Deregulated expression of circular RNA (circRNA) has been determined to be important in carcinogenesis and progression; however, in the most common type of primary malignant bone tumor osteosarcoma, the roles of circRNA in cancer development still remain to be elucidated. Here, we found that circRNA UBAP2 (circUBAP2) expression is significantly increased in human osteosarcoma tissues as compared to those in matched controls. Increased circUBAP2 expression was significantly correlated with human osteosarcoma progression and prognosis. Furthermore, increased circUBAP2 could promote osteosarcoma growth and inhibit apoptosis both in vitro and in vivo. Mechanistically, circUBAP2 was found to inhibit the expression of microRNA-143 (miR-143), thus enhancing the expression and function of anti-apoptotic Bcl-2, which is a direct target of miR-143. Together, our results suggest the roles of circUBAP2 in osteosarcoma development and implicate its potential in prognosis prediction and cancer therapy.

The Chinese tongue sole (Cynoglossus semilaevis) is a typical female heterogamete species that exhibits female-biased sexual size dimorphism, which has severely hindered the sustainable development of the species in aquaculture. In the present study, four important somatotropic and reproductive tissues including brain, pituitary, liver, and gonad from 15 females and 15 males were used for transcriptome analysis via RNA-seq. A mean of 37,533,991 high-quality clean reads was obtained from each library and 806, 1482, 818, and 14,695 differentially expressed genes in female and male were identified from the brain, pituitary, liver, and gonad, respectively (fold change ≥ 2 and q < 0.05). Enrichment analyses of GO terms and KEGG pathways showed that nucleic acid-binding transcription factor activity, G-protein-coupled receptor activity, MAPK signaling pathway, steroid biosynthesis, and neuroactive ligand-receptor interaction may be involved in the sexual growth differences. Furthermore, via weighted gene co-expression network analyses, two modules (yellowgreen and salmon4) were identified to be significantly positive-correlated with female-biased sexual size dimorphism. An illustrated network map drawn by these two modules enabled the identification of a series of hub genes, including nipped-B-like protein A (nipbla), transcriptional activator protein Pur-beta-like (purb), and BDNF/NT-3 growth factors receptor (ntrk2). Detailed functional investigation of these networks and hub genes will further improve our understanding of the underlying molecular mechanism of sexual size dimorphism in fish.

The regulation of bone formation and detailed mechanisms are still largely elusive, and the roles of microRNAs in this process have attracted much attention. Recently, a specific subtype of CD31 hi endomucin hi (CD31 hi EMCN hi) endothelium has been identified to promote bone formation, together with osteoblast development. However, the role of microRNA143 in the generation of CD31 hi EMCN hi endothelium and bone formation remains unknown. In this study, we found that miR-143 was expressed both in osteoblast cells and CD31 hi EMCN hi endothelial cells. Serum miR-143 level was negatively correlated with age in humans. Overexpression of miR-143 promoted osteoblast formation and angiogenic effects. Furthermore, CD31 hi Emcn hi vessels and osteoblast formation were significantly inhibited in miR-143 knockout mice. Mechanistically, inhibitor HDAC7 was directly targeted by miR-143 and knockdown of HDAC7 was found to rescue the function of miR-143 deficiency. Thus, miR-143 promotes angiogenesis coupling with osteoblast differentiation by targeting HDAC7, which may serve as a potential target in angiogenic and osteogenic diseases.

The sex of Chinese tongue sole (Cynoglossus semilaevis) is determined by both genetic sex determination (GSD) and environmental sex determination (ESD), making it an ideal model to study the relationship between sex-determination and temperature. In the present study, transcriptomes of undifferentiated gonads from genetic females and males, as well as differentiated gonads from males, females, and pseudomales under high and normal temperature treatments were generated for comparative transcriptomic analysis. A mean of 68.24 M high-quality clean reads was obtained for each library. Differentially expressed genes (DEGs) between different sexes and environmental treatments were identified, revealing that the heat shock protein gene family was involved in the high temperature induced sex reversal. The Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were enriched in pseudomale and genetic female comparison included neuroactive ligand-receptor interaction, cortisol synthesis and secretion, and steroid hormone biosynthesis. Furthermore, weighted gene co-expression network analyses were conducted on all samples, and two modules were positive correlated with pseudomale under high temperature. An illustrated protein-protein interaction map of the module identified a hub gene, hsc70. These findings provide insights into the genetic network that is involved in sex determination and sexual differentiation, and improve our understanding of genes involved in sex reversal under high temperature.

No abstract

The bone marrow microenvironment is composed primarily of immune and stromal cells that play important roles in fracture healing. Although immune cells have been identified in mouse bone marrow, variations in their numbers and type during the fracture healing process remain poorly defined. In this study, single-cell RNA sequencing was used to identify immune cells in fracture tissues, including neutrophils, monocytes, T cells, B cells, and plasma cells. The number of B cells decreased significantly in the early stage of fracture healing. Furthermore, B cells in mice fracture models decreased significantly during the epiphyseal phase and then gradually returned to normal during the epiphyseal transformation phase of fracture healing. The B-cell pattern was opposite to that of bone formation and resorption activities. Notably, B-cell–derived exosomes inhibited bone homeostasis in fracture healing. In humans, a decrease in the number of B cells during the epiphyseal phase stimulated fracture healing. Then, as the numbers of osteoblasts increased during the callus reconstruction stage, the number of B cells gradually recovered, which reduced additional bone regeneration. Thus, B cells are key regulators of fracture healing and inhibit excessive bone regeneration by producing multiple osteoblast inhibitors.

Albinism, a phenomenon characterized by pigmentation deficiency on the ocular side of Japanese flounder (Paralichthys olivaceus), has caused significant damage. Limited mRNA and microRNA (miRNA) information is available on fish pigmentation deficiency. In this study, a high-throughput sequencing strategy was employed to identify the mRNA and miRNAs involved in P. olivaceus albinism. Based on P. olivaceus genome, RNA-seq identified 21,787 know genes and 711 new genes by transcripts assembly. Of those, 235 genes exhibited significantly different expression pattern (fold change ≥2 or ≤0.5 and q-value≤0.05), including 194 down-regulated genes and 41 up-regulated genes in albino versus normally pigmented individuals. These genes were enriched to 81 GO terms and 9 KEGG pathways (p≤0.05). Among those, the pigmentation related pathways-Melanogenesis and tyrosine metabolism were contained. High-throughput miRNA sequencing identified a total of 475 miRNAs, including 64 novel miRNAs. Furthermore, 33 differentially expressed miRNAs containing 13 up-regulated and 20 down-regulated miRNAs were identified in albino versus normally pigmented individuals (fold change ≥1.5 or ≤0.67 and p≤0.05). The next target prediction discovered a variety of putative target genes, of which, 134 genes including Tyrosinase (TYR), Tyrosinase-related protein 1 (TYRP1), Microphthalmia-associated transcription factor (MITF) were overlapped with differentially expressed genes derived from RNA-seq. These target genes were significantly enriched to 254 GO terms and 103 KEGG pathways (p<0.001). Of those, tyrosine metabolism, lysosomes, phototransduction pathways, etc., attracted considerable attention due to their involvement in regulating skin pigmentation. Expression patterns of differentially expressed mRNA and miRNAs were validated in 10 mRNA and 10 miRNAs by qRT-PCR. With high-throughput mRNA and miRNA sequencing and analysis, a series of interested mRNA and miRNAs involved in fish pigmentation are identified. And the miRNA-mRNA regulatory network also provides a solid starting point for further elucidation of fish pigmentation deficiency.

In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300 days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24 °C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.