RNAi-mediated inhibition of MSP58 decreases tumour growth, migration and invasion in a human glioma cell line

MSP58, a 58-kD nuclear microspherule protein, is an evolutionarily conserved nuclear protein implicated in the regulation of gene transcription as well as in malignant transformation. An analysis of mRNA expression by real-time PCR revealed that MSP58 was significantly up-regulated in 29% of high-grade glioblastoma tissues as well as in four glioblastoma cell lines. In the present study, we further evaluated the biological functions of MSP58 in U251 glioma cell proliferation, migration, invasion and tumour growth in vivo by specific MSP58 knockdown using short hairpin RNA (shRNA). We found that MSP58 depletion inhibited glioma cell growth, primarily by inducing cell cycle arrest rather than apoptosis. MSP58 depletion also decreased the invasive capability of glioma cells and anchorage-independent colony formation in soft agar. Moreover, suppression of MSP58 expression significantly impaired the growth of glioma xenografts in nude mice. Finally, a cell cycle-associated gene array revealed potential molecular mechanisms contributing to cell cycle arrest in MSP58-depleted glioma cells. In summary, our data highlight the importance of MSP58 in glioma progression and provided a biological basis for MSP58 as a novel candidate target for treatment of glioma.

. These interactions led to the characterization of MSP58 as a participant in transcriptional regulation. Moreover, TOJ3, an avian homologue of MSP58, was a target of the transcription factor v-Jun. Ectopic expression of TOJ3 in avian fibroblasts led to anchorage-independent growth, strongly suggesting that TOJ3 plays an important role in Jun-induced cell transformation and tumourigenesis [8]. In another study, an isoform of MSP58, p78, was found to bind to centrosomal proteins, such as Nde1 and Su48, and have an essential role in centrosome dynamics. Thus, MSP58 could be a component of the centrosomal protein complex [9]. We have shown previously that MSP58 interacts with N-myc downstream regulated gene 2 (Ndrg2), a protein intimately involved in regulation of proliferation and differentiation, and that the interaction between these two proteins is important in controlling the cell cycle of Hela cell [10]. In addition, ectopic expression of Ndrg2 inhibits glioma cell proliferation [11]. We have also found that MSP58 is expressed in both normal brain tissue and tumour tissue from glioma patients, but its expression is significantly up-regulated in high-grade glioblastoma tissues as well as in the four glioblastoma cell lines we examined. Based upon all these studies, we hypothesized that reducing MSP58 expression may inhibit the malignant behaviour of glioma cells.
RNA interference (RNAi), a novel mechanism of post-transcriptional gene silencing, has been found in many eukaryotes. Recently, small interfering RNA (siRNA) has been shown to attenuate the expression of specific proteins both in vitro and in vivo by sequence-specific, double-stranded RNA (dsRNA) molecules [12,13]. Chemically synthesized small RNAs targeting the mRNA of interest could produce RNAi, but the effects were transient. The development of siRNA vectors engineered to express short hairpin RNA (shRNA) in mammalian cells has led to extensive gene-specific silencing [14][15][16]. In the present study, a plasmid expressing an shRNA directed against human MSP58 was generated to block the expression of MSP58 in the human glioma cell line U251. The siRNA-induced suppression of MSP58 significantly inhibited glioma tumour proliferation, invasion as well as migration. Furthermore, we used a cDNA microarray to detect potential signal transduction pathways in which MSP58 could be involved. Our results demonstrated that MSP58 participated in multiple malignant cellular processes of human glioma cells, and suggested a potential use for highly specific RNAi-based gene-silencing therapeutics in gliomas.

Tissue samples
Intracranial tissue samples were obtained from 29 17 as Grade III (anaplastic astrocytoma) and 4 as Grade IV (glioblastoma), respectively. Three samples from human normal brain tissues were used as control. All of the tumour tissues were obtained at primary resection, and none of the patients had been subjected to chemotherapy or radiation therapy before resection. The samples were snap frozen in liquid nitrogen and stored at Ϫ80ЊC until analysis. The diagnosis of glioma was confirmed histologically in all cases. For the experimental use of the surgical specimens, informed consent was obtained from the patients according to the hospital ethical guidelines.

