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RAP80 expression in breast cancer and its relationship with apoptosis in breast cancer cells

Background RAP80 is a member of BRCA1-A complex, which plays an important role in regulating the cell cycle checkpoint and DNA damage repair in the nucleus. Method We investigated RAP80 expression in breast cancer and its paired normal breast tissues to further analyze its role in the biological behavior of breast cancer cells. Results RAP80 expression in breast cancer (62.3%, 101/162) was significantly lower than that in adjacent normal breast tissues (P<0.05). RAP80 expression was related to tumor size, lymph node metastasis, TNM stage, and molecular subtype (P<0.05). RAP80 mRNA expression was significantly lower in triple-negative breast cancer than other types. The mRNA and protein of RAP80 were obvious in MCF-7 and very weak in ZR-75 or MDA-MB-231, so we picked MCF-7 to be transfected with RAP80 siRNA. The survival rate of both cells decreased in a dose-dependent manner and the IC50 value for cisplatin in MCF-7 RAP80 siRNA cells was 0.83 µg/mL, and 1.69 µg/mL in wild-type MCF-7 according to MTT. RAP80 siRNA transfection upregulated the apoptosis and downregulated invasive or migrating ability of MCF-7. RAP80 siRNA also upregulated the protein expression of Caspase-3, cleaved Caspase-3, Apaf-1, Cytochrome C, Bax, and Fas, and downregulated the protein expression of Bcl-2. Conclusion RAP80 expression was related to ER or PR activity. Inhibition of RAP80 expression can induce apoptosis in breast cancer cells and improve chemosensitivity to cisplatin. Tumor cells can activate protective responses to inhibit cell cycle progression, which may be related to RAP80, and repair cisplatin-induced DNA damage. RAP80 is related to BRCA1’s effect, which can be used as an interesting target for pharmacological modulation that can increase the efficiency of cisplatin chemotherapy.


Introduction
RAP80 is a member of BRCA1-A complex, which regulates the cell cycle checkpoint and DNA damage repair in the nucleus. 1 DNA damage, mutations of tumor suppressor genes, and defects in DNA damage response (DDR) are critical events in carcinogenesis. 2,3 RAP80 is a key regulator of DDR, which is crucial for the recruitment of BRCA1 complex in DDR. 4 Breast cancer carcinogenesis and advancement associated with DDR, and most of the breast cancer susceptibility genes are involved in DNA repair. 5,6 RAP80 works upstream of BRCA1 and is essential for the localization of BRCA1 to the site of damaged DNA. A previous study indicated that RAP80 and BRCA1 mRNA expression can be predictive markers in advanced non-small-cell lung cancer patients treated with platinum-based chemotherapy. 7 What about RAP80's role in breast cancer? In the present study, we sought to investigate the expression of RAP80 in invasive breast cancer and its paired normal breast tissues, and further analyzed its role in the biological behavior of breast cancer cells.
Fresh breast tissue samples were collected from Department of Breast Surgery, the First Affiliated Hospital of China Medical University between February 2010 and September 2011, including ductal carcinoma in situ, invasive breast ductal cancer tissues and its paired normal breast tissues (with .2 cm distance from the primary cancer tissue). None of the patients underwent chemotherapy, radiotherapy or adjuvant treatment before surgery. Patients' ages ranged from 24 to 79, with an average age of 51.8 years old. Clinicopathological information was reviewed using the hospital medical records. All patients enrolled in this study signed an informed consent form agreeing to participate in this study and for publication of the results. The study protocol was reviewed and approved by the ethics committee of China Medical University (Shenyang, People's Republic of China) and the participating hospital.
immunohistochemistry Formalin-fixed paraffin-embedded specimens (4 µm thick) were incubated with RPA80 monoclonal antibody (no 14466, diluted 1:200, Cell Signaling Technology, Danvers, MA, USA). Immunohistochemical staining was performed using UltraSensitiveTM S-P kits (Maixin-Bio, Fujian, People's Republic of China) according to the manufacturer's instructions and using the reagent supplied with the kit. PBS was used in place of the primary antibodies for negative control. The staining intensity and area extent were scored by the German semiquantitative scoring system as previously described. 8 Breast cancer cell lines and cell culture conditions Breast cancer cell lines including MCF-7 (chemotherapy responsive, ER+, PR+/-, Her-2-, Ki67 low), ZR-75 (usually endocrine responsive, variable response to chemotherapy, ER+, PR+/-, HER2+, Ki67 high), and MDA-MB-231 (intermediate response to chemotherapy, ER-, PR-, HER2-) were chosen for this study and maintained under recommended culture conditions. They were obtained from the American Type Culture Collection (Manassas, VA, USA) and stored in the laboratory of Pathology Department, the First Affiliated Hospital and College of Basic Medical Sciences of China Medical University (Shenyang, People's Republic of China). All the cells were cultured in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) or DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (Sigma-Aldrich, Buchs, Switzerland) in a 5% CO 2 humidified atmosphere at 37°C.
Western blot and quantitative real-time polymerase chain reaction (qrT-Pcr) analysis of raP80 in breast cancer cell lines or breast tissues The tissue or cells of breast were washed with ice-cold PBS and then lysed in lysis buffer containing 10 mM Tris (pH 7.5), 1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate, and a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/mL pepstatin A, 2 µg/mL aprotinin). The lysates were sonicated for 10 seconds, centrifuged for 20 minutes at 20,000× g and then stored at -70°C. Equal amounts (25 µg) of the cell lysates were resolved by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking, blots were incubated with mouse anti-RAP80 monoclonal antibody (no 14466, diluted 1:400, Cellsignal) or β-actin (1:1,000, Zhongshan Golden Bridge Biotechnology, Beijing, People's Republic of China) overnight at 4°C, followed by each corresponding second antibody at room temperature for 1 hour at 37°C. Then, the results were obtained by enhanced chemiluminescence (Pierce Biotechnology, Waltham, MA, USA). The protein bands were then analyzed using the BioImaging System (UVP, Upland, CA, USA). The grayscale values of the RAP80 were normalized to the values of the corresponding β-actin band to determine the expression level of the protein. The experiments were repeated at least three times independently. Total RNA from breast cancer cell lines was extracted with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and the quality of RNA was analyzed by A260/A280 ratio by Nano-Photometer (IMPIEN, Gttingen, Germany). The ratios were between 1.6 and 1.8. The reverse transcription was performed with RNA PCR Kit (AMV Ver.3.0, Takara, Kyoto, Japan) according to the manufacturer's protocols. qRT-PCR was performed by SYBR ® Premix Ex TaqTM II Kit (Takara) using 7500 Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific). An amount of 2 µL template cDNA was added to the final volume of 20 µL of reaction mixture. Expression of the selected genes was normalized to GADPH, which was used as an internal housekeeping control. The experiments were repeated at least three times independently. The results showed that mRNA and protein expression of RAP80 were obvious in MCF-7 and very weak in ZR-75 or MDA-MB-231. So we picked MCF-7 to be transfected with RAP80 siRNA, to further investigate the role of RAP80 in MCF-7.

