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MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer
Biochemical and Biophysical Research Communications xxx (2016) 1e8
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MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer Suhong Xie a, b, Hui Zheng a, b, Xuemei Wen b, Jiajun Sun a, b, Yanchun Wang a, b, Xiang Gao a, b, Lin Guo a, b, Renquan Lu a, b, * a b
Department of Clinical Laboratory, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2016 Accepted 28 May 2016 Available online xxx
The dysfunction of DNA damage repair (DDR) pathway contributes to tumorigenesis and drug-resistance in cancer. MUS81 is a member of the conserved xeroderma pigmentosum group F (XPF) family protein of endonucleases, which is important to the DDR pathway. However, the role of MUS81 in the development of ovarian cancer remains uncertain. To explore the expression of MUS81 and its association to serous ovarian cancer (SOC), 43 biopsies of SOC patients were detected by qRT-PCR, and 29 specimens were further performed by immunohistochemistry analysis. Here, we observed that MUS81 was overexpressed in SOC tissues at both transcript and protein levels, and the expression level of MUS81 protein in ovarian cancer cell lines was also higher than that in human normal ovarian surface epithelial cell line (HOSEpiC). We also found that down-regulation of MUS81 expression in ovarian cancer cells inhibited cell proliferation and colony formation ability, and influenced cell cycle progression. Moreover, inhibition of MUS81 expression induced cellular senescence and enhanced the antitumor effect of cisplatin. Down-regulation of MUS81 expression could suppress the growth and development of SOC. These results indicate that MUS81 might play important roles in the progression of SOC and influence the antitumor effect of cisplatin. © 2016 Elsevier Inc. All rights reserved.
Keywords: Ovarian cancer MUS81 Proliferation Senescence Drug sensitivity
1. Introduction Ovarian cancer is the leading cause of morbidity among malignant gynecologic diseases. The most frequent subtype among ovarian cancer is serous ovarian cancer (SOC), which presents with 96% TP53 mutations and results in chromosomal instability [1,2]. The standard treatment for ovarian carcinoma is aggressive surgical debulking accompanied by cytotoxic chemotherapy. However, disease that initially responds well to this treatment frequently relapses in a more aggressive form and becomes drug resistant. Therefore, it is necessary to make further research on new therapies and treatment targets. MUS81 has been originally identified in yeast as a member of the XPF family of endonucleases [3,4], which prefers to cleave 30 flap structures with its partners EME1 or EME2 [5,6]. Further studies confirmed that the complex plays important roles in DNA
* Corresponding author. 270 DongAn Road, Xuhui District, Shanghai, 200032, China. E-mail address: [email protected] (R. Lu).
repair, including the repair and restart of stalled replication forks (RFs) [7,8], the repair of interstrand cross-links [9], and the resolution of the recombination of intermediates [10,11]. These findings have indicated that MUS81 plays a key role in the maintenance of genetic stability. Meanwhile, some works demonstrated that MUS81 interacts with checkpoint proteins, such as Checkpoint kinase 1 [12] and Checkpoint kinase 2 [13], suggesting the association between MUS81 and cell cycle progression. Interestingly, McPherson et al. constructed the first MUS81Dex34/Dex34 mouse model and showed that 73% of MUS81/ mice and 50% of MUS81/þ mice died of various spontaneous tumors, such as breast cancer and lymphoma [14], implicating MUS81 as a candidate tumor suppressor gene. However, Dendouga N and his team showed a different result in another MUS81Dex912/Dex912 mouse model [15]. They indicated that Mus81/mice and cells are hypersensitive to DNA cross-linking agents, while Mus81/mice do not show increased predisposition to lymphoma or any other malignancy. Down-regulation of MUS81 has been observed in human hepatocellular carcinoma [16] and gastric cancer [17]. However, the role of MUS81 in ovarian
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Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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S. Xie et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e8
2. Materials and methods
pathologists. The percentage of positive cells was scored as: 0, 0e5%; 1, 6e25%; 2, 26e50%; 3, 51e75%; 4, 76e100%. The intensity score was given as follows: samples with no staining were given a score of 0; samples with weak/equivocal, moderate, strong staining were given a score of 1, 2, 3, respectively. A final staining score was calculated by adding the score corresponding to the percentage of positive cells and the intensity score, and the scores ranged from 0 to 7.
2.1. Patients and tissues
2.4. Cell lines
The study was approved by the Ethics Committee of Shanghai Cancer Center, Fudan University (Certification no. 050432-41212B). Prior written informed consent was obtained from all patients. Tissues were obtained from patients who had undergone an operation at Gynecologic Surgery, Fudan University Shanghai Cancer Center (Shanghai, China) between 2013 and 2015, including 43 SOC tissues (FIGO stage Ⅱ-Ⅳ, median age 55 years) and 43 control tissues (ovary tissues obtained from 43 patients who underwent ovariohysterectomy for benign gynecological diseases, median age 58 years). All specimens were collected and frozen in liquid nitrogen immediately after surgery and then stored at 80 C until analysis. The diagnoses of all the patients were confirmed by histopathological examination.
Human ovarian cancer OVCAR3, A2780, SKOV3, HO8910, HEYA8 cells were acquired from the Chinese Academy of Sciences Committee (Shanghai, China). All ovarian cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cisplatin-resistant SKOV3 cells (SKOV3-cis) was induced in our lab using cisplatin (SigmaAldrich, MO, USA) over a period of 16 months by intermittent incremental method. Normal Human Ovarian Surface Epithelial cells (HOSEpiC) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and were cultured on poly-L-lysineecoated dishes in ovarian epithelial cell medium (ScienCell, Carlsbad, USA), supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were incubated at 37 C in a humidified atmosphere with 5% CO2.
cancer still remains unknown. In this study, we first confirmed that MUS81 transcript and protein were spontaneously over-expressed in SOC tissues. To further explore the association between MUS81 and ovarian cancer, experiments in vitro and in vivo were performed to clarify its potential functions in the development of SOC.
