miRanda Software Research Articles

Comprehensive Analysis of circRNA and mRNA Revealing Potential Mechanism Underlying Neuroinflammation in BV2 Cells

Background: The significance of circular RNAs (circRNAs) in diabetic complications has been established. However, their role in basal and diabetic states, as well as cognitive dysfunction, requires further investigation. Methods: BV-2 microglial cells were exposed to high glucose (50mM) and insulin (2μM) for 48 hours. The levels of interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factoralpha (TNF-α) were assessed through quantitative polymerase chain reaction (qPCR), western blot, and ELISA. CircRNA and messenger RNA (mRNA) sequencing were performed, and the data were analyzed. Differentially expressed circRNAs and mRNAs were identified using qPCR. The circRNA-miRNA interaction was predicted using Miranda and TargetScan software, and their levels were quantified by qPCR. Results: The results demonstrated a significant increase in mRNA and protein levels of IL-1β, IL-6, and TNF-α in BV2 cells treated with glucose and insulin. Five circRNAs (four upregulated and one downregulated) were identified in both glucose and insulin groups compared to the control. Further qPCR analysis revealed marked increases in the levels of chr17:40159331- 40159711+ and chr2:72800499-72801858- (mmu_circ_0010164) in both treatment groups. Competitive endogenous RNA networks showed significant upregulation of mRNA levels of mitochondrial transcription termination factor 1b (Mterf1b) and G protein subunit gamma 4 (Gng4), accompanied by a decrease in mmu-miR-6918-3p and mmu-miR-7043-3p levels in the glucose and insulin groups compared to the control. Knockdown of mmu_circ_0010164 significantly inhibited the inflammatory response induced by glucose and insulin in BV-2 microglial cells. Conclusion: These findings indicate that both glucose and insulin can elicit inflammatory responses in BV2 cells through the modulation of mmu_circ_0010164 levels. The underlying mechanism may involve potential downstream targets of mmu_circ_0010164, specifically mmu-miR-7043-3p/Gng4 and mmu-miR-6918-3p/Mterf1b. This provides novel insights into the treatment of glucose-induced neuroinflammation.

Pre-eclampsia intronic polyadenylation enriched in VEGFA-VEGFR2 signaling pathway

AimsThis study aims to reveal transcriptome-wide intronic polyadenylation (IPA) events associated with Pre-eclampsia (PE). BackgroundPre-eclampsia (PE) is a potentially life-threatening complication of pregnancy, affecting both maternal and fetal health. However, our understanding of the underlying molecular mechanisms of PE remains limited. ObjectiveIn this study, we conducted a transcriptome-wide analysis of gene expression levels and intronic polyadenylation (IPA) events in samples of patients with PE. We also conducted motif analysis and scanned the microRNA targeting IPA network. MethodWe collected 90 PE-related samples from GEO database. IPA events were analyzed using IPAfinder software from hg38 alignment files from STAR. Miranda software was used to perform miRNA target gene prediction. Differentially expressed genes (DEG) were evaluated using edgeR with log2 fold change >1 and adjusted p-value <0.05. Function enrichment was performed through Clusterprofiler. ResultOur analysis revealed that genes in the VEGFA-VEGFR2 signaling pathway were functionally enriched with IPA events related to PE. We observed a negative correlation between gene expression levels and IPA events in VEGFA-VEGFR2 pathway. We identified LIN28B and AGO2 as the most significantly binding motifs to IPA sites. Furthermore, our analysis of miRNA binding sites associated with these IPA events revealed the central regulatory roles of miR-193b and miR-365a in IPA genes. ConclusionOur findings suggest that transcriptome data-mining might be a useful approach in future studies aimed at identifying potential biomarkers for PE.

CircRNA expression profiling of the rat thalamus in temporomandibular joint chronic inflammatory pain

Orofacial pain (OFP) induced by temporomandibular disorders (TMDs) is prevalent, affecting approximately 4.6 % of the population. One specific type of TMD is temporomandibular osteoarthritis (TMJOA), a common degenerative disease that significantly impacts patients’ quality of life. Differentially expressed circular RNAs (DEcircRNAs) in the thalamus, which serves as a relay station in the orofacial pain transmission pathway, may play a crucial role and serve as potential target markers for inflammation and the progression of inflammatory pain in TMJOA. The aim of this study was to investigate the expression profile of circRNAs in the thalamus of TMJOA. We obtained the circRNA expression profile from the thalamus of a rat model of TMJOA through high-throughput sequencing (HT-seq) and further validated their expression using reverse transcription real-time polymerase chain reaction (RT-qPCR), followed by bioinformatics analysis of the expression data. A total of 425 circRNAs (DESeq2 p- value < 0.05, |log2FoldChange| > 0.0) were identified as significantly differentially expressed by RNA-Seq, comprising 188 up-regulated and 237 down-regulated circRNAs. After validation via RT-qPCR, we employed miRanda software to predict the binding sites of miRNAs for the identified circRNAs to further explore the functions of DEcircRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that DEcircRNAs were primarily enriched in pathways and functions related to synapse development, protein signaling and modification, ’Circadian entertainment’, the ’MAPK signaling pathway’, and ’Glutamatergic synapse’. These findings suggest that DEcircRNAs in the thalamus play a significant role in the progression of TMJOA and may serve as promising candidate molecular targets for gene therapy.

