Genome-wide patterns of noncoding and protein-coding sequence variation in the major fungal pathogen Aspergillus fumigatus.

Aspergillus fumigatus is a deadly fungal pathogen, responsible for >400,000 infections/year and high mortality rates. A. fumigatus strains exhibit variation in infection-relevant traits, including in their virulence. However, most A. fumigatus protein-coding genes, including those that modulate its virulence, are shared between A. fumigatus strains and closely related nonpathogenic relatives. We hypothesized that A. fumigatus genes exhibit substantial genetic variation in the noncoding regions immediately upstream to the start codons of genes, which could reflect differences in gene regulation between strains. To begin testing this hypothesis, we identified 5,812 single-copy orthologs across the genomes of 263 A. fumigatus strains. In general, A. fumigatus noncoding regions showed higher levels of sequence variation compared with their corresponding protein-coding regions. Focusing on 2,482 genes whose protein-coding sequence identity scores ranged between 75 and 99%, we identified 478 total genes with signatures of positive selection only in their noncoding regions and 65 total genes with signatures only in their protein-coding regions. Twenty-eight of the 478 noncoding regions and 5 of the 65 protein-coding regions under selection are associated with genes known to modulate A. fumigatus virulence. Noncoding region variation between A. fumigatus strains included single-nucleotide polymorphisms and insertions or deletions of at least a few nucleotides. These results show that noncoding regions of A. fumigatus genes harbor greater sequence variation than protein-coding regions, raising the hypothesis that this variation may contribute to A. fumigatus phenotypic heterogeneity.

M-F noncoding region sequences of H1 genotype measles virus provide higher resolution for virus transmission tracing.

As countries and regions move toward measles elimination, extended sequence window including noncoding region located between the matrix and fusion protein genes (M - F NCR) was considered to be used in molecular surveillance. The molecular resolution of M - F NCR was evaluated with 192 genotype H1 strains circulating during 2011-2018 in China. Phylogenetic analyses of the N450 and M - F NCR targets indicated that both two targets could confirm epi-linked outbreak, while M - F NCR target could further improve resolution of the molecular characterization: (1) it could differentiate the strains with identical N450 circulated in one county within one month of disease onset; (2) different transmission chains could be distinguished for strains with identical N450; (3) better spatial-temporal consistency with topology could be provided among sporadic cases with inconsistent N450. Accordingly, M - F NCR could be used to complement the information from N450 to address the specific questions in tracking the virus transmission chains.

Predicting functional UTR variants by integrating region-specific features.

The untranslated region (UTR) of messenger ribonucleic acid (mRNA), including the 5'UTR and 3'UTR, plays a critical role in regulating gene expression and translation. Variants within the UTR can lead to changes associated with human traits and diseases; however, computational prediction of UTR variant effect is challenging. Current noncoding variant prediction mainly focuses on the promoters and enhancers, neglecting the unique sequence of the UTR and thereby limiting their predictive accuracy. In this study, using consolidated datasets of UTR variants from disease databases and large-scale experimental data, we systematically analyzed more than 50 region-specific features of UTR, including functional elements, secondary structure, sequence composition and site conservation. Our analysis reveals that certain features, such as C/G-related sequence composition in 5'UTR and A/T-related sequence composition in 3'UTR, effectively differentiate between nonfunctional and functional variant sets, unveiling potential sequence determinants of functional UTR variants. Leveraging these insights, we developed two classification models to predict functional UTR variants using machine learning, achieving an area under the curve (AUC) value of 0.94 for 5'UTR and 0.85 for 3'UTR, outperforming all existing methods. Our models will be valuable for enhancing clinical interpretation of genetic variants, facilitating the prediction and management of disease risk.

Polymorphisms of the PD-L1 gene 3'-untranslated region are associated with the expression of PD-L1 in non-small cell lung cancer.

