Abstract: 149 ISJ 15: 149-157, 2018 ISSN 1824-307X RESEARCH REPORT Identification of BmBrat gene in silkworm, Bombyx mori XX Ke1#, H Gao1#, K Chen2, L Zhang1, S Chen1, X Wang1, Z Wei1, L Yang1, H Cui1* #These authors contributed equally to this work 1State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, P.R. China 2Department 6 of Research Institute of Surgery, State Key Laboratory of Trauma, Burns and Combined Injury, Daping Hospital, Army Medical University, Chongqing 400042, P.R. China Accepted April 7, 2018 Abstract NHL protein family is evolutionarily conserved in either vertebrates or invertebrates. It has been extensively studied in mammals for the function of regulating cell proliferation and differentiation. Brat, known as the homologue of TRIM in insects, may have the same function. In this paper, we identified and characterized BmBrat in silkworm. The gene expression profile indicated that BmBrat was expressed in all tissues and the expression was especially high in ovary and head at larval stage. The expression profile was also detected at different stages of embryo development, and reached a peak at 4th and 5th days of the embryonic period. Subcellular localization of BmBrat in hemocytes revealed that it was specifically expressed in cytoplasm. Over-expression of BmBrat in silkworm significantly reduced the rate of cell proliferation by BrdU staining. This finding suggested that BmBrat played an important role in cell processes. Key Words: silkworm, BmBrat, gene expression profile, subcellular localization Introduction The name NHL originates from three members, which were earlier discovered: NCL-1, HT2A and LIN-41. These three factors have relationship with the RNA metabolism, and may be involved in post-transcriptional regulation of gene expression (Loedige et al., 2009). Researches showed that family members of the NHL contain three conserved motifs: a RING, one or two B-box and a coiled-coil domain (Petrera et al., 2012; Tocchini et al., 2015). NHL domain plays a vital role in regulating the protein activity of Brat (Brain tumor) (Marchetti et al., 2014). Brat itself as a member of the NHL protein family is a kind of tumor suppressor genes, but it is also a candidate gene controlling cell proliferation (Loedige et al., 2014). In the Drosophila nervous system, the expression of Brat largely limits the peripheral nervous system, ventral nerve cord and brain development during embryonic stage (Bello et al., 2006). In stem cells development system, Brat, Prospero and Numb work as cell fate determination factors, promoting the changes of cell fate (Lee et al., ___________________________________________________________________________ Corresponding author: Hongjuan Cui Cell Biology Laboratory State Key Laboratory of Silkworm Genomics Biology Southwest University Chongqing 400716, P.R.China E-mail: hongjuan.cui@gmail.com; hcui@swu.edu.cn 2006). Numb is a membrane-bound protein involved in the regulation of Notch signaling pathway (Cotton et al., 2013). During neural precursor cell division, Numb gathered to one end of the cell to combine with the membrane, distributed into the ganglion mother cells through asymmetric cell division (Hutterer et al., 2005). Then Numb interacts with Sanpodo, which is α-ubiquitin mediated protein activity regulating factor, to inhibit the Notch protein (Berdnik et al., 2002). The inhibition of Notch signaling pathway, which plays a crucial role in proliferation and differentiation of neuroblastoma cells, would result in a cell fate transformation, which means self-renewing cells can gradually begin to differentiate (Wang et al., 2011). Prospero is a transcriptional regulatory protein, which exists both in the embryo and larvae neuroblast, and was continuously expressed in the differentiated glial cells (Choksi et al., 2006; Reichert, 2011). Prospero functions as a downstream regulator of Brat, and plays a key role in cell fate determination and cell proliferation regulation. Thus studying Brat in cell differentiation and proliferation can provide theoretical basis for differentiation of stem cells. The homolog of Brat in human and mammalian is the TRIM protein family, and they all play a role in the development, differentiation, host cell antiviral defense and cancer. TRIM family is a subgroup of the NHL domain, such as TRIM2, TRIM3, TRIM32 150 Fig. 1 Complete cloning of silkwrom BmBrat. (A) PCR of BmBrat. (B) The diagram of the complete BmBrat cDNA. (C) The location of BmBrat on chromosome 14 and TRIM71 in mammalian, Brat, Mei-p26, Abba and Wech in Drosophila, and Ncl-1, Nhl-1, Nhl-1, Nhl-3, Lin41 in Caenorhabditis elegans (Nisole et al., 2005; Neumuller et al., 