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The long noncoding RNA HOTAIRM1 reportedly plays important roles in acute myeloid leukemia, gastric cancer and colorectal cancer. Here, we analyzed potential function of HOTAIRM1 in glioma and asked whether it participates in long-range chromatin interactions. We monitored expression of HOTAIRM1 in glioma tissues and correlated levels with patient survival using the TCGA dataset. HOTAIRM1 was highly expressed in glioma tissue, with high levels associated with shortened patient survival time. We then suppressed HOTAIRM1 activity in the human glioblastoma U251 line using CRISPR-cas9 to knock in a truncating polyA fragment. Reporter analysis of these and control cells confirmed that the HOTAIRM1 locus serves as an active enhancer. We then performed Capture-C analysis to identify target genes of that locus and applied RNA antisense purification to assess chromatin interactions between the HOTAIRM1 locus and HOXA cluster genes. HOTAIRM1 knockdown in glioma cells decreased proliferation and reduced expression of HOXA cluster genes. HOTAIRM1 regulates long-range interactions between the HOTAIRM1 locus and HOXA genes. Our work suggests a new mechanism by which HOTAIRM1 regulates glioma progression by regulating high-order chromatin structure and could suggest novel therapeutic targets to treat an intractable cancer.

Temozolomide (TMZ) is a frequently used chemotherapy for glioma; however, chemoresistance is a major problem limiting its effectiveness. Thus, knowledge of mechanisms underlying this outcome could improve patient prognosis. Here, we report that deletion of a regulatory element in the HOTAIR locus increases glioma cell sensitivity to TMZ and alters transcription of multiple genes. Analysis of a combination of RNA-seq, Capture Hi-C, and patient survival data suggests that CALCOCO1 and ZC3H10 are target genes repressed by the HOTAIR regulatory element and that both function in regulating glioma cell sensitivity to TMZ. Rescue experiments and 3C data confirmed this hypothesis. We propose a new regulatory mechanism governing glioma cell TMZ sensitivity.

Synpolydactyly is a congenital limb malformation characterized by incomplete separation and duplication in fingers and/or toes, which is mainly caused by mutations in the homeobox D13 (HOXD13) gene. Here, a four-generation family with variant phenotypes of synpolydactyly was analyzed, in which the proband had bilateral preaxial synpolydactyly in toes with normal fingers, the father had clinodactyly in the fifth fingers, while the mother and grandma was normal. Trio whole-exome sequencing (trio-WES) is a high throughput sequencing targeting whole genome for detecting exonic variants from the proband and the parents in a family. Through trio-WES followed by Sanger sequencing and enzyme digestion, a heterozygous nonsense mutation (c.859 C>T/p.Gln287Ter) was newly identified in the homeodomain of the HOXD13 gene from the proband and the affected father, but not from the unaffected mother, the unaffected grandma, or the normal control. Mutation Taster, Human Splicing Finder and EX-SKIP predicted that the heterozygous mutation (c.859 C>T) would result in haploinsufficiency of HOXD13 protein through nonsense-mediated mRNA decay (NMD) and splicing abnormality, which might disrupt the integrity and reduce the expression level of the HOXD13 protein (loss-of-function). In short, a heterozygous nonsense mutation in the HOXD13 gene was newly identified in two patients with mild phenotypes of synpolydactyly, which extends the mutation spectrum in HOXD13 gene. Moreover, the findings we presented here deepen our understanding of the clinical consequences of non-syndromic synpolydactyly and may provide a new clue for further studies of the pathogenic mechanism of the mutation that causes aberrant splicing of HOXD13 gene.

