Analysis Of miRNA Expression Profiles Research Articles

Vitamin D boosts immune response of macrophages through a regulatory network of microRNAs and mRNAs

Vitamin D is associated with the stimulation of innate immunity, inflammation, and host defense against pathogens. Macrophages express receptors of Vitamin D, regulating the transcription of genes related to immune processes. However, the transcriptional and post-transcriptional strategies controlling gene expression in differentiated macrophages, and how they are influenced by Vitamin D are not well understood. We studied whether Vitamin D enhances immune response by regulating the expression of microRNAs and mRNAs. Analysis of the transcriptome showed differences in expression of 199 genes, of which 68% were up-regulated, revealing the cell state of monocyte-derived macrophages differentiated with Vitamin D (D3-MDMs) as compared to monocyte-derived macrophages (MDMs). The differentially expressed genes appear to be associated with pathophysiological processes, including inflammatory responses, and cellular stress. Transcriptional motifs in promoter regions of up- or down-regulated genes showed enrichment of VDR motifs, suggesting possible roles of transcriptional activator or repressor in gene expression. Further, the microRNA-Seq analysis indicated that there were 17 differentially expressed miRNAs, of which, seven were up-regulated and 10 down-regulated, suggesting that Vitamin D plays a critical role in the regulation of miRNA expression during macrophages differentiation. The miR-6501-3p, miR-1273h-5p, miR-665, miR-1972, miR-1183, miR-619-5p were down-regulated in D3-MDMs compared to MDMs. The integrative analysis of miRNA and mRNA expression profiles predicts that miR-1972, miR-1273h-5p, and miR-665 regulate genes PDCD1LG2, IL-1B, and CD274, which are related to the inflammatory response. Results suggest an essential role of Vitamin D in macrophage differentiation that modulates host response against pathogens, inflammation, and cellular stress.

Analysis of miRNA Expression Profiling of RIP2 Knockdown in Chicken HD11 Cells When Infected with Avian Pathogenic E. coli (APEC).

Colibacillosis is an acute and chronic avian disease caused by avian pathogenic E. coli (APEC). Previous studies have demonstrated that RIP2 plays a significant role in APEC infection. Moreover, increasing evidence indicates that microRNAs (miRNAs) are involved in host–pathogen interactions and the immune response. However, the role of miRNAs in the host against APEC infection remains unclear. Herein, we attempted to reveal new miRNAs potentially involved in the regulation of the immune and inflammatory response against APEC infection, with a particular focus on those possibly correlated with RIP2 expression, via miRNA-seq, RT-qPCR, Western blotting, dual-luciferase reporter assay, and CCK-8. The results showed that a total of 93 and 148 differentially expressed (DE) miRNAs were identified in the knockdown of RIP2 cells following APEC infection (shRIP2+APEC) vs. knockdown of RIP2 cells (shRIP2) and shRIP2 vs. wild-type cells (WT), respectively. Among those identified DE miRNAs, the biological function of gga-miR-455-5p was investigated. It was found that gga-miR-455-5p regulated by RIP2 was involved in the immune and inflammatory response against APEC infection via targeting of IRF2 to modulate the expression of type I interferons. Additionally, RIP2 could directly regulate the production of the type I interferons. Altogether, these findings highlighted the crucial role of miRNAs, especially gga-miR-455-5p, in host defense against APEC infection.

GATA1 regulates the microRNA‑328‑3p/PIM1axis via circular RNA ITGB1 to promote renal ischemia/reperfusion injury in HK‑2 cells.

Acute kidney injury (AKI) is caused by renal ischemia/reperfusion injury (IRI) during kidney transplantation. The levels of both circular RNAs (circRNAs) and microRNAs (miRNAs/miR) appear to be critical for AKI detection. While several RNA interactions in AKI have been found, the regulatory mechanisms between the molecules remain to be fully elucidated. In the present study, miRNA expression profiling analysis was conducted using an online dataset to identify the differentially expressed miRNAs in rats with IRI. miR-328-3p was also found to be downregulated in human kidney-2 (HK-2) cells subjected to hypoxia/reperfusion (H/R), and its overexpression targeting pim-1 proto-oncogene (PIM1) resulted in an increased viability and a reduced apoptosis, as well as in the decreased expression of inflammatory factors upon H/R exposure. Putative targets and circRNAs of miR-328-3p were identified using publically available databases. The inhibition of circRNA integrin beta 1 (ITGB1; circITGB1) suppressed the inflammatory response induced by H/R by sponging miR-328-3p in HK-2 cells. Furthermore, a sequence of the functional ITGB1 promoter was studied for transcription factor GATA binding protein 1 (GATA1) binding sites. GATA1 binds to the ITGB1 promoter, leading to the expression of circITGB1. On the whole, the findings of the present study revealed a regulatory pathway modulating miR-328-3p in IRI, demonstrating that the GATA1-mediated regulation of circITGB1 enhanced the H/R-induced inflammatory response via the miR-328-3p/PIM1 axis.