2008; Sardiello et al., 2008). Among these, TRIM3, known as a tumor suppressor, can inhibit cancer development by binding to CDK inhibitor p21WAF1/CIP1 to prevent the accumulation of Cyclin D1-CDK4 (Boulay et al., 2009; Liu et al., 2014). Reducing TRIM3 expression in mice can increase and accelerate the platelet-derived growth factor (PDGF) induced glioma incidence (Boulay et al., 2009; Chen et al., 2014). However, there is almost no report on the BmBart gene in silkworm, Bombyx mori at present, and the exact function of BmBart gene remains unknown. In this study, we reported the identification, characterization, expression analysis and functional study of the BmBart gene in silkworm, B. mori, which is not only an important economical insect for silk production, but also an excellent research model of invertebrate. Materials and Methods Biological materials Bombix mori strain Dazao (P50) used in our study is provided by the Gene Resource Library of Domesticated Silkworm of Southwest University. The larvae was fed with fresh mulberry leaves under standard conditions. Different tissues were extracted in 1× DEPC-treated PBS, flash-frozen and stored at -80 °C until needed. The cultured silkworm embryo-derived cell line was established by our laboratory, and it was cultured in Grace' medium supplemented with 10% (V/V) FBS. RNA extraction and cDNA synthesis Total RNA was extracted from the head, midgut, epidermis, silk gland, fat body, malpighian tubules, testis, ovary, haemocytes and embryo with TRIzol reagent referring to the protocol provided by the manufacturer. After being treated with RNase-free DNase I, the first-strand cDNA was synthesized with M-MLV reverse transcriptase, and the products were stored at -20 °C. Bioinformatic and phylogeny analysis The ORF was verified with the ORF Finder software (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The BmBrat protein domain was predicted using SMART (http://smart.embl-heidelberg.de/). The deduced amino acid sequences of BmBrat was aligned using the Clustal X program, and a phylogenetic tree was constructed by the neighbor-joining method (Tamura et al., 2007). The sequences used in this study were downloaded from GeneBank (http://www.ncbi.nlm.nih.gov/). Quantitative real-time PCR (qRT-PCR) For qRT-PCR analysis, a StepOne PlusTM Real-Time PCR system (Applied Biosystems, USA) and the SYBR Master Mix (Promega, USA) were used under the following conditions: 95 °C for 10 min, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. The housekeeping gene BmGAPDH was used as an internal control. All reactions were performed in triplicates in a total volume of 20 μL. Sequences of qRT-PCR primers were designed as follows: BmBrat-F, 5’-CGTGCGTTACTTGTCGTGT-3’; BmBrat-R, 5’-GCAGTTGGGGCAGAGGT-3’; GAPDH-F, 5’-CATTCCGCGTCCCTGTTGCTAAT-3’; GAPDH-R, 5’-GCTGCCTCCTTGACCTTTTGC-3’. Relative mRNA expression levels were calculated by using the standard 2-ΔΔCT method (Livak et al., 2001). The online t-test software GraphPad Software (http://www.graphpad.com/quickcalcs/ttest1.cfm) was used to evaluate the statistical significance (P<0.05). 151 Fig. 2 Bioinformatic analysis of BmBrat. (A) Multiple protein domain alignment of BmBrat in several species. BmBrat protein has a conserved RING domain, two B-BOX domain and a BBC domain. (B) Multiple sequence alignment of BmBrat in several species, and BmBrat gene has high homology in these species 152 Fig. 3 The phylogenetic tree of BmBrat. The phylogenetic tree of the BmBrat based on the full-length amino acid by the neighbor-joining method. The number closed to individual branches represents the percentage of 1000 bootstrap iterations supporting the branch, and values below 60% were omitted. The silkworm BmBrat are labeled with circles Cell transfection and BmBrat overexpression The BmBrat-Flag fragment for overexpression was generated by PCR using Dazao (P50) head cDNA template. Then it was ligated into the pSL1180-A4-DsRed plasmid. 2×105 BmE cells were seeded into 24-well plates for 24 h, and transiently transfected with pSL1180-A4-DsRed-BmBrat-Flag vector with X-tremeGENE HP DNA Transfection Reagent (Roche). After 72h transfection, cells were processed for western blot and immunofluorescence assay. Observation of the fluorescent signal was performed by using the confocal fluorescence microscope (Olympus FV1000, Olympus Corporation, Tokyo, Japan). BrdU staining After transfection with the overexpression vector, cells were grown on coverslips, and incubated with 10 μg /ml BrdU (Sigma) for 3 h, then washed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) for 20 min. Subsequently, cells were pre-treated with 1 mol/L HCl, and blocked with 10% goat serum for 1 h, followed by a monoclonal rat primary antibody against BrdU 153 (1:200, ab6326, Abcam, Cambridge, MA, USA) for 1 h and Alexa FluorR® 488 goat anti-rat IgG secondary antibody, (H+L; Invitrogen). DAPI (300 nM) was used for nuclear staining; the percentage of BrdU was calculated at least from 10 microscopic fields (Nikon 80i, Nikon Corporation, Tokyo, Japan). Flow cytometry For cell cycle analysis, flow cytometry was performed in BmE cells overexpressing BmBrat. 