Recent results show that polymorphisms of programmed death ligand 1 (PD-L1, also known as CD274 or B7-H1) might be used as a possible marker for effectiveness of chemotherapy and cancer risk. However, the effect of PD-L1 gene variations on PD-L1 expression remain unclear. Given the post-transcriptional machinery in tumor PD-L1 expression, we investigated single nucleotide polymorphisms (SNPs) in the 3'-untranslated region (3'-UTR) of the PD-L1 gene, rs4143815 and rs4742098, using formalin-fixed paraffin-embedded sections of 154 patients with non-small cell lung cancers (NSCLCs). In rs4143815, the GG genotype showed significant association with PD-L1 expression (P = 0.032). In rs4742098, the AA genotype was significantly associated with histology and PD-L1 expression (P = 0.022 and P = 0.008, respectively). In multivariate logistic regression analysis, the AA genotype in rs4742098 was correlated with PD-L1 expression (odds ratio 0.408, P = 0.048). Interestingly, approximately 10% of the NSCLC cases showed somatic mutation when we compared genotypes of these SNPs between NSCLC tissues and non-tumor tissues from the same patients. In addition, cases with somatic mutation showed higher levels of PD-L1 expression than cases with germline mutation in rs4143815 GG. In conclusion, we demonstrated that the rs4143815 and rs4742098 SNPs in the 3'-UTR of PD-L1 were associated with tumor PD-L1 expression in NSCLCs.

Predictive modeling of oocyte maternal mRNA features for five mammalian species reveals potential shared and species-restricted regulators during maturation.

Oocyte maturation is accompanied by changes in abundances of thousands of mRNAs, many degraded and many preferentially stabilized. mRNA stability can be regulated by diverse features including GC content, codon bias, and motifs within the 3'-untranslated region (UTR) interacting with RNA binding proteins (RBPs) and miRNAs. Many studies have identified factors participating in mRNA splicing, bulk mRNA storage, and translational recruitment in mammalian oocytes, but the roles of potentially hundreds of expressed factors, how they regulate cohorts of thousands of mRNAs, and to what extent their functions are conserved across species has not been determined. We performed an extensive in silico cross-species analysis of features associated with mRNAs of different stability classes during oocyte maturation (stable, moderately degraded, and highly degraded) for five mammalian species. Using publicly available RNA sequencing data for germinal vesicle (GV) and MII oocyte transcriptomes, we determined that 3'-UTR length and synonymous codon usage are positively associated with stability, while greater GC content is negatively associated with stability. By applying machine learning and feature selection strategies, we identified RBPs and miRNAs that are predictive of mRNA stability, including some across multiple species and others more species-restricted. The results provide new insight into the mechanisms regulating maternal mRNA stabilization or degradation.NEW & NOTEWORTHY Conservation across species of mRNA features regulating maternal mRNA stability during mammalian oocyte maturation was analyzed. 3'-Untranslated region length and synonymous codon usage are positively associated with stability, while GC content is negatively associated. Just three RNA binding protein motifs were predicted to regulate mRNA stability across all five species examined, but associated pathways and functions are shared, indicating oocytes of different species arrive at comparable physiological destinations via different routes.

Functional Consequences of Shifting Transcript Boundaries in Glucose Starvation.

Glucose is a major source of carbon and essential for the survival of many organisms, ranging from yeast to human. A sudden 60-fold reduction of glucose in exponentially growing fission yeast induces transcriptome-wide changes in gene expression. This regulation is multilayered, and the boundaries of transcripts are known to vary, with functional consequences at the protein level. By combining direct RNA sequencing with 5'-CAGE and short-read sequencing, we accurately defined the 5'- and 3'-ends of transcripts that are both poly(A) tailed and 5'-capped in glucose starvation, followed by proteome analysis. Our results confirm previous experimentally validated loci with alternative isoforms and reveal several transcriptome-wide patterns. First, we show that sense-antisense gene pairs are more strongly anticorrelated when a time lag is taken into account. Second, we show that the glucose starvation response initially elicits a shortening of 3'-UTRs and poly(A) tails, followed by a shortening of the 5'-UTRs at later time points. These result in domain gains and losses in proteins involved in the stress response. Finally, the relatively poor overlap both between differentially expressed genes (DEGs), differential transcript usage events (DTUs), and differentially detected proteins (DDPs) highlight the need for further study on post-transcriptional regulation mechanisms in glucose starvation.

Natural recombination of the torque teno canis virus within the ORF1, -2, and -3 genes and the untranslated region.

