bmo-miR-275 down-regulates expression of Bombyx mori sericin gene 2 in vitro
bmo-miR-275 down-regulates expression of Bombyx mori sericin gene 2 in vitro
Ping Qian 0 1 2
Tao Jiang 0 1 2
Xin Wang 0 1 2
Fei Song 0 1 2
Chen Chen 0 1 2
Xingjia Shen 0 1 2
0 Nature Science Foundation of China (Grant Nos. 31402143 to PQ, 31672490 to XS), the Nature Science Foundation of JiangSu Province (Grant No. BK20141285 to XS), and the Doctoral Scientific Research Foundation of Jiangsu University of Science and Technology (Grant No. 1732931702 to PQ). The funders had no role in study design , data
1 Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology , Zhenjiang, Jiangsu , China , 2 Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences , Zhenjiang, Jiangsu , China
2 Editor: Erjun Ling, Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences , CHINA
We hypothesized that bmo-miR-275 has a potential regulatory function regarding the expression of sericin gene 2 (BmSer-2). First, we examined the expression of bmo-miR-275 and its target gene BmSer-2 in seven different tissues from 5th instar day-3 silkworm larvae. The results showed that they were both specifically expressed in the middle silk gland, implying that spatio-temporal conditions are required for bmo-miR-275 to regulate the expression of BmSer-2. To test this hypothesis, we constructed a pri-bmo-miR-275 expressing plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] and BmSer-2-3ÂUTR recombinant reporter plasmids pGL3.0 [A3-luc-Ser-2-30 UTR-SV40]. Finally, BmN cells were harvested and luciferase activity was detected. Results showed that luciferase activity was reduced significantly (P<0.05) in BmN cells co-transfected with pcDNA3.0 [ie1-egfp-pri-bmo-miR275-SV40] and pGL3.0 [A3-luc-Ser-2-3'UTR-SV40], suggesting that bmo-miR-275 can down-regulate the expression of BmSer-2 in vitro. Our results improve the understanding of the regulatory function of Bombyx mori miRNA on the expression of genes regulating silk formation.
Data Availability Statement: All relevant data are
within the paper.
MicroRNAs (miRNAs) are a class of endogenous noncoding regulatory RNAs with a length of
19±22 nucleotides. Mature miRNAs are sheared from longer primary transcripts through a
series of nuclease cleavage and modification processes and are then assembled into
RNAinduced silencing complexes. Mature miRNAs recognize target mRNAs through
complimentary base-pairing and regulate silencing complexes according to the degree of complementarity
with the target mRNA to degrade target mRNA or repress translation[1±4]. miRNAs are
mostly found in clusters in the genome[
], and most sequences are located in intergenic
regions, indicating that the transcription of miRNA is independent of other gene transcription
and that these species have their own mechanisms for transcriptional regulation. Studies have
shown that animal and plant miRNAs are located in the 30 and 50 untranslated regions (UTRs)
of the target mRNA[6±8]. miRNAs exhibit time-dependent, tissue-specific expression, which
is highly conserved[
]. Additionally, miRNAs are thought to regulate more than 30% of all
collection and analysis, decision to publish, or
preparation of the manuscript.
genes, blocking the expression of genes involved in many physiological and pathological
Bombyx mori is an important model used for studying animal development, differentiation,
growth, immune regulation, genetics, and mutations[
]. The silk gland of B. mori can
synthesize silk proteins with high efficiency and specificity and has been the focus of research for
many years. The silk gland of B. mori includes three parts: the anterior silk gland, middle silk
gland, and posterior silk gland. The silk protein sericin is synthesized mainly in the middle silk
gland. BmSer-2 in 11 chromosome is a specific gene coding sericin protein expressing in 120
cells of front of middle silk gland and is consist of 12 exon and 11 intron. Huang et al[
showed that bmo-miR-2b can suppress the expression of the fibroin protein gene BmP25 as
well as bmo-miR-965 and bmo-miR-1926 can suppress the expression of the fibroin light
chain gene (BmFib-L). Song Fei et al demonstrated that bmo-miR-2739 can up-regulatethe
fibroin heavy chain gene (BmFib-H). Additionally, we recently used Solexa to sequence the
sRNA of the middle silk gland of wild-type (P50) and fifth instar, 3-day larvae and obtained
several differentially expressed known miRNAs. Thus, we speculated that these miRNAs
might be associated with the synthesis of the silk protein sericin.
