Endogenous miRNAs Research Articles

Expression and Secretion of Circular RNAs in the Parasitic Nematode, Ascaris suum.

Circular RNAs (circRNAs) are a recently identified RNA species with emerging functional roles as microRNA (miRNA) and protein sponges, regulators of gene transcription and translation, and modulators of fundamental biological processes including immunoregulation. Relevant to this study, circRNAs have recently been described in the parasitic nematode, Haemonchus contortus, suggesting they may have functionally important roles in parasites. Given their involvement in regulating biological processes, a better understanding of their role in parasites could be leveraged for future control efforts. Here, we report the use of next-generation sequencing to identify 1,997 distinct circRNAs expressed in adult female stages of the gastrointestinal parasitic nematode, Ascaris suum. We describe spatial expression in the ovary-enriched and body wall muscle, and also report circRNA presence in extracellular vesicles (EVs) secreted by the parasite into the external environment. Further, we used an in-silico approach to predict that a subset of Ascaris circRNAs bind both endogenous parasite miRNAs as well as human host miRNAs, suggesting they could be functional as both endogenous and exogenous miRNA sponges to alter gene expression. There was not a strong correlation between Ascaris circRNA length and endogenous miRNA interactions, indicating Ascaris circRNAs are enriched for Ascaris miRNA binding sites, but that human miRNAs were predicted form a more thermodynamically stable bond with Ascaris circRNAs. These results suggest that secreted circRNAs could be interacting with host miRNAs at the host-parasite interface and influencing host gene transcription. Lastly, although we have previously found that therapeutically relevant concentrations of the anthelmintic drug ivermectin inhibited EV release from parasitic nematodes, we did not observe a direct effect of ivermectin treatment on Ascaris circRNAs expression or secretion.

Circulating exosome miRNA, is it the novel nutrient molecule through cross-kingdom regulation mediated by food chain transmission from microalgae to bivalve?

MicroRNAs (miRNAs) can efficiently regulate gene expression at intracellular and extracellular levels. Plant-derived miRNAs are highly enriched in animal haemolymph and regulate mammalian gene expression. However, evidence for food-derived miRNAs in Mollusca species is lacking. In this study, we fed the microalga Nannochloropsis oculata to the pearl oyster Pinctada fucata martensii and detected dietary miRNAs in exosomes isolated from the haemolymph by RNA-seq. In total, 273 endogenous miRNAs were identified in all biological replicates. We identified 23 microalgae-derived miRNAs in the exosomes of pearl oyster haemolymph. Most microalgae-derived miRNAs showed high expression levels in both exosomes and microalgae and exhibited apparent variation among individuals. These food-derived miRNAs were predicted to participate in endocytosis, apoptosis, signal transduction, energy metabolism, and biomineralization by targeting multiple genes. These findings demonstrated the cross-kingdom transport of miRNAs from microalgae to bivalves and provide insights into novel nutrient transmission through the food chain.

LncRNA LINC00665 Promotes Ovarian Cancer Cell Proliferation and Inhibits Apoptosis via Targeting miR-181a-5p/FHDC.

Previous reports indicate that long intergenic non-coding RNA LINC00665 naturally occurred vital effects in various cancers. Herein, the role of LINC00665 in ovarian cancer progress was explored. We found that LINC00665 was upregulated in ovarian cancer cell lines. Besides, a series of assays including flow cytometry, wound-healing, transwell, cell counting Kit-8 (CCK-8), and EdU assay confirmed that the knockdown of LINC00665 could reduce the viability, proliferation, and migration of SKOV-3 and OVCAR-3 cells. Accumulating evidence indicates that many lncRNAs can function as endogenous miRNA sponges by competitively binding common miRNAs. In this study, the bioinformatics analysis suggests that LNC00665 specifically binds to miR-181a-5p. LINC00665 downregulated the miR-181a-5p in SKOV-3 and OVCAR-3 cells. The knockdown of miR-181a-5p evidently reverses the inhibitory effect of sh-LINC00662. Besides, FH2 domain containing 1 (FHDC1) has been proved to deed as an effective target of miR-181a-5p. The results reveal the knockdown of LINC00665 facilitates ovarian cancer via development by sponging miR-181a-5p and up-regulating FHDC1 expression. These may contribute to ovarian cancer therapy.