The torque teno canis virus (TTCaV) was first reported in 2001 and it shares similarities with the known Torque teno virus (TTV) in terms of genomic organization and putative transcriptional features. It is a single-stranded DNA virus characterized by high rates of recombination and nucleotide substitution, like RNA viruses. Studies reported recombination events in torque teno virus; however, there is limited reporting of TTCaV reorganization events. This study screened fecal samples from domestic dogs in Henan Province. There was a positivity rate of 16.5% (19/115) for TTCaV. Four nearly complete TTCaV genomes, namely Canine/HeNan/4, 5, 6, and 13/2019, were obtained from the 19 positive fecal samples, whose genome sequence was obtained using gap-filling PCR. Sequence analysis revealed two unique amino acid mutation sites in the TTCaV strains, K278Q (compared with the first isolate Cf-TTV10 in Japan) and V/L268I (compared with the TTCaV strain from southern China). Subsequently, 17 near full-length TTCaV genome sequences were subjected to phylogenetic and recombination detection program analyzes. We obtained evidence supporting recombination events in the Chinese TTCaV strains. These findings suggest that mutation and recombination occurred in the three individual gene segments (ORF1, ORF2, ORF3) and the untranslated region, an area of major recombination in the Chinese TTCaV strain GX265 genome. Interestingly, the TTCaV strain (Canine/HeNan/6/2019) was a major parent involved in the genetic recombination of the GX265 strain. This study provides insights into the genetic variability and evolution of TTCaV.

Chromosome-level genome assembly of Chouioia cunea Yang, the parasitic wasp of the fall webworm.

Chouioia cunea Yang 1989 is a parasitic wasp of many lepidopteran insects during their pupal stage, and has been successfully used to control pests such as the fall webworm Hyphantria cunea. Here we reported the chromosome-level genome of C. cunea by using short (MGI-SEQ), long (Oxford Nanopore), chromatin-linked (Hi-C) sequencing reads and transcriptomic data, representing the first chromosome-level genome of parasitic wasps of the family Eulophidae. The total assembly length is 171.99 Mb, containing 6 pesudo-chromosomes with a GC content of 36.89% and the scaffold/contig N50 length of 31.70/26.52 Mb. The BUSCO completeness of the assembly was estimated to be 98.7%. A total of 12,258 protein-coding genes (PCGs), 10,547 3'-UTRs, and 10,671 5'-UTRs were annotated. This high-quality genome is an important step toward a better understanding of the genomes of the Eulophidae (Chalcidoidea), and will serve as a valuable resource for analyses of phylogenetic relationships and the evolution of Hymenoptera.

N6-methyladenosine profiling reveals that Xuefu Zhuyu decoction upregulates METTL14 and BDNF in a rat model of traumatic brain injury.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese herbal formula Xuefu Zhuyu decoction (XFZYD) is a classic formula in the category of invigorating blood circulation and resolving blood stasis. It has been proven to improve the neurological and ethological prognosis of traumatic brain injury. XFZYD promotes synaptic and axonal regeneration after traumatic brain injury, which is functionally modulated by the N6-methyladenosine (m6A) modification of RNA. However, the epigenetic effects of XFZYD on m6A modification remain unknown. AIM OF THE STUDY: To explore how XFZYD protects against traumatic brain injury induced by controlled cortical impact (CCI) injury by altering RNA m6A modification. MATERIALS AND METHODS: The modified neurological severity scoring and Morris water maze were performed to evaluate the neuroprotective effects of XFZYD for 14 days and screen the dose. Then, dot blot, western blotting, and methylated RNA immunoprecipitation sequencing (MeRIP-Seq) were used to explore changes in RNA m6A modification in the perilesional cortex. The Metascape platform was used to analyze the Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway of the differential m6A-tagged genes. Furthermore, MeRIP-qPCR was conducted to quantify differences in the hub differential m6A modification gene brain-derived neurotrophic factor (Bdnf). RESULTS: XFZYD significantly ameliorated the neurological deficits, spatial learning, and memory impairments in rats post-CCI on day 14. XFZYD enhanced the m6A level, and the expression of METTL14 and YTHDC2 in the perilesional cortex of CCI rats. In all three groups, the 3'-untranslated regions and coding sequence were primarily enriched for m6A peaks. XFZYD reversed the increased proportion of 3'-untranslated regions, and the decreased proportion of coding sequence and 5'-untranslated regions post-CCI. Moreover, XFZYD markedly downregulated 41 elevated m6A-tagged transcripts and upregulated 119 decreased m6A-tagged transcripts following CCI. Gene ontology and KEGG pathway analysis revealed that XFZYD-regulated m6A-tagged transcripts were predominantly enriched in synapse assembly, synaptic plasticity, learning or memory, and MAPK signaling pathway. Then, the hub-regulated m6A-tagged gene BDNF was identified. Both the m6A methylation level and the protein level of BDNF were ascended by XFZYD treatment. CONCLUSION: XFZYD improves neurological deficits, spatial learning and memory impairments in rats post-TBI probably through increasing the expression of METTL14 and BDNF in the cortex. Our study highlights a novel post-transcriptional regulation mechanism mediated by herbal medicine for traumatic brain injury treatment.