Study on the mechanism of miRNAs on liver injury in the condition of Protoscocephalus alveolarus transhepatic portal vein infection.

To explore the role of miRNA in liver damage caused by Echinococcus multilocularis infection. Six female C57BL mice were randomly divided into two groups, the control group and the infection group. Mice in the control group were injected with 100 μL PBS through the hepatic portal vein, and mice in the infection group were infected with E. multilocularis via the hepatic portal vein to establish a mouse model of infection. Small RNA sequencing was performed for detecting the expression of miRNAs in the liver of mice infected with 2000 E. multilocularis after 3 months of infection, screen out miRNAs related to liver damage, and verify by RT-PCR. Seventy-one differentially expressed miRNAs were found in the liver in comparison with control, and a total of 36 mouse miRNAs with |FC| >0.585 were screened out, respectively. In addition, Targetscan (V5.0) and miRanda (v3.3a) software were used to predict differential miRNAs target genes and functional enrichment of target genes. Functional annotation showed that "cytokine-cytokine interaction," "positive regulation of cytokine production," "inflammatory response," and "leukocyte activation" were enriched in the liver of E. multilocularis-infected mice. Moreover, the pathways "human cytomegalovirus infection," "cysteine and methionine metabolism," "Notch signaling pathway," and "ferroptosis" were involved in liver disease. Furthermore, four miRNAs (mmu-miR-30e-3p, mmu-miR-203-3p, mmu-miR-125b-5p, and mmu-miR-30c-2-3p) related to liver injury were screened and verified. This study revealed that the expression profiling of miRNAs in the livers was changed after E. multilocularis infection, and improved our understanding of the transcriptomic landscape of hepatic echinococcosis in mice.

Acting mechanism and clinical significance of hsa_circ_0005927 in the invasion and metastasis of gastric cancer.

Background: An increasing number of studies have demonstrated that differentially expressed circular RNAs (circRNAs) play critical roles in carcinogenesis. However, the biological function and clinical significance of hsa_circ_0005927 during gastric carcinogenesis remain unclear. The aim of this study was to investigate the acting mechanism and clinical significance of hsa_circ_0005927 in the invasion and metastasis of gastric cancer (GC). Methods: Hsa_circ_0005927 was detected in GC tissues, plasma and gastric juice from patients with GC, and its correlations with clinicopathological parameters were investigated. Receiver operating characteristic curves, Kaplan-Meier survival curves and a prognostic nomogram model were generated to analyze the diagnostic and prognostic value. Real-time cell analyzer, plate colony formation, and Transwell migration and invasion assays were utilized to assess GC cell proliferation, migration and invasion, respectively. Nucleoplasmic separation was applied to determine the distribution of hsa_circ_0005927 in cells. TargetScan and miRanda software were used for target microRNA (miRNA) prediction. Transcriptome sequencing and bioinformatics analysis were performed to annotate the functions of hsa_circ_0005927 in gastric carcinogenesis and metastasis from an RNomic perspective. Key target genes and immune cell infiltrations were analysed. Results: Hsa_circ_0005927 was found downregulated in high-grade intraepithelial neoplasia (HGIEN) tissues and GC tissues. Hsa_circ_0005927 levels in GC tissues were negatively correlated not only with lymphatic metastasis and distal metastasis but also with overall survival and disease-free survival. As a screening biomarker for GC, plasma hsa_circ_0005927 levels significantly increased in the early stages of GC, with a sensitivity and specificity of 52.38% and 76.19%, respectively. Hsa_circ_0005927 was mainly distributed in the cytoplasm, and structurally, it possesses multiple miRNA response elements (MREs) that interact with five miRNAs. A total of 421 downstream target genes of hsa_circ_0005927 were identified by transcriptome sequencing; and bioinformatics analysis suggested that these genes were involved mainly in the negative regulation of the T-cell apoptotic process, the interleukin-27-mediated signaling pathway, growth factor activity, guanylate cyclase activity, transcriptional misregulation in cancer, the cGMP-PKG signaling pathway, and the GnRH signaling pathway during gastric carcinogenesis and metastasis. GUCY1A2 and STK32A are key target genes significantly associated with immune infiltration. Conclusion: Our study revealed that hsa_circ_0005927 is a new player related to the invasion and metastasis of GC and is a potential indicator for early GC screening.

