Enriched RNA Research Articles

Lack of Targetable FGFR2 Fusions in Endemic Fluke-Associated Cholangiocarcinoma.

PURPOSECholangiocarcinoma (CCA) remains a disease with poor prognosis and limited therapeutic options. Identification of driver genetic alterations may lead to the discovery of more effective targeted therapies. CCAs harboring FGFR2 fusions have recently demonstrated promising responses to FGFR inhibitors, highlighting their potential relevance as predictive biomarkers. CCA incidence is high in the northeast of Thailand and its neighboring countries because of chronic infection with the liver fluke Opisthorchis viverrini (Ov). However, there are currently no available data on the prevalence of FGFR alterations in fluke-associated CCA in endemic countries.MATERIALS AND METHODSIn this study, we performed anchored multiplex polymerase chain reaction target enrichment RNA sequencing of FGFR1-3, validated by fluorescence in situ hybridization and Sanger sequencing, in 121 Ov-associated and 95 non–Ov-associated CCA tumors.RESULTSCompared with non–fluke-associated CCA (11/95; 11.6%), FGFR2 fusions were significantly less common in fluke-associated CCA (1/121; 0.8%; P = .0006). All FGFR fusions were detected exclusively in intrahepatic CCAs and were mutually exclusive with KRAS/ERBB2/BRAF/FGFR mutations, pointing to their potential roles as oncogenic drivers.CONCLUSIONFGFR2 fusions are rare in fluke-associated CCA, underscoring how distinct etiologies may affect molecular landscapes in tumors and highlighting the need to discover other actionable genomic alterations in endemic fluke-associated CCA.

A Novel Microfluidic Device Integrated with Chitosan-Modified Capillaries for Rapid ZIKV Detection.

The outbreak of Zika virus (ZIKV) has posed a great challenge to public health in recent years. To address the urgent need of ZIKV RNA assays, we integrate the microfluidic chip embedded with chitosan-modified silicon dioxide capillaries, smartphone-based detection unit to be a C3-system for the rapid extraction and detection of ZIKV RNA. The C3-system is characterized by: (1) four chitosan-modified silicon dioxide capillaries integrated in the microfluidic chip for target ZIKV RNA enrichment and “in situ PCR” (polymerase chain reaction) amplification; (2) smartphone-based point of care (POC) device consisting of a pneumatic subsystem for controlling the nucleic acid extraction processes in the microfluidic chip, a heating subsystem for sample lysis and PCR amplification, and an optical subsystem for signal acquisition. The entire detection processes including sample lysis, ZIKV RNA enrichment, and reverse-transcription polymerase chain reaction (RT-PCR) is achieved in the microfluidic chip. Moreover, PCR buffers can be directly loaded into the chitosan-modified silicon dioxide capillaries for “in situ PCR”, in which the captured ZIKV RNA is directly used for downstream PCR without any loss. ZIKV RNA extracted by the C3-system can be successfully recovered at very low concentrations of 50 transducing units (TU)/mL from crude human saliva. This means that our method of detecting viremia in patients infected with ZIKV is reliable.

A 6 lncRNA-Based Risk Score System for Predicting the Recurrence of Colon Adenocarcinoma Patients