Integrative Analysis of Expression Profiles of mRNA and MicroRNA Provides Insights of Cotton Response to Verticillium dahliae.

Cotton Verticillium wilt, caused by the notorious fungal phytopathogen Verticillium dahliae (V. dahliae), is a destructive soil-borne vascular disease and severely decreases cotton yield and quality worldwide. Transcriptional and post-transcriptional regulation of genes responsive to V. dahliae are crucial for V. dahliae tolerance in plants. However, the specific microRNAs (miRNAs) and the miRNA/target gene crosstalk involved in cotton resistance to Verticillium wilt remain largely limited. To investigate the roles of regulatory RNAs under V. dahliae induction in upland cotton, mRNA and small RNA libraries were constructed from mocked and infected roots of two upland cotton cultivars with the V. dahliae-sensitive cultivar Jimian 11 (J11) and the V. dahliae-tolerant cultivar Zhongzhimian 2 (Z2). A comparative transcriptome analysis revealed 8330 transcripts were differentially expressed under V. dahliae stress and associated with several specific biological processes. Moreover, small RNA sequencing identified a total of 383 miRNAs, including 330 unique conserved miRNAs and 53 novel miRNAs. Analysis of the regulatory network involved in the response to V. dahliae stress revealed 31 differentially expressed miRNA–mRNA pairs, and the up-regulation of GhmiR395 and down-regulation of GhmiR165 were possibly involved in the response to V. dahliae by regulating sulfur assimilation through the GhmiR395-APS1/3 module and the establishment of the vascular pattern and secondary cell wall formation through GhmiR165-REV module, respectively. The integrative analysis of mRNA and miRNA expression profiles from upland cotton lays the foundation for further investigation of regulatory mechanisms of resistance to Verticillium wilt in cotton and other crops.

Comparative Analysis of miRNA Expression Profiles in Skeletal Muscle of Bian Chickens at Different Embryonic Ages.

Simple SummaryThe development of skeletal muscle during the embryonic period has an important impact on the meat production of poultry, and miRNAs are vital regulators of skeletal muscle development. In this study, through the RNA-seq analysis of leg muscles from 14- and 20-day Bian chicken embryos, we screened out 127 and 131 differentially expressed miRNAs (DEMs) in a fast-growing group (F14 vs. F20) and a slow-growing group (S14 vs. S20), respectively. Based on functional enrichment analysis and interaction network analysis, we further screened out 13 miRNAs and 5 hub genes, which potentially play important roles in skeletal muscle development. This study provides data support for future studies on the regulatory mechanisms of miRNAs in the skeletal muscle development of Bian chickens.MicroRNAs (miRNAs) are widely involved in the growth and development of skeletal muscle through the negative regulation of target genes. In order to screen out the differentially expressed miRNAs (DEMs) associated with skeletal muscle development of Bian chickens at different embryonic ages, we used the leg muscles of fast-growing and slow-growing Bian chickens at the 14th and 20th embryonic ages (F14, F20, S14 and S20) for RNA-seq. A total of 836 known miRNAs were identified, and 121 novel miRNAs were predicted. In the F14 vs. F20 comparison group, 127 DEMs were screened, targeting a total of 2871 genes, with 61 miRNAs significantly upregulated and 66 miRNAs significantly downregulated. In the S14 vs. S20 comparison group, 131 DEMs were screened, targeting a total of 3236 genes, with 60 miRNAs significantly upregulated and 71 miRNAs significantly downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the predicted target genes were significantly enriched in 706 GO terms and 6 KEGG pathways in the F14 vs. F20 group and 677 GO terms and 5 KEGG pathways in the S14 vs. S20 group. According to the interaction network analysis, we screened five coexpressed DEMs (gga-miR-146a-3p, gga-miR-2954, gga-miR-34a-5p, gga-miR-1625-5p and gga-miR-18b-3p) with the highest connectivity degree with predicted target genes between the two comparison groups, and five hub genes (HSPA5, PKM2, Notch1, Notch2 and RBPJ) related to muscle development were obtained as well. Subsequently, we further identified nine DEMs (gga-let-7g-3p, gga-miR-490-3p, gga-miR-6660-3p, gga-miR-12223-5p, novel-miR-327, gga-miR-18a-5p, gga-miR-18b-5p, gga-miR-34a-5p and gga-miR-1677-3p) with a targeting relationship to the hub genes, suggesting that they may play important roles in the muscle development of Bian chickens. This study reveals the miRNA differences in skeletal muscle development between 14- and 20-day embryos of Bian chickens from fast- and slow-growing groups and provides a miRNA database for further studies on the molecular mechanisms of the skeletal muscle development in Bian chickens.