Characterization of A-to-I Editing in Pigs under a Long-Term High-Energy Diet.

Long-term high-energy intake has detrimental effects on pig health and elevates the risk of metabolic disease. RNA editing modifying RNA bases in a post-transcriptional process has been extensively studied for model animals. However, less evidence is available that RNA editing plays a role in the development of metabolic disorders. Here, we profiled the A-to-I editing in three tissues and six gut segments and characterized the functional aspect of editing sites in model pigs for metabolic disorders. We detected 64,367 non-redundant A-to-I editing sites across the pig genome, and 20.1% correlated with their located genes' expression. The largest number of A-to-I sites was found in the abdominal aorta with the highest editing levels. The significant difference in editing levels between high-energy induced and control pigs was detected in the abdominal aorta, testis, duodenum, ileum, colon, and cecum. We next focused on 6041 functional A-to-I sites that detected differences or specificity between treatments. We found functional A-to-I sites specifically involved in a tissue-specific manner. Two of them, located in gene SLA-DQB1 and near gene B4GALT5 were found to be shared by three tissues and six gut segments. Although we did not find them enriched in each of the gene features, in correlation analysis, we noticed that functional A-to-I sites were significantly enriched in gene 3'-UTRs. This result indicates, in general, A-to-I editing has the largest potential in the regulation of gene expression through changing the 3'-UTRs' sequence, which is functionally involved in pigs under a long-term high-energy diet. Our work provides valuable knowledge of A-to-I editing sites functionally involved in the development of the metabolic disorder.

mRNA 3' -UTR-mediate translational control through PAS and CPE in sheep oocyte.

In oocytes, the cytoplasmic polyadenylation and maternal mRNAs translation is regulated by cis-elements, including polyadenylation signal (PAS) and cytoplasmic polyadenylation element (CPE) in 3'-UTR. Recent studies illustrate non-canonical polyadenylation mechanisms of translational regulation in mouse oocytes, which is different from that in Xenopus oocytes. However, it is still unclear if this regulation in rodent oocytes functions in the domestic animal oocyte. Here, by using sheep as an animal model, we cloned the 3'-UTRs of Cpeb1 or Btg4 and ligated it into the pRK5-Flag-Gfp vector. Variant numbers and positions of PASs and CPEs within the 3'-UTRs were constructed to detect their effects on translational control. After in vitro-transcription and microinjection into sheep fully grown germinal vesicle stage oocytes, the expression efficiency of mRNAs was detected by the GFP and flag expression. Our results show that: (i) PAS located at the proximal end of 3'-UTR can mediate the translation of the maternal mRNAs, as long as they locate far from CPEs; (ii) The proximal PAS has higher efficiency in regulating transcription than the distal one; (iii) increase of PAS number can promote the translational activity more efficiently; (iv) a single CPE located close to PAS (<50 bp) in 3'-UTRs of Cpeb1 or Btg4 could partially repress translation. In 3'-UTRs of Btg4, two CPEs have a higher inhibitory effect, and three CPEs can completely inhibit mRNA translation. These results confirm the existence of the non-canonical mechanism in domestic animal oocytes.

