Cancer Chemother Pharmacol DOI 10.1007/s00280-014-2379-y

Original Article

shRNA‑mediated silencing of ZFX attenuated the proliferation of breast cancer cells Hongjian Yang · Yue Lu · Yabing Zheng · Xingfei Yu · Xianghou Xia · Xiangming He · Weiliang Feng · Lei Xing · Zhiqiang Ling 

Received: 20 October 2013 / Accepted: 8 January 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Purpose  Breast cancer is the cause for highest number of cancer-related death among women worldwide. This study was focused on investigating the role of zinc-finger protein X-linked (ZFX) in human breast cancer. Methods Expression levels of ZFX were analyzed in 99 patients and four breast cancer cell lines. Lentivirus-mediated RNA interference was applied to silence ZFX expression, and the effects of ZFX knockdown on the growth of breast cancer cells were investigated. Results The immunohistochemical expression of ZFX was higher in more advanced tumor tissues. ZFX was also overexpressed in multiple breast cancer cell lines. Knockdown of ZFX inhibited cell proliferation and colony formation of MCF-7 and MDA-MB-231 cells. Moreover, ZFX silencing resulted in cell cycle arrest at G0/G1 phase. Depletion of ZFX decreased the phosphorylation level of Hongjian Yang and Yue Lu have contributed equally to this work. H. Yang (*) · X. Yu · X. Xia · X. He · W. Feng · L. Xing  Department of Breast Surgery, Zhejiang Cancer Hospital, 38 Guangji Road, Hangzhou 310022, China e-mail: [email protected] Y. Lu  Cancer Center, The Medical School, Hospital of Qingdao University, No. 16, Jiangsu Road, Qingdao 266003, People’s Republic of China Y. Zheng (*)  Department of Internal Oncology, Zhejiang Cancer Hospital, 38 Guangji Road, Hangzhou 310022, China e-mail: [email protected] Z. Ling  Zhejiang Cancer Research Institute, Zhejiang Cancer Hospital, 38 Guangji Road, Hangzhou 310022, China

AKT and increased the phosphorylation level of ERK2 and the expression of cyclin D1, which is involved in cell survival and cell cycle regulation. Conclusions These findings suggest that ZFX plays an important role in breast cancer development and could be a potential therapeutic target for breast cancer. Keywords  Breast cancer · ZFX · RNAi · Proliferation

Introduction Breast cancer is the primary cause of death among women worldwide despite the advancement in chemotherapy [1]. It was found that the initiation of breast cancer is closely related to genetic alterations that facilitate cancer progression [2]. Over the years, more than 100 oncogenes related to breast cancer have been found, and they could protect cancer cells from normal physiological regulation [3]. Targeted silencing of oncogenes has been identified as a potent therapeutic possibility to treat various cancers. Therefore, use of RNA interference (RNAi) to target oncogenes would be a successful strategy for the treatment of breast cancer. Previous studies also have reported the possibilities of using RNAi to control c-Myc expression [4] and KLF8 expression [5] in breast cancer cells. Short hairpin RNA (shRNA) guides this process and results in a base-pairingdependent down regulation of gene expression [6]. Moreover, specific delivery system should be chosen for efficient use of RNAi technique. In this regard, lentivirus-based vectors have been used to deliver shRNA successfully and efficiently with long-term silencing effects [7]. Zinc-finger proteins are involved in numbers of physiological processes such as DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis,

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protein folding and assembly, and lipid binding [8]. Among them, zinc-finger protein X-linked (ZFX) is a member of the krueppel C2H2-type zinc-finger protein family and is located on the X chromosome [9]. ZFX is required as a transcriptional regulator for self-renewal of embryonic and adult hematopoietic mouse stem cells [10]. Recently, it has been shown that ZFX is related with several human cancers. ZFX was upregulated in cancer stemlike cells in esophageal carcinoma cell lines [11]. Furthermore, ZFX was overexpressed in prostate adenocarcinoma [12], diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) [13]. Nikpour et al. [14] demonstrated that ZFX was differentially expressed in gastric cancer. Moreover, it was found that depletion of ZFX inhibited the self-renewal and survival of hematopoietic stem cells, causing a severe block in cell development [15]. The above evidence suggests that ZFX regulates cell survival and may serve as a therapeutic target in cancers. With this background, the present study was designed to investigate the effects of shRNA-mediated silencing of ZFX on the growth of breast cancer cells.