raP80 sirna transfection
The human RAP80 gene sequence was obtained from GeneBank. According to the design principle of siRNA, an siRNA Target Designer (GenePharma, Shanghai, People's Republic of China) was used to design siRNAs and synthesized targeting the specific RAP80. The MCF-7 cells were cultured in a 24-well plate for 24 hours before the experiment of transient transfection. The cells were transfected with Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Following the transfection, the cells were harvested at 48 hours to measure the protein levels. Control RAP80 siRNA (sc-92007, 1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and negative control siRNA (sc-36869, 1:500, Santa Cruz Biotechnology Inc.) were used in Western blot analysis to verify the downregulation of RAP80. A control study using non-specific siRNA was carried out under the same conditions.

MTT assay
Cell proliferation was assessed at various time points by MTT assay. Briefly, 2,000 cells were seeded in each well of a 96-well plate (eight repeats) and allowed to adhere for 8 hours. Then, 5 mg/mL MTT (Sigma-Aldrich Co., St Louis, MO, USA) was added to each well and incubated for 24 hours. Then, cells were further treated with different concentrations of cisplatin for 72 hours. The cells were lysed by adding 150 µL/well of dimethyl sulfoxide and absorbance was read at 570 nm wavelength in a microplate reader. The experiments were repeated at least three times independently. IC50 values were calculated for cisplatin from the respective sigmoidal dose-response curves.

Flow cytometry apoptosis assay
Analysis of apoptosis was carried out using an Annexin V-FITC/propidium iodide (PI) double staining kit (Beyotime, Nanjing, People's Republic of China) following the manufacturer's protocols. In brief, each group was plated in six wells. An amount of 1 µM of brivudine was treated. Cells were continuously cultured for 48 hours and 72 hours, and then harvested. Before flow cytometry analysis, cell suspensions were washed in PBS, resuspended with a 1× binding buffer, and exposed to 5 µL of Annexin V-FITC (20 µg/mL) and 10 µL of PI (50 µg/mL). After incubation for 20 minutes in the dark, the samples were subjected to FACScan flow cytometry (CellQuest and ModFITLT for Mac V1.01 software [BD Biosciences, San Jose, CA, USA]). The experiments were repeated at least three times independently.