Upconversion Nanoparticle@Au Core-Satellite Assemblies for In Situ Amplified Imaging of MicroRNA in Living Cells and Combined Cancer Phototherapy.

Stimuli-responsive therapy of cancer with spatial and temporal control is crucial in improving the treatment efficacy and minimizing the side effects. MicroRNA (miRNA) as an important biomarker has become one of the most promising endogenous stimuli for cancer therapy. However, the therapy efficacy is often impeded by the low expression amount of miRNA. Herein, the upconversion nanoparticle@Au (UCNP@Au) core-satellite nanostructures are rationally fabricated for isothermal amplification detection and in situ imaging of microRNA-21 (miR-21) in living cells based on the toehold-mediated strand displacement (TMSD) reaction, which is further applied to miRNA-responsive combined photothermal and photodynamic therapy of breast cancer. The UCNP@Au are constructed by linking AuNPs to photosensitizers Rose Bengal (RB)-loaded UCNPs through DNA hybridization. The upconversion luminescence (UCL) is quenched by AuNPs, resulting in the attenuation of singlet oxygen generation of RB. Once UCNP@Au are internalized into MCF-7 cells, the overexpressed intracellular miR-21 trigger the cyclic disassembly of UCNP@Au through cascade TMSD reactions, which facilitate the restoration of UCL for in situ imaging of miR-21 with signal amplification. Moreover, the released AuNPs are aggregated for photothermal therapy (PTT), while the singlet oxygen generated by RB is enhanced for photodynamic therapy (PDT). Compared with single-mode therapy, the miRNA-activated combinational phototherapy has demonstrated a greatly improved therapeutic efficacy for breast cancer. Therefore, our proposed core-satellite nanostructures cannot only achieve in situ amplified imaging of endogenous miRNA but also provide an effective nanoplatform for stimuli-responsive combinational phototherapy, which hold great prospects in early diagnosis and treatment of cancers.

Verticillium dahliae Secretes Small RNA to Target Host MIR157d and Retard Plant Floral Transition During Infection.

Bidirectional trans-kingdom RNA silencing [or RNA interference (RNAi)] plays a key role in plant-pathogen interactions. It has been shown that plant hosts export specific endogenous miRNAs into pathogens to inhibit their virulence, whereas pathogens deliver small RNAs (sRNAs) into plant cells to disturb host immunity. Here, we report a trans-kingdom fungal sRNA retarding host plant floral transition by targeting a miRNA precursor. From Arabidopsis plants infected with Verticillium dahliae, a soil-borne hemibiotrophic pathogenic fungus that causes wilt diseases in a wide range of plant hosts, we obtained a number of possible trans-kingdom V. dahliae sRNAs (VdsRNAs) by sequencing AGO1-immunoprecipitated sRNAs. Among these, a 24-nt VdsRNA derived from V. dahliae rRNA, VdrsR-1, was shown to be an actual trans-kingdom VdsRNA that targets the miR157d precursor MIR157d, resulting in increased rather than reduced miR157d accumulation in V. dahliae-infected plants. Consistent with the miR157 family in the regulation of vegetative and floral transitions by targeting SPL genes in several plant species, we detected two SPL genes, SPL13A/B, that were notably reduced in V. dahliae-infected and VdrsR-1-expressing plants compared with control plants. Furthermore, V. dahliae-infected and VdrsR-1-expressing plants also displayed delayed vegetative phase change and floral transition compared to control plants. Taken together, we disclosed a novel mode of action for a trans-kingdom fungal sRNA, VdrsR-1, which was secreted into host cells to modulate plant floral transition by employing the miR157d/SPL13A/B regulatory module, leading to prolonged host vegetative growth that would undoubtedly benefit fungal propagation.

Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles

Engineered signaling networks can impart cells with new functionalities useful for directing differentiation and actuating cellular therapies. For such applications, the engineered networks must be tunable, precisely regulate target gene expression, and be robust to perturbations within the complex context of mammalian cells. Here, we use bacterial two-component signaling proteins to develop synthetic phosphoregulation devices that exhibit these properties in mammalian cells. First, we engineer a synthetic covalent modification cycle based on kinase and phosphatase proteins derived from the bifunctional histidine kinase EnvZ, enabling analog tuning of gene expression via its response regulator OmpR. By regulating phosphatase expression with endogenous miRNAs, we demonstrate cell-type specific signaling responses and a new strategy for accurate cell type classification. Finally, we implement a tunable negative feedback controller via a small molecule-stabilized phosphatase, reducing output expression variance and mitigating the context-dependent effects of off-target regulation and resource competition. Our work lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks.