MicroRNA PC-5p-3991_515 mediates triflumezopyrim susceptibility in the small brown planthopper through regulating the post-transcriptional expression of P450 CYP417A2.

Cytochrome P450 monooxygenases (P450s) are recognized as a major contributor to metabolic resistance in insects to most insecticides, through gene overexpressions and protein mutations. MicroRNA (miRNA), an important post-transcriptional regulator, has been reported to promote insecticide resistance by mediating the expression of detoxification enzyme genes. In the present study, we reported that a novel microRNA PC-5p-3991_515 was involved in the post-transcriptional regulation of CYP417A2 and mediated the triflumezopyrim susceptibility in the small brown planthopper (SBPH), Laodelphax striatellus (Fallén). The tissue expression profiles showed that CYP417A2 was highly expressed in fat body. CYP417A2 was significantly up-regulated at 12, 36, 60, 84 and 108 h after the triflumezopyrim treatment. RNA interference (RNAi) against CYP417A2 significantly increased triflumezopyrim susceptibility in SBPH. According to the prediction by miRanda and TargetScan software, three miRNAs were indicated to bind to CYP417A2. However, when oversupply of agomir, only two miRNAs, PC-3p-625_4405 and PC-5p-3991_515, significantly increased the susceptibility to triflumezopyrim and decreased CYP417A2 levels. Furthermore, PC-5p-3991_515 was confirmed to be involved in the post-transcriptional regulation of CYP417A2 by dual luciferase reporter assay. Meanwhile, PC-5p-3991_515 was co-localized with CYP417A2 in the midgut in situ hybridization. Our findings revealed that the novel microRNA, PC-5p-3991_515, post-transcriptionally regulated CYP417A2 expression, which then mediated the triflumezopyrim susceptibility in SBPH. © 2023 Society of Chemical Industry.

The identification of regulatory ceRNA network involved in Drosophila Toll immune responses

Non-coding RNAs play important roles in the innate immunity of Drosophila, with various lncRNAs and miRNAs identified to maintain Drosophila innate immune homeostasis by regulating protein functions. However, it remains unclear whether interactions between lncRNAs and miRNAs give rise to a ceRNA network. In our previous study, we observed the highest differential expression levels of lncRNA-CR11538, lncRNA-CR33942, and lncRNA-CR46018 in wild-type flies after Gram-positive bacterial infection, prompting us to investigate their role in the regulation of Drosophila Toll immune response through RNA-seq analysis. Herein, our comprehensive bioinformatics analysis revealed that lncRNA-CR11538, lncRNA-CR33942, and lncRNA-CR46018 are involved in defense mechanisms and stimulus response. Moreover, lncRNA-CR11538 and lncRNA-CR46018 can also participate in the metabolic recovery processes following Gram-positive bacterial infection. Subsequently, we employed GSEA screening and RT-qPCR to identify seven miRNAs (miR-957, miR-1015, miR-982, miR-993, miR-1007, miR-193, and miR-978) that may be regulated by these three lncRNAs. Furthermore, we predicted the potential target genes in the Toll signaling pathway for these miRNAs and their interaction with the three lncRNAs using TargetScan and miRanda software and preliminary verification. As a result, we established a potential ceRNA regulatory network for Toll immune responses in Drosophila, comprising three lncRNAs and seven miRNAs. This study provides evidence of a ceRNA regulatory network in Drosophila Toll immune responses and offers novel insights into understanding the regulatory networks involved in the innate immunity of other animals.

LncRNA AC125982.2 regulates apoptosis of cardiomyocytes through mir-450b-3p/ATG4B axis in a rat model with myocardial infarction

BackgroundThe occurrence and disability of myocardial infarction (MI) are on the rise globally, making it a significant contributor to cardiovascular mortality. Irreversible myocardial apoptosis plays a crucial role in causing MI. Long non-coding RNAs (LncRNAs) are key regulators of the cardiac remodeling process. Therefore, it is necessary to explore the effect of LncRNAs on cardiomyocyte apoptosis in MI. MethodsThe rat-MI model was constructed, LncRNA-Seq and qPCR analyses were used to determine differentially expressed genes obtained from heart tissue of rats in the MI and sham groups. The miRanda software was used to predict the binding sites of LncRNA-miRNA and miRNA-mRNA, which were futhrer verified by dual luciferase assay. The LncRNA-miRNA-apoptosis pathway was further validated using hypoxia-exposed primary cardiomyocytes. ResultsCompared to the sham group, 412 LncRNAs were upregulated and 501 LncRNAs were downregulated in MI-rat heart tissues. Among them, LncRNA AC125982.2 was most significantly upregulated in MI-rat heart tissues and hypoxic cardiomyocytes. Knockdown of AC125982.2 and ATG4B expression reversed hypoxia-induced apoptosis. In addition, transfection of mir-450b-3p inhibitor attenuated the protective effect of AC125982.2 knockdown. Moreover, we found that AC125982.2 modulated ATG4B expression by acting as a sponge for miR-450b-3p. ConclusionUpregulated AC125982.2 expression regulates ATG4B by sponging miR-450b-3p, promoting cardiomyocyte apoptosis and contributing to rat MI development.