1×106 cells were harvested and fixed with 70% ethanol, stained with propidium iodide (PI) (BD Biosciences), then incubated with RNaseA for 30 min at room temperature. Finally, the cell samples were analyzed by Flow Cytometry using a FACS C6 (BD BioSciences), and the data was analyzed with CellQuest Pro software. Results Cloning and identification of BmBrat in silkworm BmBrat full-length sequence was obtained and identified (Fig. 1 A and B), which was located on chromosome 14 (Fig. 1 C), containing 859 amino acid residues. There is a conserved RING domain, two B-BOX domain and a BBC domain in Brat protein sequence of silkworm (Fig. 2 A). The homology of Brat from silkworm and other species was explored via multiple sequence alignment using clustal X (Fig. 2 B). Results of multiple protein domain alignment (Fig. 2 A) showed some differences between silkworm and other species in the genetic structure, especially in the sequence similarity among species. Although a relatively conserved NHL domain was found in the C-terminal region in all analyzed species, there were some differences between invertebrates and mammals. It was found that the sequence conservation of each domain was rather low, especially the conservation of the RING domain. However, the homology of the NHL domains in invertebrates and mammals, respectively, was very high. In addition, the cysteine in the BBOX domain was highly conserved, the hydrophobic amino acid was relatively conserved in the coiled-coil domain. Furthermore, phylogenetic analysis of BmBrat from several species using MEGA6 software generated a phylogenetic tree (Fig. 3). And results showed that, in evolution, BmBrat had far relationship with vertebrate homologous gene family TRIM. However, Danaua plexippus had the closest relationship with silkworm. BmBrat is highly expressed in ovary and head The mRNA expression levels of BmBrat over the embryo period from day 1 to day 9 and in various tissues at day 3 of 5th instar larvaes (L5D3) were analyzed by qRT-PCR, the results of which were shown in Fig. 4. We found that the BmBrat was highly expressed at day 4 and day 5, especially in day 5, and the expression level went down rapidly after day 5. We collected and compared most of organs in silkworm, including testis, ovary, epidermis, silk gland, malpighian tubule, midgut, head, fat body, and hemocytes. The data showed that BmBrat was expressed in various organizations, including the highest in the ovary, followed by the head. Fig. 4 The expression profile of BmBrat. (A) The temporal and spatial expression profiles of BmBrat in silkworm embryos. cDNAs were obtained from embryos at different stages. (B) Tissue-specific expression of BmBrat in silkworm larvae. cDNAs were obtained from the head, whole body, midgut, epidermis, fat body, silk gland, testis, ovary, malpighian tubule of L5D3 larvae. qRT-PCR was used to analyse BmBrat mRNA expression levels, and BmGAPDH was used as an internal control. All reactions were performed in triplicate BmBrat is specifically located in cytoplasm For further analysis of BmBrat in expression characteristics silkworm, full length expression vector pSL1180-A4-BmBrat-FLAG-Dsred was constructed (Fig. 5A). Then we overexpressed BmBrat in BmE cell line, pSL1180-A4-Dsred empty vector was used as control. Results showed that in the control group, the red signal of Dsred evenly spread out in cytoplasm and nucleus. On the other hand in the experimental group, the BmBrat signal only existed in cytoplasm but not in nucleus. In addition, we observed that the position of BmBrat signal is different in the prophase, metaphase, early telophase, and late telophase (Fig. 5C). This result suggested that the BmBrat was contributed in different positions in different period of cell division. 