Colon adenocarcinoma (COAD) is a common type of colon cancer, and post-operative recurrence and metastasis may occur in COAD patients. This study is designed to build a risk score system for COAD patients. The Cancer Genome Atlas (TCGA) dataset of COAD (the training set) was downloaded, and GSE17538 and GSE39582 (the validation sets) from Gene Expression Omnibus database were obtained. The differentially expressed RNAs (DERs) were analyzed by limma package. Using survival package, the independent prognosis-associated long non-coding RNAs (lncRNAs) were selected for constructing risk score system. After the independent clinical prognostic factors were screened out using survival package, a nomogram survival model was constructed using rms package. Furthermore, competitive endogenous RNA (ceRNA) regulatory network and enrichment analyses separately were performed using Cytoscape software and DAVID tool. Totally 404 DERs between recurrence and non-recurrence groups were identified. Based on the six independent prognosis-associated lncRNAs (including H19, KCNJ2-AS1, LINC00899, LINC01503, PRKAG2-AS1, and SRRM2-AS1), the risk score system was constructed. After the independent clinical prognostic factors (Pathologic M, pathologic T, and RS model status) were identified, the nomogram survival model was built. In the ceRNA regulatory network, there were three lncRNAs, four miRNAs, and 77 mRNAs. Additionally, PPAR signaling pathway and hedgehog signaling pathway were enriched for the mRNAs in the ceRNA regulatory network. The risk score system and the nomogram survival model might be used for predicting COAD recurrence. Besides, PPAR signaling pathway and hedgehog signaling pathway might affect the recurrence of COAD patients.

Exosomes Transmit Viral Genetic Information and Immune Signals may cause Immunosuppression and Immune Tolerance in ALV-J Infected HD11 cells

Avian leukosis virus (ALV) is oncogenic retrovirus that not only causes immunosuppression but also enhances the host's susceptibility to secondary infection. Exosomes play vital role in the signal transduction cascades that occur in response to viral infection. We want to explore the function of exosomes in the spread of ALV and the body's subsequent immunological response. RNA-sequencing and the isobaric tags for relative and absolute quantitation (iTRAQ) method were used to detect differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) in exosomes secreted by macrophage cells in response to injection with ALV subgroup J (ALV-J). RNA-sequencing identified 513 DEGs in infected cells, with specific differential regulation in mRNA involved in tight junction signaling, TNF signaling, salmonella infection response, and immune response, among other important cellular processes. Differential regulation was observed in 843 lncRNAs, with particular enrichment in those lncRNA targets involved in Rap1 signaling, HTLV-I infection, tight junction signaling, and other signaling pathways. A total of 50 DEPs were identified in the infected cells by iTRAQ. The proteins enriched are involved in immune response, antigen processing, the formation of both MHC protein and myosin complexes, and transport. Combined analysis of the transcriptome and proteome revealed that there were 337 correlations between RNA and protein enrichment, five of which were significant. Pathways that were enriched on both the RNA and protein levels were involved in pathways in cancer, PI3K-Akt signaling pathway, Endocytosis, Epstein-Barr virus infection. These data show that exosomes are transmitters of intercellular signaling in response to viral infection. Exosomes can carry both viral nucleic acids and proteins, making it possible for exosomes to be involved in the viral infection of other cells and the transmission of immune signals between cells. Our sequencing results confirme previous studies on exosomes and further find exosomes may cause immunosuppression and immune tolerance.

Isolating Multiple Extracellular Vesicles Subsets, Including Exosomes and Membrane Vesicles, from Bovine Milk Using Sodium Citrate and Differential Ultracentrifugation.

Milk is a complex fluid that contains various types of proteins and extracellular vesicles (EVs). Some proteins can mingle with EVs, and interfere with their isolation. Among these proteins, caseins form micelles of a size comparable to milk EVs, and can thus be co-isolated with EVs. Preliminary steps that affect milk are crucial for EV isolation and impact the purity and abundance of isolated EVs. In the course of our previous works on cow's milk EVs, we found that sodium citrate (1% final), which is a biocompatible reagent capable of breaking down casein micelles into 40-nm monomers, allowed the isolation of high quantities of EVs with low coprecipitation of caseins or other contaminating proteins. Using this protocol, we successfully separated different EV subsets, characterized in depth their morphology, protein content and small RNA enrichment patterns. We were also able to describe their biological function in a mouse model of intestinal inflammation. We, hereby, detail the differential ultracentrifugation procedure that leads to high quantify, medium specificity, isolation of different milk EV subsets from the same sample. More specifically, we highlight the use of sodium citrate as a standardized approach to isolate and study milk EVs and its potential for isolation techniques other than differential ultracentrifugation.

Messenger RNA enrichment using synthetic oligo(T) click nucleic acids.