Untranslated regions of brain-derived neurotrophic factor mRNA control its translatability and subcellular localization.

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival and growth during development. In the adult nervous system, BDNF is important for synaptic function in several biological processes such as memory formation and food intake. In addition, BDNF has been implicated in development and maintenance of the cardiovascular system. The Bdnf gene comprises several alternative untranslated 5' exons and two variants of 3' UTRs. The effects of these entire alternative UTRs on translatability have not been established. Using reporter and translating ribosome affinity purification analyses, we show that prevalent Bdnf 5' UTRs, but not 3' UTRs, exert a repressive effect on translation. However, contrary to previous reports, we do not detect a significant effect of neuronal activity on BDNF translation. In vivo analysis via knock-in conditional replacement of Bdnf 3' UTR by bovine growth hormone 3' UTR reveals that Bdnf 3' UTR is required for efficient Bdnf mRNA and BDNF protein production in the brain, but acts in an inhibitory manner in lung and heart. Finally, we show that Bdnf mRNA is enriched in rat brain synaptoneurosomes, with higher enrichment detected for exon I-containing transcripts. In conclusion, these results uncover two novel aspects in understanding the function of Bdnf UTRs. First, the long Bdnf 3' UTR does not repress BDNF expression in the brain. Second, exon I-derived 5' UTR has a distinct role in subcellular targeting of Bdnf mRNA.

Upregulation of miR-184 and miR-19a-3p induces endothelial dysfunction by targeting AGO2 in Kawasaki disease.

BACKGROUND: Endothelial dysfunction is a marked feature of Kawasaki disease during convalescence, but its pathogenesis is currently unclear. Circulating microRNAs (miRNAs) are associated with the progression of Kawasaki disease. However, the role and mechanism of circulating miRNAs in endothelial dysfunction are largely unknown. Kawasaki disease patients were found to have a unique circulating miRNA profile, including upregulation of miRNA-210-3p, miR-184 and miR-19a-3p, compared to non-Kawasaki disease febrile controls. This study aimed to investigate the effects of these three miRNAs on endothelial function. METHODS: Overexpression of miRNAs in human umbilical vein endothelial cells was done by transfection of miRNA mimics. The tube formation assay was used to evaluate the function of human umbilical vein endothelial cells. The potential binding sites of miRNAs on 3'untranslated regions were predicted by using TargetScan database and validated by dual luciferase reporter assay. The protein expression of AGO2, PTEN and VEGF in human umbilical vein endothelial cells was detected by Western blot. Overexpression of AGO2 in human umbilical vein endothelial cells was done by transfection of AGO2 expression plasmids. RESULTS: Overexpression of miRNA-184 and miRNA-19a-3p, but not miR-210-3p, impaired the function of human umbilical vein endothelial cells. Mechanistically, miR-184 and miR-19a-3p could target the 3'untranslated regions of AGO2 mRNA to downregulate its expression and subsequently impede the AGO2/PTEN/VEGF axis. To be noted, the rescue of the expression of AGO2 remarkably recovered the function that was impaired by overexpression of miRNA-184 and miRNA-19a-3p. CONCLUSIONS: This study suggested that miR-184 and miR-19a-3p could target AGO2/PTEN/VEGF axis to induce endothelial dysfunction in Kawasaki disease.

Digoxigenin-labeled RNA probes for untranslated regions enable the isoform-specific gene expression analysis of myosin heavy chains in whole-mount in situ hybridization.