Differentiated expressed miRNAs in splenic monocyte induced by burn injury in mice.

To find potential biomarkers based on miRNA and their potential targets in splenic monocytes in burn-injured mice. Male Balb/c mice were subjected to sham or scalding injury of 15% total body surface area. Spenic CD11b+ monocytes were purified with magnetic beads. The monocytes were cultured in the presence of lipopolysaccharide. The proliferation of monocytes was detected by MTT assay, and the cytokines in the supernatant were examined by enzyme linked immunosorbent assay. The purified monocytes were also under total RNA extraction. The differential monocytic miRNAs expression between the sham and burn-injured mice was analysed by miRNA microarray. The activity of monocytes was comparable between the two groups (p > 0.05). However, monocytes from burn-injured mice secreted higher levels of tumour necrosis factor (TNF)-α and transforming growth factor-β, but lower level of monocyte chemoattratctant protein-1. A total of 54 miRNAs were differentially expressed in monocytes from burn relative to sham-injured mice (fold >3). Further quantitative reverse transcription polymerase chain reaction confirmed that the expression of miR-146a was significantly down-regulated, while miR-3091-6p was up-regulated after burn injury. Using the combination of Miranda and TargetScan softwares, we found that mir-146a may regulate 180 potential target genes including TNF receptor related factor 6 (TRAF6), interleukin-1 receptor related kinase 1 (IRAK1) and CD28. Mir-3091-6p may regulate 39 potential targets, including SOCS7 (cytokine signal transduction inhibitor 7) and ARRB2 (arrestin, β 2). The miRNAs expressed by monocytes after burn injury may be involved in the regulation of innate immune response in burn injury.

Analysis the mechanism of cadmium-induced cytotoxicity in mouse testicular mesenchymal cells with miRNA transcriptomic

To analyze the mechanism of cadmium-induced cytotoxicity in mouse testicular mesenchymal cells(TM3) with transcriptome sequencing and bioinformatics techniques. TM3 cells were selected as the cell model and divided into control group(no cadmium treatment) and cadmium-treated group(20 μmol/L CdCl_2). After 24 hours of administration, cells were harvested to extract total RNA, and then miRNA expression profiles were obtained by sequencing program after RNA quality detection. The fold change(FC) &gt;2, P&lt;0.05 was used as the standard to screen for differentially expressed miRNAs. The quantitative real-time polymerase chain reaction(qRT-PCR) was used to verify the differentially expressed miRNAs. Then, their target genes were predicted by miRanda software to construct miRNA-target gene interaction network, and their target genes were enriched by gene ontology(GO) and Kyoto encyclopedia of genes and genomes(KEGG) pathway function. A total of 26 differentially expressed miRNAs were identified which may be related to cadmium-induced TM3 cytotoxicity, including 19 up-regulated and 7 down-regulated miRNAs. The result of qRT-PCR were consistent with the miRNA sequencing result. Meanwhile, bioinformatics analysis result showed that the 26 differentially expressed miRNAs predicted 657 target genes. GO enrichment was mainly classified into biological regulation, metabolic process, protein binding and catalytic activity. KEGG pathway analysis showed that target genes were significantly involved in mitogen-activated protein kinase(MAPK) and tumor necrosis factor(TNF) signal pathways closely related to inflammatory response and apoptosis. Cadmium can lead to the differential expression of miRNAs in TM3 cells, and its target genes may be involved in Cd-induced TM3 cytotoxicity through signaling pathways such as MAPK and TNF.

Construction of circRNA-miRNA-mRNA Network Reveal Functional circRNAs and Key Genes in Acute Myeloid Leukemia.