Enrichment of mRNA is a key step in a number of molecular biology techniques, particularly in the rapidly growing field of transcriptomics. Currently, mRNA is isolated using oligo(thymine) DNA (oligo(dT)) immobilized on solid supports, which binds to the poly(A) tail of mRNA to pull the mRNA out of solution through the use of magnets or centrifugal filters. Here, a simple method to isolate mRNA by complexing it with synthetic click nucleic acids (CNAs) is described. Oligo(T) CNA bound efficiently to mRNA, and because of the insolubility of CNA in water, >90% of mRNA was readily removed from solution using this method. Simple washing, buffer exchange, and heating steps enabled mRNA's enrichment from total RNA, with a yield of 3.1 ± 1.5% of the input total RNA by mass, comparable to the yield from commercially available mRNA enrichment beads. Further, the integrity and activity of mRNA after CNA-facilitated pulldown and release was evaluated through two assays. In vitro translation of EGFP mRNA confirmed the translatability of mRNA into functional protein and RT-qPCR was used to amplify enriched mRNA from total RNA extracts and compare gene expression to results obtained using commercially available products.

Comparison of RNA isolation procedures for analysis of adult murine brain and spinal cord astrocytes

BackgroundMolecular analyses of cell populations and single cells have been instrumental in the advancement of our understanding of the physiology and pathologic processes of the nervous system. However, the limitation of these methods is the dependence on a gentle, efficient and specific enrichment procedure for the target cell population. In particular, this has been challenging for tightly interconnected cells, for example central nervous system (CNS) endogenous cells such as astrocytes. New methodHere we adopted one of the most common methods of cell extraction, namely, enzymatic tissue digestion followed by fluorescence-activated cell sorting (FACS) of individual cells. We evaluated different enzymatic/mechanical tissue dissociation procedures and analyzed different astrocyte lineage transgenic models. Furthermore, we compared the cell extraction efficiency from spinal cord vs. brain. ResultsEnzymatic digestion of CNS tissue of Glast-CreERT2x tdTomatofl/fl or Aldh1l1-CreERT2x tdTomatofl/fl followed by FACS resulted in highly purified astrocytes. Automated tissue digestion strongly improved the isolated cell numbers. Aldh1l1-CreERT2 identified more astrocytes than Glast-CreERT2; isolation from brain yields higher numbers than from spinal cord. Comparison with existing methodsWe compared the efficiency and purity of the enzymatic dissociation/FACS approach with a more modern procedure consisting of tissue homogenization followed by translating ribosome affinity purification (TRAP). ConclusionWe found that both methods result in highly enriched astrocytic RNA. However, only TRAP isolation resulted in reliably detectable RNA concentrations from spinal cord tissue on a single animal level. Depending on the aim of the study both methods have advantages and disadvantages but both are acceptable for astrocytic RNA analysis.

Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions

Fungal infections, ranging from superficial to life-threatening infections, represent a major public health problem that affects 25% of the worldwide population. In this context, the study of host-pathogen interactions within the host is crucial to advance antifungal therapy. However, since fungal cells are usually outnumbered by host cells, the fungal transcriptome frequently remains uncovered. We compared three different methods to selectively lyse human cells from in vitro mixes, composed of Candida cells and peripheral blood mononuclear cells. In order to prevent transcriptional modification, the mixes were stored in RNAlater. We evaluated the enrichment of fungal cells through cell counting using microscopy and aimed to further enrich fungal nucleic acids by centrifugation and by reducing contaminant nucleic acids from the host. We verified the enrichment of fungal DNA and RNA through qPCR and RT-qPCR respectively and confirmed that the resulting RNA has high integrity scores, suitable for downstream applications. The enrichment method provided here, i.e., lysis with Buffer RLT followed by centrifugation, may contribute to increase the proportion of nucleic acids from fungi in clinical samples, thus promoting more comprehensive analysis of fungal transcriptional profiles. Although we focused on C. albicans, the enrichment may be applicable to other fungal pathogens.