Matrigel invasion assay
Cell invasive ability was examined using a 24-well Transwell with 8.0 µm pore polycarbonate membrane inserts (Corning Incorporated, Corning, NY, USA) according to the manufacturer's protocol. The Matrigel (100 µL/mL) was applied to the upper surface of the membranes. After the transfection for 48 hours and 72 hours, cells of each group were seeded on the upper chamber (5×104 cells/well) and incubated for 18 hours. Cells that had invaded the surface of the membrane were fixed with methanol and stained with hematoxylin. The cells that invaded and moved onto the lower surface of the filter membrane were counted in ten random high-power fields (400×) by an inverted microscope. The experiment was repeated five times and the data were shown as mean ± SD. statistical analyses SPSS version 13.0 for Windows was used for all analyses. The Pearson's chi-squared test was used to analyze the relationship between RAP80 expression with clinicopathological factors in breast cancer. One-way ANOVA was performed to compare data from the densitometry analysis of MTT, flow cytometry analysis, Western blotting, RT-PCRq, and Matrigel invasion assay. Statistical significance in this study was set at P,0.05. All reported P-values are two-sided.

Result expression of raP80 in invasive ductal breast cancer tissues and paired normal breast tissues by immunohistochemistry
The results of immunohistochemistry revealed mainly nucleus staining of the RAP80 protein in the mammary epithelium of paired normal breast tissues, and the breast cancer cells showed staining on the nucleus (Figure 1). Total positive rate of RAP80 expression was 62.3% in invasive ductal breast cancer, whereas it was 86.75% in its paired normal breast tissues. RAP80 expression in breast cancer (62.3%, 101/162) was significantly lower than that in adjacent normal breast tissues (P,0.05). The relationship between RAP80 expression and different clinicopathological factors in breast cancer is shown in Table 1

expression of raP80 mrna expression in invasive ductal breast cancer by qrT-Pcr
The results of qRT-PCR revealed that RAP80 mRNA was significantly lower in triple-negative breast cancer than other types, as shown in Table 2. RAP80 mRNA expression was correlated with ER or PR expression, lymph node metastasis, and Ki67 status (P.0.05). RAP80 mRNA expression was not associated with age, TNM stage, C-erbB-2 status or histology grade (P.0.05).
The raP80 expression in breast cancer cell lines and raP80 sirna in McF-7 As shown in Figure 2, the mRNA and protein expression of RAP80 were obvious in MCF-7 and very weak in ZR-75 or MDA-MB-231 ( Figure 2). So we picked MCF-7 to be transfected with RAP80 siRNA, and to further investigate the role of RAP80 in MCF-7's biological behavior. Using Western blot and RT-PCRq analysis, RAP80 protein and mRNA expression was obviously observed in RAP80 siRNA-normal control (NC) transfection group or wild-type MCF-7, and it was very weak in RAP80 siRNA transfection group ( Figure 2). This indicates the effective downregulation of RAP80 in MCF-7 by RAP80 siRNA.

effect of raP80 sirna transfection on cell proliferation by MTT
The IC50 value of cisplatin in wild-type MCF-7 or MCF-7 RAP80 siRNA was shown in Figure 3. The survival rate of both cell lines decreased in a dose-dependent manner and the IC50 value for cisplatin in MCF-7 RAP80 siRNA cells was 0.83 µg/mL, and 1.69 µg/mL in wild-type MCF-7.  group was 46.8% at 48 hours and 52.7% at 72 hours after transfection, which was higher than the siRNA-NC transfection group (13.1% at 48 hours after transfection, 10.7% at 72 hours after transfection) or the group of MCF-7 without treatment (15.1% at 48 hours after transfection, 12.9% at 72 hours after transfection) (P,0.05).

raP80 sirna transfection decreased invasive ability of McF-7 cells
Matrigel invasion and migration assays showed that the invasive and migrating ability of the MCF-7 cells was decreased with RAP80 siRNA transfection after the treatment for 72 hours compared to that in MCF-7 cells without any additive or control cells with scrambled siRNA (P,0.05; Figure 5).

effect of raP80 sirna on the expression of apoptosis-related proteins
Western blot results showed that siRNA transfection upregulated the protein expression of Caspase-3, cleaved Caspase-3, Apaf-1, Cytochrome C, and Bax (P,0.05, Figure 6), and it downregulated the mRNA and protein expression of Bcl-2 (P,0.05, Figure 6). Protein levels analyzed by Western blot also confirmed this result ( Figure 6).