Myosin heavy chains (MyHCs), which are encoded by myosin heavy chain (Myh) genes, are the most abundant proteins in myofiber. Among the 11 sarcomeric Myh isoform genes in the mammalian genome, seven are mainly expressed in skeletal muscle. Myh genes/MyHC proteins share a common role as force producing units with highly conserved sequences, but have distinct spatio-temporal expression patterns. As such, the expression patterns of Myh genes/MyHC proteins are considered as molecular signatures of specific fiber types or the regenerative status of mammalian skeletal muscles. Immunohistochemistry is widely used for identifying MyHC expression patterns; however, this method is costly and is not ideal for whole-mount samples, such as embryos. In situ hybridization (ISH) is another versatile method for the analysis of gene expression, but is not commonly applied for Myh genes, partly because of the highly homologous sequences of Myh genes. Here we demonstrate that an ISH analysis with the untranslated region (UTR) sequence of Myh genes is cost-effective and specific method for analyzing the Myh gene expression in whole-mount samples. Digoxigenin (DIG)-labeled antisense probes for UTR sequences, but not for protein coding sequences, specifically detected the expression patterns of respective Myh isoform genes in both embryo and adult skeletal muscle tissues. UTR probes also revealed the isoform gene-specific polarized localization of Myh mRNAs in embryonic myofibers, which implied a novel mRNA distribution mechanism. Our data suggested that the DIG-labeled UTR probe is a cost-effective and versatile method to specifically detect skeletal muscle Myh genes in a whole-mount analysis.

UTRdb 2.0: a comprehensive, expert curated catalog of eukaryotic mRNAs untranslated regions.

The 5' and 3' untranslated regions of eukaryotic mRNAs (UTRs) play crucial roles in the post-transcriptional regulation of gene expression through the modulation of nucleo-cytoplasmic mRNA transport, translation efficiency, subcellular localization, and message stability. Since 1996, we have developed and maintained UTRdb, a specialized database of UTR sequences. Here we present UTRdb 2.0, a major update of UTRdb featuring an extensive collection of eukaryotic 5' and 3' UTR sequences, including over 26 million entries from over 6 million genes and 573 species, enriched with a curated set of functional annotations. Annotations include CAGE tags and polyA signals to label the completeness of 5' and 3'UTRs, respectively. In addition, uORFs and IRES are annotated in 5'UTRs as well as experimentally validated miRNA targets in 3'UTRs. Further annotations include evolutionarily conserved blocks, Rfam motifs, ADAR-mediated RNA editing events, and m6A modifications. A web interface allowing a flexible selection and retrieval of specific subsets of UTRs, selected according to a combination of criteria, has been implemented which also provides comprehensive download facilities. UTRdb 2.0 is accessible at http://utrdb.cloud.ba.infn.it/utrdb/.

A ferritin gene in the marine copepod Acartia tonsa as a highly sensitive biomonitor for nano-contamination.

Toxicology is not only for eco-risk assessments, but also for the real-time environmental monitoring based on the quick response of specific biomarkers. Ferritin gene (ftn) is a potential biomarker involving in crucial protective responses in biota. However, little information is available concerning the ftn in marine copepod Acartia tonsa (A. tonsa), a model organism widely applied in toxicology assessments. Our study for the first time identified and characterized the ftn in A. tonsa, along with its time-dependent transcriptional response to the reproductive toxicity of two newly emerged nanomaterials. The full-length cDNA of ftn contains a 114-bp 5'-untranslated region (UTR), a 236-bp 3'-untranslated region, and a 510-bp open reading frame which encodes an 18.51 kDa polypeptide composed of 169 amino acids. The ftn sequence has an iron binding signature and a potential phosphorylation site, which is closely-related to the ftn of Calanus sinicus and Pseudodiaptomus annandalei genes at the phylogenetical level. The ftn showed a quick and highly sensitive response to nanomaterial exposures, even at no observed effect concentrations. In detail, after exposure to nickel nanomaterials (up to 17.0 mg/L), the ftn was significantly upregulated immediately at 0.5 h and peaked at 9.5-fold in adults within 48 h, along with a significant reduction of egg hatching rate. When exposed to CdSe/ZnS quantum dots (up to 135 mg/L), no significant change in egg productions or hatching rates was observed, while the expression of ftn still significantly increased to over 3.0-fold in the initial 48 h. After that, the upregulation of ftn induced by CdSe/ZnS quantum dots or nickel nanoparticles both gradually returned back within 96 h. These findings demonstrate the highly sensitive response of this new cloned ftn to nanomaterial exposures, and highlight the suitability of ftn in A. tonsa as a promising biomonitor for nano-contamination in marine environments.

Locus-Specific Enrichment Analysis of 5-Hydroxymethylcytosine Reveals Novel Genes Associated with Breast Carcinogenesis.

