Prmt2β, a C-Terminal Splice Variant of PRMT2, Inhibits the Growth of Breast Cancer Cells

ONCOLOGY REPORTS 38: 1303-1311, 2017 PRMT2β, a C-terminal splice variant of PRMT2, inhibits the growth of breast cancer cells JING ZHONG1, YA-JUN CHEN1,2, LING CHEN1, YING-YING SHEN1, QING-HAI ZHANG1, JING YANG3, REN-XIAN CAO1,3, XU-YU ZU1 and GE-BO WEN1,3 1Institute of Clinical Medicine, The First Affiliated Hospital of University of South China; 2Department of Metabolism and Endocrinology, The Second Affiliated Hospital of University of South China; 3Department of Metabolism and Endocrinology, The First Affiliated Hospital of University of South China, Hengyang, Hunan 421001, P.R. China Received January 5, 2017; Accepted June 27, 2017 DOI: 10.3892/or.2017.5786 Abstract. Our previous study reported several alternative the transcription activity of the cyclin D1 promoter, and splicing variants of arginine N-methyltransferase 2 (PRMT2), PRMT2β was also found to inhibit cyclin D1 expression via which lose different exons in the C-terminals of the wild-type the suppression of Akt/GSK-3β signaling in breast cancer PRMT2 gene. Particularly, due to frame-shifting, PRMT2β cells. Clinically, it was revealed that PRMT2β expression was encodes a novel amino acid sequence at the C-terminus of negatively correlated with human epidermal growth factor the protein, the function of which is not understood. In the receptor 2 (HER2) (p=0.033) in breast tumors. Our results present study, we determined the role of PRMT2β in breast revealed that PRMT2β, a novel splice variant of PRMT2, plays cancer cell proliferation, apoptosis and its effect on the Akt potential antitumor effect by suppressing cyclin D1 expression signaling pathway. Stable breast cancer MCF7 cell line with and inhibiting Akt signaling activity. This also opens a new lentivirus-mediated PRMT2β overexpression was obtained avenue for treating breast cancer. after selection by puromycin for 2 weeks. The effect of lentivirus-mediated PRMT2β overexpression on breast cancer Introduction cellular oncogenic properties was evaluated by MTT, colony formation, cell cycle analysis and apoptosis assays in MCF7 Alternative splicing is a sophisticated and ubiquitous nuclear cells. Luciferase activity assay and western blot analysis were process in eukaryotic cells by which a single gene may yield performed to characterize the effects of PRMT2β on cyclin D1 various mRNAs, leading to products of multiple protein promoter activities and the Akt signaling pathway. Tissue isoforms. In humans, more than 70% of genes are alterna- microarray was performed to investigate the association of tively spliced (1,2), which greatly increases the diversity of PRMT2β with breast cancer progression. Lentivirus-mediated proteins that can be encoded by the surprisingly low number PRMT2β overexpression suppressed the cell proliferation of human genes. Alternative splicing is known to play a role and colony formation of breast cancer MCF7 cells. PRMT2β in the alteration of the binding properties of proteins, their overexpression induced cell cycle arrest and apoptosis of enzymatic activity, their intracellular localization, or their MCF7 cells. Furthermore, PRMT2β was revealed to suppress stability and post-translational modifications (3,4). Recently increasing evidence has revealed that alternative splicing plays an important role in tumorigenesis and tumor progression, and splice variants represent potentially useful targets for the Correspondence to: Professor Ge-Bo Wen or Dr Xu-Yu Zu, Institute development of novel therapeutic strategies (5,6). of Clinical Medicine, The First Affiliated Hospital of University Human protein arginine N-methyltransferase 2 (PRMT2, of South China, 69 Chuanshan Road, Hengyang, Hunan 421001, HRMT1L1) is a protein that belongs to the arginine methyl- P. R. Ch i na transferase family and has weak methyltransferase activity on E-mail: [email protected] a histone H4 substrate. However, its optimal substrates have not E-mail: [email protected] yet been identified (7,8). Subsequent studies have revealed that PRMT2 is clearly involved in a variety of cellular processes, Abbreviations: PRMT2, arginine N-methyltransferase 2; AR, androgen receptor; ER, estrogen receptor; PR, progesterone receptor; including lung function, the inflammatory response, apoptosis HER2, human epidermal growth factor receptor 2; AP-1, activator promotion, and Wnt and leptin signaling regulation (9-13), protein-1; Dox, doxycycline hyclate; FBS, fetal bovine serum; GFP, suggesting that PRMT2 has diverse roles in transcriptional green fluorescent protein regulation through different mechanisms depending on its binding partners. Key words: PRMT2β, antitumor, proliferation, breast cancer Our previous study demonstrated that the PRMT2 gene produces several isoforms through complex alternative