Discussion
DDR is a series of regulatory events including DNA damage, regulation of DNA replication, cell cycle arrest, and repair or bypass of DNA damage to ensure the maintenance of genomic stability and cell viability. 9 The deregulation   12,13 What about RAP80's role in breast cancer? In the present study, we sought to investigate the expression of RAP80 in invasive breast cancer and its paired normal breast tissues. Our results indicated that RAP80 expression in breast cancer (62.3%, 101/162) was significantly lower than that in adjacent normal breast tissues (P,0.05). And the low expression of RAP80 was related to positive lymph node metastasis, TNM stage, negative ER or PR expression, and high level of Ki67 proliferation index in breast cancer patients. A previous study indicated that RAP80 is a wellknown DNA repair protein related to DDR -the existence of crosstalk mechanisms linking the DDR mechanism and hormone signaling pathways cooperate to influence both cancer progression and therapeutic response. 14 Recent evidence has suggested that the suppression effect of DDR by estrogen altered the response of cancers to antihormone treatment or chemotherapy that induces DNA damage. 15 Downregulation of endogenous RAP80 expression by siRNA reduced ERα protein level, and RAP80 may be an important modulator of ERα activity. 16 Ki67 proliferation index is an important prognostic factor in breast cancer, it is expressed in all phases of cell cycle except G0 and serves as a prognostic and predictive marker for breast cancer. 17,18 Our results suggested that low RAP80 expression was correlated with high Ki67 levels. What about its role in breast cancer? Is it related to breast cancer cell proliferation? We found that mRNA and protein expression of RAP80 were obvious in MCF-7 and very weak in ZR-75 or MDA-MB-231. So we transfected MCF-7 with  in Hela cells treated with ionizing radiation showed a defective G2/M checkpoint control. 19 Our results indicated that RAP80 can significantly inhibit cell growth, participate in G2/M checkpoint regulation, and promote cell apoptosis in MCF-7. RAP80 siRNA also promoted expression of apoptosis-related proteins and decreased invasive ability. If   Previous research indicated that RAP80 interacts with BRCA1 and is critical for efficient repair through DNA damage-induced homology-directed recombination and in cell cycle checkpoint control. They found that UV irradiation can induce the RAP translocation to DNA damage foci, which is dependent on the UIMs of RAP80 and that the UV-induced phosphorylation of RAP80 at Ser205 is mediated by ATR. 22 RAP80 opposes homologous recombination by inhibiting DNA end-resection and sequestering BRCA1 into the BRCA1-A complex. 23 BRCA1 can reduce apoptosis caused by chemotherapy drugs, and it can be used as a marker for predicted chemoresistance. Cisplatin is an antitumor drug widely used to treat breast cancer, especially triple negative breast cancer. Its primary biological target is genomic DNA, and it causes a plethora of DNA lesions that block transcription and replication. 24 DNA repair and DNA damage tolerance play crucial roles in the response to cisplatin treatment, making DDR and repair proteins tempting therapeutic targets to modulate chemoresistance, sensitize tumor cells to cisplatin, and enhance chemotherapeutic efficiency. 25 Our results showed that the apoptosis of MCF-7 was significantly higher after RAP80 siRNA combined with cisplatin than after treatment with control siRNA with cisplatin. Tumor cells can activate protective responses to inhibit cell cycle progression, which may be related to RAP80, and repair cisplatin-induced DNA damage. RAP80 is related to BRCA1's effect, which can be used as an interesting target for pharmacological modulation that can increase the efficiency of cisplatin chemotherapy.

Conclusion
RAP80 expression is related to ER or PR activity. RAP80 siRNA can also increase the chemosensitivity of MCF-7 to cisplatin. It is related to carcinogenesis of ER negative breast cancer, and it may be a potential target for future breast cancer therapy. More clinical investigations are required to determine a drug which targets RAP80 which can be used in breast cancer therapy.

Ethics statement
The clinicopathological information of each patient was reviewed. All the patients who enrolled in this study had signed an informed consent form agreeing to participate in this study and for publication of the results. The study was reviewed and approved by the Ethics Committee of China Medical University (Shenyang, People's Republic of China) and the participating hospital, the First Hospital of China Medical University. The study was carried out in accordance with the principles of the Declaration of Helsinki.