Integrated analysis of expression profiles with meat quality traits in cattle

MicroRNAs (miRNAs) play a vital role in improving meat quality by binding to messenger RNAs (mRNAs). We performed an integrated analysis of miRNA and mRNA expression profiling between bulls and steers based on the differences in meat quality traits. Fat and fatty acids are the major phenotypic indices of meat quality traits to estimate between-group variance. In the present study, 90 differentially expressed mRNAs (DEGs) and 18 differentially expressed miRNAs (DEMs) were identified. Eighty-three potential DEG targets and 18 DEMs were used to structure a negative interaction network, and 75 matching target genes were shown in this network. Twenty-six target genes were designated as intersection genes, screened from 18 DEMs, and overlapped with the DEGs. Seventeen of these genes enriched to 19 terms involved in lipid metabolism. Subsequently, 13 DEGs and nine DEMs were validated using quantitative real-time PCR, and seven critical genes were selected to explore the influence of fat and fatty acids through hub genes and predict functional association. A dual-luciferase reporter and Western blot assays confirmed a predicted miRNA target (bta-miR-409a and PLIN5). These findings provide substantial evidence for molecular genetic controls and interaction among genes in cattle.

Suppression of bone remodeling associated with long-term bisphosphonate treatment is mediated by microRNA-30a-5p

ABSTRACT Oral bisphosphonates (BPs) are a first-line treatment for osteoporosis. It is becoming a hot topic to identify new indicators for the early prediction of therapeutic effects and adverse reactions during the long-term use of BPs. To determine whether microRNA (miRNA) expression is modulated by long-term BPs treatment, we performed miRNA expression profiling analysis in patients receiving long-term BP treatment for postmenopausal OP. To assess the effect of BPs on miRNA expression, we used an Affymetrix Genechip miRNA array to analyze serum samples obtained from postmenopausal OP patients on long-term BP treatment and healthy controls. MiRNAs affected by BPs and their predicted targets were examined. We also investigated the effects of miRNA on osteoblast differentiation in vitro and on ovariectomy-induced bone loss in vivo. We observed that the level of miR-30a-5p was significantly increased in patients receiving long-term BP treatment for postmenopausal OP. Furthermore, miR-30a-5p was negatively correlated with bone formation. Consistent with this, in vitro osteoblast activity and matrix mineralization were increased by an antagomir of miR-30a-5p and decreased by an agomir of miR-30a-5p. We also found that miR-30a-5p directly targeted RUNX1 to inhibit osteoblastic differentiation. Consistent with the in vitro results, miR-30a-5p antagomir administration promoted bone formation in ovariectomized mice. Our findings identified miR-30a-5p as a novel mediator of long-term BP treatment that regulates bone formation in postmenopausal OP patients.

Whole-transcriptome sequencing analysis of placental differential miRNA expression profile in Down syndrome

To identify new biomarkers and molecular pathogenesis of Down syndrome (DS) by analyzing differentially expressed miRNAs in the placentas and their biological pathways. Whole transcriptome sequencing was used to identify the differentially expressed miRNAs in DS (n=3) and normal placental samples (n=3) diagnosed by prenatal diagnosis. The target genes were predicted using miRWalk, Targetscan and miRDB, and GO and KEGG pathway analyses were performed for gene enrichment studies. We identified a total of 82 differentially expressed miRNAs in the placental tissues of DS, including 29 up-regulated miRNAs (fold change ≥2, P < 0.05) and 15 down-regulated miRNAs (fold change ≥2, P < 0.05), among which 10 miRNAs with relatively high expression abundance were selected for further analysis, including 4 up-regulated and 6 down-regulated miRNAs. These selected miRNAs shared the common target genes BTBD3 and AUTS2, both of which were associated with neurodevelopment. GO analysis showed that the target genes of the selected miRNAs were mainly enriched in protein binding, hydrolytic enzymes, metal ion binding protein combining, transferase activity, nucleotide, cytoplasmic constituents, nucleus composition, transcriptional regulation, RNA metabolism regulation, DNA-dependent RNA polymerase Ⅱ promoter transcriptional regulation, eye development, and sensory organ development. KEGG enrichment analysis showed that the target genes of these differentially expressed miRNAs were involved in the signaling pathways including tumor-related signaling pathway, PI3K-Akt signaling pathway, Ras signaling pathway, Rap1 signaling pathway, cytoskeletal regulatory signaling pathway, purine metabolization-related signaling pathway and P53 signaling pathway. The differentially expressed miRNAs may play important roles in placental damage and pregnancy pathology in DS and provide clues for the prevention and treatment of mental retardation-related diseases.