A Genetically Encoded Diazirine Analogue for RNA-Protein Photo-crosslinking.

Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA–protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA‐binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA–protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA‐binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA‐binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine‐based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA–protein interactions.

Isolation of enriched small RNA from cell-lysate using on-chip isotachophoresis.

In spite of the growing interest in the roles and applications of small RNAs (sRNAs), sRNA isolation methods are inconsistent, tedious, and dependent on the starting number of cells. In this work, we employ ITP to isolate sRNAs from the cell-lysate of K562 (chronic myelogenous leukemia) cells in a polydimethylsiloxane (PDMS) mesofluidic device. Our method specifically purifies sRNA of <60 nucleotides from lysate of a wide range of cell number spanning from 100 to 1000000 cells. We measured the amount of sRNA using the Agilent Bioanalyzer and further verified the extraction efficiency by reverse transcription quantitative PCR. Our method was shown to be more efficient in sRNA extraction than commercial sRNA isolation kits, especially when using smaller numbers of starting cells. Our assay presents a simple and rapid sRNA extraction method with 20min assay time and no intermediate transfer steps.

P4460microRNA-17-5p and microRNA-20a-5p downregulation by atorvastatin in Hepg2 cells: a new mechanism involved in the lipid-lowering therapy

Abstract Background Hypercholesterolemia increases the risk of coronary artery disease and its pharmacological treatment has demonstrated, besides reducing cholesterol levels, a decrease in the incidence and mortality from coronary events. The treatment of hypercholesterolemia is mainly driven by using statins. However, the response to pharmacological therapy shows high inter-individual variability, resulting in a variable effect in both lipid lowering and risk reduction. Thus, a better understanding of lipid-lowering mechanism and response variability at molecular level is required. Previously, we demonstrated a deregulation of microRNA (miR) profile in HepG2 cells after atorvastatin treatment, including the downregulation of miR-17-5p and miR-20a-5p which potentially targets the LDL receptor gene (LDLR), suggesting that it might be involved in allowing the LDLR overexpression on the surface of hepatic cells to subsequently capture circulating LDL and to reach the expected lipid-lowering effect. Purpose To determine the role of miR-17-5p and miR-20a-5p on the regulation of LDLR gene expression in HepG2 cells. Methods Cells HepG2 were treated with atorvastatin 10μM for 24 hours. RNA extraction and enrichment of smallRNAs were performed. The gene expression of miR-17-5p, miR-20a-5p, miR-24-3p, miR-93-5p, miR-106a-5p and LDLR were evaluated. To evaluate the effect of miR-17-5p and miR-20a-5p on LDLR gene expression, both miRs were overexpressed or repressed by transfection of mimics or inhibitors respectively into HepG2 cells for 24, 48 and 72 Hours. The gene expression of LDLR was quantified by real time PCR using RPL27 gene as reference gene. The protein expression of LDLR and beta actin were evaluated using western blot and quantified using the ImageJ software. Results Our data showed that atorvastatin significantly repressed the expression of miR-17-5p (P&lt;0.0001) and miR-20a-5p (P=0.0456) in HepG2 cells. In silico studies showed that miR-17-5p interact with the 3'-UTR region of the LDLR. Consistently, when miR-17-5p or miR-20a-5p were overexpressed by using mimics, we observed that gene and protein expression of LDLR decreased significantly (P&lt;0.0001 and P&lt;0.05 respectively). Consistently, when miR-17-5p or miR-20a-5p were repressed by the use inhibitors, we observed that the gene and protein expression of LDLR increases significantly (P&lt;0.005). Conclusions In conclusion, we demonstrate that atorvastatin induces a significant down-regulation of the miR-17-5p and miR-20a-5p in HepG2 cells. The overexpression or repression regulate the gene and protein expression of LDLR. Acknowledgement/Funding FONDECYT N°3160567, FONDECYT N°1171765 and DIUFRO DI19-0094