Neonicotinoid insecticides are now the most widely used insecticides in the world. Previous studies have indicated that sublethal doses of neonicotinoids impair learning, memory capacity, foraging, and immunocompetence in honey bees (Apis mellifera, Linnaeus) (Hymenoptera: Apidae). Despite these, few studies have been carried out on the molecular effects of neonicotinoids. In this study, we focus on the second-generation neonicotinoid thiamethoxam, which is currently widely used in agriculture to protect crops. Using high-throughput RNA-Seq, we investigated the transcriptome profile of honey bees after subchronic exposure to 10 ppb thiamethoxam over 10 d. In total, 609 differentially expressed genes (DEGs) were identified, of which 225 were upregulated and 384 were downregulated. Several genes, including vitellogenin, CSP3, defensin-1, Mrjp1, and Cyp6as5 were selected and further validated using real-time quantitative polymerase chain reaction assays. The functions of some DEGs were identified, and Gene Ontology-enrichment analysis showed that the enriched DEGs were mainly linked to metabolism, biosynthesis, and translation. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that thiamethoxam affected biological processes including ribosomes, the oxidative phosphorylation pathway, tyrosine metabolism pathway, pentose and glucuronate interconversions, and drug metabolism. Overall, our results provide a basis for understanding the molecular mechanisms of the complex interactions between neonicotinoid insecticides and honey bees.

Amitraz is an acaricide that is widely used in apiculture. Several studies have reported that in honeybees (Apis mellifera Linnaeus; Hymenoptera: Apidae), amitraz affects learning, memory, behavior, immunity, and various other physiological processes. Despite this, few studies have explored the molecular mechanisms underlying the action of amitraz on honeybees. Here, we investigated the transcriptome of honeybees after exposure to 9.4 mg/L amitraz for 10 d, a subchronic dose. Overall, 279 differentially expressed genes (DEGs) were identified (237 upregulated, 42 downregulated). Several, including Pla2, LOC725381, LOC413324, LOC724386, LOC100577456, LOC551785, and P4504c3, were validated by quantitative PCR. According to gene ontology, DEGs were mainly involved in metabolism, biosynthesis, and translation. Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that amitraz treatment affected the relaxin signaling pathway, platelet activation, and protein digestion and absorption.

Porcine circovirus type 2 (PCV2) is one of the major threats to pig farms worldwide. Although PCV2 has been identified to promote IL-10 production, the detailed regulatory roles of PCV2 Rep for IL-10 production remain unclear. Herein, we first found that PCV2 Rep, rather than PCV1 Rep, enhanced IL-10 expression at the later phase of PCV2 infection in porcine alveolar macrophages (PAMs). Furthermore, we found that PCV2 Rep directly activated the p38-MAPK pathway to promote transcription factors NF-&kappa, B p50 and Sp1 binding to the il10 promoter, but PCV1 Rep did not. During PCV2 infection, however, PCV2 Rep promoted the binding activities of NF-&kappa, B p50 and Sp1 with the il10 promoter only at the later phase of PCV2 infection, since Rep proteins only expressed at the later phase of the infection. Moreover, silence of the thymine DNA glycosylase (TDG), a Rep-binding protein, significantly reduced the binding activities of NF-&kappa, B p50 and Sp1 with il10 promoter, resulting in the reduction of IL-10 production in PCV2-inoculated PAMs at the later phase of infection. Taken together, our results demonstrate that Rep proteins enhance IL-10 production during PCV2 infection of PAMs via activation of p38-MAPK pathways, in which host TDG is a critical mediator.

