.280–39.716

P < 0.05

24 42

42.049 25.309

39.126–44.972 21.456–29.162

P < 0.05

Table 2 – Expression of Hiwi in normal brain (NB) and different grades of gliomas. Group

NB Grade I–II Grade III Grade IV

Case

12 24 30 12

Score of Hiwi expression −

+

++

+++

10 10 6 0

2 9 5 2

0 4 15 3

0 1 4 7

The Hiwi positive rate was significantly correlated with WHO classification of glioma grades, P < 0.01.

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Fig. 3 – Kaplan–Meier survival curves for Hiwi expression. The patients with high expression of Hiwi had a significant worse outcome than the patients with low expression of Hiwi, P<0.01.

and found significant differences between low positive group and high positive group (P<0.001). The Hiwi high positive cases showed a marked worse outcome than the low positive cases (Fig. 3).

3.

it has been reported that Hiwi was significantly enhanced in seminomas, but not in nonseminomatous tumors, spermatocytic seminomas, or nongerm cell tumors of the adult testis, indicating that Hiwi may participate in germ cell malignancy (Qiao et al., 2002). Recently, a high level expression of Hiwi has been found in human esophageal squamous cell carcinoma, and has significantly associated with histological grade. Furthermore, patients with high Hiwi expression tumors had a poorer outcome than those with low expression tumors (He et al., 2009). In our study, we showed that Hiwi was highly expressed in majority of malignant gliomas and glioblastoma cell lines, and increased obviously with the ascending of tumor grades both in protein and mRNA level. In addition, statistically significant differences of clinical outcome in patients with low Hiwi expression and high Hiwi expression were observed. The results suggest that Hiwi plays an important role in gliomagenesis. Recently, Liu et al. showed that a knockdown of Hiwi by antisense adenovirus or RNA inference induced the cell cycle to halt in G2/M phase in gastric cancer cells with Hiwi high expression (Liu et al., 2006). In summary, we have demonstrated that Hiwi is overexpressed in gliomas, and increased with ascending tumor grades. The higher level of Hiwi expression was significantly associated with poorer overall survival. Our results suggest that Hiwi could be an intrinsic regulator of progression in glioma cells and used as potential target and predictor of survival in this dismal disease. However, the mechanism responsible for Hiwi in tumorigenesis and the biological functions merit further evaluation.

Discussion

Here we report the expression of Hiwi which was associated with stem cell in gliomas and glioma cell lines, and access the correlation of Hiwi expression and the clinical prognosis of glioma patients. Previous study has showed that overexpression of Piwi increases the number of germline stem cells and their rate of mitosis, indicating that Piwi is a critical regulator in germline stem cell (Cox et al., 2000). Further analysis of the expression of Hiwi demonstrated that Hiwi gene overexpressed in human CD34+ hematopoietic progenitor cells, but in the differentiation and maturation of hematopoietic cells did not detect Hiwi expression, suggesting that Hiwi functions as an intrinsic regulator of stem cell self-replication, and could serve as a genetic marker of progenitor and stem cells, which is consistent with the effects of Piwi (Sharma et al., 2001). Cancer stem cells, a small number of cells with self-renewal capacity cells, have been isolated in leukocythemia, multiple myeloma, breast cancer and gliomas (Al-Hajj et al., 2003; Blair et al., 1997; Matsui et al., 2004; Soltysova et al., 2005), and were essential to the development and proliferation of human malignant tumors. Till now, glioma stem cells, which originated from transformed neural stem cells, are considered to be responsible for glioma initiation, recurrence and resistance to chemotherapy and radiotherapy (Fan et al., 2007; Lesniak, 2006). Therefore, these findings attract us to explore the stem cellassociated gene Hiwi expression profile in glioma tissues as well as glioblastoma cells. Emerging evidence suggested that elevated expression of Hiwi mRNA was found in most patients with soft-tissue sarcomas (STS), and overexpression of Hiwi mRNA identified STS patients at high risk of tumor related death (Taubert et al., 2007). In addition,

4.

Experimental procedures

4.1.

