RNA Transport Pathways Research Articles

Abstract 12684: Transcriptional Features Of Biological Age Maintained In Cultured Cardiac Interstitial Cells

Introduction: Ex vivo expansion of cells is necessary in regenerative medicine to generate large populations for therapeutic use. Adaptation to culture conditions prompt an increase in transcriptome diversity and decreased population heterogeneity in cKit+ cardiac interstitial cells (cCICs). Hypothesis: Age and/or condition of origin is impacts the transcriptional phenotype as well as population heterogeneity characteristics of cCICs undergoing in vitro expansion. Methods: cCICs isolated from neonatal and adult cardiac tissue (Left Ventricular Assist Device; LVAD). Single cell libraries from in vitro expanded cells were prepared following five passages. “Transcriptional memory” was surveyed via bioinformatic analysis including unsupervised clustering, differential expression analysis, gene ontology and pathway analysis. Transitional states were assessed through pseudotime analysis. Results: Cell cycle imprint associated with biological age after culture was observed in Neonatal cCICs via upregulation of G2M genes. LVAD derived cCICs retained a widespread senescent profile, in particular high expression of interleukins and elements of the senescence associated secretory phenotype (SASP). Nuclear pore complex TPR and UBC9 and translation initiation factors, displayed age-associated downregulation of elements in the RNA transport and processing pathway. Pathway and co-expression analysis of fibroblast markers Ddr2, Tcf21, Vimentin, Periostin and Collagen deposition markers indicated a primed fibrotic phenotype in senescent cells. A small subset of cCICs exist in a transcriptional continuum between “youthful” phenotype and the damaged microenvironment of adult tissue in which they were embedded. Conclusions: The influence of age, pathology and the cellular stress associated to the in vivo tissue microenvironment persist after culture adaptation, influencing targets of 1) cell cycle, 2) senescence associated secretory phenotype (SASP), 3) RNA transport, and 4) ECM-receptor/fibrosis. The connate transcriptional phenotypes offer fundamental biological insight and highlights cellular input as a consideration in culture expansion and adoptive transfer protocols.

Profiling and Bioinformatics Analysis Revealing Differential Circular RNA Expression about Storage Lesion Regulatory in Stored Red Blood Cells

Introduction: Circular RNA (circRNA) plays an important role in regulating metabolism of red blood cells (RBCs) and their storage lesions, but the study of how circRNA expression changes in stored RBCs has rarely been conducted. Methods: The expression change of circRNA was systemically evaluated via high-throughput sequencing on healthy RBCs on day 0, 20, and 40. And then we confirmed the reliability of the high-throughput sequencing analysis by RT-qPCR characterization on selected circRNAs. A higher parental gene enrichment was used to explore circRNA function in pathways. In addition, we deciphered a dysregulated circRNA-related ceRNAs network, and identified three circRNA-miRNA-mRNA regulatory axes related to storage lesion. Results: We identified 2,586 known and 6,216 putative novel circRNAs, more than 100 circRNAs expression levels were shifted, and the number of downregulated circRNAs was greater with longer storage time. Furthermore, a higher parental gene enrichment related to circRNA was found in pathways, including cAMP signaling pathway, ubiquitin-mediated proteolysis, apoptosis, adhesion, MAPK signaling pathway, cystine methionine metabolism, RNA degradation, RNA transport, TGF-β, and actin regulatory pathway. hsa_circ_0007127-miR-513a-5p-SMAD4, hsa_circ_0000033-miR-19a-3p-VAMP3, and hsa_circ_0005546-miR-4720-CCND3 regulatory axes related to storage lesion was found. Conclusions: Through investigation in circRNAs profile and circRNA-miRNA-mRNA interactions, this study provides insights on stored RBC circRNA expression changes, which closely relate to the storage lesion of RBCs and their physiological functions.