An imbalance in DNA methylation is a hallmark epigenetic alteration in cancer. The conversion of 5-methylcytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC), which causes the imbalance, results in aberrant gene expression. The precise functional role of 5-hydroxymethylcytosine in breast cancer remains elusive. In this study, we describe the landscape of 5-mC and 5-hmC and their association with breast cancer development. We found a distinguishable global loss of 5-hmC in the localized and invasive types of breast cancer that strongly correlate with TET expression. Genome-wide analysis revealed a unique 5-mC and 5-hmC signature in breast cancer. The differentially methylated regions (DMRs) were primarily concentrated in the proximal regulatory regions such as the promoters and UTRs, while the differentially hydroxymethylated regions (DhMRs) were densely packed in the distal regulatory regions, such as the intergenic regions (>-5 kb from TSSs). Our results indicate 4809 DMRs and 4841 DhMRs associated with breast cancer. Validation of nine 5-hmC enriched loci in a distinct set of breast cancer and normal samples positively correlated with their corresponding gene expression. The novel 5-hmC candidates such as TXNL1, and CNIH3 implicate a pro-oncogenic role in breast cancer. Overall, these results provide new insights into the loci-specific accumulation of 5-mC and 5-hmC, which are aberrantly methylated and demethylated in breast cancer.

Specificity of oligonucleotide gene therapy (OGT) agents.

Oligonucleotide gene therapy (OGT) agents (e. g. antisense, deoxyribozymes, siRNA and CRISPR/Cas) are promising therapeutic tools. Despite extensive efforts, only few OGT drugs have been approved for clinical use. Besides the problem of efficient delivery to targeted cells, hybridization specificity is a potential limitation of OGT agents. To ensure tight binding, a typical OGT agent hybridizes to the stretch of 15-25 nucleotides of a unique targeted sequence. However, hybrids of such lengths tolerate one or more mismatches under physiological conditions, the problem known as the affinity/specificity dilemma. Here, we assess the scale of this problem by analyzing OGT hybridization-dependent off-target effects (HD OTE) in vitro, in animal models and clinical studies. All OGT agents except deoxyribozymes exhibit HD OTE in vitro, with most thorough evidence of poor specificity reported for siRNA and CRISPR/Cas9. Notably, siRNA suppress non-targeted genes due to (1) the partial complementarity to mRNA 3'-untranslated regions (3'-UTR), and (2) the antisense activity of the sense strand. CRISPR/Cas9 system can cause hundreds of non-intended dsDNA breaks due to low specificity of the guide RNA, which can limit therapeutic applications of CRISPR/Cas9 by ex-vivo formats. Contribution of this effects to the observed in vivo toxicity of OGT agents is unclear and requires further investigation. Locked or peptide nucleic acids improve OGT nuclease resistance but not specificity. Approaches that use RNA marker dependent (conditional) activation of OGT agents may improve specificity but require additional validation in cell culture and in vivo.

Overexpression of MiR-633 Suppresses the Tumorigenicity of Gastric Cancer Cells and Induces Apoptosis by Targeting MAPK1.