Abstract 409: Regulation of Cardiomyocyte Maturation by an RNA Splicing Regulator Rbfox1

The maturation of stem cell derived cardiomyocytes (iCMs) is incomplete relative to the fully matured adult myocytes. Lack of maturation represents a major limitation to the applications of iCMs as heart disease models or heart failure therapies. Current attempts to promote iCMs maturation, such as prolonged culture, mechanical stretching and electronic pacing, are often based on empirical methods with poor reproducibility or little mechanistic basis. In order to better understand the molecular mechanisms driving cardiomyocyte maturation, we performed extensive transcriptome analyses in neonatal vs. adult hearts. In addition to metabolic and cell cycle regulatory pathways, Gene Ontology analysis revealed RNA splicing regulation is significantly enriched in the transcriptome reprogramming during postnatal maturation in heart. Specifically, we find a cardiomyocyte enriched RNA splicing factor Rbfox1 is dramatically induced in the perinatal maturating mouse hearts. Ectopic expression of Rbfox1 in neonatal cardiomyocytes markedly promotes the cellular and molecular features of adult cardiomyocyte, including contractility, calcium handling, sarcomere organization, morphology, electrophysiology and gene expression. Most remarkably, expression of RBFox1 in human iPSC derived cardiomyocytes promotes similar maturation process as observed in the neonatal rat myocytes. At mechanistic level, RBFox1 expression in the iCMs enhances transcriptome maturation as indicated by targeted RNA splicing in genes involved in muscle contraction, gene expression, RNA processing and sarcomere organization. In summary, we have uncovered a novel molecular path towards neonatal myocyte maturation in perinatal murine hearts by targeted modulation of cardiomyocyte transcriptome via RNA-splicing. This approach has potential to be employed as a molecular approach to promote human iCMs maturation.

189 Comparative transcriptomic analysis between Total RNA and mRNA isolated from feces of neonatal dairy calves

Abstract The fecal RNA method can be used to evaluate biological adaptations of the gastrointestinal tract of dairy calves through gene expression analysis. The process of RNA isolation from fecal samples presents several challenges, including the potential enrichment of prokaryotic (bacterial), RNA which can dilute the targeted eukaryotic RNA and consequently affect the sensitivity of the fecal RNA method to low expressed genes. Therefore, our objective in this study was to determine the differential eukaryotic RNA enrichment in total RNA vs mRNA from feces of healthy neonatal dairy calves. To test this, a comparative transcriptomic profiling of genes specific for epithelial cells, including cytokeratin 8 (KRT8) and aquaporin (AQP3), as well as inflammatory-related genes (TLR4 and IL1B) was performed in fecal samples collected from 6 pre-weaned Holstein calves. The total RNA was isolated from 200 mg of feces, using a Trizol based method along with the RNeasy Plus Mini Kit (Qiagen). Then, 45 μg of fecal total RNA was used to isolate mRNA through magnetic selection using Dynabeads® Oligo (dT)25 (Invitrogen). The standard curve was composite from all samples including cDNA from total RNA and mRNA. The internal control genes used in this experiment were B2M, ACTB, GAPDH, RPS9, and PPIA. Normalized gene expression data were log-transformed prior to statistical analysis using the Proc Mixed of SAS (SAS 9.4). The expression of KRT8 was greater (P = 0.03) in fecal mRNA than in fecal total RNA. A trend (P = 0.09) was observed for greater expression of TLR4 in fecal total RNA than in fecal mRNA. The expression of AQP3 and IL1B was not different. These preliminary data further confirms that fecal RNA method has potential to be used as a tool to evaluate gastrointestinal tract health in dairy calves, but further adjustments are needed to improve accuracy and robustness.

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy.