154 Fig. 5 The over-expression and localisation of BmBrat in silkworm BmE cells. (A) Construction of the pSL1180-A4-BmBrat-Dsred vector. BmBrat cloned into pSL1180-A4-Dsred vector with FLAG tag. (B) Western blot analyses of over-expression of BmBrat gene in BmE cells. Over-expression of BmBrat gene in BmE cells was obtained by transfection of BmBrat-FLAG-Dsred plasmid. (C) Localisation of BmBrat protein in BmE cells by immunofluorescence assay. FLAG (green) showed the distribution of BmBrat-FLAG; Dsred (red) showed the distribution of BmBrat-Dsred; DAPI (blue) showed the cell nuclei; Scale bar: 5 µm 155 Fig. 6 Over-expression of BmBrat inhibits cell proliferation in silkworm BmE cells. (A) BrdU staining was used to investigate cell proliferation in BmBrat up-regulation cells. Immunofluorescence detected by Anti-BrdU antibody. Scale bar: 20 µm. (B) Statistical analysis of BrdU positive cells ratio. Statistical analysis were performed using two-tailed student’s t-test, *p < 0.01. (C) FACS analysis were performed to investigate BmBrat overexpression influence cell cycle of BmE cells Over-expression of BmBrat inhibits the cell proliferation in BmE cells In order to figure out the role of BmBrat in cell process, BrdU staining was utilized after it was up-regulated in BmE cell line. First we use anti-Flag antibody to make sure that the BmBrat was successfully overexpressed (Fig. 5B). The BrdU staining result revealed that the BrdU positive cells decreased significantly (Fig. 6 A and B), which means the cell proliferation was surpressed after overexprssion of BmBrat. Moreover, FACS anaylsis was performed to check the changes of cell cycle in BmE cells which were overexpressed of BmBrat. And the result shown in Figure 6 C clarified that the cell cycle of BmE cells was arrested in G1 phase, and the S phase was significantly decreased, when overexpression of BmBrat was performed. It is suggested that the function of BmBrat may be to inhibit cell proliferation. Discussion NHL, particularity TRIM family, has been commonly studied in mammals for the function in cell proliferation especially in cancer development (Petrera et al., 2012). Bioinformatic analysis of BmBrat showed the gene encoding protein has four relatively conservative structure domain from the N terminal to C terminal, respectively a RING, two B-Box and a BBC structure domain, which were typical NHL structure domains. indicating it might function in the cell proliferation and differentiation (Tocchini et al., 2015). Expression spectrum analysis of BmBrat in silkworm embryonic showed that the amount of gene expression reached the peak at 4th and 5th day and went down after that. Silkworm eggs need about 10 days to incubate, and this period can be divided into divers stages according to the embryonic development process. The earlier 6 days of the incubation period is time for organ formation, and 4th and 5th day is the very summit of that, and the gene was highly expressed at this time. The organ development completed after the earlier 6 days, and the gene expression was downregulated significantly at the same time. This suggested that BmBrat might be associated with silkworm organ formation process in the embryonic development. L5D3 larvae 156 expression spectrum analysis illustrated that the gene was almost expressed in all tissues, especially in ovary and head, which means that the BmBrat was a tissue-specific gene and might have some certain functions that are still unknown. Results of localization analysis showed that the gene expression in embryonic cells BmBrat exists in cytoplasm, as part of the cells to start dividing BmBrat protein in the cells due to the role of cell polarity factors together to the bottom, asymmetrical distribution in the daughter cells. Symmetrical division of cells, the protein will average distribution into the daughter cells. To sum up, BmBrat genes in silkworm growth process plays a very important role. Speculation BmBrat genes involved in cell division, and is associated with the cell cycle, and the inhibition of cell proliferation. It has been reported the Brat has a variety of functions in Drosophila differentiating germ cells. It is known the maintenance of germline stem cell is regulated by the Dpp signaling pathway (Xie et al., 1998; Chen et al., 2003) and translation inhibitory factor Nanos (Nos)/Pumilio (Pum) (Forbes et al., 1998). In the ovary, Pum-Nos adjust the mRNA level of the Brat, limiting its expression in the cystoblasts (Muraro et al., 2008). Up-regulation of Dpp signal can promote the inhibition on Brat mRNA generation by Pum-Nos (Li et al., 2009). When cells divide, Dpp signal come into silence, the loss of Dpp signal downregulated Nos, which accelerate expression of the Brat. In return, Brat formed new compounds with Pum to inhibit mRNA levels of Mad and dMyc which works in Dpp signaling pathway (Ohlstein et al., 1997), reducing the response capacity of cystoblasts to Dpp signal, promoting cell differentiation (Harris et al., 2011). Therefore, whether BmBrat has the similar function in silkworm remains to be further studied. Competing interests The authors disclose no potential conflicts of interest. Acknowledgement Our work was supported by the National Natural Science Foundation of China (grant no. 81502574, 31672496), the Chongqing Research Program of Basic Research and Frontier Technology (grant no. cstc2017jcyjAX0304), the Fundamental Research Funds for the Central Universities (grant no. XDJK2016C007 and SWU118033), the Natural Science Foundation of Chongqing (grant no. cstc2016jcyjA0425), and the Chongqing University Innovation Team Building Special Fund (grant no. CXTDX201601010). Reference Bello B, Reichert H, Hirth F. The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila. Development. 