2.2. RNA extraction and quantitative real-time PCR (qRT-PCR) RNA was extracted from collected ovary tissues using a DNA/ RNA isolation kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. Total RNA (2 mg) was reverse transcribed into cDNA using the PrimeScript® RT reagent Kit (TaKaRa, Shiga, Japan). The qRT-PCR was performed in a 20 mL reaction solution containing 20 ng cDNA, 0.2 mmol/L primer (Table 1), and 10 mL 2 SYBR Premix Ex Taq (TaKaRa, Shiga, Japan). The PCR amplification was carried out at 95 C for 5 min, 40 cycles at 95 C for 15 sec, and 65 C for 40 sec, using the Mastercycler® ep realplex (Eppendorf, Hamburg, Germany). The abundance of the MUS81 transcript was expressed relative to the control, i.e. b-actin. 2.3. Immunohistochemical staining Immunohistochemistry (IHC) was performed using anti-MUS81 mouse monoclonal antibody, diluted 1:50 (Santa Cruz, Texas, USA). In brief, each section (4-mm-thick) was dewaxed and hydrated, followed by inhibition of endogenous peroxidase activities with methanol containing 0.3% H2O2. After antigen retrieval and cooling down, the sections were blocked with 1% BSA and incubated overnight at 4 C with primary antibody. On the second day, the sections were incubated with HRP-conjugated secondary antibody (Shanghai Long Island Biotech, Shanghai, China) for 1 h at room temperature, followed by a reaction with diaminobenzidine and counterstained with hematoxylin. The evaluation of the immunohistochemical staining was carried out double-blinded by two
2.5. Establishment of MUS81 down-regulation transduced cells To generate lentivirus expressing RNAi specific for MUS81 gene, the RNA interference sequences (Table 1) were designed with the manufacturer’s RNAi Designer program. The segments of nucleotides were cloned into the AgeІ and EcoRІ sites of the pLKO.1-puro vector (Addgene, MA, USA) to generate pLKO.1-puro-MUS81 (shMUS81-1 and shMUS81-2) and pLKO.1-puro-control (shCtrl) recombination vectors. Lentiviruses were packaged by transfecting HEK-293T cells with above lentivirus recombination vectors, as well as the packing plasmid psPAX2 and the envelop vector pMD2.G (addgene, MA, USA) using Lipofectamine 2000 transfection reagent (invitrogen, CA, USA). After 48 h, the medium containing the lentivirus particles was collected. Ovarian cancer cells were transfected with 1 106 IFU/mL of lentivirus in 8 mg/mL of polybrene (Sigma-Aldrich, MO, USA) for 24 h. Stably transduced cells were screened using 1 mg/mL puromycin (Sigma-Aldrich, MO, USA) for 3e5 days, and the knockdown efficiency of the RNAi was determined by western blot analysis. 2.6. Western blot analysis Cells were lysed for total protein extraction using RIPA lysis buffer. The primary antibodies included a mouse monoclonal antibody against human MUS81 (dilution: 1:200; Santa Cruz, Texas, USA) and a mouse monoclonal antibody against b-actin (Abcam, MA, USA). 2.7. Assessment of proliferation
Table 1 Sequences of primers and targets. Primers/targets
Sequences
MUS81 qRT-PCR forward MUS81 qRT-PCR reverse b-actin qRT-PCR forward b-actin qRT-PCR reverse shMUS81-1 shMUS81-2 shCtrl (scrambled sequence)
50 -CTGAAGCGCTGTGGTCTG-30 50 -AGTGTTGGTGACAGCCTG-3 50 - AAGGTGACAGCAGTCGGTT-30 50 - TGTGTGGACTTGGGAGAGG-30 50 -ACACTGCTGAGCACCATTAAG-30 50 -GCAGCCCTGGTGGATCGATAC-30 50 -CCTAAGGTTAAGTCGCCCTCG-30
CCK-8 (cell counting kit-8, Dojindo, Japan) cell viability assay was performed as previously described [18]. Assay was performed at 1, 2, 3, 4 days, absorbance of each well was determined with a microplate reader (Synergy H4, Bio-Tek) at a 450 nm wave length. Cell proliferation was also assessed using 5-ethynyl-2’-deoxyuridine (EdU) assay (RiboBio, Guangzhou, China) according to manufacturer’s instructions. Cells were seeded at 3 105 cells/well in 6-well plates. The following day, EdU (50 mM) was added to the plates, and the cells were cultured for an additional 2 h before
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
S. Xie et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e8
harvesting. The cells were then stained and analyzed by flow cytometry (Beckman Cytomics FC 500 BD FACSCanto II). The EdUþ cells were determined based on the negative control whose cells were stained in parallel by the panel of antibody and Apollo®643 azide but cultured without EdU before. 2.8. Colony-formation assay For the colony formation assay, 1000 cells per well were seeded into 6 well-plates and incubated for 10e14 days until visible clones appeared. Cell colony formation was stained with 0.5% crystal violet. The number of colonies was viewed under a microscope. The assays were repeated in triplicate. 2.9. Cell cycle and senescence assay For cell cycle analysis, transduced cells were harvested and fixed in 70% alcohol overnight at 4 C, then were incubated with 500 mL PI (BD Pharmingen™, USA) for 15 min in the dark. Finally, cells were analyzed by flow cytometry. For the senescence assay, b-galactosidase (SA-b-gal) activity, a characterized marker for senescence, was used to assess the status of cellular senescence. In brief, one day before the assay, the treated cells were plated in 6 well-plates, rinsed three times with PBS and fixed in 1 Fixing Solution at room temp for 15 min. The cells were then washed again as described above and stained overnight in a solution containing 1 mg/mL X-gal, 40 mM citric acid/sodium phosphate (pH 6), 5 mM potassium ferrocyanide, 150 mM NaCl and 2 mM MgCl2. The staining was then removed and viewed under a microscope. 2.10. Cisplatin sensitivity assay Single-cell suspension was prepared and seeded in 96-well plates (1 104 cells/well). After treatment with serial dilutions of cisplatin ranging from 0.8 to 100 mM for 48 h, cell viability was assessed using CCK-8 kit. The cell viability was calculated as follows: Viability of cells (%) ¼ (drug groupeblank) OD450/(no drug groupblank) OD450 100%. 2.11. In vivo experiment The animal study was approved by the Institutional Animal Care and Use Committee of Shanghai Medical College, Fudan University (LASFDI-20140187). Nude mice (BALB/c-nu, 5 weeks old) were purchased from Chinese Academy of Science, and randomly divided into two groups. For transplantation tumor experiment, a suspension of stable transduced SKOV3 cells (5 106) were injected subcutaneously into the left flank of each group. Tumor volumes were measured every 4 days using a caliper. For metastasis formation in the mouse experiments, we performed this experiment by intraperitoneal injection of transduced SKOV3 cells, and MicroMagnetic Resonance imaging system (Micro-MRI, Simens Inc., Germany) was used to determine metastasis formation. All surgery was conducted under carbon dioxide euthanasia. 2.12. Statistical analysis All statistical analyses were performed using SPSS 16.0 software. The differences of MUS81 expression in tissues between groups were analyzed using ManneWhitney U test and Fisher exact test. One-way ANOVA was performed to evaluate the results of other experiments between groups. Differences were considered significant as P < 0.05.