Integrated Analysis of miRNA and mRNA Expression Profiles Reveals the Molecular Mechanism of Posttraumatic Stress Disorder and Therapeutic Drugs

PurposePost-traumatic stress disorder (PTSD) is a result of trauma exposure and is related to psychological suffering as a long-lasting health issue. Further analysis of the networks and genes involved in PTSD are critical to the molecular mechanisms of PTSD.MethodsIn this study, we aimed to identify key genes and molecular interaction networks involved in the pathogenesis of PTSD by integrating mRNA and miRNA data.ResultsBy integrating three high-throughput datasets, 5606 differentially expressed genes (DEGs) were detected, including five differentially expressed miRNAs (DEmiRNAs) and 5525 differentially expressed mRNAs (DEmRNAs). Nineteen upregulated and 46 downregulated DEmRNAs were identified in both GSE64813 and GSE89866 datasets, while five upregulated DEmiRNAs were found in the GSE87768 dataset. Functional annotations of these DEmRNAs indicated that they were mainly enriched in blood coagulation, cell adhesion, platelet activation, and extracellular matrix (ECM)-receptor interaction. Integrated protein-protein and miRNA-protein interaction networks among the DEGs were established with the help of 65 nodes and 121 interactions. Finally, 286 small molecules were obtained based on the Drug-Gene Interaction database (DGIdb). Three genes, prostaglandin-endoperoxide synthase 1 (PTGS1), beta-tubulin gene (TUBB1), and cyclin-dependent kinase inhibitor 1A (CDKN1A), were the most promising targets for PTSD therapy. Additionally, the present study also provided a higher performance diagnostic model for PTSD based on 17 DEmRNAs, which was validated in two independent datasets, GSE109409 and GSE63878.ConclusionOur data provides a new molecular aspect that ECM-receptor interaction and the platelet activation process could be the potential molecular mechanism of PTSD, and the genes involved in this process may be promising therapeutic targets. A higher-performance diagnostic model for PTSD has also been identified.

Insights into the identification of a molecular signature for amyotrophic lateral sclerosis exploiting integrated microRNA profiling of iPSC-derived motor neurons and exosomes

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by progressive degeneration of motor neurons (MNs). Most cases are sporadic, whereas 10% are familial. The pathological mechanisms underlying the disease are partially understood, but it is increasingly being recognized that alterations in RNA metabolism and deregulation of microRNA (miRNA) expression occur in ALS. In this study, we performed miRNA expression profile analysis of iPSC-derived MNs and related exosomes from familial patients and healthy subjects. We identified dysregulation of miR-34a, miR-335 and miR-625-3p expression in both MNs and exosomes. These miRNAs regulate genes and pathways which correlate with disease pathogenesis, suggesting that studying miRNAs deregulation can contribute to deeply investigate the molecular mechanisms underlying the disease. We also assayed the expression profile of these miRNAs in the cerebrospinal fluid (CSF) of familial (fALS) and sporadic patients (sALS) and we identified a significant dysregulation of miR-34a-3p and miR-625-3p levels in ALS compared to controls. Taken together, all these findings suggest that miRNA analysis simultaneously performed in different human biological samples could represent a promising molecular tool to understand the etiopathogenesis of ALS and to develop new potential miRNA-based strategies in this new propitious therapeutic era.

Downregulation of Three Novel miRNAs in the Lymph Nodes of Sheep Immunized With the Brucella suis Strain 2 Vaccine.