Comparison of Data Normalization Strategies for Array-Based MicroRNA Profiling Experiments and Identification and Validation of Circulating MicroRNAs as Endogenous Controls in Hypertension.

Introduction: MicroRNAs are small noncoding RNAs with potential regulatory roles in hypertension and drug response. The presence of many of these RNAs in biofluids has spurred investigation into their role as possible biomarkers for use in precision approaches to healthcare. One of the major challenges in clinical translation of circulating miRNA biomarkers is the limited replication across studies due to lack of standards for data normalization techniques for array-based approaches and a lack of consensus on an endogenous control normalizer for qPCR-based candidate miRNA profiling studies. Methods: We conducted genome-wide profiling of 754 miRNAs in baseline plasma of 36 European American individuals with uncomplicated hypertension selected from the PEAR clinical trial, who had been untreated for hypertension for at least one month prior to sample collection. After appropriate quality control with amplification score and missingness filters, we tested different normalization strategies such as normalization with global mean of imputed and unimputed data, mean of restricted set of miRNAs, quantile normalization, and endogenous control miRNA normalization to identify the method that best reduces the technical/experimental variability in the data. We identified best endogenous control candidates with expression pattern closest to the mean miRNA expression in the sample, as well as by assessing their stability using a combination of NormFinder, geNorm, Best Keeper and Delta Ct algorithms under the Reffinder software. The suitability of the four best endogenous controls was validated in 50 hypertensive African Americans from the same trial with reverse-transcription–qPCR and by evaluating their stability ranking in that cohort. Results: Among the compared normalization strategies, quantile normalization and global mean normalization performed better than others in terms of reducing the standard deviation of miRNAs across samples in the array-based data. Among the four strongest candidate miRNAs from our selection process (miR-223-3p, 19b, 106a, and 126-5p), miR-223-3p and miR-126-5p were consistently expressed with the best stability ranking in the validation cohort. Furthermore, the combination of miR-223-3p and 126-5p showed better stability ranking when compared to single miRNAs. Conclusion: We identified quantile normalization followed by global mean normalization to be the best methods in reducing the variance in the data. We identified the combination of miR-223-3p and 126-5p as potential endogenous control in studies of hypertension.

Construction of a HOXA11-AS-Interact Ed Network in Keloid Fibroblasts Using Integrated Bioinformatic Analysis and in Vitro Validation.

Background: Expression of the long noncoding RNA (lncRNA) HOXA11-AS significantly increased in keloids by unclarified molecular regulation mechanisms. Methods: Using successfully primary cultured keloid-derived fibroblasts from central region of chronic keloid tissues (sample 0), small interfering RNAs were designed and transfected into two keloid fibroblast samples (samples 1 and 2) to knockdown HOXA11-AS. One nonspecific transfection control (sample 3) and one blank control (sample 4) were used to remove nonspecific overlap from the studied group. The lncRNAs, messenger RNAs (mRNAs), and microRNAs (miRNAs) of five samples were sequenced to identify differentially expressed (DE) profiles in HOXA11-AS-knockdown keloid fibroblasts in samples 1 and 2 (by intersection), which facilitated removal of overlap with the nonspecific controls (samples 3 and 4, by union). Using stepwise bioinformatic analysis, a HOXA11-AS-interacted competing endogenous network (ceRNA) was screened based on three DE profiles. Results: Keloid fibroblasts with or without HOXA11-AS as well as with or without nonspecific interferences were successfully constructed respectively. A total of 1,396 mRNAs and 39 lncRNAs were significantly changed in keloid fibroblast with HOXA11-AS knockdown. Simultaneously, 1,626 mRNAs and 99 lncRNAs were significantly changed in keloid fibroblast with nonspecific interference. With removal of nonspecific overlap, a lncRNA–mRNA interactive network characterized by close natural/intronic antisense relationship was initially constructed in keloid fibroblast with HOXA11-AS knockdown. Based on this network, a lncRNA–mRNA–protein interaction network was extended by integration of the human protein–protein interaction network. Significant functional genes were screened using PageRank algorithm in the extended network. Three genes, including SNED1, NIPAL3, and VTN, were validated by real-time PCR in HOXA11-AS-knockdown keloid fibroblasts. Only NIPAL3 was predicted to be a target gene for HOXA11-AS via three competing endogenous miRNAs (hsa-miRNA-19a-3p, hsa-miR-141-3p, and hsa-miR-140-5p). Conclusion: An interactive network of HOXA11-AS–three miRNAs–NIPAL3 was predicted in keloid fibroblasts by integrative bioinformatic analysis and in vitro validation.