3
3. Results 3.1. MUS81 is up-regulated in SOC tissues and ovarian cancer cell lines To investigate the expression level of MUS81 in ovarian cancer tissues, 43 SOC tissues and 43 ovarian control tissues were enrolled. As shown in Fig 1A, MUS81 transcript level was significantly higher in SOC tissues than that in control tissues (P ¼ 0.016). We extended this observation to five ovarian cancer cell lines (HEY-A8, A2780, OVCAR3, HO8910, SKOV3) using Western blot analysis. Compared to human normal ovarian surface epithelial cell line (HOSEpiC), the protein expression of MUS81 was markedly elevated in ovarian cell lines (Fig. 1B). Then, we confirmed this finding in 15 SOC tissues and 14 control tissues by immunohistochemical staining. Table 2 summarizes the MUS81 expression in different groups. In control tissues, 8 of 14 were negative for MUS81 expression, however most of SOC tissues (12/15) showed positive to different extent (P ¼ 0.018). Examples of MUS81 expression patterns are depicted in Fig. 1C. These results showed that the MUS81 expression in SOC was increased abnormally. 3.2. Down-regulation of MUS81 inhibits cell proliferation To explore the potential functions of MUS81 in ovarian cancer cells, we down-regulated the expression of MUS81 using Lentivirus-mediated RNAi. The stable transduced SKOV3 and A2780 cells were obtained. The western blot analysis showed a satisfactory evidence of the knockdown efficiency of the RNAi (Fig. 2A). The data suggested that the stable transduced cells (shCtrl, shMUS81-1 and shMUS81-2) were successfully constructed. We then investigated the effect of MUS81 on cell proliferation. Compared with control group shCtrl, the stable transduced shMUS81-1 and shMUS81-2 cells had statistically decreased viability at 2e4 days (Fig. 2B) in both transduced SKOV3 and A2780 cells (P < 0.05). 5-Ethynyl-20 -deoxyuridine (EdU) incorporation is becoming the gold standard method for in vitro and in vivo visualization of proliferating cells [19], thus we further performed this experiment to verify the proliferation of transduced cells. As expected, compared with shCtrl group, the percentage of EdUþ cells in shMUS81-1/shMUS81-2 group decreased significantly (Fig. 2C, P < 0.05). Furthermore, we performed colony formation assays. As Fig. 2D showed, the colony number of stable transduced shMUS81-1/ shMUS81-2 cells was significantly less than that of shCtrl cells (P < 0.05). As the knockdown efficiency of shMUS81-1 was higher than that of shMUS81-2 (Fig. 2A) and the effect of shMUS81-1 on cell proliferation was also stronger, we chose transduced with shMUS81-1 (abbreviated to shMUS81 in following text) for following experiments. 3.3. Down-regulation of MUS81 induces cell cycle arrest and premature cellular senescence Stable transduced cells were analyzed by flow cytometry to determine the cell cycle progression (Fig. 3A). The results demonstrated that the stable transduced shMUS81 SKOV3 and A2780 cells were arrested in G0/G1 phase (shMUS81 vs. shCtrl: 53.17 vs. 38.67% in SKOV3 cells and 48.91 vs. 32.10% in A2780 cells, P < 0.05), and the percentage of S phase cells decreased obviously (shMUS81 vs. shCtrl: 29.13 vs. 42.30% in SKOV3 cells, P < 0.05, and 37.48 vs. 46.46% in A2780 cells, P ¼ 0.154). Given that MUS81 has been shown to be required for the survival of telomerase-negative cancer cells by alternative lengthening
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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Fig. 1. Overexpression of MUS81 in SOC tissues and ovarian cancer cells. A. The transcript level of MUS81 in SOC tissues (n ¼ 43) and control tissues (n ¼ 43) were determined by quantitative real-time PCR and normalized to the internal control b-actin gene. The bars in the figure indicate the median of the relative expression of MUS81. B. The expression level of MUS81 protein was up-regulated in five ovarian cancer cell lines (HEY-A8, A2780, OVCAR3, HO8910 and SKOV3) compared to human normal ovarian surface epithelial cells (HOSEpiC). *P < 0.05, **P < 0.01. Data are presented as the mean ± SD of three independent experiments. C. Samples with different staining intensities against MUS81 protein by immunohistochemical analysis. (a): Weak MUS81 expression in control tissue. (bed): Representative images of weak, moderate and strong MUS81 expression in SOC tissues respectively. Bar ¼ 20 mm.
Table 2 The comparison of MUS81 expression between SOC tissues and control tissues. MUS81 expression
SOC
Control
P-value
Negative Weak Moderate Strong
3 1 5 6
8 4 1 1
0.018
of the telomeres pathway [20], we investigated whether downregulation of MUS81 induces cellular senescence in SOC cells. bgalactosidase (SA-b-gal), an associated biomarker of senescence, was detected by histochemical staining of cells using the artificial substrate X-gal. SA-b-gal biomarker was confirmed to distinguish the senescent cells from quiescent cells independent of DNA synthesis [21]. As seen in Fig. 3B, knockdown of MUS81 in SKOV3 and A2780 cells increased the emergence of premature senescence cells.
3.4. Inhibition of MUS81 enhances the sensitivity to cisplatin and inhibits tumor growth in vivo To explore the impact of MUS81 on cisplatin sensitivity in ovarian cells, we performed special experiments in pair of parental SKOV3 cells and cisplatin-resistant SKOV3 cells (SKOV3-cis). As shown in Fig. 4A, the expression level of MUS81 SKOV3-cis was higher than that in parental SKOV3 cells. Then, the MUS81 expression of SKOV3-cis cells was knockdown by Lentivirusmediated RNAi, and the cell viability assay was performed to assess the cisplatin sensitivity. In Fig. 4B, after treated with various concentration of cisplatin, the cell viability of SKOV3-cis cells was obviously higher than that in parental SKOV3 cells, while after knockdown of MUS81 in SKOV3-cis (SKOV3-cis-shMUS81), the cell viability dramatically decreased. These results indicated that MUS81 was increased in cisplatin-resistant cells and might affect the sensitivity to cisplatin. In vivo experiment, stable transduced SKOV3 cells were
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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Fig. 2. Knockdown of MUS81 inhibits cell proliferation. A. The expression level of MUS81 protein in transduced SKOV3 and A2780 cells was determined by western blot assay. B. The cell viability of stable transduced SKOV3 and A2780 cells was assessed by CCK-8 assay. C. The proliferation of stable transduced SKOV3 (a) and A2780 (b) cells was analyzed using 5-ethynyl-20 -deoxyuridine (EdU) assay. D. The stable transduced SKOV3 (a) and A2780 (b) cells were assessed by colony formation assay. *P < 0.05, **P < 0.01, shMUS81-1 or shMUS81-2 vs shCtrl, data are presented as the mean ± SD of three independent experiments.