In this study, we performed bioinformatics analysis to identify potential targets of
bmomiR-275 and subsequently analyzed the role of bmo-miR-275 in the regulation of BmSer-2.
Our data provide important insights into miRNA functions in B. mori and the molecular
mechanisms regulating the expression of genes encoding silk proteins.
Materials and methods
Total RNA extraction from tissues of B. mori larva
An RNAiso Plus kit (TaKaRa, Shiga, Japan) was used to extract total RNA from various tissues
and organs of wild-type silkworms (P50) at 3-day fifth instar larvae provided by the
Sericultural Research Institute, Chinese Academy of Agricultural Sciences (CAAS; Zhenjiang,
China). The following tissues and organs were collected: skin, fat body, Malpighian tubules,
midgut, anterior silk gland, middle silk gland, posterior silk gland, and head. Electrophoresis
was used to determine the quality of total RNA.
Prediction of miRNAs in B. mori
Differentially expressed miRNAs obtained from Solexa-based high-throughput sequencing of
the middle silk gland in wild-type silkworms (P50) at fifth instar 3-day larvae in preliminary
experiments were used for comparisons with the 30UTR nucleic acid sequence of BmSer-2 in
B. mori, as downloaded from the NCBI database. Sequences of B. mori mature miRNAs from
miRBase (http://www.mirbase.org/) were analyzed by RNAhybrid software (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 (7-nucleotide
sequence from bases 2 to 8 at the 5' end of the miRNAs) and the 3'UTR of target 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[
Identification of bmo-miR-275. The specific reverse transcription primers for
bmo-miR275 were designed in accordance with a previous studies[
]. Total RNA from the middle silk
gland of the fifth instar 3-day larvae was used as the template, and cDNA synthesis was
performed using a reverse transcription kit (TaKaRa) following the manufacturer's protocol. The
cDNA was then used as a template for PCR. The PCR conditions were as follows: denaturation
at 94ÊC for 5 mins, 30 cycles of 94ÊC for 30 s, 64ÊC for 30 s, and 72ÊC for 30 s,with a final
extension at 72ÊC for 10 mins. PCR products were detected by electrophoresis using 4%
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agarose gels. The sequences of interest were collected and ligated into the pMD18-T vector at
16ÊC overnight. The ligation products were transformed into E.coli Top10 competent cells.
The plasmid DNA was then extracted and sequenced. Sequencing results were compared
using ClustalX 1.83 software.
Semiquantitative RT-PCR for bmo-miR-275 and its predicted target genes in B. mori
Primer design and synthesis. The upstream/downstream primers for BmSer-2, primers
for semiquantitative RT-PCR, and upstream/downstream primers for bmo-miR-275,
pri-miR275 (the primary of bmo-miR275) and the internal control U6 a housekeeping gene in B.mori
were designed using Oligo 6.0 software. Primers were synthesized by Shanghai Biological
Engineering Technical Services Corporation (Sangon, China).
cDNA synthesis and semiquantitative RT-PCR. Total RNA, extracted from different
organs of B. mori larvae, was reverse transcribed into a cDNA template for bmo-miR-275,
primiR-275, BmSer-2, and U6 amplification using specific primers and a reverse transcription kit
(TaKaRa), according to the manufacturer's protocol. The cDNA concentration of each sample
was diluted to 500ng/μL as the template for PCR. The PCR conditions were as follows:
denaturation at 94ÊC for 5 mins, 28 cycles of 94ÊC for 30 s,64ÊC for 30 s and 72ÊC for 30 s, with a
final extension at 72ÊC for 10 mins.