Multiple osteochondromas (MO) is one of the most common benign bone tumors in humans with an autosomal dominant hereditary mode. MO is a genetic heterogeneity disease with variable number and size of osteochondromas, as well as changeable number and location of diseased bones. Mutations in Exostosin-1/Exostosin-2 (EXT1/EXT2) genes are the main molecular basis of MO. EXT1 and EXT2 genes encode exostosin 1 and exostosin 2, respectively, both of which are transmembrane glycosyltransferases that elongate the chains of heparin sulfate (HS) at HS proteoglycans (HSPGs). HSPGs are considered to be involved in regulating the proliferation and differentiation of chondrocytes. Owing to large size of EXT1/EXT2 genes and lack of mutation hotspots, molecular diagnosis of MO is challenging. Here, we applied targeted next-generation sequencing (t-NGS) in mutation screening of EXT1/EXT2 genes for 10 MO patients. The results were compared and validated with Sanger sequencing. Overall, nine mutations identified by t-NGS were confirmed with Sanger sequencing, excluding two variants of false positive, suggesting the reliability of mutation screening by t-NGS. The nine mutations identified by t-NGS include two missense mutations (EXT1: c.1088G>A and c.2120C>T), one splicing mutation (EXT2: c.744-1G>T), and six nonsense mutations (EXT1: c.351C>G, c.1121G>A, and c.1843_1846dup; EXT2: c.67C>T, c.561delG, and c.575T>A). In summary, our paper provides the primary data of the application of t-NGS in MO molecular diagnosis, including six newly identified mutations (EXT1: c.1843_1846dup, c.1088G>A, c.351C>G, and c.2120C>T and EXT2: c.744-1G>T and c.575T>A), which further enrich the mutation database of MO from the Chinese population.

Background Glioblastoma multiforme (GBM) is the most malignant central nervous system tumor. Alkylating agent, temozolomide (TMZ), is currently the first-line chemotherapeutic agent for GBM. However, the sensitivity of GBM cells to TMZ is affected by many factors. And, several clinic trials, including co-administration of TMZ with other drugs, have failed in successful treatment of GBM. We have previously reported that Netrin-4 (NTN4), a laminin-like axon guidance protein, plays a protective role in GBM cell senescence upon TMZ-triggered DNA damage. However, the master regulator of NTN4 needs further elucidation. Epidermal growth factor/Epidermal growth factor receptor (EGF/EGFR) can modulate the expression of various extracellular matrix related molecules, and prevent DNA damage in GBM cells. In this study, we investigated the relationship between EGF/EGFR signaling and NTN4, and explored their effect on therapeutic efficacy in GBM cells upon TMZ treatment. Methods Co-expression analysis were performed by using the RNA sequencing data from NIH 934 cell lines and from single cell RNA sequencing data of GBM tumor. The co-expressing genes were used for GO enrichment and signaling pathway enrichment. mRNA expression of the target genes were quantified by qPCR, and cell senescence were investigated by Senescence-Associated Beta-Galactosidase Staining. Protein phosphorylation were observed and analyzed by immunoblotting. The RNA sequencing data and clinical information of TMZ treated patients were extracted from TCGA-glioblastoma project, and then used for Kaplan-Meier survival analysis. Results Analysis of RNA sequencing data revealed a potential co-expression relationship between NTN4 and EGFR. GO enrichment of EGFR-correlated genes indicated that EGFR regulates GBM cells in a manner similar to that in central nervous system development and neural cell differentiation. Pathway analysis suggested that EGFR and its related genes contribute to cell adhesion, extracellular matrix (ECM) organization and caspase related signaling. We also show that EGF stimulates NTN4 expression in GBM cells and cooperates with NTN4 to attenuate GBM cell senescence induced by DNA damage, possibly via AKT and ERK. Clinical analysis showed that co-expression of EGFR and NTN4 significantly predicts poor survival in TMZ-treated GBM patients. Conclusions This study indicates that EGF/EGFR regulates and cooperates with NTN4 in DNA damage resistance in GBM. Therefore, our findings provide a potential therapeutic target for GBM.