Phosphoproteomic Analysis of Primary Myeloma Patient Samples Identifies Distinct Phosphorylation Signatures Correlating with Chemo-Sensitivity Profiles in an Ex Vivo Drug Sensitivity Testing Platform

Introduction: Multiple myeloma (MM) is characterized by the clonal expansion of plasma cells in the bone marrow resulting in end-organ damage. Despite an extensive increase in the five-year survival rate in recent years, MM is still considered an incurable disease as patients will repeatedly relapse and develop resistance to current chemotherapies. A key focus for the personalization of myeloma therapy is understanding the biological mechanisms of drug resistance and identifying clinically useful biomarkers of therapeutic response. Highly efficient techniques for the enrichment of phosphorylated peptides followed by high resolution mass spectrometry facilitates the quantitation of thousands of site-specific phosphorylation events. Here, we have performed a phosphoproteomic analysis on MM cell lysates stratified based on their ex vivo drug response profiles to advance our understanding of drug resistance mechanisms.Materials and Methods: CD138 + plasma cells were isolated from 20 adult MM patient bone marrow aspirates at diagnosis (n=7) or relapse (n=13). Samples were grouped based on ex vivo drug sensitivity and resistance testing (DSRT) as follows: highly sensitive (Group I), sensitive (Group II), resistant (Group III), highly resistant (Group IV) [1]. For the phosphoproteomic analysis, peptides were generated and purified using the filter aided sample preparation (FASP) protocol. Peptide tandem mass tag (TMT) labelling, Fe 3+ immobilized metal ion affinity chromatography (IMAC), synchronous precursor selection (SPS), and triple stage tandem mass spectrometry (MS3) was performed. Nonenriched peptides were used for proteomic analysis. Resulting data was analysed using MaxQuant, followed by normalization of phosphosite intensities using the internal reference scale (IRS) method, and statistical analysis using Perseus. Functional enrichment and kinase enrichment analyses were performed on significant phosphoproteins using g:profiler and KEA2, respectively.Results: Our quantitative MS-based phosphoproteomic analysis identified 2,945 phosphorylation sites on 2,232 phosphopeptides from 690 phosphoproteins. Of these phosphorylation sites, 176 were significantly changed between all four DSRT groups and 267 were significantly changed between Group I and Group IV (False Discovery Rate (FDR) < 0.05). Hierarchical clustering was performed to highlight the distinct phosphoproteomic profiles associated with each DSRT group, of which the very sensitive (Group I) and very resistant (Group IV) subgroups demonstrated a well-defined separation (Fig. 1A, 1B). KEGG pathway analysis and gene ontology (GO) analysis of significantly increased phosphorylated proteins in Group IV compared to Group I MM patients demonstrated an increased phosphorylation of proteins associated with tight junctions, the Rap1 signalling pathway and the phosphatidylinositol signalling system; indicating an upregulation of cell adhesion associated processes in drug resistant MM. Phosphoproteins increased in abundance in Group I compared to Group IV MM patients revealed an increased phosphorylation of proteins involved in translation and RNA processing including the spliceosome, RNA transport and RNA binding pathways (Fig. 1C). We identified filamin A serine 2152, RAS guanyl-releasing protein 2 serine 576 and proto-oncogene tyrosine-protein kinase Src serine 17 as increased in Group IV MM, and nuclease-sensitive element-binding protein 1 (YBX1) serine 165, CD44 serine 697 and Bcl2-associated agonist of cell death (BAD) serine 99 as increased in Group I MM. KEA of the upregulated phosphoproteome in Group IV revealed an enrichment of cyclin dependent kinase 1 (CDK1) and ribosomal s6 kinases (RPS6K) whereas casein kinase 2 (CK2) and the glycolysis-associated kinases were enriched in Group I (Fig. 1D).Conclusion: Our study has generated a phosphoproteomic dataset demonstrating distinct phosphorylation signatures associated with drug sensitivity in clinical MM plasma cells. The identification of phosphorylation events associated with drug resistance provides a basis for further exploration of these events and associated signalling pathways to further understand drug resistance mechanisms in MM and identify potential biomarkers of therapeutic response and targets for drug re-sensitization in MM.

Phosphorylated protein modification analysis on normal liver and Exo-celiac liver of Glyptosternum maculatum.