CircRNA is closely correlated with a wide variety of processes of acute myeloid leukemia (AML), whereas the novel circRNAs, their molecular mechanism and the specific function they played in AML should be explored in depth. The microarray chip data of AML patients and normal samples in the Gene Expression Omnibus (GEO) database were selected to differentially expressed (DE) circRNA, miRNA, and mRNA genes. The miRNA gene was the intersection of the circRNA target gene predicted using CSCD and the miRNA gene screened from AML patients, while the mRNA gene was the intersection of the target gene mRNA of miRNA predicted using miRanda and miRTarBase software and the mRNA gene screened from AML patients. The hub mRNAs related to survival were further screened through Cox proportional hazard regression. CircRNA/miRNA/mRNA interaction network was constructed by using Cytoscape software.10 circRNAs and 6 miRNAs in bone marrow mononuclear cells (BMMNCs) of AML patients (n=43) and healthy controls (n=35) were determined by RT-qPCR. Correlations between them were analyzed by Pearson correlation coefficient. 10 circRNAs, 6 miRNAs, and 33 mRNAs were identified. Subsequently, the network of circRNAs, miRNAs, and hub genes was built using Cystoscope. Four key circRNAs, seven hub genes and their regulatory pathways were identified. The result of RT-qPCRs showed that hsa_circ_0009581 and hsa_circ_0005273 were significantly upregulated in AML patients while hsa_circ_0000497 and hsa_circ_0001947 were significantly downregulated. Hsa-miR-150-5p was significantly downregulated; hsa-miR-454-3p was upregulated in AML patients. Hsa_circ_0009581 and hsa-miR-150-5p; hsa_ circ_0001947 and hsa-miR-454-3p were inversely correlated using Pearson's correlation coefficient. This study suggests that differentially expressed circRNAs take on a critical significance to AML development and may be the effective therapeutic targets. We suppose that hsa_circ_0009581 promotes leukemia development through hsa-miR-150-5p and hsa_circ_0001947 through hsa-miR-454-3p. hsa_circ_0001947 and hsa_circ_0009581 may provide new directions in the pathogenesis of AML.

Identification of target genes and pathways related to heat tolerance in Chinese Holstein cows

Heat stress has detrimental effects on the production and reproduction of dairy cows. The present work reveals the role of differentially expressed (DE) miRNAs on the heat stress tolerance of bovine blood using RNA sequencing. Twelve dairy cows were divided into heat-resistant and heat-stressed groups according to rectal temperature and reduction of milk yield. Using the ACGT101-miR program, 1262 known miRNAs and 719 novel miRNAs were identified. Of the 719 novel miRNAs, 144 significantly DE miRNAs (67 up-regulated and 77 down-regulated) (P < 0.05) were screened between heat-resistant and heat-stressed cows using edgeR software. Eleven high DE miRNAs (bta-miR-455–3p, bta-miR-455–5p, mmu-mir-6240, bta-miR-106b, bta-miR-11,971, bta-miR-29a, bta-miR-142–5p, bta-miR-15b, bta-miR-342, bta-miR-32, and bta-miR-6524) were verified by qRT-PCR, an outcome which was consistent with the sequencing results. The target genes related to heat tolerance of the heat shock protein (HSP) family, malonyl CoA-acyl carrier protein transacylase (MACT), and prolactin receptor (PRLR), were predicted using the TargetScan and miRanda software. Furthermore, KEGG analyses with the R package OmicStudio and on the basis of DE miRNAs revealed an enriched MAPK signaling pathway, endocytosis, Th17 cell differentiation, cellular senescence, peroxisome, and Fc gamma R-mediated phagocytosis. These findings could help in screening for heat tolerance genes for improved dairy cow heat tolerance.

LncRNA ANRIL promotes HR repair through regulating PARP1 expression by sponging miR-7-5p in lung cancer

BackgroundRadiotherapy is an important treatment for lung cancer, mainly by triggering DNA double-strand breaks to induce cell death. Blocking DNA damage repair can increase the radiosensitivity of tumor cells. Recent studies have identified long noncoding RNAs as key regulators in DNA damage repair. The lncRNA ANRIL was previously shown to be involved in homologous recombination (HR) repair, but its specific mechanism has not been fully elucidated.MethodsThe downstream interacting miRNAs of ANRIL were predicted according to miRanda software. Fluorescence quantitative PCR was used to detect the expression levels of ANRIL and candidate miRNAs. Clone formation experiment and cell viability assays detect cell viability after ionizing radiation. Apoptosis assay was used to detect the apoptosis of cells after 8 h of ionizing radiation. Western blot analysis and immunofluorescence assays verified the protein expression levels of the downstream target molecule PARP1 of miR-7-5p and key molecules in the HR pathway. Fluorescent reporter gene experiments were used to verify the interaction between ANRIL and miR-7-5p and between miR-7-5p and PARP1.ResultsBioinformatics analysis and qPCR validation suggested that miR-7-5p might be a downstream molecule of ANRIL. The expression of miR-7-5p was up-regulated after knockdown of ANRIL, and the expression of miR-7-5p was down-regulated after overexpression of ANRIL. Meanwhile, there was a negative correlation between ANRIL and miR-7-5p expression changes before and after ionizing radiation. The luciferase reporter gene assay confirmed the existence of ANRIL binding site with miR-7-5p, and found that transfection of miR-7-5p inhibitor can reduce the radiation sensitivity of ANRIL-KD cells. A downstream target molecule of miR-7-5p related to HR repair, PARP1, was screened through website prediction. Subsequently, it was confirmed by Western blot and luciferase reporter assays that miR-7-5p could down-regulate the expression of PARP1, and there was a miR-7-5p binding site on the 3'UTR of PARP1 mRNA. This suggests that ANRIL may act as a competitive endogenous RNA to bind miR-7-5p and upregulate the expression of PARP1. Western blot and immunofluorescence staining were used to detect the expression changes of HR repair factors in ANRIL-KD cells after ionizing radiation, and it was found that knockdown of ANRIL can inhibit the expression of PARP1, BRCA1 and Rad51, hinder radiation-induced HR repair, and eventually result in resensitizing ANRIL-KD cells to ionizing radiation.ConclusionsOur findings provide evidence that ANRIL targets the miR-7-5p/PARP1 axis to exert its regulatory effect on HR repair, suggesting that altering ANRIL expression may be a promising strategy to overcome radiation resistance.