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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Fig. 3. The effect of MUS81 on cell cycle distribution and premature senescence. A. Knockdown of MUS81 in SKOV3 and A2780 cells increased the percentage of G0/G1 phase and decreased the percentage of S phase. B. Down-regulation of MUS81 in SKOV3 and A2780 cells increased the number of b-galactosidase (a marker for senescence) positive cells. *P < 0.05, shMUS81 vs shCtrl, data are presented as the mean ± SD of three replicate experiments.
transplanted in the subcutis of nude mice. As shown in Fig. 4C, compared with the shCtrl group, growth of the subcutaneous transplanted tumors in shMUS81 group decreased markedly, and tumor volume was significantly smaller. For metastasis formation in the mouse experiment, we have performanced this experiment by intraperitoneal injection. The metastasis formation was observed using Micro-MRI. As seen in Fig. 4C, compared with shCtrl
group, the size and number of liver metastasis were significantly decreased in shMUS81 group. These data suggested that downregulation of MUS81 might inhibit SOC growth and development. 4. Discussion MUS81 plays an important role in preserving genomic integrity
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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Fig. 4. Knockdown of MUS81 sensitizes SKOV3 cells to cisplatin and inhibits tumor growth. A. Expression of MUS81 in parental cells and cisplatineresistance cells (SKOV3-cis) was determined by western blot. B. The cell viability was assessed by CCK-8 assay after exposure to various concentration of cisplatin for 48 h. Data represent mean ± SD from three independent experiments. SKOV3-cis: cisplatin-resistance SKOV3 cells, SKOV3-cis-shMUS81: cisplatin-resistance cells knockdown of MUS81. C. Knockdown of MUS81 inhibited tumor growth in vivo. (a) Transplanted tumor volume was measured every four days (n ¼ 8, per group). *P < 0.05, shMUS81 vs shCtrl. (b) Representative images of transplanted tumors in both groups. Bar ¼ 1 cm. (c) Metastasis formation in the mouse experiments (intraperitoneal injection) was determined by Micro-MRI. The liver metastases were noted by red lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
by involving in DDR pathway, especially promoting homologous recombination (HR) repair and the resolution of DNA lesions [13,22,23]. However, its role in tumorigenesis is ambiguous in a few cancers. In this study, our results showed that the transcript and protein levels of MUS81 were significantly up-regulated in SOC tissues, although the samples size is limited in our study (Fig.1). Wu F and his team showed that MUS81 was down-regulated in hepatocellular carcinoma, gastric and colorectal cancer tissues [16,17,24]. At this point, the expression level of MUS81 in different cancers presents many fluctuations, suggesting that MUS81 would play versatile roles in various cancers. To further explore the function of MUS81 in ovarian cancer cells, transduced cells with down-regulation of MUS81 expression were constructed. Our data indicated that knockdown of MUS81 in ovarian cancer cells significantly inhibited proliferation and colony formation ability (Fig. 2). This finding was similar to a study on human colon cancer cell lines HCT116, which showed that the growth rate of MUS81 deficient cells was significantly lower than that of wild-type cells and these deficient cells promoted to activate
the intra-S-phase checkpoint [23]. Our results demonstrated that deficiency of MUS81 induced an accumulation of cells in G0/G1 phase (Fig. 3A), suggesting that MUS81 might play an important role in G1/S transformation. We deduced that cellular senescence may be a mechanism to prevent cells from cancerization. However, it has also been supposed that the accumulation of premature cellular senescence may conduce to persistent double strand DNA breaks, mitochondrial oxygen radical injury and abnormal proliferation [25]. Thus we investigated whether MUS81 had an effect on SOC cell senescence. Our data indicated that cells deficient in MUS81 were more likely to accelerate aging (Fig. 3B). Thus, it reveals that MUS81would involve in the mechanism of cellular senescence and abnormal proliferation. MUS81-deficient embryonic stem cells and mice were found to be hypersensitive to mitomycin C (MMC) [14], and disruption of MUS81 gene would increase the sensitivity to MMC and cisplatin in mouse embryonic fibroblasts (MEFs) [15]. Since cisplatin is a chemotherapy reagent used commonly in treatment of ovarian cancer, we investigate whether MUS81 impacts on the sensitivity to
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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cisplatin. Our data showed that the expression level of MUS81 in cisplatin-resistance cells (SKOV3/Cis) was higher than that in parental cells (Fig. 4A); on the other hand, the cell viability assay indicated that knockdown of MUS81 in SKOV3/Cis cells could markedly restore the sensitivity to cisplatin (Fig. 4B). These data suggest that down-regulation of MUS81 could enhance the sensitivity of cisplatin in ovarian cancer cells. Recently, Mus81 was reported to be associated with chemosensitivity in human malignancies such as colon cancer [22], breast cancer [26] and hepatocellular carcinoma [27]. All the data suggest that MUS81 would contribute to chemosensitization. Furthermore, in vivo experiment confirmed that down-regulation of MUS81 expression could suppress the growth and development of SOC (Fig. 4C). Therefore, we infer that MUS81 might involve the development and chemosensitivity of SOC through the cell cycle or cellular senescence pathway. In this study, our work first reveals that MUS81 is up-regulated in SOC tissues, knockdown of MUS81 inhibits the SOC cells proliferation and enhance the antitumor effect of cisplatin. Based on the above evidences, future investigations will be imperative to provide precise mechanism. Funding This work is supported by grants from the National Natural Science Foundation of China (Grant No. NSF-81572552), Shanghai Municipal Commission of Heath and Family Planning (Grant No. 20154Y0137), and the Foundation of Shanghai Cancer Center (Grant No. YJ201311). Competing interests The authors have declared that no competing interests exist. Author contributions
[6] [7]
[8]
[9]
[10]
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[12]
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[14]
[15]
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[18]
[19]
Conceived and designed the experiments: RL and LG. Performed the experiments: SX and JS. Analyzed the data: HZ and RL. Contributed reagents/materials/analysis tools: XW and XG. Wrote the paper: SX and RL.