Materials and methods Reagents and plasmids Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 medium, and fetal bovine serum (FBS) were obtained from Hyclone (Logan, UT). Short hairpin (shRNA) expression vector pFH-L, lentiviral packaging aid vectors pVSVG-I, and pCMVΔR8.92 were purchased from Shanghai Hollybio (China). RNeasy MidiKit was from Qiagen (Valencia, CA, USA). AgeI, EcoRI, and SYBRGreen Master Mix Kits were purchased from TaKaRa (Dalian, China). Lipofectamine2000, TRIzol, and Super-scriptII reverse transcriptase were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were obtained from Sigma (St. Louis, MO, USA). The antibodies used were as follows: anti-ZFX (1:800 dilution; Sigma-Aldrich); anti-GAPDH (glyceraldehyde3-phosphate dehydrogenase, 1:2,000 dilution; Abcam); anti-AKT (1:1,000 dilution; Santa Cruz Biotechnology); Phospho-AKT antibody (p-AKT Ser473, 1:500 dilution; Santa Cruz Biotechnology); anti-cyclin B1, anti-cyclin D1, anti-ERK2, anti-p44/42 MAP kinase (1:2,000 dilution; Abcam). Immunohistochemistry We studied 99 patients suffering from breast cancer at different stages, treated in Hospital of Qingdao University Medical School, China. For immunohistochemistry, 90

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Cancer Chemother Pharmacol

patients with invasive ductal carcinoma (IDC) and nine with ductal carcinoma in situ breast cancer were fixed in 10 % formal insolution and embedded in paraffin. Histological slices of 3-mm were deparaffinized in xylene and then rehydrated with incremental ethanol concentrations. Endogenous peroxidase was blocked with 0.3 % H2O2 in methanol for 20 min at room temperature. Following the antigen retrieval, the sections were blocked with 5 % BSA for 20 min at room temperature and then treated with rabbit anti-ZFX (1:300 dilution) at 4 °C overnight. After the incubation, the sections were stained with biotinylatedgoat anti-rabbit immunoglobulins at room temperature for 1 h and visualized using the peroxidase-conjugated streptavidin and diaminobenzidine. Then, the sections were counterstained with Mayer’s hematoxylin. The results were evaluated with pathologist blinded to all clinical data. Samples were identified as positive when more than 10 % of the cells have been reacted with the anti-ZFX antibody and presented cytoplasm staining. Cell culture Human breast cancer cells (MDA-MB-231, BT474, MCF-7, and T47D) and human embryonic kidney cells (HEK293T) were obtained from cell bank of Shanghai institute of cell biology. The cells were maintained in DMEM supplemented with 10 % heat-inactivated FBS and penicillin/streptomycin. All cells were incubated at 37 °C in humidified atmosphere containing 5 % CO2. Construction and infection of lentivirus containing ZFX shRNA Small interfering RNA (siRNA) targeting human ZFX gene (5′-GTCGGAAATTGATCCTTGTAA-3′) was designed. The control non-silencing siRNA was 5′-TTCTCCGAAC GTGTCACGT-3′. The stem-loop-stem oligos (shRNAs) were synthesized, annealed, and ligated into the pL-GFP vector (Hollylab, Shanghai, China). The lentivirus-based shRNA-expressing vectors were confirmed by DNA sequencing. For the transfection, HEK293T cells (1 × 107) were seeded in 10-cm dishes and cultured for 24 h to reach 70–80 % confluence. Two hours before transfection, the medium was replaced with serum-free DMEM. The plasmid mixture contained 20 μg pL-GFP-shZFX or 20 μg pL-GFP-shCon, 15 μg packaging vector Helper 1.0 and 10  μg VSVG expression plasmid Helper 2.0 in 200 μl opti-MEM, and 15 μl of Lipofectamine 2000 was added to the cells and incubated for 5 h before replacing the medium with 10 ml of DMEM with 10 % FBS. Lentiviral particles were harvested at 48 h after transfection and purified by ultra-centrifugation. As the lentivirus carries green fluorescence protein (GFP), the viral titer was determined