This study aimed to reveal the biological function and molecular mechanism of phosphorylated proteins in the normal liver (NG) and Exo-celiac liver (WG) of Glyptosternum maculatum and potential plateau-adaption mechanisms of G. maculatum. A multivariate analysis was performed on proteomic quantitative data (label-free group) and phosphorylated proteome data (phosphorylation group) to reveal protein characteristics. The differentially expressed proteins (DEPs) between NG and WG in the two groups were analysed. Enrichment analysis of these DEPs was performed prior to the protein-protein interaction (PPI) analysis. Finally, an integrated interaction network was constructed to reveal the biological mechanism of the DEP-mediated signal transduction process. The NG and WG samples in the phosphorylation group were well distinguished compared to the label-free group. A total of 49 and 313 DEPs were identified in the label-free and phosphorylation groups, respectively. These DEPs, including LIM and calponin homology domains-containing protein 1 (LIMCH1) and DEAD(Asp-Glu-Ala-Asp)-Box Helicase 51 (DDX51), were mainly assembled in functions such as cell adhesion. Two PPI networks were constructed using DEPs in the two groups. Finally, an integrated interaction network was constructed using co-DEP Ferredoxin 1 (FDX1) and associated pathways, including RNA transport. LIMCH1 and DDX51 might play important roles in the organogenesis of normal liver and Exo-celiac liver in G. maculatum via the cell adhesion function. Moreover, FXD1 might be associated with the plateau-adaption mechanisms of G. maculatum via participation in the RNA transport pathway.

Insights from label free-based proteomic analysis into inhibitory effects ε-Poly-lysine against Vibrio parahaemolyticus

Vibrio parahaemolyticus is one of the most common pathogenic bacteria that pose a threat to human health. The purpose of this study was to investigate antibacterial mechanisms of ε-poly-lysine (ε-PL) against V. parahaemolyticus using a lable free-based proteomic analysis. The differentially expressed proteins (DEPs) were subjected to bioinformatics analysis. The results indicated that a total of 196 DEPs, including 118 up-regulated and 78 down-regulated, were identified in the ε-PL-treated cells compared with control group. Upon Go functional enrichment, 13, 9, and 8 specific Go terms in biological processes, molecular functions and cellular components were identified, respectively. KEGG pathways analysis indicated that the DEPs were mainly involved in bacterial chemotaxis, RNA transport and two-component system, which were significantly enriched (P < 0.05). In PPI analysis, Che R and Che V, both involved in bacterial chemotaxis and RNA transport pathways, are closely related to other DEPs. Therefore, the down-regulation of Che R and Che V in ε-PL-treated cells resulted in the reduction or even loss of bacterial adaptability, and they were the critical action sites of ε-PL to inactivate V. parahaemolyticus.

The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions.

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two-color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3′UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA, suggesting a possible feedback loop acting at the level of mRNA transport.

Abstract P347: Transcriptional Features Of Biological Age Maintained In Cultured Cardiac Interstitial Cells

Introduction: Ex vivo expansion of cells is necessary in regenerative medicine to generate large populations for therapeutic use. Adaptation to culture conditions prompt an increase in transcriptome diversity and decreased population heterogeneity in cKit+ cardiac interstitial cells (cCICs). The “transcriptional memory” influenced by cellular origin remains unexplored and is likely to differ between neonatal versus senescent input cells undergoing culture expansion. Approach: cCICs isolated from neonatal and adult cardiac tissue (Left Ventricular Assist Device; LVAD). Single cell libraries from in vitro expanded cells were prepared following five passages as previously demonstrated by our group to promote transcriptional homogeneity. “Transcriptional memory” was surveyed via bioinformatic analysis including unsupervised clustering, differential expression analysis, gene ontology and pathway analysis. Transitional states were assessed through pseudotime analysis. Results: Cell cycle imprint associated with biological age after culture was observed in Neonatal cCICs via upregulation of G2M genes. LVAD derived cCICs retained a widespread senescent profile, in particular high expression of interleukins and elements of the senescence associated secretory phenotype (SASP). Nuclear pore complex TPR and UBC9 and translation initiation factors, displayed age-associated downregulation of elements in the RNA transport and processing pathway. Pathway and co-expression analysis of fibroblast markers Ddr2, Tcf21, Vimentin, Periostin and Collagen deposition markers indicated a primed fibrotic phenotype in senescent cells. A small subset of cCICs exist in a transcriptional continuum between “youthful” phenotype and the damaged microenvironment of adult tissue in which they were embedded. Conclusion: The influence of age, pathology and the cellular stress associated to the in vivo tissue microenvironment persist after culture adaptation, influencing targets of 1) cell cycle, 2) senescence associated secretory phenotype (SASP), 3) RNA transport, and 4) ECM-receptor/fibrosis. The connate transcriptional phenotypes offer fundamental biological insight and highlights cellular input as a consideration in culture expansion and adoptive transfer protocols.