Background The behavioural transition from nurses to foragers in honey bees is known to be affected by intrinsic and extrinsic factors, including colony demography, hormone levels, brain chemistry and structure, and gene expression in the brain. However, the molecular mechanism underlying this behavioural transition of honey bees is still obscure. Results Through RNA sequencing, we performed a comprehensive analysis of lncRNAs and mRNAs in honey bee nurses and foragers. Nurses and foragers from both typical colonies and single-cohort colonies were used to prepare six libraries to generate 49 to 100 million clear reads per sample. We obtained 6863 novel lncRNAs, 1480 differentially expressed lncRNAs between nurses and foragers, and 9308 mRNAs. Consistent with previous studies, lncRNAs showed features distinct from mRNAs, such as shorter lengths, lower exon numbers, and lower expression levels compared to mRNAs. Bioinformatic analysis showed that differentially expressed genes were mostly involved in the regulation of sensory-related events, such as olfactory receptor activity and odorant binding, and enriched Wnt and FoxO signaling pathways. Moreover, we found that lncRNAs TCONS_00356023, TCONS_00357367, TCONS_00159909 and mRNAs dop1, Kr-h1 and HR38 may play important roles in behavioural transition in honey bees. Conclusion This study characterized the expression profile of lncRNAs in nurses and foragers and provided a framework for further study of the role of lncRNAs in honey bee behavioural transition. Electronic supplementary material The online version of this article (10.1186/s12864-019-5664-7) contains supplementary material, which is available to authorized users.

The cyano-substituted neonicotinoid insecticide, thiacloprid, is nowadays widely used in agriculture for controlling insect pests. However, it also simultaneously has adverse effects on the health of important pollinators, such as honey bees. Previous studies have reported that sublethal doses of neonicotinoids impaired immunocompetence, learning and memory performance, and homing behaviour in honey bees. In the present study, using LC-MS-based combined with GC–MS-based metabolomic approaches, we profiled the metabolic changes that occur in the head of honey bee after subchronic exposure to 2 mg/L thiacloprid over 3 days. The estimated total dose of thiacloprid fed to each bee was 0.12 μg. The results showed that there were 115 metabolites significantly affected in thiacloprid-treated bees compared to control. The metabolites with high level of abundance enriched to wide range pathways associated with oxidative stress and detoxification suggest that the honey bees have activated their detoxification system to resistant toxicity of thiacloprid. While, the reduction of serotonin suggest thiacloprid may hinder the brain activity implicated in learning and behaviour development. Our study expand the understanding of the molecular basis of the complex interactions between neonicotinoids and honey bees.

MicroRNAs (miRNAs) are small endogenous noncoding single-stranded RNAs regulating gene expression in eukaryotes. They play important roles in regulating caste differentiation, behavior development, and immune defences in the honey bee, Apis mellifera (Linnaeus) (Hymenoptera: Apidae). In this study, we explored the effect of the neonicotinoid insecticide, thiamethoxam, on miRNA expression in this species using deep small RNA sequencing. The results showed that seven miRNAs were significantly differentially expressed (q-value 1) upon exposure to 10 ppb thiamethoxam over 10 d. Some candidate target genes were related to behavior, immunity, and neural function. Several miRNAs, including ame-miR-124, ame-miR-981, ame-miR-3791, and ame-miR-6038, were selected and further validated using real-time quantitative PCR analysis. The findings expand our understanding of the effects of neonicotinoid insecticides on honey bees at the molecular level.

The Mount Huang eastern honey bees (Apis cerana) are an endemic population, which is well adapted to the local agricultural and ecological environment. In this study, the genetic structure of seven eastern honey bees (A. cerana) populations from Mount Huang in China were analyzed by SSR (simple sequence repeat) markers. The results revealed that 16 pairs of primers used amplified a total of 143 alleles. The number of alleles per locus ranged from 6 to 13, with a mean value of 8.94 alleles per locus. Observed and expected heterozygosities showed mean values of 0.446 and 0.831 respectively. UPGMA cluster analysis grouped seven eastern honey bees in three groups. The results obtained show a high genetic diversity in the honey bee populations studied in Mount Huang, and high differentiation among all the populations, suggesting that scarce exchange of honey bee species happened in Mount Huang. Our study demonstrated that the Mount Huang honey bee populations still have a natural genome worth being protected for conservation.