Xingjia Shen

A study of miRNAs targets prediction and experimental validation

AbstractmicroRNAs (miRNAs) are 20–24 nucleotide (nt) RNAs that regulate eukaryotic gene expression post-transcriptionally by the degradation or translational inhibition of their target messenger RNAs (mRNAs). To identify miRNA target genes will help a lot by understanding their biological functions. Sophisticated computational approaches for miRNA target prediction, and effective biological techniques for validating these targets now play a central role in elucidating their functions. Owing to the imperfect complementarity of animal miRNAs with their targets, it is difficult to judge the accuracy of the prediction. Complexity of regulation by miRNA-mediated targets at protein and mRNAs levels has made it more challenging to identify the targets. To date, only a few miRNAs targets are confirmed. In this article, we review the methods of miRNA target prediction and the experimental validation for their corresponding mRNA targets in animals.

Construction and detection of expression vectors of microRNA-9a in BmN cells

MicroRNAs (miRNAs) are small endogenous RNAs molecules, approximately 21–23 nucleotides in length, which regulate gene expression by base-pairing with 3′ untranslated regions (UTRs) of target mRNAs. However, the functions of only a few miRNAs in organisms are known. Recently, the expression vector of artificial miRNA has become a promising tool for gene function studies. Here, a method for easy and rapid construction of eukaryotic miRNA expression vector was described. The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a) precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid. The enhanced green fluorescent protein (EGFP) gene was used as reporter gene. The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection. Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot. Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.

Expression of a vitelline membrane protein, BmVMP23, is repressed by bmo-miR-1a-3p in silkworm, Bombyx mori

Previous studies have shown that the 3′ UTR of BmVMP23 was destroyed by an inserted fragment, and the BmVMP23 expression was downregulated in the lethal egg of “Ming” (l‐em ). In this study, we found a miRNA (bmo‐miR‐1a‐3p) that matches the 3′ UTR sequence of BmVMP23 perfectly. The relationship between bmo‐miR‐1a‐3p and the BmVMP23 expression was examined by the co‐transfection in vitro. The luciferase assay result showed that luc expression was strongly repressed. This result inferred that bmo‐miR‐1a‐3p may downregulate BmVMP23 expression via complementary interaction with the target sites at the 3′ UTR.

Entry of Bombyx mori nucleopolyhedrovirus into BmN cells by cholesterol-dependent macropinocytic endocytosis.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen of silkworm, and no drug or specific protection against BmNPV infection is available at present time. Although functions of most BmNPV genes were depicted in recent years, knowledge on the mechanism of BmNPV entry into insect cells is still limited. Here BmNPV cell entry mechanism is investigated by different endocytic inhibitor application and subcellular analysis. Results indicated that BmNPV enters BmN cells by clathrin-independent macropinocytic endocytosis, which is mediated by cholesterol in a dose-dependent manner, and cholesterol replenishment rescued the BmNPV infection partially.

Mutation of a Cuticle Protein Gene, BmCPG10, Is Responsible for Silkworm Non-Moulting in the 2nd Instar Mutant

In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2nd instar (nm2). Genetic analysis indicated that the nm2 mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, nm2 was located in a region approximately 275 kb on the 5th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the BmCPG10 gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene’s structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The BmCPG10 mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The BmCPG10 mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of nm2 larvae was lower than that of the wild-type larvae. The nm2 mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7—dehydrocholesterol (7dC), but the rescued nm2 only developed to the 4th instar and subsequently died. The moulting time of silkworms could be delayed by BmCPG10 RNAi. Thus, we speculated that the mutation of BmCPG10 was responsible for the silkworm mutant that did not moult in the 2nd instar.

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.

Molecular cloning and characterization of hatching enzyme-like geneII (BmHELII) in the silkworm, Bombyx mori.

Hatching enzyme (HE) is an enzyme that digests an egg envelop at the time of embryo hatching. Previously, we have reported a kind of Bombyx mori hatching enzyme-like gene (BmHEL). In this paper, the full length of another BmHEL cDNA sequence (BmHELII, GenBank ID: JN627443) was cloned from bluish-silkworm-eggs. The cDNA was 977 bp in length with an open reading frame of 885 bp which encodes a polypeptide of 294 amino acids including a putative signal peptide of 16 amino acid residues and a mature protein of 278 amino acids. The deduced BmHELII had a predicted molecular mass of 33.62 kDa, isoelectric point of 5.44 and two conserved signature sequences of astacin family. Bioinformatic analysis results showed that the deduced protease domain amino acid sequence of BmHELII had 29.5-87.0% identities to that of HE identified in the other species. The BmHELII gene structure was 6-exon-5-intron, and the promoter region harbored some basal promoter elements and some embryo development related transcription factor binding sites. Semi-quantitative RT-PCR analysis revealed that the relative level of BmHELII transcripts at different stages during egg incubation increased with the development of embryos and reached to a maximum just before hatching, hence declined gradually after hatching. The spatio-temporal expression pattern of BmHELII basically resembled that of hatching enzyme gene. Moreover, the BmHELII transcript was detected in testis of the silkworm, and semi-quantitative RT-PCR analysis showed that it kept at the high level in testis of silkworm from larvae to moth, which suggested that BmHELII might take part in the development of sperm. These results will be helpful to provide a molecular basis for understanding the mechanism underlying silkworm hatching as well as spermatogenesis.

Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

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.

BmNPV-miR-415 up-regulates the expression of TOR2 via Bmo-miR-5738

MicroRNAs (miRNAs) have emerged as key players in host–pathogen interaction and many virus-encoded miRNAs have been identified (computationally and/or experimentally) in a variety of organisms. A novel Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNA miR-415 was previously identified through high-throughput sequencing. In this study, a BmNPV-miR-415 expression vector was constructed and transfected into BmN cells. The differentially expressed protein target of rapamycin isoform 2 (TOR2) was observed through two-dimensional gel electrophoresis and mass spectrometry. Results showed that TOR2 is not directly a target gene of BmNPV-miR-415, but its expression is up-regulated by BmNPV-miR-415 via Bmo-miR-5738, which could be induced by BmNPV.