Construction of recombinant vectors expressing bmo-miR-275 and its predicted target genes
The pGL3.0-Basic plasmid was used as the template vector. The BmSer-2 30UTR region was
cloned downstream of the luciferase-encoding gene, luc, and the A3 promoter to generate the
recombinant vector pGL3.0 expressing a fusion gene containing the BmSer-2 30UTR and the
luc gene (A3-luc-Ser-2-30UTR-SV40). The pcDNA3 plasmid was used as the template vector.
The fragment containing the enhanced green fluorescent protein-encoding gene, egfp, and the
nucleotide sequences of precursors of the bmo-miRNA-275 and their up and down-stream
flanking regions which were downloaded from SilkBase
(http://silkbase.ab.a.u-tokyo.ac.jp/cgibin/index.cgi) were cloned into the vector with BmNPV ie1 as the promoter to construct the
recombinant vector expressing bmo-miR-275 pcDNA3 (ie1-egfp-pri-miR-275-SV40).
Plasmid transfection for verification of the regulation of predicted target gene expres
sion by bmo-miR-275. Healthy growing BmN cells were seeded in 12-well cell culture plates
1 day before transfection. Then, 1 mL of cell suspension, gently pipetted from the flasks, was
added per well for cell culture to allow cell attachment. The cell density for transfection was
approximately 1 × 105 cells/mL, and the plasmid concentration was approximately 400 ng/μL.
BmN cells were co-transfected as follows. The Renilla luciferase reporter plasmid pRL-CMV
was used as an internal control. Two solutions were prepared in 1.5-mL centrifuge tubes;
solution A contained 3 μL reporter plasmid DNA in 50 μL medium without antibiotics or fetal calf
serum and solution B contained 1 μL transfection reagent Per-Fect (UCallM, China) dissolved
in 50 μL medium without antibiotics or fetal calf serum. Solutions A and B were evenly mixed
and incubated at room temperature for 30 min. The original cell culture solution in 12-well
cell culture plates was discarded, and the cells were washed with 1 mL medium without serum
or antibiotics. Washing medium was then discarded and 800 μL cell culture medium without
antibiotics or fetal calf serum was added to the mixture of solutions A and B. The solutions
were mixed well and then added to cell culture plates. Cells were incubated in a 37ÊC incubator
for 5 h, after which the mixture of solutions A and B was removed, and 1 mL medium
containing fetal calf serum and antibiotics was added. Cells were cultured at 37ÊC for 72 h and were
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then collected for luciferase assays. Each treatment was repeated three times. A LuminoMeter
20/20n fluorescence spectrophotometer (Turner Biosystems, USA) was used to measure
activity of luciferase and Renilla luciferase[
]. After correction using the negative control and
blank control, the ratio of the two measurements was calculated to analyze the regulation of
Ser-2 expression by bmo-miR-275. Data were analyzed for statistical significance using SPSS
Evaluation of the quality of total RNA from B. mori larvae tissues and the bmo-miR-275 precursor
The bmo-miR-275 mature sequence of B. mori was obtained through sequencing conducted
by Liu et al[
]. Total RNA from tissues of the fifth instar 3-day larvae was extracted and
determined to be of sufficiently good quality for subsequent use by electrophoresis using 2% agarose
gels. All RNA samples showed three clear bands (data not shown).
The cDNA templates for amplification were generated by reverse transcription from the
total RNA of the middle silk gland of the fifth instar 3-day larvae. Electrophoresis showed a
fragment with a length of 60±80 bp, which was consistent with the theoretical length of the
bmo-miR-275 precursor fragment (Fig 1). The fragment of interest was collected and cloned
into the pMD18-T vector, and the plasmid was then transformed into E. coli Top10 competent
cells. The recombinant plasmid was extracted and sequenced. The size of the sequenced
product coincided with that of the expected sequence.
Prediction of bmo-miR-275 targeting BmSer-2 3'UTR
Target prediction software RNAhybrid was used to assess the scores and the complementarity
between the miRNA seed regions and the target site. bmo-miR-275 was predicted to have the
potential to target BmSer-2 3'UTR. Moreover, bmo-miR-275 binds BmSer-2 3'UTR starting
from the 39th base and the folding energy is −16.3 kcal/mol, suggesting that bmo-miR-275
function independently (Fig 2).