Phosphorylation-Mediated Molecular Pathway Changes in Human Pituitary Neuroendocrine Tumors Identified by Quantitative Phosphoproteomics.

To investigate the biological role of protein phosphorylation in human nonfunctional pituitary neuroendocrine tumors (NF-PitNETs), proteins extracted from NF-PitNET and control tissues were analyzed with tandem mass tag (TMT)-based quantitative proteomics coupled with TiO2 enrichment of phosphopeptides. A total of 595 differentially phosphorylated proteins (DPPs) with 1412 phosphosites were identified in NF-PitNETs compared to controls (p < 0.05). KEGG pathway network analysis of 595 DPPs identified nine statistically significant signaling pathways, including the spliceosome pathway, the RNA transport pathway, proteoglycans in cancer, SNARE interactions in vesicular transport, platelet activation, bacterial invasion of epithelial cells, tight junctions, vascular smooth muscle contraction, and protein processing in the endoplasmic reticulum. GO analysis revealed that these DPPs were involved in multiple cellular components (CCs), biological processes (BPs), and molecule functions (MFs). The kinase analysis of 595 DPPs identified seven kinases, including GRP78, WSTF, PKN2, PRP4, LOK, NEK1, and AMPKA1, and the substrate of these kinases could provide new ideas for seeking drug targets for NF-PitNETs. The randomly selected DPP calnexin was further confirmed with immunoprecipitation (IP) and Western blot (WB). These findings provide the first DPP profiling, phosphorylation-mediated molecular network alterations, and the key kinase profiling in NF-PitNET pathogenesis, which are a precious resource for understanding the biological roles of protein phosphorylation in NF-PitNET pathogenesis and discovering effective phosphoprotein biomarkers and therapeutic targets and drugs for the management of NF-PitNETs.

The Expression Profiles of mRNAs and lncRNAs in Buffalo Muscle Stem Cells Driving Myogenic Differentiation.

Buffalo breeding has become an important branch of the beef cattle industry. Hence, it is of great significance to study buffalo meat production and meat quality. However, the expression profiles of mRNA and long non-coding RNAs (lncRNA) molecules in muscle stem cells (MuSCs) development in buffalo have not been explored fully. We, therefore, performed mRNA and lncRNA expression profiling analysis during the proliferation and differentiation phases of MuSCs in buffalo. The results showed that there were 4,820 differentially expressed genes as well as 12,227 mRNAs and 1,352 lncRNAs. These genes were shown to be enriched in essential biological processes such as cell cycle, p53 signaling pathway, RNA transport and calcium signaling pathway. We also identified a number of functionally important genes, such as MCMC4, SERDINE1, ISLR, LOC102394806, and LOC102403551, and found that interference with MYLPF expression significantly inhibited the differentiation of MuSCs. In conclusion, our research revealed the characteristics of mRNA and lncRNA expression during the differentiation of buffalo MuSCs. This study can be used as an important reference for the study of RNA regulation during muscle development in buffalo.

Defining the Caprin-1 Interactome in Unstressed and Stressed Conditions.

Cytoplasmic stress granules (SGs) are dynamic foci containing translationally arrested mRNA and RNA-binding proteins (RBPs) that form in response to a variety of cellular stressors. It has been debated that SGs may evolve into cytoplasmic inclusions observed in many neurodegenerative diseases. Recent studies have examined the SG proteome by interrogating the interactome of G3BP1. However, it is widely accepted that multiple baits are required to capture the full SG proteome. To gain further insight into the SG proteome, we employed immunoprecipitation coupled with mass spectrometry of endogenous Caprin-1, an RBP implicated in mRNP granules. Overall, we identified 1543 proteins that interact with Caprin-1. Interactors under stressed conditions were primarily annotated to the ribosome, spliceosome, and RNA transport pathways. We validated four Caprin-1 interactors that localized to arsenite-induced SGs: ANKHD1, TALIN-1, GEMIN5, and SNRNP200. We also validated these stress-induced interactions in SH-SY5Y cells and further determined that SNRNP200 also associated with osmotic- and thermal-induced SGs. Finally, we identified SNRNP200 in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) spinal cord and motor cortex. Collectively, our findings provide the first description of the Caprin-1 protein interactome, identify novel cytoplasmic SG components, and reveal a SG protein in cytoplasmic aggregates in ALS patient neurons. Proteomic data collected in this study are available via ProteomeXchange with identifier PXD023271.

