bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro

bmo-miR-0001 and bmo-miR-0015 down-regulate expression of Bombyx mori fibroin light chain gene in vitro 家蚕miR-0001和miR-0015体外下调 BmFib-L 基因的表达 1 2 first-author 1 2 1 2 1 2 1 2 1 2 1 2 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:shenxjsri@163.com †E-mail:shenxjsri@163.com shenxjsri@163.com Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212003, China Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212003, China Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmo-miR-0015, from high-throughput sequencing of the Bombyx mori larval posterior silk gland (PSG). Firstly, we examined the expression of bmo-miR-0001 and bmo-miR-0015 in 12 different tissues of the 5th instar Day-3 larvae of the silkworm. The results showed that the expression levels of both bmo-miR-0001 and bmo-miR-0015 were obviously higher in the PSG than in other tissues, implying there is a spatio-temporal condition for bmo-miR-0001 and bmo-miR-0015 to regulate the expression of BmFib-L . To test this hypothesis, we constructed pri-bmo-miR-0001 expressing the plasmid pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0001-SV40] and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0015-SV40]. Finally, the BmN cells were harvested and luciferase activity was detected. The results showed that luciferase activity was reduced significantly ( P < 0.05) in BmN cells co-transfected by pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0001-SV40] or pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0015-SV40] with pGL3.0 [ A 3 -luc-Fib-L -3'UTR-SV40], suggesting that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro. 目的 2 探究新发现的两个miRNA对家蚕丝素轻链基因 BmFib-L 的调控作用。 创新点 2 在家蚕后部丝腺中发现两个新的miRNA，并首次证明它们对家蚕丝素蛋白轻链基因 BmFib-L 有负调控作用。 方法 2 本实验通过生物信息学分析，从家蚕后部丝腺miRNA高通量测序获得的新miRNA中，筛选出两个可能对家蚕丝素蛋白轻链基因 BmFib-L 有调控作用的miRNA，即bmo-miR-0001和bmo-miR-0015。设计茎环引物，采用反转录聚合酶链反应（RT-PCR）方法对家蚕5龄3 d头部、表皮、脂肪体、马氏管、精巢/卵巢、丝腺（前、中、后）、气管、中肠和血淋巴细胞等12个不同组织的bmo-miR-0001和bmo-miR-00015进行半定量表达分析。并采用双荧光报告基因检测系统进一步在细胞水平上验证bmo-miR-0001和bmo-miR-0015对 BmFib-L 表达的调控作用。 结论 2 本实验中RT-PCR结果显示，这两个新miRNA在家蚕后部丝腺中表达量最高（图4）。双荧光报告基因检测结果显示，报告基因荧光素酶活性明显低于阳性对照组（图7），转染bmo-miR-0001和bmo-miR-0015表达载体的细胞，报告基因和荧光素酶活性分别只及对照组60%和69%。 t 检验分析结果显示两个实验组与对照组之间差异都达到显著水平（ P < 0.05）。由此可见，bmo-miR-0001和bmo-miR-0015在体外对 BmFib-L 的表达具有显著的抑制作用。 Bombyx mori MicroRNA bmo-miR-0001 bmo-miR-0015 BmFib-L Regulation of expression 家蚕 miRNA bmo-miR-0001 bmo-miR-0015 BmFib-L 功能验证 Introduction 1 MicroRNAs (miRNAs) are small non-coding RNA molecules about 19 to 22 nucleotides long, which mostly inhibit the expression or translation of target genes by complementary base pairing to the target mRNA 3' untranslated region (3'UTR) (Lee et al., 2004 2004 ). They are involved in a lot of biological processes, including development, hematopoietic lineage differentiation, organ formation, proliferation, apoptosis, host viral interactions, and tumorigenesis (Lim et al., 2003 2003 ; Poy et al., 2004 2004 ; Bushati and Cohen, 2007 2007 ; Yuan et al., 2014 2014 ). Research on miRNAs has been conducted on a range of organisms including animals (Dai et al., 2014 2014 ; Lu et al., 2014 2014 ), plants (Mallory and Vaucheret, 2006 2006 ), microbes (Liu et al., 2016 2016 ), and viruses (Jopling et al., 2005 2005 ). Studies on miRNAs will provide a theoretical basis for post-transcriptional regulation of genes and benefit our understanding of gene regulatory mechanisms. The silkworm ( Bombyx mori ) is an insect that undergoes complete metamorphosis. It not only has the biological traits common to other insects, but also has its own characteristic properties making it an excellent model for the study of insect genetics and molecular biology (Xia et al., 2004 2004 ; 2009 2009 ). With the completion of sequencing of the silkworm genome, the study of B. mori miRNA has entered a new development stage that has led to the discovery of novel miRNAs (Yu et al., 2008 2008 ; 2009 2009 ; Cai et al., 2010 2010 ; Yang et al., 2012 2012 ), prediction of miRNA targets (He et al., 2008 2008 ; Zeng et al., 2009 2009 ; Huang et al., 2012 2012 ; Chen et