Temporal and spatial characteristics of the expression of bmo-miR-275 and
its target gene in BmSer-2
The expression levels of bmo-miR-275 and its target gene BmSer-2 in different tissues and
organs of the fifth instar, 3-day larvae were detected using semi-quantitative reverse
transcription polymerase chain reaction (RT-PCR)(The primers are list in Table 1). BmSer-2 was
expressed only in the middle silk gland, but was not expressed in other tissues. In contrast,
bmo-miR-275 was expressed only in the middle silk gland (Fig 3). These results suggest that
the expression of bmo-miR-275 and BmSer-2 in the fifth instar 3-day larvae was highly tissue
Regulation of BmSer-2 expression by bmo-miR-275
Construction of expression vectors. The expression vector pcDNA3.0
[ie1-egfp-pri-bmomiR-275-SV40] for miR-275 expression was successfully constructed to contain the BmNPV
ie1 promoter and an egfp reporter gene. The inserted gene fragments were tested by
electrophoresis using agarose gels after double digestion with HindIII and BamHI (Fig 4) and
sequencing by Shanghai Biological Engineering Technical Services Corporation (Sangon,
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Fig 1. Electrophoresis pattern of RT-PCR products ofbmo-miR-275. (M: 20 bp marker;1: bmo-miR-275).
Co-transfection efficiency of recombinant expression vectors
In the treatment group, BmN cells were co-transfected with a mixture of pcDNA3.0
[ie1-egfppri-bmo-miR-275-SV40], pGL3.0 [A3-luc-Ser-2-3'UTR-SV40], and pRL-CMV. In the control
group, BmN cells were co-transfected with a mixture of pcDNA3.0 [ie1-egfp-SV40], pGL3.0
[A3-luc-Ser-2-3'UTR-SV40], and pRL-CMV.
Observations were performed using an inverted fluorescence microscope. Results showed
that recombinant plasmids had been transfected into cells and that the majority of cells emitted
green fluorescence (Fig 5).
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Fig 2. Prediction of the binding sites of bmo-miR-275 in BmSer-2 mRNA using RNAhybrid software. The folding energy was −16.3 kcal/mol. The initiation
binding site at the target gene was the 39th base of the 3'UTR.
Primer sequences (50-30)
Regulation of BmSer-2 by bmo-miR-275, as determined by luciferase activity assays.
BmN cells were co-transfected with the bmo-miR-275 recombinant expression vector and the
recombinant expression vector containing the 30UTR sequence of the target gene. Subsequent
luciferase assays showed that luciferase activity in cells co-transfected with the bmo-miR-275
Fig 3. Expression analyses of bmo-miR-275, BmSer-2, and U6 in different tissues of 5th instar,3-day larvae of B. mori. (A) Expression of bmo-miR-275; (B)
Expression of BmSer-2; (C) Expression of internal gene U6. 1, head; 2, fat body; 3,anterior silk gland;4,middle silk gland; 5, post silk gland; 6, testis;7, ovary.
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Fig 4. Double enzyme digestion of recombinant plasmid pcDNA3.0 [ie1-egfp-pri-bmo-miR-275-SV40] with
HindIII and BamHI. M1: DL 2000 DNA marker; M2:λ-HindШ Marker; 1: pcDNA3.0
[ie1-egfp-pri-bmo-miR275-SV40] after double digestion with HindIII and BamHI.
recombinant expression vector and the expression vector containing the 30UTR of BmSer-2
fused with the reporter gene was significantly decreased by nearly 29% (p < 0.05) compared to
that in the control group (Fig 6).
From the above results, we conclude thatBmSer-2is one of the target genes of
bmo-miR275, and that bmo-miR-275 can down-regulate the expression of the BmSer-2 gene by
interacting with the 30UTR of its mRNA in vitro.
miRNAs participate in a variety of biological processes including cell proliferation, embryonic
development, individual growth, disease initiation, and cell apoptosis[21±23]. Currently, there
are 563 mature miRNA sequences in B. mori recorded in the miRBase database, indicating
that miRNAs play important roles in the regulation of gene expression during the growth and
development of silkworms. In this study, we used bioinformatics analysis and found that
BmSer-2 mRNA is a target of bmo-miR-275. We showed, for the first time, that this miRNA
significantly inhibited the expression of BmSer-2 using a dual luciferase reporter gene
detection system. Our results not only enrich the understanding of the B. mori miRNA database,
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Fig 5. Expression of enhanced green fluorescent proteinin BmN cells transfected with recombinant plasmids.
