BTG1 potentiates apoptosis and suppresses proliferation in renal cell carcinoma by interacting with PRMT1

B-cell translocation gene 1 (BTG1) is a member of the BTG/transducer of Erb family. BTG1 regulates cell cycle progression, inhibits proliferation, promotes apoptosis and stimulates cellular differentiation in multiple cell types. However, the functions of BTG1 in renal cell carcinoma (RCC) remain unclear. Therefore, the present study investigated the role of BTG1 in RCC tissue samples and 786-O RCC cells. RCC tissues and cells exhibited significantly weaker BTG1 protein and mRNA expression compared with para-carcinoma control tissues (P<0.05). Upregulated BTG1 expression induced significant G0/G1 cell cycle arrest, apoptosis and inhibition of cell proliferation in 786-O cells (P<0.05). Furthermore, BTG1 interacted with protein arginine N-methyltransferase 1 (PRMT1), and blocking the action of PRMT1 in 786-O cells resulted in inhibition of BTG1 function. These findings indicate that BTG1 may inhibit cell growth and promote apoptosis by interacting with PRMT1 in RCC; the identification of this mechanism may aid in the production of novel therapies for RCC.


Introduction
Renal cell carcinoma (RCC) is among the most common types of cancer and accounts for ~2-3% of all malignancies (1). The incidence of RCC continuously increases each year, with an estimated 61,560 new cases of RCC and 14,080 RCC-associated mortalities predicted to occur in the United States in 2015 (2). Despite advances in diagnostic techniques, 25-30% of patients present with metastatic disease (3). The prognosis is poor for such patients, with subsequent chemotherapy and radiotherapy treatment regimes yielding ineffective results (4). Tumourigenesis and progression are multistep processes that are affected by changes in gene expression. Therefore, understanding the gene expression changes that occur in RCC may improve the diagnosis, treatment and prevention of RCC.
B-cell translocation gene 1 (BTG1) is a member of the BTG/transducer of Erb (TOB) family. This family comprises six members; BTG1, BTG2/TIS21/PC3, BTG3, BTG4/PC3B, TOB1 and TOB2. The BTG/TOB family proteins are composed of two highly conserved and characteristic domains, Box A and Box B, in the N-terminal region. In addition, these proteins are involved in regulating cell cycle progression, inhibiting proliferation, promoting apoptosis and stimulating cellular differentiation in multiple cell types (5). As BTG1 exhibits these characteristics, it is considered to be a tumour suppressor gene (6). Previous studies have identified that BTG1 enhances the antiproliferative function of homeobox B9-mediated transcription (7), while overexpression of BTG1 induces increased apoptosis in NIH 3T3 cells (8). However, the functions of BTG1 and its precise molecular mechanisms in RCC remain unclear. It has been shown that BTG1 interacts with protein arginine N-methyltransferase 1 (PRMT1) in vitro (9). PRMT1 then catalyses the formation of ω-monomethylarginine and asymmetric dimethylarginine. This arginine methylation regulates transcription or affects cytokine signalling pathways (10). However, whether BTG1 functions in RCC via its effect on PRMT1 remains unknown.
Therefore, the present study examined BTG1 expression in RCC tissues and cells, and investigated the function of BTG1 in cell proliferation, cell cycle distribution and apoptosis in vitro. In addition, it was investigated whether the functions of BTG1 are attributable to interactions with PRMT1.

Materials and methods
Tissues. RCC and corresponding para-carcinoma tissue samples were obtained from 20 patients, who underwent nephrectomy at the Affiliated Zhongda Hospital of Southeast University (Nanjing, China) between June 2007 and June 2010. Histological diagnoses were established following analysis of standard hematoxylin and eosin-stained sections by two senior