al., 2013 2013 ), and identification and expression of miRNAs (Zhang et al., 2009 2009 ; Liu et al., 2010 2010 ; Li et al., 2014 2014 ). The mechanism of silk protein biosynthesis has been a popular focus of research among a large numbers of studies. The silk glands, a special organ in the silkworm that synthesizes and secretes the silk protein, can be divided into the anterior silk gland (ASG), middle silk gland (MSG), and posterior silk gland (PSG) according to their morphological specificity and function (Inoue et al., 2000 2000 ). The ASG is a catheter of silk protein with no function in silk protein biosynthesis and the MSG mainly synthesizes and secretes sericin proteins, whereas the PSG synthesizes and secretes silk fibroin, which accounts for more than 70% of the total silk protein. Fibroin comprises the fibroin heavy chain protein (Fib-H), fibroin light chain protein (Fib-L), and P25/fibrohexamerin protein (P25) (Sprague, 1975 1975 ; Yamaguchi et al., 1989 1989 ). Studies have shown that B. mori miRNAs are involved in regulation of fibroin gene expression. Four bmo-miRNAs have been predicted to have potential binding sites in the BmFib-L gene and might play important roles in the regulation of silk protein biosynthesis (Cao et al., 2008 2008 ). The miRNA bmo-miR-2b also regulates the expression of BmP25 (Huang et al., 2011 2011 ). Both bmo-miR-965 and bmo-miR-1926 inhibit the expression of BmFib-L in vitro (Huang et al., 2012 2012 ). However, bmo-miR-2739 has been shown to up-regulate the expression of BmFib-H (Song et al., 2014 2014 ). In the previous high-throughput sequencing of PSG small RNAs in our laboratory, 35 novel bmo-miRNAs were obtained, among which two, bmo-miR-0001 and bmo-miR-0015, are predicted to function in regulating the expression of BmFib-L . To validate this prediction, the expressions of bmo-miR-0001, bmo-miR-0015, and their potential target gene BmFib-L were examined by reverse transcription polymerase chain reaction (RT-PCR) at the mRNA level. The regulatory function of these two miRNAs was also tested using a dual luciferase reporter (DLR) system in BmN cells. Materials and methods 1 Materials 2 The domesticated silkworm ( B. mori ) used in this experiment was of the strain P50, provided by the Sericultural Research Institute, Chinese Academy of Agricultural Sciences (CAAS; Zhenjiang, China). Plasmids pcDNA3.0 (Invitrogen, Shanghai, China), pRL-CMV (containing a Renilla luciferase gene), and pGL3.0 [ A 3- luc - Fib-L- 3'UTR-SV40], and BmN cells were preserved or constructed by the Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, China. Restriction enzymes, T4 DNA ligase, PCR reagents, and a reverse transcription kit were purchased from TaKaRa (Dalian, China). Primers were synthesized by Sangon Biotech (Shanghai, China). Fetal bovine serum (FBS) and TC-100 cell culture medium were purchased from Invitrogen. Cell perfect transfection reagent was purchased from UcallM (Wuxi, China). A Dual-Luciferase® Reporter (DLR™) Assay System kit was purchased from Promega (Madison, USA). Methods 2 Screening of B. mori miRNAs regulating BmFib-L expression 3 To screen B. mori miRNAs for those that may regulate the expression of BmFib-L , the BmFib-L sequence from the National Center for Biotechnology Information (NCBI) ( http://www.ncbi.nlm.nih.gov/gene http://www.ncbi.nlm.nih.gov/gene ) and sequences of B. mori mature miRNAs from Solexa sequencing were analyzed by RNAhybrid software ( http://bibiserv.techfak.uni-bielefeld.de/rnahybrid http://bibiserv.techfak.uni-bielefeld.de/rnahybrid ). The potential targets of miRNAs were identified as having perfect sequence complementarity between the seed region of the miRNA (a 7-nucleotide sequence from bases 2 to 8 at the 5' end of the miRNAs) and the 3'UTR of target ( BmFib-L ) mRNAs, and less than −20.0 kcal/mol (1 kcal/mol=4.182 kJ/mol) free energy in the secondary structure of the miRNA/mRNA duplex (Huang et al., 2010 2010 ). Cloning of novel miRNAs and prediction of the secondary structures of their precursors 3 Total RNA was extracted from the PSG of Day-3 5th instar larvae using TRIzol (Invitrogen) according to the manufacturer’s instructions. The complementary DNAs (cDNAs) were synthesized from total RNA using miRNA specific stem-loop primers according to the method of Kramer ( 2011 2011 ). Subsequently, PCR was carried out with the following cycling conditions: initial denaturation at 94 °C for 5 min, 34 cycles of 94 °C for 30 s, 55 °C for 25 s, and 72 °C for 30 s, and final extension at 72 °C for 10 min. The PCR products were separated by electrophoresis in 4% (0.04 g/ml) agarose gels. After purification, the gene fragments were cloned into a pMD18-T vector and sequenced. Using ClustalX 1.83, multiple sequence alignment software was applied for further confirmation of sequencing, and secondary structures were predicted using RNAfold ( http://rna.tbi.univie http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi.ac.at/cgi-bin/RNAfold.cgi . ac.at/cgi-bin/RNAfold.cgi). The sequences of the primers are listed in Table 1 1 . Primer 2 Sequence (5'→3') bmo-miR-0001 RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTGTAC Forward GCCCGATTTATTTATATAGATAGT Reverse GTGCAGGGTCCGAGGT bmo-miR-0015 RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACTTC Forward GTGCAGGGTCCGAGGT Reverse CCCAAGCTTAAGTCCATTCCAATAGC U6 RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACG Forward CCTGCGCAAGGATGAC Reverse GTGCAGGGTCCGAGGT pri-bmo-miR-0001 Forward CGGGATCCAGCTCACCTACATGTTA Reverse CCCAAGCTTAAGTCCATTCCAATAGC pri-bmo-miR-0015 Forward CGGGATCCACAAGTACGCAAAAGCT Reverse CCCAAGCTTAAGGCGCACCATCAATT Detection of expression by RT-PCR 3 To investigate the possibility of spatio-temporal effects on the predicted function of bmo-miRNAs in regulating the expression of BmFib-L , the expression levels of potential bmo-miRNAs and BmFib-L in the silk gland of the silkworm larvae were analyzed. Firstly, total RNAs were extracted from different tissues including the head, epidermis, fat body, Malpighian tubule, ASG, MSG, PSG, trachea, testis, ovary, midgut, and haemolymph of the 5th instar Day-3 larva using TRIzol (Invitrogen) according to the manufacturer’s instructions. cDNAs were synthesized from total RNAs using a cDNA reverse transcription kit (TaKaRa). Subsequently, PCR was carried out and the products were detected as described above and photographed under UV light. Three parallel experiments were carried out and the B. mori U6 gene served as an internal control. Finally, the relative expression levels of the potential bmo-miRNAs and BmFib-L in different tissues were determined using Gel-Pro Analyzer software (Media Cybernetics, USA). Construction of expression vectors 3 To validate the function of potential bmo-miRNAs in the regulation of BmFib-L expression in vitro, bmo-miRNA expression vectors were constructed as described previously (Huang et al., 2011 2011 ). The luciferase gene ( luc ) fused BmFib-L 3'UTR expression vectors pGL3.0 [ A 3 -luc-Fib-L -3'UTR-SV40] constructed previously in our laboratory (Huang et al., 2012 2012 ) were used. The nucleotide sequences of precursors of the potential bmo-miRNAs and their up- and down-stream flanking regions were downloaded from SilkBase ( http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi ). Then primers designed using Oligo 7 software (molecular Biology Insights, Inc., Cascade, CO, USA) were constructed for PCR of the potential bmo-miRNAs and the PCR products were cloned into the pET-18 vector for sequence determination. The pET-18-pri-miR-0001 and pET-18-pri-miR-0015 constructs were double digested with Hin dIII and Bam HI, separately. The nucleotide sequences of the mature bmo-miRNAs, bmo-miR-0001, and bmo-miR-0015, and their flanking regions (±100 bp) were cloned into the pCDNA3.0 plasmid downstream of the egfp gene controlled by the B. mori nucleopolyhedrovirus (BmNPV) ie 1 promoter for construction of the bmo-miRNA expression vectors pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0001-SV40] and [ ie 1- egfp -pri-bmo-miR-0015-SV40]. The structure of these constructs was confirmed by sequencing (Sangon Biotech). Cell culture, transfection, and transient expression assay 3 The method of Zhou et