Exploring the genomic resources of seven domestic Bactrian camel populations in China through restriction site-associated DNA sequencing.

The domestic Bactrian camel is a valuable livestock resource in arid desert areas. Therefore, it is essential to understand the roles of important genes responsible for its characteristics. We used restriction site-associated DNA sequencing (RAD-seq) to detect single nucleotide polymorphism (SNP) markers in seven domestic Bactrian camel populations. In total, 482,786 SNPs were genotyped. The pool of all remaining others were selected as the reference population, and the Nanjiang, Sunite, Alashan, Dongjiang, Beijiang, Qinghai, and Hexi camels were the target populations for selection signature analysis. We obtained 603, 494, 622, 624, 444, 588, and 762 selected genes, respectively, from members of the seven target populations. Gene Ontology classifications and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed, and the functions of these genes were further studied using Genecards to identify genes potentially related to the unique characteristics of the camel population, such as heat resistance and stress resistance. Across all populations, cellular process, single-organism process, and metabolic process were the most abundant biological process subcategories, whereas cell, cell part, and organelle were the most abundant cellular component subcategories. Binding and catalytic activity represented the main molecular functions. The selected genes in Alashan camels were mainly enriched in ubiquitin mediated proteolysis pathways, the selected genes in Beijiang camels were mainly enriched in MAPK signaling pathways, the selected genes in Dongjiang camels were mainly enriched in RNA transport pathways, the selected genes in Hexi camels were mainly enriched in endocytosis pathways, the selected genes in Nanjiang camels were mainly enriched in insulin signaling pathways, while the selected genes in Qinghai camels were mainly enriched in focal adhesion pathways; these selected genes in Sunite camels were mainly enriched in ribosome pathways. We also found that Nanjiang (HSPA4L and INTU), and Alashan camels (INO80E) harbored genes related to the environment and characteristics. These findings provide useful insights into the genes related to the unique characteristics of domestic Bactrian camels in China, and a basis for genomic resource development in this species.

The prognostic value of an autophagy-related lncRNA signature in hepatocellular carcinoma

BackgroundlncRNA may be involved in the occurrence, metastasis, and chemical reaction of hepatocellular carcinoma (HCC) through various pathways associated with autophagy. Therefore, it is urgent to reveal more autophagy-related lncRNAs, explore these lncRNAs’ clinical significance, and find new targeted treatment strategies.MethodsThe corresponding data of HCC patients and autophagy genes were obtained from the TCGA database, and the human autophagy database respectively. Based on the co-expression and Cox regression analysis to construct prognostic prediction signature.ResultsFinally, a signature containing seven autophagy-related lncRNAs (PRRT3-AS1, RP11-479G22.8, RP11-73M18.8, LINC01138, CTD-2510F5.4, CTC-297N7.9, RP11-324I22.4) was constructed. Based on the risk score of signature, Overall survival (OS) curves show that the OS of high-risk patients is significantly lower than that of low-risk patients (P = 2.292e−10), and the prognostic prediction accuracy of risk score (AUC = 0.786) is significantly higher than that of ALBI (0.532), child_pugh (0.573), AFP (0.5751), and AJCC_stage (0.631). Moreover, multivariate Cox analysis and Nomogram of risk score are indicated that the 1-year and 3-year survival rates of patients are obviously accuracy by the combined analysis of the risk score, child_pugh, age, M_stage, and Grade (The AUC of 1- and 3-years are 0.87, and 0.855). Remarkably, the 7 autophagy-related lncRNAs may participate in Spliceosome, Cell cycle, RNA transport, DNA replication, and mRNA surveillance pathway and be related to the biological process of RNA splicing and mRNA splicing.ConclusionIn conclusion, the 7 autophagy-related lncRNAs might be promising prognostic and therapeutic targets for HCC.