133: 2639-2648, 2006. Berdnik D, Torok T, Gonzalez-Gaitan M, Knoblich JA. The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila. Dev. Cell. 3: 221-231, 2002. Boulay JL, Stiefel U, Taylor E, Dolder B, Merlo A, Hirth F. Loss of heterozygosity of TRIM3 in malignant gliomas. BMC Cancer. 9: 71, 2009. Chen D, McKearin D. Dpp signaling silences bam transcription directly to establish asymmetric divisions of germline stem cells. Curr. Biol. 13: 1786-1791, 2003. Chen G, Kong J, Tucker-Burden C, Anand M, Rong Y, Rahman F, et al. Human Brat ortholog TRIM3 is a tumor suppressor that regulates asymmetric cell division in glioblastoma. Cancer Res. 74: 4536-4548, 2014. Choksi SP, Southall TD, Bossing T, Edoff K, de Wit E, Fischer BE, et al. Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells. Dev. Cell. 11: 775-789, 2006. Cotton M, Benhra N, Le Borgne R. Numb inhibits the recycling of Sanpodo in Drosophila sensory organ precursor. Curr. Biol. 23: 581-587, 2013. Forbes A, Lehmann R. Nanos and Pumilio have critical roles in the development and function of Drosophila germline stem cells. Development. 125: 679-690, 1998. Harris RE, Pargett M, Sutcliffe C, Umulis D, Ashe HL. Brat promotes stem cell differentiation via control of a bistable switch that restricts BMP signaling. Dev. Cell. 20: 72-83, 2011. Hutterer A, Knoblich JA. Numb and alpha-Adaptin regulate Sanpodo endocytosis to specify cell fate in Drosophila external sensory organs. EMBO Rep. 6: 836-842, 2005. Lee CY, Wilkinson BD, Siegrist SE, Wharton RP, Doe CQ. Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal. Dev. Cell. 10: 441-449, 2006. Li Y, Minor NT, Park JK, McKearin DM, Maines JZ. Bam and Bgcn antagonize Nanos-dependent germ-line stem cell maintenance. Proc. Natl. Acad. Sci. U S A. 106: 9304-9309, 2009. Liu Y, Raheja R, Yeh N, Ciznadija D, Pedraza AM, Ozawa T, et al. TRIM3, a tumor suppressor linked to regulation of p21(Waf1/Cip1.). Oncogene. 33: 308-315, 2014. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25: 402-408, 2001. Loedige I, Filipowicz W. TRIM-NHL proteins take on miRNA regulation. Cell. 136: 818-820, 2009. Loedige I, Stotz M, Qamar S, Kramer K, Hennig J, Schubert T, et al. The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation. Genes Dev. 28: 749-764, 2014. Marchetti G, Reichardt I, Knoblich JA, Besse F. The TRIM-NHL protein Brat promotes axon maintenance by repressing src64B expression. J. Neurosci. 34: 13855-13864, 2014. Muraro NI, Weston AJ, Gerber AP, Luschnig S, Moffat KG, Baines RA. Pumilio binds para mRNA and requires Nanos and Brat to regulate sodium current in Drosophila motoneurons. J. Neurosci. 28: 2099-2109, 2008. Neumuller RA, Betschinger J, Fischer A, Bushati N, Poernbacher I, Mechtler K, et al. Mei-P26 regulates microRNAs and cell growth in the 157 Drosophila ovarian stem cell lineage. Nature. 454: 241-245, 2008. Nisole S, Stoye JP, Saib A. TRIM family proteins: retroviral restriction and antiviral defence. Nat. Rev. Microbiol. 3: 799-808, 2005. Ohlstein B, McKearin D. Ectopic expression of the Drosophila Bam protein eliminates oogenic germline stem cells. Development. 124: 3651-3662, 1997. Petrera F, Meroni G. TRIM proteins in development. Adv. Exp. Med. Biol. 770: 131-141, 2012. Reichert H. Drosophila neural stem cells: cell cycle control of self-renewal, differentiation, and termination in brain development. Results. Probl. Cell. Differ. 53: 529-546, 2011. Sardiello M, Cairo S, Fontanella B, Ballabio A, Meroni G. Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties. BMC Evol. Biol. 8: 225, 2008. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596-1599, 2007. Tocchini C, Ciosk R. TRIM-NHL proteins in development and disease. Semin. Cell. Dev. Biol. 47-48: 52-59, 2015. Wang W, Liu W, Wang Y, Zhou L, Tang X, Luo H. Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development. Dev. Biol. 350: 414-428, 2011. Xie T, Spradling AC. Decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary. Cell. 94: 251-260, 1998.