Quantitative real-time RT-PCR
Total RNA was isolated from human brain and brain tumour tissues or cultured glioma cells using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Total RNA (2 g

Transfection of synthetic siRNA
According to what was described previously [9], target siRNA sequences for MSP58 were synthesis as follows: antisense: GAAGUUCGAUGAU-GAGCUG; sense: CAGCUCAUCAUCGAACUUC. The synthetic siRNA duplex oligonucleotides were obtained from Shanghai GenePharma Co., Ltd, Shanghai, China. U251 cells (5 ϫ 10 4 ) were seeded into six-well plates. After 24 hrs, cells in each well were transfected with 100 nM siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR)
The stable transfected clones and cells transfected with siRNA were harvested for RT-PCR analyses. According to the manufacturer's instructions, total RNA was extracted with TRIZOL reagent (Invitrogen) and cDNAs were generated from 2 g of total RNA using reverse transcriptase (Fermentas Vilnius, Lithuania). The primers for human MSP58 were: 5Ј-ACGCCCT-GCTCTACGAT-3Ј and 5Ј-TCATGCCTGTGATGCTGTC-3Ј; and the predicted PCR product was 483 bp. The primers for GAPDH, used as an internal control, were: 5Ј-AGGTCCACCACTGACACGTT-3Ј and 5Ј-GCCTCAAGAT- CATCAGCAAT

Western blot analysis
The U251 cells were collected at the indicated time points, and washed twice with cold phosphate-buffered saline (PBS) (Gibco/BRL, Grand Island, USA) before being lysed in lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 50 mM EDTA, 1% NonIdet P-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 2 g/ml pepstatin A]. The protein samples (20 g/lane) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes (Sigma-Aldrich Co.). After blocking overnight at 4ЊC in a buffer containing PBS, 0.1% Tween 20 and 5% low fat milk powder, the resulting blots were incubated with a rabbit anti-human MSP58 serum (1:600) as described previously [17], and then with peroxidase-conjugated secondary antibodies (1:4000 dilution; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) and an enhanced chemiluminescence's (ECL) detection solution was applied (Pierce, Rockford, IL, USA). Finally, the blots were stripped as described previously [18]. The membranes were reprobed with a ␤-actin rabbit polyclonal antibody (1:1000; Boster, Wu Han, China) to normalize for loading and allow comparisons of target protein expression among all groups of cells. Densitometric analysis was performed using Scion Image

Flow cytometric analysis of the cell cycle
The cells were seeded in 25 ml flasks and incubated until they were 80-85% confluent in DMEM containing 10% FBS. Then the cells were harvested, washed twice with ice-cold PBS, fixed with 70% ethanol overnight at 4ЊC, washed and resuspended in 100 l of PBS containing a final concentration of 50 g/ml RNase A for 30 min. at room temperature. Finally, the cells were stained with 20 g/ml PI in a final volume of 300 l for 20 min. DNA content and cell cycle were analysed by flow cytometry (FACSCalibur, Becton Dickinson), using the software MODFIT and CEL-LQUEST. All of the samples were assayed in triplicate. U87 cells, which were treated with 5 g/ml Cisplatin (Sigma) for 48 hrs, were used as positive control for apoptosis as described previously [19].

Monolayer wound-healing assay
Wound-healing assay was performed as previously described with minor modification [20,21]

Transwell invasion assay
The invasiveness of cells was measured by using the Matrigel invasion assay as described previously [22].