Comparative genome-wide DNA methylation analysis reveals epigenomic differences in response to heat-humidity stress in Bombyx mori

DNA methylation is an important epigenetic modification and has been shown to be involved in the response to abiotic stress. However, there are few studies on DNA methylation in insect response to environmental signals. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles between two silkworm strains with significantly different resistance to heat and humidity by whole-genome bisulfite sequencing (WGBS). We identified, in total, 2934 differentially methylated regions (DMRs) between RT_48h (resistant strain with high-temperature/humidity treatment for 48 h) and ST_48h (sensitive strain with high-temperature/humidity treatment for 48 h) under cytosine context (CG), which corresponded to 1230 DMR-related genes (DMGs), and the DMRs were primarily located in the gene body (exon and intron) region. Gene ontology (GO) and KEGG analysis showed that these DMGs were most significantly enriched in binding, cellular metabolic process, and RNA transport pathways. Moreover, 10 DMGs have been revealed to be involved in the heat-humidity stress response in the silkworm. The results of this study indicated that DNA methylation plays crucial roles in silkworm response to environmental stressors and provides important clues to identify key resistance genes in silkworm under high-temperature/humidity stress response.

Genome-Wide Identification and Characterization of UTR-Introns of Citrus sinensis.

Introns exist not only in coding sequences (CDSs) but also in untranslated regions (UTRs) of a gene. Recent studies in animals and model plants such as Arabidopsis have revealed that the UTR-introns (UIs) are widely presented in most genomes and involved in regulation of gene expression or RNA stability. In the present study, we identified introns at both 5′UTRs (5UIs) and 3′UTRs (3UIs) of sweet orange genes, investigated their size and nucleotide distribution characteristics, and explored the distribution of cis-elements in the UI sequences. Functional category of genes with predicted UIs were further analyzed using GO, KEGG, and PageMan enrichment. In addition, the organ-dependent splicing and abundance of selected UI-containing genes in root, leaf, and stem were experimentally determined. Totally, we identified 825 UI- and 570 3UI-containing transcripts, corresponding to 617 and 469 genes, respectively. Among them, 74 genes contain both 5UI and 3UI. Nucleotide distribution analysis showed that 5UI distribution is biased at both ends of 5′UTR whiles 3UI distribution is biased close to the start site of 3′UTR. Cis- elements analysis revealed that 5UI and 3UI sequences were rich of promoter-enhancing related elements, indicating that they might function in regulating the expression through them. Function enrichment analysis revealed that genes containing 5UI are significantly enriched in the RNA transport pathway. While, genes containing 3UI are significantly enriched in splicesome. Notably, many pentatricopeptide repeat-containing protein genes and the disease resistance genes were identified to be 3UI-containing. RT-PCR result confirmed the existence of UIs in the eight selected gene transcripts whereas alternative splicing events were found in some of them. Meanwhile, qRT-PCR result showed that UIs were differentially expressed among organs, and significant correlation was found between some genes and their UIs, for example: The expression of VPS28 and its 3UI was significantly negative correlated. This is the first report about the UIs in sweet orange from genome-wide level, which could provide evidence for further understanding of the role of UIs in gene expression regulation.

RNA-Seq reveals differential expression profiles and functional annotation of genes involved in retinal degeneration in Pde6c mutant Danio rerio

BackgroundRetinal degenerative diseases affect millions of people and represent the leading cause of vision loss around the world. Retinal degeneration has been attributed to a wide variety of causes, such as disruption of genes involved in phototransduction, biosynthesis, folding of the rhodopsin molecule, and the structural support of the retina. The molecular pathogenesis of the biological events in retinal degeneration is unclear; however, the molecular basis of the retinal pathological defect can be potentially determined by gene-expression profiling of the whole retina. In the present study, we analyzed the differential gene expression profile of the retina from a wild-type zebrafish and phosphodiesterase 6c (pde6c) mutant.ResultsThe datasets were downloaded from the Sequence Read Archive (SRA), and adaptors and unbiased bases were removed, and sequences were checked to ensure the quality. The reads were further aligned to the reference genome of zebrafish, and the gene expression was calculated. The differentially expressed genes (DEGs) were filtered based on the log fold change (logFC) (±4) and p-values (p < 0.001). We performed gene annotation (molecular function [MF], biological process [BP], cellular component [CC]), and determined the functional pathways Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway for the DEGs. Our result showed 216 upregulated and 3527 downregulated genes between normal and pde6c mutant zebrafish. These DEGs are involved in various KEGG pathways, such as the phototransduction (12 genes), mRNA surveillance (17 genes), phagosome (25 genes), glycolysis/gluconeogenesis (15 genes), adrenergic signaling in cardiomyocytes (29 genes), ribosome (20 genes), the citrate cycle (TCA cycle; 8 genes), insulin signaling (24 genes), oxidative phosphorylation (20 genes), and RNA transport (22 genes) pathways. Many more of all the pathway genes were down-regulated, while fewer were up-regulated in the retina of pde6c mutant zebrafish.ConclusionsOur data strongly indicate that, among these genes, the above-mentioned pathways’ genes as well as calcium-binding, neural damage, peptidase, immunological, and apoptosis proteins are mostly involved in the retinal and neural degeneration that cause abnormalities in photoreceptors or retinal pigment epithelium (RPE) cells.