In plant mitochondria, some steady-state transcripts have 5' triphosphate derived from transcription initiation (primary transcripts), while the others contain 5' monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5' triphosphate. However, the triphosphate at primary 5' termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5' polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5' triphosphate. In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5' polyphosphatase, which converts 5' triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5' polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5' end and is insensitive to 5' polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot. By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

Abstract 1682: Non-V600 BRAF mutants in melanoma: Multiple MAPK mutations and distinctive RNA expression

Abstract Large discovery sequencing analyses conducted by our group and others enabled classification of melanomas into major and minor subtypes, helping to elucidate the role and diversity of activating mutations within the MAPK pathway. We sequenced 482 melanoma PDX, cell lines and patient biopsies, of which 20 (4.1%) carry non-V600 BRAF mutations. Of the 20 mutations, 14 are pathogenic, 5 are likely pathogenic and 1 is a variant of unknown significance. Interestingly, 78.5% of tumors with pathogenic non-V600 mutants and all likely pathogenic or VUS non-V600 mutants carry additional mutations within the MAPK pathway. To explore this pattern, we queried publicly available melanoma sequencing data from six previously published sequencing studies of melanoma. Consistent with our findings, nonV600-BRAF mutations were identified in 0 - 9% of melanoma sequencing studies. Of the tumors carrying non-V600 BRAF mutants, 26 of 42 (62%) carry additional mutations in the MAPK pathway. Given the co-occurrence of MAPK mutations with non-V600 mutants, we considered whether these mutations are found together within the same cell or if mutants are encoded in distinct cell populations and reflect admixture of a heterogenous tumor. We compared the variant allele frequency (VAF) of co-occurring MAPK mutants with their non-V600 BRAF counterparts in 41 available samples. The VAF between mutants was discordant (&amp;gt;0.10 change in VAF between mutants) in 27 of 41 (68%) of the samples. To more precisely assess variants at single-cell resolution, we established a PCR based method for variant calling using single-cell-derived cDNA libraries. Pilot studies supported distinct expression patterns and we are working to generate robust and reproducible sequencing data. We also compared RNA expression data between BRAFV600 melanomas and non-V600 melanomas. This analysis revealed distinct transcriptomes that were easily distinguished based on previously established quaternary structure and kinase activity of various BRAF mutants. Interestingly, MAPK-pathway genes did not account for most of the differential expression, and an AXL-high/MITF-low expression program was highly enriched in non-V600 BRAF tumors relative to their V600 BRAF counterpart (FDR &amp;lt;0.01). Taken together, non-V600 BRAF mutant melanomas represent a subtype characterized by a tendency for multiple MAPK mutations and an AXL-high/MITF-low enriched RNA expression pattern differentiated based on BRAF structure and function. Further studies of this rare sub-type of melanoma are important to demonstrate the diversity and interplay of MAPK dysregulation in cancer and may reveal mechanisms of tumorigenesis. Citation Format: Elizabeth Kiernan, Paul Bastian, Liza Dorfman, Ioannis N. Anastopoulos, Brad Wubbenhorst, Clemens Krepler, Vito Rebecca, Meenhard Herlyn, Katherine Nathanson. Non-V600 BRAF mutants in melanoma: Multiple MAPK mutations and distinctive RNA expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1682.

Short communication: Comparative gene expression analysis on the enrichment of polymorphonuclear leukocytes and gastrointestinal epithelial cells in fecal RNA from nondiarrheic neonatal dairy calves