MicroRNA as a Potential Therapeutic Molecule in Cancer.

Small noncoding RNAs, as post-translational regulators of many target genes, are not only markers of neoplastic disease initiation and progression, but also markers of response to anticancer therapy. Hundreds of miRNAs have been identified as biomarkers of drug resistance, and many have demonstrated the potential to sensitize cancer cells to therapy. Their properties of modulating the response of cells to therapy have made them a promising target for overcoming drug resistance. Several methods have been developed for the delivery of miRNAs to cancer cells, including introducing synthetic miRNA mimics, DNA plasmids containing miRNAs, and small molecules that epigenetically alter endogenous miRNA expression. The results of studies in animal models and preclinical studies for solid cancers and hematological malignancies have confirmed the effectiveness of treatment protocols using microRNA. Nevertheless, the use of miRNAs in anticancer therapy is not without limitations, including the development of a stable nanoconstruct, delivery method choices, and biodistribution. The aim of this review was to summarize the role of miRNAs in cancer treatment and to present new therapeutic concepts for these molecules. Supporting anticancer therapy with microRNA molecules has been verified in numerous clinical trials, which shows great potential in the treatment of cancer.

A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals

A Cell-Anchored and Self-Calibrated DNA Nanoplatform for in Situ Imaging and Quantification of Endogenous MicroRNA in Live Cells: Introducing Two Controls to Normalize the Sensing Signals

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Arabidopsis RBV is a conserved WD40 repeat protein that promotes microRNA biogenesis and ARGONAUTE1 loading

MicroRNAs (miRNAs) play crucial roles in gene expression regulation through RNA cleavage or translation repression. Here, we report the identification of an evolutionarily conserved WD40 domain protein as a player in miRNA biogenesis in Arabidopsis thaliana. A mutation in the REDUCTION IN BLEACHED VEIN AREA (RBV) gene encoding a WD40 domain protein led to the suppression of leaf bleaching caused by an artificial miRNA; the mutation also led to a global reduction in the accumulation of endogenous miRNAs. The nuclear protein RBV promotes the transcription of MIR genes into pri-miRNAs by enhancing the occupancy of RNA polymerase II (Pol II) at MIR gene promoters. RBV also promotes the loading of miRNAs into AGO1. In addition, RNA-seq revealed a global splicing defect in the mutant. Thus, this evolutionarily conserved, nuclear WD40 domain protein acts in miRNA biogenesis and RNA splicing.

Nucleic Acid-Based COVID-19 Therapy Targeting Cytokine Storms: Strategies to Quell the Storm.

Coronavirus disease 2019 (COVID-19) has shaken the world and triggered drastic changes in our lifestyle to control it. Despite the non-typical efforts, COVID-19 still thrives and plagues humanity worldwide. The unparalleled degree of infection has been met with an exceptional degree of research to counteract it. Many drugs and therapeutic technologies have been repurposed and discovered, but no groundbreaking antiviral agent has been introduced yet to eradicate COVID-19 and restore normalcy. As lethality is directly correlated with the severity of disease, hospitalized severe cases are of the greatest importance to reduce, especially the cytokine storm phenomenon. This severe inflammatory phenomenon characterized by elevated levels of inflammatory mediators can be targeted to relieve symptoms and save the infected patients. One of the promising therapeutic strategies to combat COVID-19 is nucleic acid-based therapeutic approaches, including microRNAs (miRNAs). This work is an up-to-date review aimed to comprehensively discuss the current nucleic acid-based therapeutics against COVID-19 and their mechanisms of action, taking into consideration the emerging SARS-CoV-2 variants of concern, as well as providing potential future directions. miRNAs can be used to run interference with the expression of viral proteins, while endogenous miRNAs can be targeted as well, offering a versatile platform to control SARS-CoV-2 infection. By targeting these miRNAs, the COVID-19-induced cytokine storm can be suppressed. Therefore, nucleic acid-based therapeutics (miRNAs included) have a latent ability to break the COVID-19 infection in general and quell the cytokine storm in particular.

Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine-rich preference for virus- and non-virus-based expression vectors in plants.

SummaryArtificial microRNA (amiRNA) technology has allowed researchers to direct efficient silencing of specific transcripts using as few as 21 nucleotides (nt). However, not all the artificially designed amiRNA constructs result in selection of the intended ~21‐nt guide strand amiRNA. Selection of the miRNA guide strand from the mature miRNA duplex has been studied in detail in human and insect systems, but not so much for plants. Here, we compared a nuclear‐replicating DNA viral vector (tomato mottle virus, ToMoV, based), a cytoplasmic‐replicating RNA viral vector (tobacco mosaic virus, TMV, based), and a non‐viral binary vector to express amiRNAs in plants. We then used deep sequencing and mutational analysis and show that when the structural factors caused by base mismatches in the mature amiRNA duplex were excluded, the nucleotide composition of the mature amiRNA region determined the guide strand selection. We found that the strand with excess purines was preferentially selected as the guide strand and the artificial miRNAs that had no mismatches in the amiRNA duplex were predominantly loaded into AGO2 instead of loading into AGO1 like the majority of the plant endogenous miRNAs. By performing assays for target effects, we also showed that only when the intended strand was selected as the guide strand and showed AGO loading, the amiRNA could provide the expected RNAi effects. Thus, by removing mismatches in the mature amiRNA duplex and designing the intended guide strand to contain excess purines provide better control of the guide strand selection of amiRNAs for functional RNAi effects.

Early Brain microRNA/mRNA Expression is Region-Specific After Neonatal Hypoxic-Ischemic Injury in a Mouse Model.

Background: MicroRNAs (miRNAs) may be promising therapeutic targets for neonatal hypoxic-ischemic brain injury (HIBI) but targeting miRNA-based therapy will require more precise understanding of endogenous brain miRNA expression. Methods: Postnatal day 9 mouse pups underwent HIBI by unilateral carotid ligation + hypoxia or sham surgery. Next-generation miRNA sequencing and mRNA Neuroinflammation panels were performed on ipsilateral cortex, striatum/thalamus, and cerebellum of each group at 30 min after injury. Targeted canonical pathways were predicted by KEGG analysis. Results: Sixty-one unique miRNAs showed differential expression (DE) in at least one region; nine in more than one region, including miR-410-5p, -1264-3p, 1298-5p, -5,126, and -34b-3p. Forty-four mRNAs showed DE in at least one region; 16 in more than one region. MiRNAs showing DE primarily targeted metabolic pathways, while mRNAs targeted inflammatory and cell death pathways. Minimal miRNA-mRNA interactions were seen at 30 min after HIBI. Conclusion: This study identified miRNAs that deserve future study to assess their potential as therapeutic targets in neonatal HIBI. Additionally, the differences in miRNA expression between regions suggest that future studies assessing brain miRNA expression to guide therapy development should consider evaluating individual brain regions rather than whole brain to ensure the sensitivity needed for the development of targeted therapies.

Poly-adenine-mediated spherical nucleic acid probes for live cell fluorescence imaging of tumor-related microRNAs.