al. ( 2002 2002 ) was used for routine BmN cell culture. Cells were seeded into 12-well plates (600 μl for each) at a density of about 5×10 5 cells/ml and cultured for 24 h (Song et al., 2013 2013 ). Before transfection, the medium was removed and the cells were washed twice with serum-free TC-100 medium. In the treatment group, cells in each well were co-transfected with a 1.6-μg mixture of DNAs including 0.7 μg pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0001-SV40] or pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0015-SV40], 0.7 μg pGL3.0 [ A 3- luc - Fib-L -3'UTR-SV40], and 0.2 μg internal plasmid pRL-CMV via PerFect Cellfectin reagent (UcallM), according to the manufacturer’s manual (PerFect; UcallM). In the control group, cells in each well were co-transfected with 1.6 μg of a mixture DNAs including 0.7 μg pcDNA3.0 [ ie1-egfp -SV40], 0.7 μg pGL3.0 [ A 3- luc-Fib-L- 3'UTR-SV40], and 0.2 μg pRL-CMV. There were three replicates for each experiment. Before harvesting, cells were observed under an inverted fluorescence microscope. At 72 h post transfection (hpt) cells were harvested by centrifugation at 9000 g for 5 min at 4 °C, washed once in phosphate-buffered saline and lysed in 150 μl of passive lysis buffer (Promega). The supernatants were used for luciferase activity assay using a Luc assay kit (Promega) in a GloMax® 20/20 Luminometer (Turner Biosystems Inc., Sunnyvale, CA, USA; 2 s delay and read at 10 s) as the relative luminescence unit (RLU) (Zhao et al., 2007 2007 ). The activity of firefly was normalized by the Renilla luciferase activity of pRL-CMV. Results 1 Prediction of novel B. mori miRNAs targeting BmFib-L 3'UTR 2 Target prediction software RNAhybrid was used to assess the scores and the complementarity between the miRNA seed regions and the target site. Among 35 novel B. mori miRNAs obtained from previous Solexa sequencing (Song et al., 2015 2015 ), bmo-miR-0001 and bmo-miR-00015 were predicted to have the potential to target BmFib-L 3'UTR. Moreover, pri-bmo-miR-0001 binds BmFib-L 3'UTR starting from the 43rd base, while pri-bmo-miR-0015 starts from the 5th base, suggesting that bmo-miR-0001 and bmo-miR-00015 function independently (Fig. 1 1 ). (a) Prediction of the binding site of bmo-miR-0001 in BmFib-L mRNA. The folding energy was −16.3 kcal/mol. The initiation binding site at the target gene was the 43rd base of the 3'UTR; (b) Prediction of the binding site of bmo-miR-0015 in BmFib-L mRNA. The folding energy was −15.2 kcal/mol. The initiation binding site at the target gene was the 5th base of the 3'UTR Cloning of candidate miRNAs and prediction of secondary structures of their precursors 2 For each miRNA, RT-PCR was performed with the primers above using the total RNA of the 5 th instar Day-3 larvae of the silkworm as a template, and a band of about 60–80 bp was separated by agarose gel electrophoresis, consistent with expectations (Fig. 2a 2a ). Sequencing results revealed that the nucleotide sequences of the two cloned miRNAs were the same as those from Solexa sequencing (Figs. 2b and 2c 2b and 2c ): bmo-miR-0001, AUU UAU UUA UAU AGA UAG UAC AA; bmo-miR-0015, AAA GGA UUU AAC AGA CAU GAA GUU. The secondary structures of their precursors were typical of miRNA precursors, with stem-loops (Fig. 3 3 ). The free energies of the bmo-miR-0001 and bmo-miR-0015 precursors were −18.10 and −25.80 kcal/mol, respectively. (a) Electrophoresis pattern of RT-PCR products of bmo-miR-0001 and bmo-miR-0015 (M: 20 bp marker; 1: bmo-miR-0001; 2: bmo-miR-0015; 3: bmo-miR-26* (control); 4: U6); (b) Sequence alignment result of bmo-miR-0001; (c) Sequence alignment result of bmo-miR-0015 (a) Secondary structure of the bmo-miR-0001 precursor; (b) Secondary structure of the bmo-miR-0015 precursor Expression of bmo-miR-0001 and bmo-miR-0015 and their target gene BmFib-L 2 The RT-PCR results showed that in the 12 tissues tested bmo-miR-0001 expression was detected only in the PSG (Fig. 4a 4a ). In contrast, bmo-miR-0015 expression was detected not only throughout the silk glands (ASG, MSG, and PSG), but also in the head, fat body, Malpighian