[21]
Transparency document
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Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.05.152.
[23]
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MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer Suhong Xie a, b, Hui Zheng a, b, Xuemei Wen b, Jiajun Sun a, b, Yanchun Wang a, b, Xiang Gao a, b, Lin Guo a, b, Renquan Lu a, b, * a b
Department of Clinical Laboratory, Fudan University Shanghai Cancer Center, Shanghai, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 May 2016 Accepted 28 May 2016 Available online xxx
The dysfunction of DNA damage repair (DDR) pathway contributes to tumorigenesis and drug-resistance in cancer. MUS81 is a member of the conserved xeroderma pigmentosum group F (XPF) family protein of endonucleases, which is important to the DDR pathway. However, the role of MUS81 in the development of ovarian cancer remains uncertain. To explore the expression of MUS81 and its association to serous ovarian cancer (SOC), 43 biopsies of SOC patients were detected by qRT-PCR, and 29 specimens were further performed by immunohistochemistry analysis. Here, we observed that MUS81 was overexpressed in SOC tissues at both transcript and protein levels, and the expression level of MUS81 protein in ovarian cancer cell lines was also higher than that in human normal ovarian surface epithelial cell line (HOSEpiC). We also found that down-regulation of MUS81 expression in ovarian cancer cells inhibited cell proliferation and colony formation ability, and influenced cell cycle progression. Moreover, inhibition of MUS81 expression induced cellular senescence and enhanced the antitumor effect of cisplatin. Down-regulation of MUS81 expression could suppress the growth and development of SOC. These results indicate that MUS81 might play important roles in the progression of SOC and influence the antitumor effect of cisplatin. © 2016 Elsevier Inc. All rights reserved.
Keywords: Ovarian cancer MUS81 Proliferation Senescence Drug sensitivity
1. Introduction Ovarian cancer is the leading cause of morbidity among malignant gynecologic diseases. The most frequent subtype among ovarian cancer is serous ovarian cancer (SOC), which presents with 96% TP53 mutations and results in chromosomal instability [1,2]. The standard treatment for ovarian carcinoma is aggressive surgical debulking accompanied by cytotoxic chemotherapy. However, disease that initially responds well to this treatment frequently relapses in a more aggressive form and becomes drug resistant. Therefore, it is necessary to make further research on new therapies and treatment targets. MUS81 has been originally identified in yeast as a member of the XPF family of endonucleases [3,4], which prefers to cleave 30 flap structures with its partners EME1 or EME2 [5,6]. Further studies confirmed that the complex plays important roles in DNA
* Corresponding author. 270 DongAn Road, Xuhui District, Shanghai, 200032, China. E-mail address: [email protected] (R. Lu).
repair, including the repair and restart of stalled replication forks (RFs) [7,8], the repair of interstrand cross-links [9], and the resolution of the recombination of intermediates [10,11]. These findings have indicated that MUS81 plays a key role in the maintenance of genetic stability. Meanwhile, some works demonstrated that MUS81 interacts with checkpoint proteins, such as Checkpoint kinase 1 [12] and Checkpoint kinase 2 [13], suggesting the association between MUS81 and cell cycle progression. Interestingly, McPherson et al. constructed the first MUS81Dex34/Dex34 mouse model and showed that 73% of MUS81/ mice and 50% of MUS81/þ mice died of various spontaneous tumors, such as breast cancer and lymphoma [14], implicating MUS81 as a candidate tumor suppressor gene. However, Dendouga N and his team showed a different result in another MUS81Dex912/Dex912 mouse model [15]. They indicated that Mus81/mice and cells are hypersensitive to DNA cross-linking agents, while Mus81/mice do not show increased predisposition to lymphoma or any other malignancy. Down-regulation of MUS81 has been observed in human hepatocellular carcinoma [16] and gastric cancer [17]. However, the role of MUS81 in ovarian
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Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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2. Materials and methods
pathologists. The percentage of positive cells was scored as: 0, 0e5%; 1, 6e25%; 2, 26e50%; 3, 51e75%; 4, 76e100%. The intensity score was given as follows: samples with no staining were given a score of 0; samples with weak/equivocal, moderate, strong staining were given a score of 1, 2, 3, respectively. A final staining score was calculated by adding the score corresponding to the percentage of positive cells and the intensity score, and the scores ranged from 0 to 7.
2.1. Patients and tissues
2.4. Cell lines
The study was approved by the Ethics Committee of Shanghai Cancer Center, Fudan University (Certification no. 050432-41212B). Prior written informed consent was obtained from all patients. Tissues were obtained from patients who had undergone an operation at Gynecologic Surgery, Fudan University Shanghai Cancer Center (Shanghai, China) between 2013 and 2015, including 43 SOC tissues (FIGO stage Ⅱ-Ⅳ, median age 55 years) and 43 control tissues (ovary tissues obtained from 43 patients who underwent ovariohysterectomy for benign gynecological diseases, median age 58 years). All specimens were collected and frozen in liquid nitrogen immediately after surgery and then stored at 80 C until analysis. The diagnoses of all the patients were confirmed by histopathological examination.
Human ovarian cancer OVCAR3, A2780, SKOV3, HO8910, HEYA8 cells were acquired from the Chinese Academy of Sciences Committee (Shanghai, China). All ovarian cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cisplatin-resistant SKOV3 cells (SKOV3-cis) was induced in our lab using cisplatin (SigmaAldrich, MO, USA) over a period of 16 months by intermittent incremental method. Normal Human Ovarian Surface Epithelial cells (HOSEpiC) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and were cultured on poly-L-lysineecoated dishes in ovarian epithelial cell medium (ScienCell, Carlsbad, USA), supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were incubated at 37 C in a humidified atmosphere with 5% CO2.
cancer still remains unknown. In this study, we first confirmed that MUS81 transcript and protein were spontaneously over-expressed in SOC tissues. To further explore the association between MUS81 and ovarian cancer, experiments in vitro and in vivo were performed to clarify its potential functions in the development of SOC.