Ovine and caprine brucellosis, both caused by Brucella melitensis, lead to substantial economic losses in the animal industry and health problems in human populations. Brucella suis strain 2 (B.suis S2), as a live attenuated vaccine, is used extensively in China to prevent brucellosis. It has been proven that microRNA (miRNAs) are involved in the immunopathogenesis of brucellosis; however, the miRNA-driven mechanism of immune response to B.suis S2 in vivo remains unknown. To determine which new miRNAs are involved in the host immune response to B.suis S2 and elucidate the function of these miRNAs, we performed a comprehensive analysis of miRNA expression profiles in sheep immunized with B.suis S2 using the high-throughput sequencing approach. The submandibular lymphatic nodes from sheep seropositive for Brucella were collected at 7, 14, 21, 30, 60 and 90 days post-immunization. MiRNA sequencing analysis revealed that 282 differentially expressed miRNAs (|log2 fold-change |>0.5 and p < 0.05) were significantly enriched in the immune pathways, including the NF-kappa B signaling pathway, B cell receptor signaling pathway, p53 signaling pathway and complement and coagulation cascades. Increasing the threshold to |log2 fold change|>1 and p < 0.01 revealed 48 differentially expressed miRNAs, 31 of which were novel miRNAs. Thirteen of these novel miRNAs, which were differentially expressed for at least two time points, were detected via RT-qPCR assays. The novel_229, novel_609, novel_973 and oar-miR-181a assessed by RT-qPCR were detectable and consistent with the expression patterns obtained by miRNA sequencing. Functional analyses of these miRNAs demonstrated that their target genes participated in the immune response pathways, including the innate and adaptive immunity pathways. The immune-related target genes of novel_229 included ENSOARG00000000649 and TMED1, as well as LCN2, PDPK1 and LPO were novel_609 target genes. The immune-related target genes of novel_973 included C6orf58, SPPL3, BPIFB1, ENSOARG00000021083, MPTX1, CCL28, FGB, IDO1, OLR1 and ENSOARG00000020393. The immune-related target genes of oar-miR-181a included ENSOARG00000002722, ARHGEF2, MFAP4 and DOK2. These results will deepen our understanding of the host miRNA-driven defense mechanism in sheep immunized with B.suis S2 vaccine, and provide the valuable information for optimizing vaccines and developing molecular diagnostic targets.

HIPK3 Inhibition by Exosomal hsa-miR-101-3p Is Related to Metabolic Reprogramming in Colorectal Cancer.

BackgroundExosomes are extracellular vesicles secreted by most cells to deliver functional cargoes to recipient cells. MicroRNAs (miRNAs) constitute a significant part of exosomal contents. The ease of diffusion of exosomes renders them speedy and highly efficient vehicles to deliver functional molecules. Cancer cells secrete more exosomes than normal cells. Reports have showed that exosomal miRNAs of cancer cells facilitate cancer progression. Yet the complexity of cancer dictates that many more functional exosomal miRNAs remain to be discovered.MethodsIn this study, we analyzed miRNA expression profiles of tissue and plasma exosome samples collected from 10 colorectal cancer (CRC) patients and 10 healthy individuals. We focused on hsa-miR-101-3p (101-3p), a profoundly up-regulated miRNA enriched in plasma exosomes of patients bearing CRC. We performed target analysis of 101-3p and pursued functional studies of this microRNA in two colorectal cancer cell lines, namely HCT116 and SW480.ResultsOur results indicated that inhibiting 101-3p slowed cell growth and retarded cell migration in vivo in two colorectal cancer cell lines. Target analysis showed that Homeodomain-interacting protein kinase (HIPK3) is a target of miR-101-3p. HCT116 and SW480 cells stably overexpressing HIPK3 showed increased level of phosphorylated FADD, as well as retarded cell growth, migration, and increased sensitivity to 5-FU. In-depth analysis revealed increased mitochondrial membrane potential upon HIPK3 overexpression along with increased production of reactive oxygen species, number of mitochondria, and expression of respiratory complexes. Measurements of glycolytic parameters and enzymes revealed decreased level of glycolysis upon HIPK3 overexpression in these two cell lines. Xenograft model further confirmed a profoundly improved potency of the synergistic treatment combining both 5-FU and 101-3p inhibitor compared to 5-FU alone.ConclusionThis study unraveled an oncogenic nature of the exosomal 101-3p and suggested a relationship between the 101-3p-HIPK3 axis and metabolic homeostasis in colorectal cancer. Expression level of 101-3p is positively correlated with glycolytic capacity in CRC and therefore 101-3p itself is an oncomiR. Combining 101-3p inhibitor with chemotherapeutic agents is an effective strategy against CRC.