splicing at the 3' end of the pre-mRNA, and each has a unique C-terminal 1304 ZHONG et al: noVel Variant of PRMT2 and breast cancer sequence (14). Due to the distinct gene structure characteristics, Cell proliferation and colony formation assays. For cell each isoform is therefore predicted to be functionally different. proliferation assays, MCF7 cells were seeded in a 96-well PRMT2β is the most different isoform compared with the wild- plate (2,000 cells/well) and counted using an automated cell type. Sequence analysis revealed that PRMT2β deletes exon 7-9 counter (Nexcelom Bioscience, Lawrence, MA, USA). For in its coding sequence, and downstream frame-shifting. As a the colony formation assay, the cells were seeded in a 6-well result, PRMT2β encodes 83 new amino acids at the C-terminus plate (600 cells/well) and maintained for 8 days. The culture of the protein. We had previously demonstrated the negative medium with or without 5 µg/ml of Dox was changed every effect of PRMT2 on breast cancer cell proliferation in vitro 2 days. Each experiment was carried out in triplicate and and in vivo. Moreover, cyclin D1 (CCND1) was found to be performed at least twice. downregulated by PRMT2, and PRMT2 was further shown to suppress the estrogen receptor α (ERα) binding affinity to the Soft agar colony formation assay. The assay was performed activator protein-1 (AP-1) site in the CCND1 promoter through in 6-well plates. A bottom layer of agar (0.6%) with enriched indirect binding with the AP-1 site, resulting in the inhibition Dulbecco's modified Eagle's medium (DMEM), [final 10% of the CCND1 promoter activity in MCF-7 cells (15). These fetal bovine serum (FBS)] was poured first. After the bottom studies however, neglected to examine the specific functional agar solidified, MCF7 cells (3.0x103) were seeded in top agar contributions of PRMT2β to breast cancer progression. In the (0.3%) with enriched DMEM supplemented with 20% FBS present study, we revealed that PRMT2β suppressed the cell and incubated at 37̊C for 15 days. The culture medium with or proliferation and colony formation of breast cancer MCF7 without 5 µg/ml of Dox was changed twice weekly. Colonies cells, and induced cell cycle arrest and apoptosis of MCF7 cells. were visualized by staining for 1 h with 0.005% crystal violet. PRMT2β was revealed to suppress the transcription activity of the CCND1 promoter, and PRMT2β was also found to Cell cycle analysis by flow cytometry. After transfection, the inhibit CCND1 expression via the suppression of Akt/GSK-3β MCF7 cells were plated in 6-well plates at 2x105 cells/well. signaling in breast cancer cells. Following the designated treatments, the cells were harvested by trypsinization and washed with PBS and fixed in ice-cold Materials and methods 75% ethanol overnight at -20̊C. The fixed cells were washed, and dissolved in RNAse and subsequently incubated at 37̊C for Cell culture. MCF7 and 293T cells were purchased from 30 min. Next, the cells were stained with propidium iodide (PI) American Type Culture Collection (ATCC) (Manassas, for 30 min. The DNA content of the cells (1x104 cells/experi- VA, USA), and cultured as previously described (14). Ethics mental group) was determined using a BD Accuri C6 flow approval and consent for the use of the human tissue was cytometer (BD Biosciences, Franklin Lakes, NJ, USA). confirmed by the Ethics Committee of The First Affiliated Hospital of the University of South China. Transient transfection and luciferase activity assay. Transient gene delivery was carried out to assess the effect of PRMT2β Lentiviral vector construction and lentivirus infection. and PRMT2 on the CCND1 promoter activity in 293T cells Recombined lentiviral vector pGC-LV-GV308-PRMT2β with as previously described (14). A luciferase assay kit (Promega, the variant PRMT2β (FJ436411) gene (LV-Tet-on-PRMT2β) Madison, WI, USA) was used to assess the reporter activity and pGC-LV-GV308-PRMT2 (NM_001535) with the wild- according to the manufacturer's instructions. Luciferase type PRMT2 (LV-Tet-on-PRMT2) gene were constructed by activity was normalized using a Renilla luciferase internal the GeneChem Co. (Shanghai, China). The pGC-LV-GV308 control. vector was used as a negative control. The packaging plasmid pHelper 1.0 and pHelper 2.0 were purchased from GeneChem Western blot analysis. Total cell or tissue lysates were lysed Co. on ice for 30 min. Soluble proteins (20 µg) were probed We co-transfected the pGC-LV-GV308-PRMT2β or with anti-PRMT2, anti-Flag and anti-CCND1 antibodies pGC-LV-GV308-PRMT2 vectors with the pHelper 1.0 and (1:500; Abcam, Cambridge, MA, USA) and anti-p-AKT, pHelper 2.0 packaging plasmid into 293T cells to generate anti-AKT, anti-p-GSK-3β and anti-GSK-3β (1:800; Cell recombinant lentiviruses. Culture medium

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