2.2. RNA extraction and quantitative real-time PCR (qRT-PCR) RNA was extracted from collected ovary tissues using a DNA/ RNA isolation kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. Total RNA (2 mg) was reverse transcribed into cDNA using the PrimeScript® RT reagent Kit (TaKaRa, Shiga, Japan). The qRT-PCR was performed in a 20 mL reaction solution containing 20 ng cDNA, 0.2 mmol/L primer (Table 1), and 10 mL 2 SYBR Premix Ex Taq (TaKaRa, Shiga, Japan). The PCR amplification was carried out at 95 C for 5 min, 40 cycles at 95 C for 15 sec, and 65 C for 40 sec, using the Mastercycler® ep realplex (Eppendorf, Hamburg, Germany). The abundance of the MUS81 transcript was expressed relative to the control, i.e. b-actin. 2.3. Immunohistochemical staining Immunohistochemistry (IHC) was performed using anti-MUS81 mouse monoclonal antibody, diluted 1:50 (Santa Cruz, Texas, USA). In brief, each section (4-mm-thick) was dewaxed and hydrated, followed by inhibition of endogenous peroxidase activities with methanol containing 0.3% H2O2. After antigen retrieval and cooling down, the sections were blocked with 1% BSA and incubated overnight at 4 C with primary antibody. On the second day, the sections were incubated with HRP-conjugated secondary antibody (Shanghai Long Island Biotech, Shanghai, China) for 1 h at room temperature, followed by a reaction with diaminobenzidine and counterstained with hematoxylin. The evaluation of the immunohistochemical staining was carried out double-blinded by two
2.5. Establishment of MUS81 down-regulation transduced cells To generate lentivirus expressing RNAi specific for MUS81 gene, the RNA interference sequences (Table 1) were designed with the manufacturer’s RNAi Designer program. The segments of nucleotides were cloned into the AgeІ and EcoRІ sites of the pLKO.1-puro vector (Addgene, MA, USA) to generate pLKO.1-puro-MUS81 (shMUS81-1 and shMUS81-2) and pLKO.1-puro-control (shCtrl) recombination vectors. Lentiviruses were packaged by transfecting HEK-293T cells with above lentivirus recombination vectors, as well as the packing plasmid psPAX2 and the envelop vector pMD2.G (addgene, MA, USA) using Lipofectamine 2000 transfection reagent (invitrogen, CA, USA). After 48 h, the medium containing the lentivirus particles was collected. Ovarian cancer cells were transfected with 1 106 IFU/mL of lentivirus in 8 mg/mL of polybrene (Sigma-Aldrich, MO, USA) for 24 h. Stably transduced cells were screened using 1 mg/mL puromycin (Sigma-Aldrich, MO, USA) for 3e5 days, and the knockdown efficiency of the RNAi was determined by western blot analysis. 2.6. Western blot analysis Cells were lysed for total protein extraction using RIPA lysis buffer. The primary antibodies included a mouse monoclonal antibody against human MUS81 (dilution: 1:200; Santa Cruz, Texas, USA) and a mouse monoclonal antibody against b-actin (Abcam, MA, USA). 2.7. Assessment of proliferation
Table 1 Sequences of primers and targets. Primers/targets
Sequences
MUS81 qRT-PCR forward MUS81 qRT-PCR reverse b-actin qRT-PCR forward b-actin qRT-PCR reverse shMUS81-1 shMUS81-2 shCtrl (scrambled sequence)
50 -CTGAAGCGCTGTGGTCTG-30 50 -AGTGTTGGTGACAGCCTG-3 50 - AAGGTGACAGCAGTCGGTT-30 50 - TGTGTGGACTTGGGAGAGG-30 50 -ACACTGCTGAGCACCATTAAG-30 50 -GCAGCCCTGGTGGATCGATAC-30 50 -CCTAAGGTTAAGTCGCCCTCG-30
CCK-8 (cell counting kit-8, Dojindo, Japan) cell viability assay was performed as previously described [18]. Assay was performed at 1, 2, 3, 4 days, absorbance of each well was determined with a microplate reader (Synergy H4, Bio-Tek) at a 450 nm wave length. Cell proliferation was also assessed using 5-ethynyl-2’-deoxyuridine (EdU) assay (RiboBio, Guangzhou, China) according to manufacturer’s instructions. Cells were seeded at 3 105 cells/well in 6-well plates. The following day, EdU (50 mM) was added to the plates, and the cells were cultured for an additional 2 h before
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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harvesting. The cells were then stained and analyzed by flow cytometry (Beckman Cytomics FC 500 BD FACSCanto II). The EdUþ cells were determined based on the negative control whose cells were stained in parallel by the panel of antibody and Apollo®643 azide but cultured without EdU before. 2.8. Colony-formation assay For the colony formation assay, 1000 cells per well were seeded into 6 well-plates and incubated for 10e14 days until visible clones appeared. Cell colony formation was stained with 0.5% crystal violet. The number of colonies was viewed under a microscope. The assays were repeated in triplicate. 2.9. Cell cycle and senescence assay For cell cycle analysis, transduced cells were harvested and fixed in 70% alcohol overnight at 4 C, then were incubated with 500 mL PI (BD Pharmingen™, USA) for 15 min in the dark. Finally, cells were analyzed by flow cytometry. For the senescence assay, b-galactosidase (SA-b-gal) activity, a characterized marker for senescence, was used to assess the status of cellular senescence. In brief, one day before the assay, the treated cells were plated in 6 well-plates, rinsed three times with PBS and fixed in 1 Fixing Solution at room temp for 15 min. The cells were then washed again as described above and stained overnight in a solution containing 1 mg/mL X-gal, 40 mM citric acid/sodium phosphate (pH 6), 5 mM potassium ferrocyanide, 150 mM NaCl and 2 mM MgCl2. The staining was then removed and viewed under a microscope. 2.10. Cisplatin sensitivity assay Single-cell suspension was prepared and seeded in 96-well plates (1 104 cells/well). After treatment with serial dilutions of cisplatin ranging from 0.8 to 100 mM for 48 h, cell viability was assessed using CCK-8 kit. The cell viability was calculated as follows: Viability of cells (%) ¼ (drug groupeblank) OD450/(no drug groupblank) OD450 100%. 2.11. In vivo experiment The animal study was approved by the Institutional Animal Care and Use Committee of Shanghai Medical College, Fudan University (LASFDI-20140187). Nude mice (BALB/c-nu, 5 weeks old) were purchased from Chinese Academy of Science, and randomly divided into two groups. For transplantation tumor experiment, a suspension of stable transduced SKOV3 cells (5 106) were injected subcutaneously into the left flank of each group. Tumor volumes were measured every 4 days using a caliper. For metastasis formation in the mouse experiments, we performed this experiment by intraperitoneal injection of transduced SKOV3 cells, and MicroMagnetic Resonance imaging system (Micro-MRI, Simens Inc., Germany) was used to determine metastasis formation. All surgery was conducted under carbon dioxide euthanasia. 2.12. Statistical analysis All statistical analyses were performed using SPSS 16.0 software. The differences of MUS81 expression in tissues between groups were analyzed using ManneWhitney U test and Fisher exact test. One-way ANOVA was performed to evaluate the results of other experiments between groups. Differences were considered significant as P < 0.05.