CircRNA-miRNA-mRNA network analysis to explore the pathogenesis of abnormal spermatogenesis due to aberrant m6A methylation.

Many studies have shown that circRNAs and miRNAs play important roles in many different life processes. However, the function of circRNAs in spermatogenesis remains unknown. Here, we aimed to explore the mechanisms whereby circRNA-miRNAs-mRNAs regulate abnormal m6A methylation in GC-1spg spermatogonia. We first reduced m6A methylation in GC-1spg whole cells after knocking down the m6A methyltransferase enzyme, METTL3. Then, we performed circRNA- and miRNA-seq on GC-1spg cells with low m6A methylation and identified 48 and 50 differentially expressed circRNAs and miRNAs, respectively. We also predicted the targets of the differentially expressed miRNAs by using Miranda software and further constructed the differentially expressed circRNA-differentially expressed miRNA-mRNA ceRNA network. GO analysis was performed on the differentially expressed circRNAs and miRNA-targeted mRNAs, and an interaction network between the proteins of interest was constructed using Cytoscape. The final GO analysis showed that the target mRNAs were involved in sperm formation. Therefore, a PPI network was subsequently constructed and 2 hub genes (H2afx and Dnmt3a) were identified. In this study, we constructed a ceRNA network and explored the regulatory roles of circRNAs and miRNAs in the pathogenesis of abnormal spermatogenesis caused by low levels of methylated m6A. Also, we identified two pivotal genes that may be key factors in infertility caused by abnormal m6A methylation. This may provide some ideas for the treatment of infertility resulting from abnormal spermatogenesis.

High Throughput Sequencing Analysis of Exosomal miRNA Expressions in Early-Onset Preeclampsia Patients.

The goal of this study was to screen for differentially expressed exosomal miRNAs in peripheral blood samples of early-onset preeclampsia and normal pregnant patients using high-throughput sequencing methods and explore their effects on the pathogenesis of preeclampsia. Peripheral blood samples from 5 patients with early-onset preeclampsia and 5 normal pregnant women (control group) were enrolled. Then, exosomes were extracted from each sample, and the procedure was replicated three times. An Illumina HiSeq4000 sequencing platform was used to analyze exosomal miRNAs in all samples before comparison. The target genes and signaling pathway predictions and the biological function enrichments of significant and differentially expressed miRNAs were assessed using Miranda and Starbase software, as well as GO and KEGG databases. Compared with the control, patients in the early-onset preeclampsia group had 65 significantly and differentially expressed exosomal miRNAs in their peripheral blood samples. The results have shown that of the 65 differentially expressed miRNAs, 17, including has-miR-6855, has-miR-7151, and has-miR-6777, were up-regulated, and 48, including has-miR-1247, has-miR-29B2, and has-miR-941, were down-regulated (p < 0.05). The Miranda and TargetScanS algorithms predicted a total of 2,231 target genes from the differentially expressed miRNAs. The Go and KEGG analyses showed that the principal biological function of these target genes was the regulation of Ras protein signal transduction, histone modification, GTPase-mediated signal transduction, and transforming growth factor (TGF)-β. Additionally, the results also showed that the major pathways involved in the regulation of these functions were the PI3K Akt, MAPK, tumor necrosis factor, and EGFR tyrosine kinase inhibition signaling pathways. There are significant differences in the expression profiles of exosomal miRNAs between early-onset preeclampsia patients and normal pregnant women. These differentially expressed miRNAs may not only play an important regulatory role in the occurrence of early-onset preeclampsia but also participate in its pathophysiological process through genetic regulation of a variety of biological functions and signal pathways.