Down-regulation of MSP58 inhibited growth of U251 cells in vitro
To investigate the possible role of MSP58 in U251 cell growth, we first analysed cellular growth of U251-S cells and U251-Smix clones. The cell growth curve showed that the growth of U251-S cells and U251-Smix were inhibited notably in a time-dependent manner (Fig. 3A) (Fig. 3C). Here a well-characterized model for drug-induced apoptosis was used as a positive control. As shown in Fig. 3C, Cisplatin (5 g/ml, 48 hrs) induced apoptosis on 24.5% of U87 cells [19]. To explore the potential contribution of Fig. 1 mRNA expression Fig. 3B). Because the cells we used were stable transfectants, we wished to confirm that this effect was due to depletion of MSP58 but not random gene insertion or long-term changes induced by altered MSP58 gene expression. Therefore, we synthesized siRNA oligonucleotides against MSP58 (siRNA-MSP58) and negative control siRNA (siRNA-NC), which have been published previously [9]. These siRNAs were transiently transfected into the glioma cell line U251. To evaluate the knockdown effects of siRNA on expression of MSP58, mRNA and protein extracted from U251 cells transiently transfected with either siRNA-NC or siRNA-MSP58 were analysed by RT-PCR and Western blot. As shown in Fig. 2D and F, siRNA-MSP58 successfully knocked down MSP58 expression, whereas siRNA-NC did not. Next, we found that, consistent with the stable transfectants, transient knockdown of MSP58 did not induce apoptosis in U251 cells (Fig. 3D). We therefore concluded that MSP58 might participate in the regulation of cell cycle and the inhibited proliferation but not apoptosis caused the reduced cell number in U251 cells with MSP58 expression reduced by RNAi. Fig. 4A-D). We then evaluated the effect of MSP58 suppression on anchorage-independent colony formation in soft agar as an additional assessment of tumourigenicity in vitro.

Fig. 2 RNA interference reduced expression of MSP58 in U251 cells. (A) Down-regulation of
MSP58 protein expression in clone U251-S1, S2 and S compared with control-transfected clone. (B) Down-regulation of MSP58 protein expression in clone U251-Smix. Clone U251-S1, S2 and S were pooled together to produce a mixture clone U251-Smix. ( MSP58 down-regulation significantly impaired anchorage-independent colony growth, with both colony number and size much reduced. In contrast, no loss of colony-forming ability was observed in either U251-NC or U251-H1 neo cells compared with parental U251cells (Fig. 4E and F)

Effects of MSP58 suppression on migration and invasion of U251 cells
Wound healing involves a number of processes, including migration and the establishment of cell polarity. We performed a wound-healing assay to study whether siRNA-mediated inhi-bition of MSP58 could influence migration of U251 cells. As shown in Fig. 5A (Fig. 5B and D).
Since tumour cell invasion is an important feature of glioma cells, we next examined whether stable shRNA expression decreases cell invasion. The Matrigel invasion assay revealed that MSP58 depletion significantly suppressed the invasiveness of U251 cells (Fig. 5E) (Fig. 5F). The number of the invaded cells declined notably in U251-S cells (P Ͻ 0.01) compared with the control groups, among which no obvious difference in invasion was observed (P Ͼ 0.05). These data demonstrate that MSP58 inhibition suppresses cell migration and invasion. H1 neo and U251-NC) were plated in 0.3% soft agar and cultured for 14 days. Colony formation was photographed under microscope (E) and scored (F). Clone numbers were mean Ϯ SD of three independent experiments. *P Ͻ 0.05 versus U251, U251-H1 neo and U251-NC control cells.

Stable knockdown of MSP58 inhibited tumour growth in vivo
To test the effect of decreased MSP58 levels in glioma cells in vivo, we extended our study to evaluate the effect of MSP58 RNAi on tumour growth. Parental U251, U251-H1 neo, U251-NC and U251-S cells were injected into nude mice (Fig. 6A). Figure 6B showed the time course of the growth of tumours initiated by different U251 transfectants. By day 24, mice injected with U251-S cells showed a statistically significant decrease in average tumour size compared with all other groups (Fig. 6C). The tumour of each mouse was removed on day 24 and weighed. MSP58 RNAi significantly decreased the solid tumour mass compared with the control groups (Fig. 6D). These data demonstrate that inhibition of MSP58 expression suppresses tumour formation in vivo. To determine whether tumour growth inhibition is caused by apoptosis, we performed a TUNEL assay to assess the number of apoptotic cells in tumour from nude mice. This assay detects free ends that are generated by apoptotic DNA cleavage and results in brown staining of apoptotic cells. As shown in Fig. 6F, compared with vehicle-treated controls, there was not a significant increase in the number of apoptotic cells. We additionally assessed the expression of Ki67 antigen, a generally