Preanalytical features of the determination of circulating microRNAs as new specific biomarkers of the body’s response to physical activity

MicroRNAs are small non-coding single-stranded RNAs, 18 to 25 nucleotides long, they regulate gene expression at the post-transcriptional level through specific binding to the target mRNA, leading to its degradation. In recent decades, the development of technologies for determining the expression profiles of miRNAs has become an important part of research projects, and the role of miRNAs as potential highly informative molecular biomarkers of various physiological and pathological processes in the body is actively explored by the scientific community. In particular, physical activity is an important modifying factor for circulating miRNAs. Unlike classical blood biochemical parameters, which can change over time depending on the temperature and storage conditions of the sample, microRNAs remain stable during storage and even after multiple freeze-thaw cycles, which makes them an attractive and easily accessible target for detection. However, the determination of the microRNA expression profile in clinical practice is still difficult due to the high heterogeneity of analytical procedures used for testing. In sports medicine, the preanalytical stage is especially important, since often the conditions for sampling are not standardized and can affect the analysis result. This review shows the role of miRNAs as new sensitive biomarkers of the effectiveness of the training process and regulators of the body’s response to physical activity, and also discusses some preanalytical aspects of the analysis of miRNA expression profiles.

Comprehensive Analysis of mRNA, lncRNA, circRNA, and miRNA Expression Profiles and Their ceRNA Networks in the Longissimus Dorsi Muscle of Cattle-Yak and Yak

Cattle-yak, as the hybrid offspring of cattle (Bos taurus) and yak (Bos grunniens), demonstrates obvious heterosis in production performance. Male hybrid sterility has been focused on for a long time; however, the mRNAs and non-coding RNAs related to muscle development as well as their regulatory networks remain unclear. The phenotypic data showed that the production performance (i.e., body weight, withers height, body length, and chest girth) of cattle-yak was significantly better than that of the yak, and the economic benefits of the cattle-yak were higher under the same feeding conditions. Then, we detected the expression profiles of the longissimus dorsi muscle of cattle-yak and yak to systematically reveal the molecular basis using the high-throughput sequencing technology. Here, 7,126 mRNAs, 791 lncRNAs, and 1,057 circRNAs were identified to be differentially expressed between cattle-yaks and yaks in the longissimus dorsi muscle. These mRNAs, lncRNA targeted genes, and circRNA host genes were significantly enriched in myoblast differentiation and some signaling pathways related to muscle development (such as HIF-1 signaling pathway and PI3K-Akt signaling pathway). We constructed a competing endogenous RNA (ceRNA) network and found that some non-coding RNAs differentially expressed may be involved in the regulation of muscle traits. Taken together, this study may be used as a reference tool to provide the molecular basis for studying muscle development.

Vitamin D Regulates the Expression of Immune and Stress Response Genes in Dengue Virus-infected Macrophages by Inducing Specific MicroRNAs.

The pathogenesis associated with Dengue virus (DENV) infection is marked by the impairment of host immune response. Consequently, the modulation of immune response has emerged as an important therapeutic target for the control of DENV infection. Vitamin D has been shown to regulate the immune response in DENV infection, although the molecular mechanism remains poorly understood. Post-transcriptional regulation of mRNA by miRNAs offers an opportunity to gain insight into the immunomodulation mediated by vitamin D. Previously, it has been observed that a high dose of vitamin D (4000 IU) decreased DENV-2 infection and inflammatory response in monocyte-derived macrophages (MDMs). Here, we examine whether high or low doses of vitamin D supplements exert differential effect on miRNA expression in DENV-infected macrophages. We analyzed miRNA expression profiles in MDMs isolated from healthy individuals who were given either 1000 or 4000 IU/day of vitamin D for 10 days. MDMs before or after vitamin D supplementation were challenged with DENV-2, and miRNAs profiles were analyzed by qPCR arrays. DENV-2 infected MDMs supplemented with 4000 IU, showed up-regulation of miR-374a-5p, miR-363-3p, miR-101-3p, miR-9-5p, miR-34a-5p, miR-200a-3p, and the family of miRNAs miR-21-5p, and miR-590-p. The miRNA profile and predicted target mRNAs suggested regulatory pathways in MDMs obtained from healthy donors who received higher doses of vitamin D. These DENV-2 infected MDMs expressed a unique set of miRNAs that target immune and cellular stress response genes. The results suggest vitamin D dose-dependent differential expression of miRNAs target key signaling pathways of the pathogenesis of dengue disease.

Non-coding RNAs and related molecules associated with form-deprivation myopia in mice.