3
3. Results 3.1. MUS81 is up-regulated in SOC tissues and ovarian cancer cell lines To investigate the expression level of MUS81 in ovarian cancer tissues, 43 SOC tissues and 43 ovarian control tissues were enrolled. As shown in Fig 1A, MUS81 transcript level was significantly higher in SOC tissues than that in control tissues (P ¼ 0.016). We extended this observation to five ovarian cancer cell lines (HEY-A8, A2780, OVCAR3, HO8910, SKOV3) using Western blot analysis. Compared to human normal ovarian surface epithelial cell line (HOSEpiC), the protein expression of MUS81 was markedly elevated in ovarian cell lines (Fig. 1B). Then, we confirmed this finding in 15 SOC tissues and 14 control tissues by immunohistochemical staining. Table 2 summarizes the MUS81 expression in different groups. In control tissues, 8 of 14 were negative for MUS81 expression, however most of SOC tissues (12/15) showed positive to different extent (P ¼ 0.018). Examples of MUS81 expression patterns are depicted in Fig. 1C. These results showed that the MUS81 expression in SOC was increased abnormally. 3.2. Down-regulation of MUS81 inhibits cell proliferation To explore the potential functions of MUS81 in ovarian cancer cells, we down-regulated the expression of MUS81 using Lentivirus-mediated RNAi. The stable transduced SKOV3 and A2780 cells were obtained. The western blot analysis showed a satisfactory evidence of the knockdown efficiency of the RNAi (Fig. 2A). The data suggested that the stable transduced cells (shCtrl, shMUS81-1 and shMUS81-2) were successfully constructed. We then investigated the effect of MUS81 on cell proliferation. Compared with control group shCtrl, the stable transduced shMUS81-1 and shMUS81-2 cells had statistically decreased viability at 2e4 days (Fig. 2B) in both transduced SKOV3 and A2780 cells (P < 0.05). 5-Ethynyl-20 -deoxyuridine (EdU) incorporation is becoming the gold standard method for in vitro and in vivo visualization of proliferating cells [19], thus we further performed this experiment to verify the proliferation of transduced cells. As expected, compared with shCtrl group, the percentage of EdUþ cells in shMUS81-1/shMUS81-2 group decreased significantly (Fig. 2C, P < 0.05). Furthermore, we performed colony formation assays. As Fig. 2D showed, the colony number of stable transduced shMUS81-1/ shMUS81-2 cells was significantly less than that of shCtrl cells (P < 0.05). As the knockdown efficiency of shMUS81-1 was higher than that of shMUS81-2 (Fig. 2A) and the effect of shMUS81-1 on cell proliferation was also stronger, we chose transduced with shMUS81-1 (abbreviated to shMUS81 in following text) for following experiments. 3.3. Down-regulation of MUS81 induces cell cycle arrest and premature cellular senescence Stable transduced cells were analyzed by flow cytometry to determine the cell cycle progression (Fig. 3A). The results demonstrated that the stable transduced shMUS81 SKOV3 and A2780 cells were arrested in G0/G1 phase (shMUS81 vs. shCtrl: 53.17 vs. 38.67% in SKOV3 cells and 48.91 vs. 32.10% in A2780 cells, P < 0.05), and the percentage of S phase cells decreased obviously (shMUS81 vs. shCtrl: 29.13 vs. 42.30% in SKOV3 cells, P < 0.05, and 37.48 vs. 46.46% in A2780 cells, P ¼ 0.154). Given that MUS81 has been shown to be required for the survival of telomerase-negative cancer cells by alternative lengthening
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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Fig. 1. Overexpression of MUS81 in SOC tissues and ovarian cancer cells. A. The transcript level of MUS81 in SOC tissues (n ¼ 43) and control tissues (n ¼ 43) were determined by quantitative real-time PCR and normalized to the internal control b-actin gene. The bars in the figure indicate the median of the relative expression of MUS81. B. The expression level of MUS81 protein was up-regulated in five ovarian cancer cell lines (HEY-A8, A2780, OVCAR3, HO8910 and SKOV3) compared to human normal ovarian surface epithelial cells (HOSEpiC). *P < 0.05, **P < 0.01. Data are presented as the mean ± SD of three independent experiments. C. Samples with different staining intensities against MUS81 protein by immunohistochemical analysis. (a): Weak MUS81 expression in control tissue. (bed): Representative images of weak, moderate and strong MUS81 expression in SOC tissues respectively. Bar ¼ 20 mm.
Table 2 The comparison of MUS81 expression between SOC tissues and control tissues. MUS81 expression
SOC
Control
P-value
Negative Weak Moderate Strong
3 1 5 6
8 4 1 1
0.018
of the telomeres pathway [20], we investigated whether downregulation of MUS81 induces cellular senescence in SOC cells. bgalactosidase (SA-b-gal), an associated biomarker of senescence, was detected by histochemical staining of cells using the artificial substrate X-gal. SA-b-gal biomarker was confirmed to distinguish the senescent cells from quiescent cells independent of DNA synthesis [21]. As seen in Fig. 3B, knockdown of MUS81 in SKOV3 and A2780 cells increased the emergence of premature senescence cells.
3.4. Inhibition of MUS81 enhances the sensitivity to cisplatin and inhibits tumor growth in vivo To explore the impact of MUS81 on cisplatin sensitivity in ovarian cells, we performed special experiments in pair of parental SKOV3 cells and cisplatin-resistant SKOV3 cells (SKOV3-cis). As shown in Fig. 4A, the expression level of MUS81 SKOV3-cis was higher than that in parental SKOV3 cells. Then, the MUS81 expression of SKOV3-cis cells was knockdown by Lentivirusmediated RNAi, and the cell viability assay was performed to assess the cisplatin sensitivity. In Fig. 4B, after treated with various concentration of cisplatin, the cell viability of SKOV3-cis cells was obviously higher than that in parental SKOV3 cells, while after knockdown of MUS81 in SKOV3-cis (SKOV3-cis-shMUS81), the cell viability dramatically decreased. These results indicated that MUS81 was increased in cisplatin-resistant cells and might affect the sensitivity to cisplatin. In vivo experiment, stable transduced SKOV3 cells were
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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Fig. 2. Knockdown of MUS81 inhibits cell proliferation. A. The expression level of MUS81 protein in transduced SKOV3 and A2780 cells was determined by western blot assay. B. The cell viability of stable transduced SKOV3 and A2780 cells was assessed by CCK-8 assay. C. The proliferation of stable transduced SKOV3 (a) and A2780 (b) cells was analyzed using 5-ethynyl-20 -deoxyuridine (EdU) assay. D. The stable transduced SKOV3 (a) and A2780 (b) cells were assessed by colony formation assay. *P < 0.05, **P < 0.01, shMUS81-1 or shMUS81-2 vs shCtrl, data are presented as the mean ± SD of three independent experiments.
