Xin Wang

The regulation of silkworm fibroin L chain production by miRNA-965 and miRNA-1926 in insect cells

MicroRNAs (miRNAs) are an abundant class of approximately 22-nucleotide (nt)-short noncoding RNA molecules present in the genomes of all multicellular organisms that act through base pairing to partially complementary sequences of the 3′untranslated region (UTR) of targeted mRNAs. Using bioinformatic approach, we found that the 3′ UTR of the Fibroin L chain (Fib-L) mRNA matches perfectly the nucleotides 2–8 at the 5′ end of the miRNA-965 and miRNA-1926. These two miRNAs might act as regulators of Fib-L gene expression at the post-transcriptional level. To examine whether Fib-L is directly targeted by miRNA-965 and miRNA-1926 in vitro, miRNA expression vectors and target reporter vector with 3′UTR of Fib-L were constructed respectively. Two vectors were co∼transfected into Sf21 cells. The luciferase assay showed that miRNA-965 and miRNA-1926 may down regulate the expression of Fib-L via complementary interaction with the target sites in 3′UTR.

Overview of research on Bombyx mori microRNA.

Abstract MicroRNAs (miRNAs) constitute some of the most significant regulatory factors involved at the post-transcriptional level after gene expression, contributing to the modulation of a large number of physiological processes such as development, metabolism, and disease occurrence. This review comprehensively and retrospectively explores the literature investigating silkworm, Bombyx mori L. (Lepidoptera: Bombicidae), miRNAs published to date, including discovery, identification, expression profiling analysis, target gene prediction, and the functional analysis of both miRNAs and their targets. It may provide experimental considerations and approaches for future study of miRNAs and benefit elucidation of the mechanisms of miRNAs involved in silkworm developmental processes and intracellular activities of other unknown non-coding RNAs.

bmo-miR-275 down-regulates expression of Bombyx mori sericin gene 2 in vitro.

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-2-3´UTR recombinant reporter plasmids pGL3.0 [A3-luc-Ser-2-3′ 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-miR-275-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.

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

Based on bioinformatic analysis, we selected two novel microRNAs (miRNAs), bmo-miR-0001 and bmomiR-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 [ie1-egfp-pri-bmo-miR-0001-SV40] and pri-bmo-miR-0015 expressing the plasmid pcDNA3.0 [ie1-egfp-pribmo-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 [ie1-egfp-pri-bmomiR-0001-SV40] or pcDNA3.0 [ie1-egfp-pri-bmo-miR-0015-SV40] with pGL3.0 [A3-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.中文概要目 的探究新发现的两个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bmomiR-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。

Bmo-miR-2758 Targets BmFMBP-1 (Lepidoptera: Bombycidae) and Suppresses Its Expression in BmN Cells

MicroRNAs (miRNAs) are an abundant family of endogenous noncoding small RNA molecules. They play crucial roles on regulation of life processes both in plants and animals. Fibroin modulator binding protein-1 (FMBP-1) is a silk gland transcription factor of Bombyx mori, which is considered as a trans-activator of fibroin genes. And bioinformatics prediction showed that at the 3′ untranslated region (3′ UTR) of BmFMBP-1 there were binding sites for three bmo-miRNAs, bmo-miR-2b*, bmo-miR-305, and bmo-miR-2758, separately. In order to validate whether these bmo-miRNAs involved in the regulation of BmFMBP-1 expression, the expression levels of three bmo-miRNAs and BmFMBP-1 in the middle silk gland (MSG) and posterior silk gland (PSG) during the fourth- and fifth-larval stages of B. mori were measured by semi-quantitative reverse transcription polymerase chain reaction. The results revealed that the expression level of bmo-miR-2758 was the highest in the three, and it expressed higher in the PSG than in the MSG with a similar expression pattern as BmFMBP-1, implying that bmo-miR-2758 may involved in regulation of BmFMBP-1. To validate the regulation function of bmo-miR-2758 on BmFMBP-1, recombinant plasmids pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] and pGL3 [A3-luc-FMBP-1 3′ UTR-SV40] were constructed and co-transfected in BmN cells. The dual-luciferase reporter assay system was used for assay of transient expression. The results showed that the expression of the luciferase reporter was significantly decreased when pGL3 [A3-luc-FMBP-1 3′ UTR-SV40] co-transfected with pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] (P < 0.01). Furthermore, when the artificial antisense RNA of bmo-miR-2758 (inhibitor) was added to the above co-transfection, the expression of the luciferase reporter was recovered significantly (P < 0.01). These results suggest that bmo-miR-2758 represses the expression of BmFMBP-1 in vitro.

Characterization and profiling of MicroRNAs in posterior silk gland of the silkworm ( Bombyx mori )

MicroRNAs (miRNAs) regulate expression of genes at post-transcriptional level by binding on complementary sequences of target mRNAs and play multiple roles in biological processes. To investigate the differential expression of miRNAs in posterior silk gland (PSG) of silkworm (Bombyx mori) in different periods and regulation of miRNAs on the expression of fibroin genes, Solexa sequencing technology was used to detect miRNAs in PSGs of fourth-instar day-2 larvae and fifth-instar day-3 larvae, respectively. As a result, 466 previously reported miRNAs, and 35 novel miRNAs were detected, and 499 of these detected miRNAs are predicted to target 13,383 genes by target prediction softwares. Additionally, 29 miRNAs expressed differently between the PSG of fourth-instar day-2 larvae and fifth-instar day-3 larvae were found, and the differential expression of these miRNAs may play an important role in the expression of fibroin genes.

Comparison of six promoters for transient expression of luciferase reporter gene in cultured Bombyx mori cells (BmN)

The Luciferase (luc) reporter gene was used to construct recombinant plasmids containing six different promoters, which are the cytoplasmic actin3 promoter (A3), the fibroin heavy chain promoter (Fib-H), the fibroin light chain promoter (Fib-L), a glycoprotein (P25) promoter, Sericin-1 promoter (Ser-1) and the Bombyx mori nuclear polyhedrosis virus immediate (BmNPV) early protein promoter 1(IE-1), respectively. These recombinant plasmids, which are pGL3-A3-luc, pGL3-Fib-H-luc, pGL3-Fib-L-luc, pGL3-P25-luc, pGL3-Ser-1-luc and pGL3-IE-1-luc had been constructed successfully by restriction enzyme digestion and PCR analysis, and then were transfected into BmN cells to measure the activity of the six promoters to drive luc reporter gene transient expression in cells. Transfection and transcriptional experiment indicated that except pGL3-Fib-L-luc, pGL3-P25-luc, pGL3-Ser-1-luc, others three kinds of recombinant plasmids all transfected BmN cells obviously. The promoters of Fib-H, A3 and IE-1 enhanced the transient expression activity of luc reporter gene in the BmN cells and their activity strengthened sequentially.