Accurately detecting and quantifying tumor-related microRNAs (miRNAs) in living cells is of great value for early cancer diagnosis. Herein, we present poly-adenine (polyA)-mediated spherical nucleic acid (SNA) nanoprobes for intracellular miRNA imaging in living cells. polyA-mediated spherical nucleic acid (pASNA) nanoprobes consist of gold nanoparticles (AuNPs) anchored with fluorophore-labeled DNA molecules pre-hybridized with recognition sequences and polyA tails. The detection performance for miRNAs in vitro was studied to confirm the feasibility of pASNA nanoprobes for imaging live cell miRNAs. Before the pASNA nanoprobes were used for imaging intracellular miRNAs in MCF-7, HeLa, and LO2 cells, the stability and non-cytotoxicity were investigated using Dnase I and a standard colorimetric CCK8 assay. Flow cytometry, qRT-PCR analyses were conducted to confirm the different expression levels of miR-155 in live cells. Results showed that the pASNA nanoprobes had good detection sensitivity and specificity, excellent stability, and low toxicity. After incubating with pASNA nanoprobes, noticeable fluorescence signal enhancement could be clearly observed in MCF-7 and HeLa cells but not LO2 cells by confocal microscopy. Flow cytometry analysis and qRT-PCR indicated that MCF-7 and HeLa cells had higher miR-155 expression levels compared to LO2 cells. The pASNA nanoprobes we developed had good sensitivity and specificity, excellent nuclease stability and low toxicity, thus representing a new approach to exquisitely reveal the distribution of endogenous miRNAs in live cells.

Targeting of PFKFB3 with miR-206 but not mir-26b inhibits ovarian cancer cell proliferation and migration involving FAK downregulation.

Few studies explored the role of microRNAs (miRNAs) in the post-transcriptional regulation of glycolytic proteins and downstream effectors in ovarian cancer cells. We recently showed that the functional activation of the cytoskeletal regulator FAK in endothelial cells is fostered by the glycolytic enhancer 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). We tested the hypothesis that miR-206 and mir-26b, emerging onco-suppressors targeting PFKFB3 in estrogen-dependent tumors, would regulate proliferation and migration of serous epithelial ovarian cancer (EOC) cells via common glycolytic proteins, i.e., GLUT1 and PFKFB3, and downstream FAK. PFKFB3 was overexpressed in SKOV3, and its pharmacological inhibition with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) significantly reduced cell proliferation and motility. Both miR-206 and miR-26b directly targeted PFKFB3 as evaluated by a luciferase reporter assay. However, endogenous levels of miR-26b were higher than those of miR-206, which was barely detectable in SKOV3 as well as OVCAR5 and CAOV3 cells. Accordingly, only the anti-miR-26b inhibitor concentration-dependently increased PFKFB3levels. While miR-206 overexpression impaired proliferation and migration by downregulating PFKFB3levels, the decreased PFKFB3 protein levels related to miR-26 overexpression had no functional consequences in all EOC cell lines. Finally, consistent with the migration outcome, exogenous miR-206 and miR-26b induced opposite effects on the levels of total FAK and of its phosphorylated form at Tyr576/577. 3PO did not prevent miR-26b-induced SKOV3migration. Overall, these results support the inverse relation between endogenous miRNA levels and their tumor-suppressive effects and suggest that restoring miR-206 expression represents a potential dual anti-PFKFB3/FAK strategy to control ovarian cancer progression.

Identification of Circular RNA-Based Immunomodulatory Networks in Colorectal Cancer.

BackgroundCircular RNAs (circRNAs) have been recently proposed as hub molecules in various diseases, especially in tumours. We found that circRNAs derived from ribonuclease P RNA component H1 (RPPH1) were highly expressed in colorectal cancer (CRC) samples from Gene Expression Omnibus (GEO) datasets.ObjectiveWe sought to identify new circRNAs derived from RPPH1 and investigate their regulation of the competing endogenous RNA (ceRNA) and RNA binding protein (RBP) networks of CRC immune infiltration.MethodsThe circRNA expression profiles miRNA and mRNA data were extracted from the GEO and The Cancer Genome Atlas (TCGA) datasets, respectively. The differentially expressed (DE) RNAs were identified using R software and online server tools, and the circRNA–miRNA–mRNA and circRNA–protein networks were constructed using Cytoscape. The relationship between targeted genes and immune infiltration was identified using the GEPIA2 and TIMER2 online server tools.ResultsA ceRNA network, including eight circRNAs, five miRNAs, and six mRNAs, was revealed. Moreover, a circRNA–protein network, including eight circRNAs and 49 proteins, was established. The targeted genes, ENOX1, NCAM1, SAMD4A, and ZC3H10, are closely related to CRC tumour-infiltrating macrophages.ConclusionsWe analysed the characteristics of circRNA from RPPH1 as competing for endogenous RNA binding miRNA or protein in CRC macrophage infiltration. The results point towards the development of a new diagnostic and therapeutic paradigm for CRC.