Increased understanding of the biology of the gastrointestinal tract (GIT) in neonatal dairy calves during their adaptation to an extrauterine environment will decrease health problems such as diarrhea while increasing feed efficiency and average daily gain in preweaned dairy calves. Within this context, a noninvasive method, based on fecal RNA, to study the GIT in neonatal dairy calves through the isolation of RNA from fecal samples for quantitative reverse-transcription PCR analysis can provide valuable information on GIT biological adaptations during the preweaning period. We aimed to evaluate the potential enrichment of RNA from immune cells or GIT epithelial cells during fecal RNA isolation. Eight neonatal Holstein calves less than 3 wk old (14.9 ± 5.5 d of age at sampling ± standard deviation) and a fecal score of 2.0 ± 0.7 (mean ± standard deviation) were used. During a single sampling, fecal and blood samples were taken simultaneously from each calf before the morning feeding. Fecal samples were immediately frozen in liquid nitrogen until RNA isolation, whereas polymorphonuclear leukocytes (PMN) were isolated from blood samples before RNA isolation. An quantitative reverse-transcription PCR analysis was performed using a single standard curve composited of equal amounts of all samples including cDNA from fecal and PMN. The genes myeloperoxidase (MPO) and L-selectin (SELL) were selected for their specific known function in PMN, whereas keratin 8 (KRT8) and aquaporin 3 (AQP3) have been associated with epithelial enterocytes. Our results showed a contrasting gene expression profile between PMN and fecal RNA; whereas greater mRNA expression of SELL was observed in PMN, a greater KRT8 expression was observed in fecal RNA. The mRNA expression of AQP3 tended to be greater in PMN than fecal RNA. Additionally, MPO was not amplified in fecal RNA. Our findings suggest that under nondiarrheic conditions RNA isolated from stool samples of neonatal dairy calves will have a considerable number of GIT epithelial cells, which confirms the reliability of this method under these conditions. However, further research needs to be done to determine if the same effects are observed during diarrhea or throughout the preweaning period of dairy calves.

Serum microRNA 143 as a potential biomarker for the diagnosis of hepatitis C virus-related hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the fifth most frequently diagnosed cancer worldwide. Early recognition of the onset of HCC would help to select more effective therapies for patients leading to a better prognosis and life span. So, the development of effective markers for the diagnosis of HCC could have an impact on HCC-related cancer mortality. MicroRNAs (miRNAs) are reported as a group of small noncoding RNAs that can function as endogenous RNA interference to regulate the expression of targeted genes.AimTo study serum miR-143 expression level in patients with hepatitis C virus (HCV)-related HCC.Patients and methodsThe present study was conducted on 60 participants classified into group A: 30 patients with HCV-related cirrhosis with HCC; group B: 15 patients with HCV-related cirrhosis without HCC; and group C: healthy participants as control. Expression of miR-143 in the serum of all participants was obtained in all groups. Total serum RNA was extracted with small RNA enrichment followed by reverse transcription real-time PCR. Expression of miR-143 in the serum of all participants was obtained using the comparative cycle threshold method (2–ΔΔCT) after normalization for the expression of Syn-cel-miR-39 mi script miRNA mimic as control. MiR-143 expression levels were then compared in different groups.ResultsThe mean serum miR-143 levels were significantly higher in cirrhotic patients without and with HCC than in healthy participants (1.69±0.64, 13.0±6.23 vs. 0.56 ±0.29, P< 0.001, respectively) and in patients with HCC than in patients without HCC (P<0.001). The mean serum miR-143 levels were significantly higher in cirrhotic patients with HCC Barcelona Clinic Liver Cancer stage D than stage B (19.91±2.40, 13.41±1.80, P< 0.001, respectively) and in stage B compared with stage A (13.41±1.80, 5.68±1.86, P<0.001, respectively).ConclusionThe significantly higher serum level of miR-143 in cirrhotic patients with HCC compared with those without and to normal participants may suggest its role in hepatocarcinogenesis and may have a value as a potential biomarker for early diagnosis of HCV- related HCC.

HOT or not: examining the basis of high-occupancy target regions.

High-occupancy target (HOT) regions are segments of the genome with unusually high number of transcription factor binding sites. These regions are observed in multiple species and thought to have biological importance due to high transcription factor occupancy. Furthermore, they coincide with house-keeping gene promoters and consequently associated genes are stably expressed across multiple cell types. Despite these features, HOT regions are solely defined using ChIP-seq experiments and shown to lack canonical motifs for transcription factors that are thought to be bound there. Although, ChIP-seq experiments are the golden standard for finding genome-wide binding sites of a protein, they are not noise free. Here, we show that HOT regions are likely to be ChIP-seq artifacts and they are similar to previously proposed ‘hyper-ChIPable’ regions. Using ChIP-seq data sets for knocked-out transcription factors, we demonstrate presence of false positive signals on HOT regions. We observe sequence characteristics and genomic features that are discriminatory of HOT regions, such as GC/CpG-rich k-mers, enrichment of RNA–DNA hybrids (R-loops) and DNA tertiary structures (G-quadruplex DNA). The artificial ChIP-seq enrichment on HOT regions could be associated to these discriminatory features. Furthermore, we propose strategies to deal with such artifacts for the future ChIP-seq studies.

Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences.

Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus.

MiR‐486a‐5p Exerts Cardioprotection Against Ischemia Repefusion via Suppression of PDCD4

Extracellular vesicle (EV) derived from mesenchymal stem cells (MSCs) have been demonstrated to be a critical component for cardioprotection against myocardial infarction triggered by stem cell therapy. EVs are nano‐size phospholipid bilayer particles that carry proteins, lipids, RNA, including microRNAs (miRs). Because EVs can deliver their cargo to other cells, they hold potential application for development as clinical therapeutics. Herein we tested the hypothesis that a specific MSC‐EV miR, miR‐486a‐5p, mediates some aspect of cardioprotective effect against ischemia/reperfusion injury (I/R).Extracellular vesicles were isolated from B6/129‐derived MSCs using differential ultracentrifugation method. To characterize EV RNA, total RNA was isolated from MSC cells and EVs and used to synthesize libraries for small RNA deep sequencing. EVs and cell miRs were ranked based on enrichment in EV RNA (EV/cell levels). We found that miR‐486a‐5p is the most highly enriched miR in MSC‐derived EV RNA at level of 5853‐fold higher than in cells (Padj=1.52E‐59). The differential level of miR‐486‐5p between EVs and cell was validated by Taqman Advanced small RNA qRT‐PCR. We searched for miR‐486a‐5p gene targets using Targetscan7.0 and found 64 gene targets involving in cell death and survival pathways (Pathway Studio). Programmed cell death 4 (PDCD4) was chosen for further study based on weighted accumulative score++ (Targetscan7.0). To test whether miR‐486 can directly target PDCD4, luciferase assay was performed using a reporter vector containing PDCD4 3′‐UTR. 3′‐UTR of PTEN, a known target of miR‐486 was used as a control. When H9c2 cells transfected with the reporter vector were incubated with a range of miR‐486 mimic doses, luciferase activity was significantly reduced in a dose‐specific manner. Next, we determined whether transfection of H9c2 cells with miR‐486 mimic affected PDCD4 expression. The mimic transfection was efficient with a 200‐fold expression of miR‐486 mimic relative to the control. Whereas miR‐486 mimic transfection did not influence the expression of PDCD4 mRNA, PDCD4 protein was significantly suppressed (~60% downregulation, P&lt;0.001 vs. control).In H9c2 cardiomyocytes, in vitro experiments showed that treatment with miR‐486 mimic did not change cell survival in normoxic condition. However, treatment with 50 and 100 nM of mimic significantly reduced I/R mediated injury (increased cell viability (Hoechst‐PPI assay) and decreased lactate dehydrogenase leakage (P&lt;0.01 vs. control) after simulated I/R (12h hypoxia with ischemia mimetic solution and 2h reperfusion). Moreover, TUNEL assay revealed miR‐486 mimic treatment significantly suppressed apoptosis. Western blotting analysis of cell lysates after simulated I/R showed that improved I/R outcome was associated with the inhibition of PDCD4 protein levels, and levels of its downstream effectors, cleaved caspase‐3 and Bax. In conclusion, these findings indicate that miR‐486‐5p can ameliorate I/R mediated damage through suppression of PDCD4 protein levels and inhibition of apoptosis in cardiomyocytes. Whether miR‐486a‐5p contributes to exosome‐mediated cardioprotection in vivo will be determined using our in vivo I/R model of coronary occlusion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.