Effect of condylar chondrocyte exosomes on condylar cartilage osteogenesis in rats under tensile stress.

Background: Functional orthoses are commonly used to treat skeletal Class II malocclusion, but the specific mechanism through which they do this has been a challenging topic in orthodontics. In the present study, we aimed to explore the effect of tensile stress on the osteogenic differentiation of condylar chondrocytes from an exosomal perspective. Methods: We cultured rat condylar chondrocytes under resting and tensile stress conditions and subsequently extracted cellular exosomes from them. We then screened miRNAs that were differentially expressed between the two exosome extracts by high-throughput sequencing and performed bioinformatics analysis and osteogenesis-related target gene prediction using the TargetScan and miRanda softwares. Exosomes cultured under resting and tensile stress conditions were co-cultured with condylar chondrocytes for 24h to form the Control-Exo and Force-Exo exosome groups, respectively. Quantitative real time PCR(RT-qPCR) and western blotting were then used to determine the mRNA and protein expression levels of Runx2 and Sox9 in condylar chondrocytes. Results: The mRNA and protein expression levels of Runx2 and Sox9 in the Force-Exo group were significantly higher than those in the Control-Exo group (p < 0.05). The differential miRNA expression results were consistent with our sequencing results. Bioinformatics analysis and target gene prediction results showed that the main biological processes and molecular functions involved in differential miRNA expression in exosomes under tensile stress were biological processes and protein binding, respectively. Kyoto Gene and Genome Data Bank (KEGG) pathway enrichment analysis showed significant enrichment of differentially expressed miRNAs in the mTOR signaling pathway. The differentially expressed miRNAs were found to target osteogenesis-related genes. Conclusion: These results suggest that stimulation of rat condylar chondrocytes with tensile stress can alter the expression levels of certain miRNAs in their exosomes and promote their osteogenic differentiation. Exosomes under tensile stress culture conditions thus have potential applications in the treatment of Osteoarthritis (OA).

Prediction of Potential Biomarkers in Early-Stage Nasopharyngeal Carcinoma Based on Platelet RNA Sequencing

Early diagnosis is essential for the treatment and prevention of nasopharyngeal cancer. However, there is a lack of effective biological indicators for nasopharyngeal carcinoma (NPC). Therefore, we explored the potential biomarkers in tumour-educated blood platelet (TEP) RNA in early NPC. Platelets were isolated from blood plasma and their RNA was extracted. High-throughput sequenced data from a total of 33 plasma samples were analysed using DESeq2 to identify the differentially expressed genes (DEGs). Subsequently, the DEGs were subjected to principal component analysis (PCA), gene ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis; and Cytoscape, TargetScan, and miRanda software were used for inferring the competing endogenous RNA network. We identified 19 long non-coding (lnc) RNAs (DElncRNAs) and 248 mRNAs (DEmRNAs) that were differentially expressed in the TEP RNA. In addition, SELP gene mRNA and lncRNAs AC092135.3, AC012358.2, AL021807.1, AP001972.5, and GPX1 were found to be down-regulated DEmRNA and DElncRNAs in the early stage of NPC. Bioinformatic analysis showed that these DEmRNAs and DElncRNAs may be involved in regulating the pathogenesis of NPC. Our research may provide new insights for exploring the biological mechanisms of NPC and early diagnosis using potential biomarkers.

CircRNA/miRNA/mRNA axis participates in the progression of partial bladder outlet obstruction

BackgroundMore and more evidence showed that circRNA/miRNA/mRNA axis played a vital role in the pathogenesis of some diseases. However, the role of circRNA/miRNA/mRNA axis in partial bladder outlet obstruction (pBOO) remains unknown. Our study aimed to explore the complex regulatory mechanism of circRNA/miRNA/mRNA axis in pBOO.MethodsThe pBOO rat model was established, and the bladder tissues were collected for mRNA sequencing. The differentially expressed mRNAs were analyzed by high-throughput sequencing, and the GO and KEGG analysis of the differentially expressed mRNAs were performed. Competing endogenous RNAs (ceRNAs) analysis identified the potential regulation function of circRNA/miRNA/mRNA axis in pBOO. qRT-PCR detected the expression of circRNA/miRNA/mRNA. miRanda software was performed to predict the relationship between circRNA and miRNA, miRNA and mRNA.ResultsCompared with the sham group, a total of 571 mRNAs were differentially expressed in the pBOO group, of which 286 were up-regulated and 285 were down-regulated. GO analysis showed that the mRNAs were mainly involved in cellular process, single-organism process, and cell, etc. KEGG analysis showed that the enriched signaling pathways were metabolic pathways, cell adhesion molecules (CAMs), and HTLV-I infection, etc. Based on the previous transcriptome data and differentially expressed circRNAs, we drew the ceRNA network regulation diagram. qRT-PCR results confirmed that chr3:113195876|113197193/rno-miR-30c-1-3p/Gata4, chr1:126188351|126195625/rno-miR-153-5p/Diaph3, and chr9:81258380|81275269/rno-miR-135b-5p/Pigr axis may have ceRNA function. miRanda confirmed there have the binding sites of circRNA/miRNA/mRNA axis.ConclusionsCircRNA/miRNA/mRNA axis was involved in the progression of pBOO. Our research on the circRNA/miRNA/mRNA axis revealed new pathogenesis and treatment strategies for pBOO.