Gene expression analysis of MSP58-suppressed U251 cells
Our results and a previous study support a role for MSP58 in cell cycle regulation. To further explore the molecular mechanisms of growth inhibition caused by MSP58 suppression, we used a focused DNA microarray to assess the mRNA expression levels of cell cycle-related genes. Different gene expression profiles of the U251-S and U251-NC cells were observed (Fig. 7A)

G2/M phase in U251-S cells. These results suggest that the proliferation-inhibition effect of MSP58 shRNA on U251 cells is, at least in part, through the regulation of cell cycle.
To validate expression data obtained in the microarray analysis, the expression changes of some genes listed in Fig. 7A were tested by quantitative real-time PCR. As shown in Fig. 7B, quantitative real-time PCR results partly confirmed the regulation pattern observed on the arrays. Consistent with microarray results, expression of ATM and ATR were 1.5-and 2.4-fold greater in U251-S cells (Fig. 7B)  Expressions of other gene, such as Cyclin G1, Cyclin G2, ANAPC4 and ANAPC5, were also evaluated, and no obvious differences were found for expression of these genes between the test and control group regardless of inhibition of MSP58 in U251-S cells. Based on these data, further investigation will be performed to explore the excise mechanism of MSP58 involving in cell cycle regulation.

Discussion
MSP58 was identified as an oncogene due to its involvement in pathway of c-jun induced fibroblast cell transformation, as well as its physical interaction with the PTEN tumour suppressor, where PTEN inhibited the transformation potency of MSP58 [25]. Moreover, we found higher expression of MSP58 in nearly 29% of high-grade glioma samples as well as four glioma cell lines compared with normal brain, which suggests involvement of this gene in glioma tumourgenesis (Fig. 1). In the present study, we examined the role of MSP58 in cellular proliferation inhibition and other cellular malignant progression of glioma cells by depleting of MSP58 with a specific RNAi in U251 cells (Fig. 2). We demonstrated that depletion of MSP58 inhibited the U251 glioma cell proliferation. cDNA array analysis was also performed to try to explain the molecular mechanism under its effect on cell proliferation inhibition.
We found that specifically reducing MSP58 by RNAi negatively affected tumour cell growth in vitro (Fig. 3A). The reduced growth was the result of inhibited proliferation, not apoptosis (Fig. 3C). Because this effect could be due to long-term changes in gene expression, we also examined apoptosis in U251 cells transiently transfected with a synthetic siRNA and got the same result (Fig. 3D). These results are consistent with a previous study that overex-pression of p78, an isoform of MSP58, increased cell growth and the rate of proliferation, whereas disruption of p78 function delayed entrance into mitotic prometaphase [9]. As an isoform of MSP58, p78 has considerable homology with MSP58 at the amino acid level, and, in almost all cases, is functionally redundant. An other earlier study reported the interaction between MSP58 and p120, a proliferation-related protein expressed at high levels in most human malignant tumour cells [25], suggesting that MSP58 is involved in cell proliferation. Moreover, MSP58 is localized primarily in the nucleolus and up-regulates ribosomal gene transcription [4]. We proposed a hypothesis that inhibition of MSP58 blocked the synthesis and maturation of rRNA in glioma cells and sequencially induced nucleolar stress which caused cell cycle arrest. In agreement with this idea that ribosomal RNA synthesis and cell cycle arrest are coupled processes, we showed that MSP58 depletion reduced U251 cell proliferation and led to G1 phase cell cycle arrest (Fig. 3B). Similar results have been obtained with other nucleolar proteins [26][27][28]. Together with our previous study that MSP58 interacts with candidate tumour suppressor Ndrg2 [10], which has a similar function to the PTEN tumour suppressor, reduces the S phase cell accumulation in HeLa cells caused by MSP58 overexpression, all these data confirmed function of MSP58 in cell cycle modulation.