The role of miRNAs and its regulatory mechanism in myopia are indeterminate. Our study aimed to investigate potential myopia‐associated non‐coding RNAs and related molecules by performing a comprehensive bioinformatic analysis of miRNA expression profile of mice with form‐deprivation myopia (FDM). Differentially expressed miRNAs in two raw microarray data sets (GSE58124 and GSE84220) from Gene Expression Omnibus (GEO) database were comprehensively analysed using GEO2R. Target genes were predicted using miRDB and enriched with Metascape online tool. Protein‐protein interaction (PPI) networks were constructed utilizing STRING and Cytoscape. Significant differentially expressed miRNAs were validated by real‐time polymerase chain reaction (qRT‐PCR) using RNA extracted from monocular FDM ocular tissues. As result, we identified three upregulated miRNAs (mmu‐miR‐1936, mmu‐miR‐338‐5p, and mmu‐miR‐673‐3p) significantly associated with myopia in the two microarray data sets (p < 0.05 and |Log (Fold Change) |>1). GO functional analysis suggested these three miRNAs were targeted in genes mostly enriched in morphogenesis and developmental growth of retinal tissues. Enrichment analysis revealed top eight transcription factors, including PAX6 and Smad3, related to myopia. Ten hub genes, including Rbx1, Fbxl3, Fbxo27, Fbxl7, Fbxo4, Cul3, Cul2, Klhl5, Fbxl16 and Klhl42, associated with ubiquitin conjugation were identified. qRT‐PCR confirmed the increased expression of mmu‐miR‐1936 and mmu‐miR‐338‐5p (p < 0.05), but no statistical difference was observed in mmu‐miR‐673‐3p expression in myopic retinas. Our findings indicated mmu‐miR‐1936, mmu‐miR‐338‐5p and mmu‐miR‐673‐3p upregulation may be associated with myopia development via post‐transcriptional gene regulation, and identified potential molecules that could be further explored in future studies of the mechanism in myopia.

MiR-125b-5p Is Involved in Sorafenib Resistance through Ataxin-1-Mediated Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma.

Simple SummaryThe mechanism of resistance to multikinase inhibitors in hepatocellular carcinoma (HCC) remains unclear. We analyzed miRNA expression profiles in sorafenib-resistant HCC cell lines (PLC/PRF5-R1/R2) and parental cell lines (PLC/PRF5) to identify the responsible miRNAs and target genes involved in the mechanism of resistance. Four miRNAs were significantly upregulated. Among them, we found that miR-125-5p induced sorafenib resistance in HCC cells and in a mouse model. We also revealed that miR-125-5p suppressed ataxin-1 as a target gene and consequently induced Snail-mediated epithelial-mesenchymal transition (EMT) and cancer stemness. Moreover, we demonstrated that ataxin-1 expression has an impact on the prognosis of patients with HCCs. In the future, by comparing the expression status of miR-125b-5p/ataxin-1 and the effect of sorafenib in the clinical setting, it is expected that miR-125b-5p will be established as an effective drug selection marker for treatment selection in patients with HCC.The mechanism of resistance to sorafenib in hepatocellular carcinoma (HCC) remains unclear. We analyzed miRNA expression profiles in sorafenib-resistant HCC cell lines (PLC/PRF5-R1/R2) and parental cell lines (PLC/PRF5) to identify the miRNAs responsible for resistance. Drug sensitivity, migration/invasion capabilities, and epithelial-mesenchymal transition (EMT) properties were analyzed by biochemical methods. The clinical relevance of the target genes to survival in HCC patients were assessed using a public database. Four miRNAs were significantly upregulated in PLC/PRF5-R1/-R2 compared with PLC/PRF5. Among them, miR-125b-5p mimic-transfected PLC/PRF5 cells (PLC/PRF5-miR125b) and showed a significantly higher IC50 for sorafenib compared with controls, while the other miRNA mimics did not. PLC/PRF5-miR125b showed lower E-cadherin and higher Snail and vimentin expression—findings similar to those for PLC/PRF5-R2—which suggests the induction of EMT in those cells. PLC/PRF5-miR125b exhibited significantly higher migration and invasion capabilities and induced sorafenib resistance in an in vivo mouse model. Bioinformatic analysis revealed ataxin-1 as a target gene of miR-125b-5p. PLC/PRF5 cells transfected with ataxin-1 siRNA showed a significantly higher IC50, higher migration/invasion capability, higher cancer stem cell population, and an EMT phenotype. Median overall survival in the low-ataxin-1 patient group was significantly shorter than in the high-ataxin-1 group. In conclusion, miR-125b-5p suppressed ataxin-1 and consequently induced Snail-mediated EMT and stemness, leading to a poor prognosis in HCC patients.