Screening of plasma exosomal lncRNAs to identify potential biomarkers for obstructive sleep apnea

BackgroundObstructive sleep apnea (OSA) is highly prevalent, but frequently undiagnosed. The existing biomarkers of OSA are relatively insensitive and inaccurate. Long non-coding RNAs (lncRNAs) have no protein-coding ability but have a role in regulating gene expression. They are stably expressed in exosomes, easily and rapidly measurable. Changes in expression of exosomal lncRNAs can be useful for disease diagnoses. However, there are few reports on the association of exosomal lncRNAs with OSA. We aimed to investigate the exosomal lncRNA profiles to establish the differences between non-OSA, OSA with or without hypertension (HTN) and serve as a potential diagnostic biomarker.MethodsThis diagnostic test included 63 participants: [normal control (NC) =25], (OSA =23), and (HTN-OSA =15). Expression profiling of lncRNAs in isolated exosomes was performed through high-throughput sequencing in 9 participants. Subsequently, OSA/HTN-OSA related lncRNAs were selected for validation by droplet digital polymerase chain reaction (ddPCR), receiver operating characteristic (ROC) curves were used to determine the diagnostic value. The reliabilities of the screened gene were further validated in another independent cohort: (NC =10), (OSA mild =10), (OSA moderate =11), and (OSA severe =10), the correlation between clinical features and its expression was analyzed. The MiRanda software was used to predict the binding sites of interaction between microRNA (miRNA) and target genes regulated by screened lncRNA.ResultsWe identified the differentially expressed lncRNAs and mRNAs in plasma exosomes of the NC, OSA, HTN-OSA groups. Most pathways enriched in differentially expressed lncRNAs and mRNAs had previously been linked to OSA. Among them, ENST00000592016 enables discrimination between NC and OSA individuals [area under curve (AUC) =0.846, 95% confidence interval (CI): 0.72–0.97]. The severity of OSA was associated with changes in the ENST00000592016 expression. Furthermore, ENST00000592016 affected the PI3K-Akt, MAPK, and TNF pathways by regulating miRNA expressions.ConclusionsThis is the first report about differential expression of lncRNA in OSA and HTN-OSA exosomes. ENST00000592016 enables discrimination between NC and OSA individuals. This work enabled characterization of OSA and provided the preliminary work for the study of biomarker of OSA.

CircRNA.0007127 triggers apoptosis through the miR-513a-5p/CASP8 axis in K-562 cells.

BACKGROUND: Circular RNAs (circRNAs) are covalently closed single-stranded RNAs with multiple biological functions. CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less (CCR4-NOT) complex subunit 2 (CNOT2), which was found to regulate tumor cell apoptosis through caspase pathway. METHODS: Potential circRNA.0007127 target microRNAs (miRNAs) were analyzed by miRanda, TargetScan, and RNAhybrid software, and the miRNAs with binding sites for apoptosis-related genes were screened. The roles of circRNA.0007127 and its downstream target, microRNA (miR)‍-513a-5p, were validated by quantitative real-time polymerase chain reaction (qPCR), flow cytometry, mitochondrial membrane potential, immunofluorescence, western blot, and caspase-8 (CASP8) protein activity in vitro in H2O2-induced K-562 cells. The circRNA.‍0007127‍‒‍miR-513a-5p and CASP8‍‒‍miR-513a-5p interactions were verified by luciferase reporter assays. RESULTS: Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells. Compared with the control group, the expression of CASP8 was reduced by 50% and the 43-kD fragment of CASP8 protein was significantly reduced (P≤0.05). The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8, with extremely significant differences (P≤0.001). The overexpression of miR-513a-5p inhibited the gene expression level of CASP8 in a human myeloid leukemia cell model (75% change) and the level of a 43-kD fragment of CASP8 protein (P≤0.01). The rescue experiment showed that cotransfection with circRNA.0007127 small-interfering RNA (siRNA) and the miR-513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate, suggesting that the miR-513a-5p inhibitor is a circRNA.0007127 siRNA antagonist. CONCLUSIONS: CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis, which can serve as a novel powerful molecular target for K-562 cells.