Viral and Host Genetic and Epigenetic Biomarkers Related to SARS-CoV-2 Cell Entry, Infection Rate, and Disease Severity

Simple SummaryCOVID-19 emerged as a new disease with quick transmission and a high mortality rate at the end of 2019, caused by SARS-CoV-2. Common features of the coronavirus family helped resolve structural and entry mechanism characteristics of SARS-CoV-2. Still, rapid mutagenesis leads to the fast evolution of the virus and the emergence of new strains that differ in infectivity, morbidity, and mortality. Besides differences in the viral genome, genetic variability in the host defense and immune systems may also play a role in the outcome of virus–host interactions. Furthermore, epigenetic mechanisms may also influence the outcomes, including miRNA gene silencing and DNA methylation, which may be heavily influenced by SARS-CoV-2. Molecular biomarkers are intensively investigated as potential predictive and prognostic biomarkers of the disease course and treatment response. We reviewed new data regarding the mechanisms behind fast virus mutagenesis, infectivity, and potential human genetic and epigenetic characteristics that may lead to a more severe or lethal outcome of the disease.The rapid spread of COVID-19 outbreak lead to a global pandemic declared in March 2020. The common features of corona virus family helped to resolve structural characteristics and entry mechanism of SARS-CoV-2. However, rapid mutagenesis leads to the emergence of new strains that may have different reproduction rates or infectivity and may impact the course and severity of the disease. Host related factors may also play a role in the susceptibility for infection as well as the severity and outcomes of the COVID-19. We have performed a literature and database search to summarize potential viral and host-related genomic and epigenomic biomarkers, such as genetic variability, miRNA, and DNA methylation in the molecular pathway of SARS-CoV-2 entry into the host cell, that may be related to COVID-19 susceptibility and severity. Bioinformatics tools may help to predict the effect of mutations in the spike protein on the binding to the ACE2 receptor and the infectivity of the strain. SARS-CoV-2 may also target several transcription factors and tumour suppressor genes, thus influencing the expression of different host genes and affecting cell signalling. In addition, the virus may interfere with RNA expression in host cells by exploiting endogenous miRNA and its viral RNA. Our analysis showed that numerous human miRNA may form duplexes with different coding and non-coding regions of viral RNA. Polymorphisms in human genes responsible for viral entry and replication, as well as in molecular damage response and inflammatory pathways may also contribute to disease prognosis and outcome. Gene ontology analysis shows that proteins encoded by such polymorphic genes are highly interconnected in regulation of defense response. Thus, virus and host related genetic and epigenetic biomarkers may help to predict the course of the disease and the response to treatment.

Whole Blood Holding Time Prior to Plasma Processing Alters microRNA Expression Profile.

MicroRNAs (miRNAs) can exhibit aberrant expression under different physiological and pathological conditions. Therefore, differentially expressed circulating miRNAs have been a focus of biomarker discovery research. However, the use of circulating miRNAs comes with challenges which may hinder the reliability for their clinical application. These include varied sample collection protocols, storage times/conditions, sample processing and analysis methods. This study focused on examining the effect of whole blood holding time on the stability of plasma miRNA expression profiles. Whole blood samples were collected from healthy pregnant women and were held at 4°C for 30 min, 2 h, 6 h or 24 h prior to processing for plasma isolation. Plasma RNA was extracted and the expression of 179 miRNAs were analyzed. Unsupervised principal component analysis demonstrated that whole blood holding time was a major source of variation in miRNA expression profiles with 53 of 179 miRNAs showing significant changes in expression. Levels of specific miRNAs previously reported to be associated with pregnancy-associated complications such as hsa-miR-150-5p, hsa-miR-191-5p, and hsa-miR-29a-3p, as well as commonly used endogenous miRNA controls, hsa-miR-16-5p, hsa-miR-25-3p, and hsa-miR-223-3p were significantly altered with increase in blood holding time. Current protocols for plasma-based miRNA profiling for diagnostics describe major differences in whole blood holding periods ranging from immediately after collection to 26 h after. Our results demonstrate holding time can have dramatic effects on analytical reliability and reproducibility. This highlights the importance of standardization of blood holding time prior to processing for plasma in order to minimize introduction of non-biological variance in miRNA profiles.