OBJECTIVE: MicroRNA (miRNA/miR)-633 is dysregulated in several types of cancers and is involved in tumorigenesis. However, the function and role of this miRNA in gastric cancer (GC) are not fully understood. The aim of the present study was to evaluate miR-633 expression in GC cell lines and in GC tissue vs. adjacent normal tissue, and to determine its association with clinicopathological data. This work was extended to investigate the effects of miR-633 overexpression on tumor cells in vitro. METHODS: Reverse transcription-quantitative PCR (RT-qPCR) was used to detect and compare the expression level of miR-633 in GC cells, as well as in GC and normal adjacent tissue samples. The clinical significance of miR-633 was also analyzed. MiR-633 lentivirus (LV-miR-633) and negative control lentivirus (LV-NC) were generated and used to transduce SGC-7901 and HGC-27 GC cells in order to analyze the effect of miR-633 on their phenotype. The effects of miR-633 overexpression on GC cell proliferation, apoptosis, migration and invasion were investigated. The target gene of miR-633 was predicted, then confirmed using a dual luciferase reporter gene assay, RT-qPCR and Western blotting. RESULTS: MiR-633 was significantly downregulated in GC cell lines, as well as in GC tissue compared with adjacent normal tissue. Moreover, miR-633 expression was associated with the tumor/node/metastasis (TNM) stage, invasion depth, Borrmann classification and lymph node metastasis (P<0.05). Compared with the LV-NC group, transduction with LV-miR-633 reduced the proliferation, the number of clones, the wound healing rate, the number of invading cells and the number of cells in the G1 phase of the cell cycle (P<0.01). LV-miR-633 also increased the apoptosis rate (P<0.01). The expression level of mitogen-activated protein kinase (MAPK) 1, high-mobility group box 3 (HMGB3), claudin 1 (CLDN1) and MAPK13 were downregulated in LV-miR-633-transduced cells (P<0.01). The dual luciferase reporter assay confirmed that the 3'-untranslated region of MAPK1 was the target site of miR-633 (P<0.01). CONCLUSION: MiR-633 acts as a tumor suppressor in GC, and its expression level is associated with TNM stage, invasion depth, Borrmann type and lymph node metastasis. Overexpression of miR-633 inhibits the proliferation and migration of GC cells and induces apoptosis and cell cycle arrest at the in G1 phase. In addition, miR-633 negatively regulates the expression of MAPK1, HMGB3, CLDN1 and MAPK13 and directly targets MAPK1.

Long non­coding RNA LINC01960­201 hinders decidualization of endometrial stromal cell in endometriosis: Relevance to endometrial receptivity.

Emerging data have indicated that long non­coding RNAs (lncRNA) are associated with the pathogenesis of endometriosis. However, few are associated with endometriosis­associated infertility. In addition, to the best of our knowledge, the role of lncRNAs in decidual formation during the window of implantation with endometriosis has not been reported to date. Based on our previous results, the aim of the present study was to explore the role of lncRNA long intergenic non­protein coding RNA (LINC)01960­201 in in vitro decidualization of endometrial stromal cells in endometriosis during the window of implantation, as well as to explore the biological function of LINC01960­201, and the regulation of a disintegrin and metalloproteinase with thrombospondin motifs 7 (ADAMTS7), hsa­microRNA (miR)­760 and hsa­miR­608 in the decidualization of endometrial stromal cells with endometriosis. Using miRanda, PITA and RNAhybrid, the present study predicted which miRs share the common target gene ADAMTS7 with LINC01960­201 and the existence of regulatory targets. Dual luciferase vectors were constructed to extract the plasmids and measure the relative fluorescence values in order to estimate target regulatory association between LINC01960­201, ADAMTS7 and miRs. Mid­secretory endometrial tissues were collected from women with endometriosis­associated infertility. From these tissues, endometrial stromal cells were extracted and cultured as primary cultures. Medroxyprogesterone acetate (MPA) and 8­Bromoadenosine 3',5'­cyclic monophosphate (8­Br­cAMP) were added to induce in vitro decidualization, and to knockdown LINC01960­201 and transfect a hsa­miR­608 mimic at the same time. Reverse transcription­quantitative PCR and western blotting were conducted to compare the difference in gene expression between the experimental and negative control groups. No regulatory sites between LINC01960­201 and hsa­miR­608 were identified; however, potential regulatory sites were detected between hsa­miR­608 and the 3'­untranslated region (UTR) of ADAMTS7, whereas neither the 3'­UTR of LINC01960­201 or the 3'­UTR of ADAMTS7 had any regulatory targets with hsa­miR­760. During the process of decidualization of endometrial stromal cells by in vitro induction, the expression of hsa­miR­608 in the knockdown group was significantly higher compared with that of the negative control group after LINC01960­201­knockdown, and the expression of ADAMTS7 in the transfection group was significantly lower compared with that of the negative control group after hsa­miR­608 mimic transfection. In conclusion, it was hypothesized that LINC01960­201 played a notable regulatory role in the decidualization of endometrial stromal cells in women with endometriosis during the window of implantation, and its abnormal expression may lead to the decline of endometrial receptivity and recurrent abortions.