BTG1 potentiates apoptosis and suppresses proliferation in renal cell carcinoma by interacting with PRMT1
pathologists experienced in RCC diagnosis. All patients were diagnosed with RCC. Approval was obtained from the ethics committee of the Affiliated Zhongda Hospital of Southeast University and samples were collected following receipt of written informed consent from all patients.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted from the specimens using TRIzol ® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Complementary (c)DNA synthesis was performed using Avian Myeloblastosis Virus Reverse Transcriptase and random primers (Takara Biotechnology Co., Ltd., Dalian, China), and the RT-qPCR reactions were performed using a 7300 Real-Time RT-PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA). The amplification steps consisted of 95˚C for 1 min, 40 cycles at 95˚C for 15 s and 60˚C for 30 s, followed by 72˚C for 10 min. The primer sequences for BTG1 were as follows: F 5'-ATCTCCAAGTTTCTC-CGCACC-3' and R 5'-CAACGGTAACCCGATCCCTT-3'. Subsequently, the expression BTG1 mRNA was calculated relative to GAPDH mRNA expression levels using the 2 -ΔΔCt method.
Immunohistochemistry. All surgical samples were fixed in 10% buffered formaldehyde solution and embedded in paraffin. Paraffin sections (4-µm thick) were then reacted with monoclonal antibodies against BTG1 (mouse anti-human; 1:75 dilution; cat. no. ab50991; Abcam, Cambridge, UK). The antibody was replaced by phosphate-buffered saline (PBS) as a negative control.  Immunostaining and confocal laser microscopic analysis. Cells were grown in culture dishes with a bottom well.  . Data are presented as the mean ± standard error of the mean. All calculations were performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) and scanned images of western blots were quantified using ImageJ 2X software (National Institutes of Health, Bethesda, MD, USA). Group means were compared by performing Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Low BTG1 expression in RCC tissues and cells.
In order to investigate BTG1 expression in RCC, BTG1 mRNA expression was measured using RT-qPCR, and BTG1 protein expression was determined using immunohistochemistry, in 20 RCC and 20 corresponding para-carcinoma tissue samples. BTG1 expression was significantly lower in the RCC tissues compared with that in the corresponding para-carcinoma tissues (P<0.05; Fig. 1A and 1B). In order to construct a reliable in vitro model for investigating the mechanism of action of BTG1 in RCC, BTG1 expression was examined by western blot analysis in HK-2 (control) and 786-O (RCC) cells. BTG1 expression was significantly lower in the 786-O cells compared with that in HK-2 cells (P<0.05; Fig. 1C and 1D).

Discussion
BTG1 was initially identified in B lymphoblastic leukaemia and its expression appears to be highest in the G0/G1 phases of the cell cycle (11). Weak BTG1 expression has recently been reported in various other types of cancer, including thyroid, lung and breast (12)(13)(14)(15). In the present study, weak BTG1 expression was also demonstrated in RCC tissue samples by performing qRT-PCR, western blotting and immunohistochemistry. Tumour development and progression are associated with uncontrolled proliferation and apoptosis. Zhu et al (16) and Sun et al (17) identified that BTG1 induces G0/G1 cell cycle arrest, promotes apoptosis and inhibits cell proliferation in breast and non-small cell lung cancer (NSCLC), respectively. Furthermore, Sun et al (18) determined that BTG1 expression was significantly correlated with lymph node metastasis, clinical stage, histological grade and survival in NSCLC cells. The present study explored the functions of BTG1 in RCC, and obtained similar results, with respect to the effect of BTG1 expression on cell cycle arrest, apoptosis promotion and growth inhibition. These data indicate that BTG1 may have a common antitumour function in various types of cancer.
The antiproliferative activity of BTG1 is controlled by the conserved Box A domain (11). Doidge et al (18) demonstrated that the antiproliferative activity of BTG1 is mediated through its interactions with the Caf1a and Caf1b deadenylase enzymes, and the role of BTG1 in the regulation of mRNA abundance and translation is dependent on Caf1a/Caf1b. However, in the present study, BTG1 appeared to function by interacting with PRMT1. In mammals, PRMT1 is the primary arginine asymmetric dimethylation enzyme, accounting for >90% of asymmetric dimethylation enzymes (19). Arginine methylation is a common post-translational modification that alters the stability of chromatin and affects the binding of transcriptional factors, thereby regulating gene expression without changing the original nucleotide sequence. Arginine methylation has critical functions in gene transcription, mRNA splicing, DNA repair, protein cellular localisation and signalling (20), and PRMT1 specifically exhibits key functions in breast cancer cell apoptosis and osteosarcoma cell proliferation (21)(22)(23). The present study investigated the interaction between BTG1 and PRMT1 in RCC, and it was shown that certain functions of BTG1 are suppressed by the inhibition of PRMT1. Thus, it is hypothesized that BTG1 functions by interacting with PRMT1 in RCC, as well as through other signalling pathways.
In conclusion, the present study demonstrated weak expression of BTG1 in RCC, and showed that BTG1 may inhibit cell growth and promote cell apoptosis by interacting with PRMT1. However, the underlying mechanism of action of PRMT1 in RCC requires further investigation.