Serum exosomal miRNA from endometriosis patients correlates with disease severity

PurposeExosomes are vesicles secreted by cells that contain a wide variety of biomolecules, including proteins or nucleic acids. MicroRNAs (miRNAs), which are commonly found in exosomes, are known to play important roles in the pathophysiology of endometriosis.MethodsThis study investigated the miRNA expression profile of serum exosomes from women with endometriosis in comparison with normal controls as well as the possible role of identified miRNAs in the pathogenesis of endometriosis. Exosomes with a diameter between 60 and 100 nm were identified by their expression of exosomal marker proteins CD9 and CD63.ResultsMicroarray miRNA expression profiling analysis revealed that 26 genes were significantly up-regulated and 19 genes were significantly down-regulated in serum exosomes from endometriosis patients compared with normal controls. These differentially expressed miRNAs were mainly enriched in the regulation of cellular development, metabolism, and involved in the regulation of the MAPK and PI3k-Akt pathways. qRT-PCR analysis verified the differential expression of three miRNAs, miR-26b-5p, miR-215-5p, and miR-6795-3p.ConclusionFurther analysis indicated that these differentially expressed miRNAs in serum exosomes may be involved in the pathogenesis of endometriosis and are related to the severity and certain symptoms of endometriosis.

CancerMIRNome: an interactive analysis and visualization database for miRNome profiles of human cancer.

MicroRNAs (miRNAs), which play critical roles in gene regulatory networks, have emerged as promising diagnostic and prognostic biomarkers for human cancer. In particular, circulating miRNAs that are secreted into circulation exist in remarkably stable forms, and have enormous potential to be leveraged as non-invasive biomarkers for early cancer detection. Novel and user-friendly tools are desperately needed to facilitate data mining of the vast amount of miRNA expression data from The Cancer Genome Atlas (TCGA) and large-scale circulating miRNA profiling studies. To fill this void, we developed CancerMIRNome, a comprehensive database for the interactive analysis and visualization of miRNA expression profiles based on 10 554 samples from 33 TCGA projects and 28 633 samples from 40 public circulating miRNome datasets. A series of cutting-edge bioinformatics tools and machine learning algorithms have been packaged in CancerMIRNome, allowing for the pan-cancer analysis of a miRNA of interest across multiple cancer types and the comprehensive analysis of miRNome profiles to identify dysregulated miRNAs and develop diagnostic or prognostic signatures. The data analysis and visualization modules will greatly facilitate the exploit of the valuable resources and promote translational application of miRNA biomarkers in cancer. The CancerMIRNome database is publicly available at http://bioinfo.jialab-ucr.org/CancerMIRNome.

Comprehensive analysis of lncRNA and miRNA expression profiles and ceRNA network construction in negative pressure wound therapy.

BackgroundThis study aims to explore the molecular mechanism of negative pressure wound therapy (NPWT) at the transcriptome level through whole transcriptome sequencing and biometric analysis.MethodsA rat skin defect model was constructed and randomly divided into a NPWT group and a gauze group. The tissue in the center of the wound was used for whole transcriptome sequencing, and differentially expressed messenger RNAs (DEmRNAs), long noncoding RNAs (DElncRNAs), and microRNAs (DEmiRNAs) were identified between the two groups. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis was used to verify the sequencing results. Functional enrichment analysis, pathway analysis, and protein-protein interaction (PPI) network analysis of DEmRNAs were conducted. Through bioinformatics analysis, a lncRNA-associated competing endogenous RNA (ceRNA) network was identified and constructed.ResultsWe detected 896 DEmRNAs, 1,471 DElncRNAs, and 20 DEmiRNAs between the two groups. qRT-PCR verified the sequencing results. Functional analysis showed that DEmRNAs were mainly enriched in immune system processes and the Notch signaling pathway. Protein tyrosine phosphatase receptor type C (PTPRC) and signal transducer and activator of transcription 1 (STAT1) were the central hub nodes in the PPI analysis. The ceRNA network contained 11 mRNAs, 15 lncRNAs, and 4 miRNAs.ConclusionsWe identified several DEmRNAs, DElncRNAs, and DEmiRNAs between the NPWT treatment group and the control group. These findings may provide new insights into the pathophysiological mechanism of NPWT and wound healing.