tubule, testis, trachea, ovary, and midgut, but the expression level was highest in the PSG (Fig. 4b 4b ). BmFib-L expression was detected not only in the PSG, but also in the fat body, testis, MSG, trachea, and ovary (Fig. 4c 4c ). This is consistent with the results of Sun et al. ( 2009 2009 ) and is known as “leaking expression” (Wang and Lu, 2006 2006 ). The above experimental results show that spatio-temporal conditions affect the regulation of the expression of BmFib-L by bmo-miR-0001 and bmo-miR-0015. Together with the target prediction result, we deduced that bmo-miR-0001 and bmo-miR-0015 may be involved in the regulation of BmFib-L expression in B. mori larvae. (a) Expression of bmo-miR-0001; (b) Expression of bmo-miR-0015; (c) Expression of BmFib-L ; (d) Expression of internal gene U6. 1, head; 2, epidermis; 3, fat body; 4, Malpighian tubule; 5, testis; 6, anterior silk gland; 7, middle silk gland; 8, post silk gland; 9, trachea; 10, ovary; 11, midgut; 12, haemolymph Validating the roles of bmo-miR-0001 and bmo-miR-0015 in regulating the expression of BmFib-L 2 Construction of expression vectors 3 The expression vectors pcDNA3.0 [ ie 1 -egfp -pri-bmo-miR-0001-SV40] for miR-0001 and pcDNA3.0 [ ie 1 -egfp -pri-bmo-miR-0015-SV40] for miR-0015 were successfully constructed to contain the BmNPV ie 1 promoter and an egfp reporter gene. The inserted gene fragments were tested by electrophoresis in agarose gels after double digestion with Hin dIII and Bam HI (Fig. 5 5 ). (a) Identification of ie 1- egfp -pri-bmo-miR-0001-SV40 structure (M: DL 2000 DNA marker; 1, 2: pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0001-SV40] after double digestion with Hin dIII and Bam HI); (b) Identification of ie 1- egfp -pri-bmo-miR-0015-SV40 structure (M: DL 2000 DNA marker; 3, 4: pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0015-SV40] after double digestion with Hin dIII and Bam HI) Regulation of BmFib-L expression by bmo-miR-0001 and bmo-miR-0015 in BmN cells 3 In the treatment group, BmN cells were co-transfected with a mixture of pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0001-SV40] or pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0015-SV40], pGL3.0 [ A 3- luc - Fib-L -3'UTR-SV40], and pRL-CMV. In the control group, BmN cells were co-transfected with a mixture of pcDNA3.0 [ ie 1 -egfp -SV40], pGL3.0 [ A 3- luc-Fib-L- 3'UTR-SV40], and pRL-CMV. Observations were carried out under an inverted fluorescence microscope. Results showed that recombinant plasmids had been transfected into cells and the majority of cells emitted green fluorescence (Fig. 6 6 ). Cells were then harvested and luciferase activity was assayed using a Luc assay kit (Promega) in a Luminometer 20/20, as described above. The results showed that luciferase activity in the two experimental groups decreased to 60% and 69%, respectively, of that of the control group. The differences between the treatment groups and the control groups were significant ( P < 0.05; Fig. 7 7 ), indicating that bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of BmFib-L in vitro. (a) pcDNA3.0 [ ie 1- egfp -SV40]+pGL3.0 [ A 3- luc - Fib-L -3'UTR-SV40]+pRL-CMV, bright light; (b) pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0001-SV40]+pGL3.0 [ A 3- luc - Fib-L -3'UTR-SV40]+pRL-CMV, bright light; (c) pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0015-SV40]+pGL3.0 [ A 3- luc - Fib-L -3'UTR-SV40]+pRL-CMV, bright light; (d) pcDNA3.0 [ ie 1- egfp -SV40]+pGL3.0 [ A 3- luc - Fib-L -3'UTR-SV40]+pRL-CMV, fluorescence; (e) pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0001-SV40]+pGL3.0 [ A 3- luc - Fib-L -3'UTR-SV40]+pRL-CMV, fluorescence; (f) pcDNA3.0 [ ie 1- egfp -pri-bmo-miR-0015-SV40]+pGL3.0 [ A 3- luc - Fib-L -3'UTR-SV40]+pRL-CMV, fluorescence Data represented as the mean±standard deviation (SD) from three independent experiments. *** represents P < 0.001 in unpaired t -tests From the above results, we conclude that BmFib-L is one of the target genes of bmo-miR-0001 and bmo-miR-0015, and that both bmo-miR-0001 and bmo-miR-0015 can down-regulate the expression of the BmFib-L gene by