To further define the potential molecular mechanism underlying cell cycle arrest, we analysed genes potentially regulated by MSP58 shRNA using a cell cycle-associated gene array. Among these changed genes, tumour suppression gene, BRCA1, is a known negative regulators of cell growth [29,30]. In accordance with these observations, we found down-regulation of some proliferation-related genes, such as E2F2 and Ki67, which are used as proliferation markers in a variety of tumour types [31][32][33][34][35]. Some cell cycle-related genes, including ATM, ATR, were also increased by MSP58 shRNA treatment, all of which are known to either arrest the cell cycle or inhibit the growth of different cancer cells [36,37]. Moreover, increased expression of ANAPC2, which is responsible for the G2/M transition [38], could explain the decrease in cell percent in the G2/M phase in U251-S cells. Although, we favour the idea that depletion of MSP58 caused the cell cycle arrest by inducing nucleolar stress, we cannot role out the possibility that divergent functions might be attributed to the protein. The temporal expression profile showed that MSP58 is an S-phase-specific protein, which increased during S-phase, and decreased when the cells progressed into the G2/M phase, suggesting a potential role for MSP58 in other cellular process, such as DNA replication.
Glioma cell invasion and migration are characteristic features that distinguish them from normal cells. We therefore proposed that, as an oncoprotein, MSP58 might contribute to these tumourspecific behaviours in glioma cells. Interestingly, cell migration and invasion are inhibited in MSP58-depleted glioma cells (Fig. 5). Cell invasion is the result of degradation of extracellular matrix (ECM) proteins by specific proteases. More than one study has reported that the matrix metalloproteinase (MMP) family is activated to promote tumour cell invasion and angiogenesis [39,40].
Further studies are needed to determine the molecular mechanism where MSP58 shRNA suppresses U251 cell invasiveness.
Our results also showed that suppression of MSP58 affected the transformed morphology of the glioma cells. The inhibition of anchorage-dependent and -independent growth were observed clearly in U251-S cells compared with the control cells, which suggests that depletion of MSP58 could enhance mitogenic or survival signals (Fig. 4). Consistent with these results, a previous study showed that ectopic expression of the TOJ3 gene in quail or chicken embryo fibroblasts induced anchorage-independent growth, providing evidence that the immediate activation of TOJ3 in fibroblasts contributed to cell transformation. These results, together with the high levels of MSP58 expression found in glioma tissue, demonstrate function of MSP58 in cancer development.
Since we proved that MSP58 is important in different malignant processes involved in glioma progression in vitro, we further investigated the ability of MSP58 to affect the growth of glioma tumour xenografts in vivo. MSP58 depletion did impair the growth of glioma tumour xenografts. Given MSP58 inhibition did not induce apoptosis but specifically inhibited proliferation in vitro and in vivo (Figs 3 and 6), the inhibition of glioma growth resulting from MSP58 reduction in vivo is most likely due to decreased cell number as the result of inhibited proliferation rather than apoptosis.
Since MSP58 is up-regulated in some glioma tumours and depletion of MSP58 in U251 cells suppressed multiple tumourspecific behaviours, MSP58 could be an intriguing candidate molecular target for glioma therapy. RNAi was first discovered in the nematode Caenorhabditis elegans [41] and is conserved in a variety of organisms including plants, Drosophila and mammals.
Recently, it has been developed as a viable and more effective alternative to antisense-and ribozyme-based techniques for gene silencing, offering great potential in cancer therapeutics [42]. One advantage of RNAi in therapy is that the RNAi, once formed inside a cell, can be stable for up to a few weeks in some cell types [43]. In this study, we specifically knocked down expression of MSP58 by shRNA, and consequently impaired all the malignant processes involved in glioma progression. These results provide the biological basis for inhibition of MSP58 using RNAi as a novel therapeutic approach for glioma.
To conclude, we have shown in this study that targeted MSP58 inhibition by specific RNAi prevents proliferation and suppresses multiple tumourigenic properties of glioma cells both in vitro and in vivo. These results demonstrate great potential for developing highly specific RNAi-mediated down-regulation of MSP58 as a candidate therapeutic target for glioma.