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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Fig. 3. The effect of MUS81 on cell cycle distribution and premature senescence. A. Knockdown of MUS81 in SKOV3 and A2780 cells increased the percentage of G0/G1 phase and decreased the percentage of S phase. B. Down-regulation of MUS81 in SKOV3 and A2780 cells increased the number of b-galactosidase (a marker for senescence) positive cells. *P < 0.05, shMUS81 vs shCtrl, data are presented as the mean ± SD of three replicate experiments.
transplanted in the subcutis of nude mice. As shown in Fig. 4C, compared with the shCtrl group, growth of the subcutaneous transplanted tumors in shMUS81 group decreased markedly, and tumor volume was significantly smaller. For metastasis formation in the mouse experiment, we have performanced this experiment by intraperitoneal injection. The metastasis formation was observed using Micro-MRI. As seen in Fig. 4C, compared with shCtrl
group, the size and number of liver metastasis were significantly decreased in shMUS81 group. These data suggested that downregulation of MUS81 might inhibit SOC growth and development. 4. Discussion MUS81 plays an important role in preserving genomic integrity
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Fig. 4. Knockdown of MUS81 sensitizes SKOV3 cells to cisplatin and inhibits tumor growth. A. Expression of MUS81 in parental cells and cisplatineresistance cells (SKOV3-cis) was determined by western blot. B. The cell viability was assessed by CCK-8 assay after exposure to various concentration of cisplatin for 48 h. Data represent mean ± SD from three independent experiments. SKOV3-cis: cisplatin-resistance SKOV3 cells, SKOV3-cis-shMUS81: cisplatin-resistance cells knockdown of MUS81. C. Knockdown of MUS81 inhibited tumor growth in vivo. (a) Transplanted tumor volume was measured every four days (n ¼ 8, per group). *P < 0.05, shMUS81 vs shCtrl. (b) Representative images of transplanted tumors in both groups. Bar ¼ 1 cm. (c) Metastasis formation in the mouse experiments (intraperitoneal injection) was determined by Micro-MRI. The liver metastases were noted by red lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
by involving in DDR pathway, especially promoting homologous recombination (HR) repair and the resolution of DNA lesions [13,22,23]. However, its role in tumorigenesis is ambiguous in a few cancers. In this study, our results showed that the transcript and protein levels of MUS81 were significantly up-regulated in SOC tissues, although the samples size is limited in our study (Fig.1). Wu F and his team showed that MUS81 was down-regulated in hepatocellular carcinoma, gastric and colorectal cancer tissues [16,17,24]. At this point, the expression level of MUS81 in different cancers presents many fluctuations, suggesting that MUS81 would play versatile roles in various cancers. To further explore the function of MUS81 in ovarian cancer cells, transduced cells with down-regulation of MUS81 expression were constructed. Our data indicated that knockdown of MUS81 in ovarian cancer cells significantly inhibited proliferation and colony formation ability (Fig. 2). This finding was similar to a study on human colon cancer cell lines HCT116, which showed that the growth rate of MUS81 deficient cells was significantly lower than that of wild-type cells and these deficient cells promoted to activate
the intra-S-phase checkpoint [23]. Our results demonstrated that deficiency of MUS81 induced an accumulation of cells in G0/G1 phase (Fig. 3A), suggesting that MUS81 might play an important role in G1/S transformation. We deduced that cellular senescence may be a mechanism to prevent cells from cancerization. However, it has also been supposed that the accumulation of premature cellular senescence may conduce to persistent double strand DNA breaks, mitochondrial oxygen radical injury and abnormal proliferation [25]. Thus we investigated whether MUS81 had an effect on SOC cell senescence. Our data indicated that cells deficient in MUS81 were more likely to accelerate aging (Fig. 3B). Thus, it reveals that MUS81would involve in the mechanism of cellular senescence and abnormal proliferation. MUS81-deficient embryonic stem cells and mice were found to be hypersensitive to mitomycin C (MMC) [14], and disruption of MUS81 gene would increase the sensitivity to MMC and cisplatin in mouse embryonic fibroblasts (MEFs) [15]. Since cisplatin is a chemotherapy reagent used commonly in treatment of ovarian cancer, we investigate whether MUS81 impacts on the sensitivity to
Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152
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cisplatin. Our data showed that the expression level of MUS81 in cisplatin-resistance cells (SKOV3/Cis) was higher than that in parental cells (Fig. 4A); on the other hand, the cell viability assay indicated that knockdown of MUS81 in SKOV3/Cis cells could markedly restore the sensitivity to cisplatin (Fig. 4B). These data suggest that down-regulation of MUS81 could enhance the sensitivity of cisplatin in ovarian cancer cells. Recently, Mus81 was reported to be associated with chemosensitivity in human malignancies such as colon cancer [22], breast cancer [26] and hepatocellular carcinoma [27]. All the data suggest that MUS81 would contribute to chemosensitization. Furthermore, in vivo experiment confirmed that down-regulation of MUS81 expression could suppress the growth and development of SOC (Fig. 4C). Therefore, we infer that MUS81 might involve the development and chemosensitivity of SOC through the cell cycle or cellular senescence pathway. In this study, our work first reveals that MUS81 is up-regulated in SOC tissues, knockdown of MUS81 inhibits the SOC cells proliferation and enhance the antitumor effect of cisplatin. Based on the above evidences, future investigations will be imperative to provide precise mechanism. Funding This work is supported by grants from the National Natural Science Foundation of China (Grant No. NSF-81572552), Shanghai Municipal Commission of Heath and Family Planning (Grant No. 20154Y0137), and the Foundation of Shanghai Cancer Center (Grant No. YJ201311). Competing interests The authors have declared that no competing interests exist. Author contributions
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Conceived and designed the experiments: RL and LG. Performed the experiments: SX and JS. Analyzed the data: HZ and RL. Contributed reagents/materials/analysis tools: XW and XG. Wrote the paper: SX and RL.
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Transparency document
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Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.05.152.
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Please cite this article in press as: S. Xie, et al., MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.152