interacting with its 3'UTR of mRNA in vitro. Discussion 1 miRNAs participate in a variety of biological activities, including cell proliferation, embryonic development, individual growth, occurrence and development of disease, and apoptosis (Johnston and Hobert, 2003 2003 ; Lim et al., 2003 2003 ; Poy et al., 2004 2004 ). There are 563 silkworm mature miRNA sequences registered in the MiRBase ( http://www.mirbase.org http://www.mirbase.org ). Studies have shown that B. mori miRNAs play essential roles in silkworm development and growth. At least four bmo-miRNAs, miRNA-33, miRNA-190, miRNA-276, and miRNA-7, participate in the regulation of BmFib-L expression (Cao et al., 2008 2008 ). miR-965 and miR-1926 have been shown to down-regulate the expression of BmFib-L in vitro (Huang et al., 2012 2012 ). Based on Solexa sequencing results, we obtained two novel candidate bmo-miRNAs, bmo-miR-0001 and bmo-miR-0015, complementary to the 3'UTR of BmFib-L mRNA using RNAhybrid target prediction software. The DLR system was applied to validate the role of these two miRNAs in regulating the expression of BmFib-L in cells. For the first time, we reported that bmo-miR-0001 and bmo-miR-0015 down-regulate the expression of BmFib-L in vitro. This result is similar with that described by Huang et al. ( 2012 2012 ) and is consistent with the finding that the majority of miRNAs inhibit expression of their target gene by binding to its 3'UTR (Fabian et al., 2010 2010 ). These results will not only enrich the database of silkworm miRNAs, but also provide new experimental data for further study of miRNA function and the regulatory mechanism of silk protein biosynthesis. B. mori silk fibroin comprises BmFib-H, BmFib-L, and BmP25 proteins (Sprague, 1975 1975 ; Couble et al., 1983 1983 ; Yamaguchi et al., 1989 1989 ), which are produced abundantly in the silk glands of 5th instar larvae (Wang et al., 2011 2011 ). However, in this experiment bmo-miR-0001 and bmo-miR-0015 down-regulated the expression of the BmFib-L gene. Recent studies have identified a competing endogenous RNA (ceRNA) gene regulatory mechanism, in which ceRNAs serve as miRNA absorbing sponges to vie with miRNAs by sharing miRNA response elements (MREs) in mRNAs (Khan et al., 2009 2009 ; Ebert and Sharp, 2010 2010 ). These ceRNAs act as modulators of miRNAs by lowering the available miRNA levels for the target mRNA, resulting in increased translation (Sen et al., 2014 2014 ). BmN cells used in this experiment were obtained from B. mori ovarian tissue instead of silk gland cells and might lack corresponding interference endogenous miRNAs. This may have provided an opportunity for the exogenous miRNAs (bmo-miR-0001 and bmo-miR-0015) to bind the mRNA of the target gene ( BmFib-L ), resulting in down-regulation of target gene expression. However, it is also highly likely that there are real differences between expression in the silkworm larvae and in the in vitro gene model. Therefore, further study is needed on the regulation of BmFib-L expression by bmo-miR-0001 and bmo-miR-0015 in vivo via over expression of silkworm miRNAs following infection by a recombinant B. mori baculovirus expression vector, or injection of synthetic miRNA analogues or antisense RNA of miRNAs into the 5th instar larvae. The expression and regulation of genes producing silk protein are very complex. However, Shalgi et al. ( 2007 2007 ) have proposed the existence of hundreds of “target hub” genes, each potentially subject to massive regulation by dozens of miRNAs. Therefore, the study of individual miRNAs is insufficient to uncover the complicated and precise regulatory mechanisms of silk protein biosynthesis. Studies of other transcription factors and more information about the regulation of silk protein biosynthesis by miRNAs are needed. 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All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1500078 Project supported by the National Natural Science Foundation of China (Nos. 31172266/C1703 and 31402143/C1703) 2015-04-06 2016-01-13 2015-11-07 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 17