Transcriptional analysis of deoxynivalenol-induced apoptosis of sow ovarian granulosa cell.

Litter size is one of the most important economic traits in pig production. Recent studies identified that deoxynivalenol (DON), a widespread toxin in fodder, was associated with animal prolificacy. However, the underlying mechanisms have not yet been completely elucidated. Here, we used porcine ovary granulosa cells (pGCs) as a vector to establish DON concentration-time models and performed cell morphology and transcriptome analysis to identify and analyse the effects of DON on reproductive performance in swine. The results showed that DON can induce morphological changes and apoptosis of pGCs, while inhibiting cell proliferation. Moreover, these effects of DON on pGCs were dose-dependent. After treatment of pGCs with different concentrations of DON, the percentage of cells in S phase and G2/M phase increased. RNA-seq analyses revealed 5,937 differentially expressed genes, of which 1995 were down-regulated and 3,942 were up-regulated after DON treatment. KEGG enrichment analysis indicated important metabolic pathways such as IL-17 signalling pathway, eukaryotic ribosome synthesis pathway, RNA transport pathway and RNA degradation. Based on our results, we speculate that the effects of DON are related to the DNA damage process. Our study provides novel insights and a foundation to further understand the effect of DON on swine prolificacy.

Prognostic alternative splicing regulatory network of splicing events in acute myeloid leukemia patients based on SpliceSeq data from 136 cases.

This study aimed to create prognostic signatures to predict AML patients' survival using alternative splicing (AS) events. The AS data, RNA sequencing data, and the survival statistics of 136 AML patients were obtained from The Cancer Genome Atlas (TCGA) and TCGA SpliceSeq databases. Total 34,984 AS events generated from 8,656 genes, 2,583 of which were survival-associated AS events, were identified using univariate Cox regression. The prognostic models constructed using independent survival-associated AS events revealed that low-risk splicing better predicted patients' survival. ROC analysis indicated that the predictive efficacy of the alternate terminator model was best in the area under the curve at 0.781. Enrichment analysis revealed several important genes (TP53, BCL2, AURKB, PPP2R1B, FOS, and BIRC5) and pathways, such as the protein processing pathway in the endoplasmic reticulum, RNA transport pathway, and HTLV-I infection pathway. The splicing network of splicing events and factors revealed interesting interactions, such as the positive correlation between HNRNPH3 and CALHM2-13010-AT, which may indicate the potential splicing regulatory mechanism. Taken together, survival-associated splicing events and the prognostic signatures for predicting survival can help provide an overview of splicing in AML patients and facilitate clinical practice. The splicing regulatory network may improve the understanding of spliceosomes in AML.

Identification of CDCA8, DSN1 and BIRC5 in Regulating Cell Cycle and Apoptosis in Osteosarcoma Using Bioinformatics and Cell Biology.