Tumor specimens and glioblastoma cell lines

All the patients underwent complete or partial surgical resection at the first affiliated hospital of Nanjing medical university. 66 paraffin specimens with clinical data were collected from February 2005 to May 2006, including 24 grade I–II tumors, 30 grade III tumors, and 12 grade IV tumors (glioblastomas). Patient characteristics, including the Karnofsky performance scale (KPS) score, were collected before the initial surgery. None of the patients recruited in this study had been subjected to chemotherapy or radiation therapy before tumor resection. For the analysis of survival and follow-up, the date of surgery was used to stand for the beginning of the follow-up period. All patients who died from other disease or unexpected events were excluded from the evaluation. Clinical information was obtained by reviewing the medical records, telephone and the death certificate. 40 specimens in liquid nitrogen were collected from October 2007 to October 2008, including 20 grade I–II tumors, 10 grade III tumors, and 10 grade IV tumors (glioblastomas). Normal brain tissues were obtained from patients with traumatic brain injury for internal decompression. This study was approved by the institutional review boards of the hospitals and written informed consent was obtained from all patients. Human glioblastoma cell lines, U251, U87 and LN229 cells were purchased from the Chinese Academy of Sciences Cell Bank. These cell lines were cultured in DMEM (Gibco USA) supplemented with 10% bovine serum albumin and maintained at 37 °C in an atmosphere of 5% CO2.

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4.2.

Western blot analysis

Total proteins were extracted and the protein concentration was determined by BSA method (keyGEN, China). From each sample, 40 μg of protein lysates were subjected to SDS-PAGE in 10% acrylamide gel and transferred to PVDF membranes (Millipore Corporation, USA). The membrane was blocked in 5% nonfat milk and incubated with diluted antibodies against Hiwi (1:500, Santa Cruz Biotechnology, USA) overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibody (1:2000, Santa Cruz Biotechnology, USA). After stripping, the membrane was reprobed with GAPDH (1:2000, Kangchen Biotechnology, China) using ultra enhanced chemoluminescence Western blotting detection reagents (Multi Science Biotech, China). All Western bands were quantified by densitometry and are presented in the form of a bar graph.

4.3.

RT-PCR analysis

Total RNA was isolated using Trizol reagent (Invitrogen, USA), and converted to cDNA by a BioRT Two Step RT-PCR Kit (Bioer Technolog, China). PCR reactions were carried out using Ex Taq hot start polymerase (Takara). β-actin was used as a control for adjusting the relative of total RNA between the samples. The thermal cycles were: 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s for 35 cycles for Hiwi and β-actin. The primers (Invitrogen, Shanghai, China) used for PCR were as follows: Hiwi, 5′-TGA TGA ATC TGA TGA ACT GGT C-3′ (forward) and 5′-GGT GAT GTC CTC GTC TGT AG-3′ (reverse); β-actin, 5′-AAG ACC TGT ACG CCA ACA CAG T-3′ (forward) and 5′-AGA AGC ATT TGC GGT GGA CGA T-3′ (reverse). The PCR reactions for Hiwi and β-actin were fractionated on a 2% agarose gel containing 0.5 mg/ml ethideum bromide. Gels were visualized by Gel Doc™XR gel documentation system (Bio-Rad).

4.4.

Immunohistochemical staining

For immunohistochemical staining of Hiwi, the ABCperoxidase method was used. The tissue specimens were prepared as paraffin sections. Briefly the sections were incubated with primary antibodies (1:100 dilution) against Hiwi (Santa Cruz, CA, USA) overnight at 4 °C, then incubated with a biotinylated secondary antibody (1:200 dilution) at room temperature for 1 h, followed by incubation with ABCperoxidase reagent (Vector, USA) for 1 h, washed with PBS and stained with 3,3-diaminobenzidine (30 mg dissolved in 100 ml Tris buffer containing 0.03% H2O2) for 5 min, rinsed in water and counterstained with hematoxylin. For evaluation of Hiwi expression in different grades of tumors, ten randomly selected visual fields per section were examined by light microscope under high magnification (400×). The Hiwi immunoreactivity score was assessed by multiplication of the values for staining intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and Hiwipositive tumor cells (0, <5%; 1, 5%–30%; 2, 31%–70%; and 3, >70%). Overall, the specimens were scored as follows based on both percentage and intensity: 0–1 (−); 2–3 (+); 4–5 (++); and 6 (++).

4.5.

187

Statistical analysis

All data were analyzed using SPSS Graduate Pack 11.0 statistical software (SPSS, Chicago, IL). Descriptive statistics including mean and ± SE along with independent-sample t-test and chi-square testing were used to determine significant differences. We used log-rank analysis to evaluate the differences between groups. The Kaplan–Meier analysis was employed to assess the survival rate of different Hiwi expressions. P < 0.05 was considered as a significant difference.

Acknowledgments All authors have declared all sources of funding for the research reported in this manuscript and have no financial or other contractual agreements that might cause conflicts of interest or be perceived as causing conflicts of interest. This work was supported by the China Natural Science Foundation (Proj. No. 30872657), Natural Science Foundation of Jiangsu Province (Proj. No. 2008475), Medical Major Talent Program of Jiangsu Province (Proj. No. RC2007061) and Jiangsu Province's “333” Key Talent Foundation (Proj. No. 0508RS08).

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