Cancer Chemother Pharmacol

by end-point dilution assay through counting the numbers of GFP-expressing cells under fluorescence microscopy. RNA extraction and real‑time PCR analysis Total RNAs of cultured cells were extracted using the Trizol reagent on 6 days after transfection. cDNA was synthesized using Promega M-MLV cDNA synthesis kit according to the manufacture’s instruction. ZFX mRNA expression was evaluated by real-time PCR using the SYBR Green Master Mix Kit on a Bio-Rad connet real-time PCR platform. Each PCR reaction mixture, containing 10 μl of 2 × SYBR Green Master Mix, 2.5 μl of sense and antisense primers and 0.8 μl of cDNA (2 ng), was run for 40 cycles with initial denaturation at 95 °C for 1 min, annealing at 60 °C for 5 s, and extension at 60 °C for 20 s in a total volume of 20  μl. The forward and reverse primers used were as follows. ZFX: 5′-GGCAGTCCACAGCAAGAAC-3′ (forward) and 5′-TTGGTATCCGAGAAAGTCAGAAG-3′ (reverse); β-actin: 5′-GTGGACATCCGCAAAGAC-3′ (forward) and 5′-AAAGGGTGTAACGCAACTA-3′ (reverse). β-actin was used as an internal gene. Relative expression levels were calculated using 2−ΔΔCT analysis. Western blot analysis Lentivirus-transduced cells were washed twice with icecold PBS and lysed in radio-immune precipitation assay (RIPA) buffer [50 mM Tris (pH 7.5), 150 mM Nacl, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS]. The supernatant was collected after centrifuging (12,000×g for 15 min), and the protein content was measured by Lowry method. The protein concentration of each sample was adjusted to 2 μg/μl and electrophoresed on 12 % SDS-PAGE gel at 60 V for 4 h. The gel was transferred to polyvinylidenedifluoride membrane. The protein expression levels were detected after staining with respective antibodies using ECL detection kit (Amersham) by exposing to X-ray film. The bands obtained were quantified with an Image Quantdensitometric scanner (molecular dynamics).

Colony formation assay MCF-7 and MDA-MB-231 cells (200 cells/well) were seeded into 6-well plates 3 days after lentivirus infection. Cells were cultured for 8 days with media renewal in every 2 days. For colony formation assay, the incubated cells were washed with PBS and fixed with 4 % paraformaldehyde for 30 min at room temperature. The fixed cells were stained with freshly prepared Giemsa for 20 min and washed with PBS prior to the colony counting using light microscope and fluorescent microscope. Cell cycle analysis Cell cycle distribution was analyzed by flow cytometry following propidium iodide (PI) staining. Four days following lentivirus infection, MDA-MB-231 cells were seeded on a 6-well plate at a density of 5 × 105 cell and synchronized by serum starvation for 72 h. After washing with ice-cold PBS, cells were suspended in about 0.7 ml of 70 % cold alcohol and incubated for 30 min at 4 °C. The ethanol was discarded by centrifugation, and the PI (100 μg/ml) solution containing 10 μg/ml of DNase-free RNase A was added to stain the cells and kept for 30 min. The suspension was filtered through a 50-μm nylon mesh, and total of 1 × 104 stained cells were analyzed by a flow cytometer (FACS Calibur, BD Biosciences). Statistical analysis Statistical analyses were conducted using SPSS 13.0 software. The results of immunohistochemistry staining were evaluated by chi-squared test, and the other data were evaluated by Student’s t test and expressed as mean ± SD of three independent experiments. P