(A) pcDNA3.0 [ie1-egfp-SV40]+pGL3.0 [A3-luc-Ser-2-3'UTR-SV40]+pRL-CMV, bright light;
(B) pcDNA3.0 (ie1-egfp-SV40)+ pGL3.0 (A3-luc-Ser-2-3'UTR-SV40)+pRL-CMV, fluorescence;
(C) pcDNA3.0 (ie1-egfp-pri-bmo-miR-275-SV40)+ pGL3(A3-luc-Ser-2-3'UTR-SV40)+ pRL-CMV, brightlight;
(D) pcDNA3.0 (ie1-egfp-pri-bmo-miR-275-SV40)+ pGL3(A3-luc-Ser-2-3'UTR-SV40)+ pRL-CMV, fluorescence.
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Fig 6. Effect of bmo-miR-275 expressionon luciferase activity in transfected BmN cells. Data are represented as the mean ±standard deviation (SD) from three
(A) pcDNA3.0 (ie-1-egfp-SV40)+ pGL3 (A3-luc-Ser-2-3'UTR-SV40)+pRL-CMV
(B) pcDNA3.0 (ie-1-egfp -pri-mir-275-SV40)+ pGL3(A3-luc-Ser-2-3'UTR-SV40)+ pRL-CMV.
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but also provide new experimental data for further studies on miRNA function and to
elucidate the regulatory mechanisms of silk protein synthesis.
Some reports have shown that miRNAs can be regulated through the functions of
competitive endogenous RNAs (ceRNAs)[
]. These ceRNAs can act like ªspongesº to absorb
miRNA and reduce the concentration of miRNAs that are capable of binding to target mRNA
in cells. This dampens miRNA inhibition and thereby increases the level of mRNA translation
. Based on this concept, rather than silk gland cells, we used BmN cells in experiments;
these cells originated from the B. mori ovaries, which lack appropriate endogenous miRNA,
permitting the binding of exogenous miRNAs to target mRNAs, thereby downregulating the
expression of target genes. However, in vitro experimental models do not perfectly replicate
the in vivo situation, so additional studies are needed to further validate the functions of
miRNAs. This could be achieved by constructing recombinant baculoviral expression vectors for
overexpression of B. mori miRNA and artificially synthesizing miRNA analogs or antisense
RNA of miRNAs for transfection or injection into fifth instar larvae.
The regulatory mechanisms of silk protein expression are complicated, and the relationships
between miRNAs and target genes can vary, with miRNAs regulating more than one target
gene and a single target gene being regulated by more than one miRNA. Therefore, analysis of
transcriptional regulation through the assessment of individual miRNAs is not sufficient for
understanding the sophisticated mechanisms of silk protein gene regulation. Additional studies
involving an increased number of transcription factors are needed to obtain more information
regarding the regulation of silk protein gene expression by miRNAs. Furthermore, due to
miRNA can bind to both the 30 and 50UTRs of mRNAs, the latter of which can promote gene
expression rather than inhibit gene expression[
], more studies are needed to obtain
information regarding target sites for miRNAs in the 50UTRs of potential mRNA targets.
Conceptualization: Ping Qian.
Data curation: Ping Qian.
Formal analysis: Ping Qian.
Funding acquisition: Ping Qian, Xingjia Shen.
Investigation: Ping Qian, Tao Jiang, Xin Wang, Fei Song, Chen Chen.
Methodology: Ping Qian.
Project administration: Xingjia Shen.
Resources: Ping Qian, Xingjia Shen.
Supervision: Xingjia Shen.
Validation: Ping Qian.
Visualization: Ping Qian.
Writing ± original draft: Ping Qian.
Writing ± review & editing: Ping Qian.
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