Introduction:Osteosarcoma is the most common primary tumor of bone, although some molecular markers have been identified, the detailed molecular mechanisms underlying osteosarcoma are currently not fully understood. In the present study, we attempted to identify the potential key genes and pathways in osteosarcoma using bioinformatics analysis.Methods:GSE14359 was downloaded from the GEO database, and analyzed using Limma package. Gene Ontology and pathway enrichment analyses of the DEGs were performed in the DAVID database, followed by the construction of a protein–protein interaction (PPI) network with software Cytoscape, subnetwork modules were subsequently identified and analyzed, and further validation in human osteosarcoma tissues and osteosarcoma cells line was performed.Results:964 Differentially expressed genes (DEGs) identified, of which 222 were up-regulated and 742 were down-regulated. Among them, 10 genes (including BIRC5, MAD2L1, Bub1, DSN1, SPC24, CDCA8, STAG2, CENPA, MLF1IP and Mis12) were identified as hub genes and they were mainly enriched in pathways, including mRNA surveillance, RNA transport and PI3K-Akt signaling pathways. Further validation indicated 6 gene (DSN1, BIRC5, CDCA8, MLF1IP, MAD2L1 and SPC24) is highly expressed in osteosarcoma tissues. Among them, CDCA8, DSN1 and BIRC5 significantly promoted the proliferation of osteosarcoma cells in vitro. In terms of mechanism, DSN1 and CDCA8 were mainly involved in cell cycle regulation, while BIRC5 was mainly involved in the regulation of apoptosis pathway.Conclusions:We identified some key genes and pathways in osteosarcoma, which might be used as molecular targets or diagnostic biomarker for the diagnosis and therapy of osteosarcoma.

The RNA degradation pathway is involved in PPARα-modulated anti-oral tumorigenesis.

Background: The activation of peroxisome proliferator-activated receptor alpha (PPARα) has been shown to reprogram tumor metabolism and exhibits great potential for treating anti-oral tumorigenesis.Methods: In this study, we used a pathway-based strategy to explore possible functional pathways involved in the anticancer activity of PPARα in oral cancer cells through next-generation sequencing (NGS) and bioinformatic approaches.Results: We found that 3919 genes were upregulated and 1060 genes were downregulated through PPARα activation. These genes were mainly involved in the proteasomal, mRNA surveillance, spliceosomal, RNA transport, and RNA degradation pathways, as indicated by GO and KEGG enrichment analysis. Importantly, a total of 13 upregulated genes in the RNA degradation pathway were identified including 3 core exosome factor genes (RRP43, RRP42, and CSL4), 2 TRAMP complex genes (TRF4 and Mtr4), 2 exosome cofactor genes (RRP6 and MPP6), 2 CCR4-NOT complex genes (CNOT2 and CNOT3), 2 Ski complex genes (SKI2 and Ski3), 1 decapping complex gene (EDC4), and 1 gene involved in 5’ exoribonuclease activity (XRN1).Conclusion: Our findings suggest that the activation of PPARα to upregulate the RNA degradation pathway might provide a new strategy for oral cancer treatment.

Identification of key genes, regulatory factors, and drug target genes of recurrent implantation failure (RIF)

Objective: Recurrent implantation failure (RIF) exacerbates the physical trauma of infertile women that undergone in vitro fertilization-embryo transfer (IVF-ET). We aimed to identify the key genes, regulatory factors, and drug target genes involved in the RIF.Methods: The dataset GSE58144 that obtained from the Gene Expression Omnibus mainly contained 43 RIF and 72 control endometrial samples. Differently expressed genes (DEGs) between RIF and control groups were firstly analyzed, followed by the pathway and Gene Ontology (GO) enrichment analysis. Then, protein-protein interaction (PPI) network and miRNA-transcript factor (TF)-DEGs network were established. Finally, a drug-target interaction network was constructed.Results: A total of 399 DEGs were identified between the RIF and controls. In the PPI and key module network, UBE2I, PLK4, XPO1, AURKB, and NUP107 were identified as the hub genes, which mainly enriched in RNA transport and cell division cycle-related pathways and GO items. In the miRNA-TF-DEGs network, E2F4, SIN3A, miRNA489, miRNA199A, miRNA369-3P, miRNA422, and miRNA522 were considered as the key regulatory factors during RIF. In addition, HTR1A, NR3C1, and GABRA3 were the main targets of the drugs annotated in DrugBank.Conclusion: The effects of PLK4, XPO1, AURKB, and NUP107 on the RIF may be via affecting the proliferation and differentiation of endometrial stromal cells. Besides, SIN3A and miRNA199A may be crucial for embryo implantation. In addition, NR3C1 may be used as a possible target for the clinical therapy of RIF.