miRNA Levels Research Articles

The Use of Tissue-on-Chip Technology to Focus the Search for Extracellular Vesicle miRNA Biomarkers in Thyroid Disease.

Small extracellular vesicles (sEVs) contain microRNAs (miRNAs) which have potential to act as disease-specific biomarkers. The current study uses an established method to maintain human thyroid tissue ex vivo on a tissue-on-chip device, allowing the collection, isolation and interrogation of the sEVs released directly from thyroid tissue. sEVs were analysed for differences in miRNA levels released from benign thyroid tissue, Graves' disease tissue and papillary thyroid cancer (PTC), using miRNA sequencing and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to identify potential biomarkers of disease. Thyroid biopsies from patients with benign tissue (n = 5), Graves' disease (n = 5) and PTC (n = 5) were perfused with medium containing sEV-depleted serum for 6 days on the tissue-on-chip device. During incubation, the effluents were collected and ultracentrifuged to isolate sEVs; miRNA was extracted and sequenced (miRNASeq). Out of the 15 samples, 14 passed the quality control and miRNASeq analysis detected significantly higher expression of miR-375-3p, miR-7-5p, miR-382-5p and miR-127-3p in the sEVs isolated from Graves' tissue compared to those from benign tissue (false discovery rate; FDR p < 0.05). Similarly, miR-375-3p and miR-7-5p were also detected at a higher level in the Graves' tissue sEVs compared to the PTC tissue sEVs (FDR p < 0.05). No significant differences were observed between miRNA in sEVs from PTC vs. those from benign tissue. These results were supported by Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). The novel findings demonstrate that the tissue-on-chip technology is a robust method for isolating sEVs directly from the tissue of interest, which has permitted the identification of four miRNAs, with which further investigation could be used as biomarkers or therapeutic targets within thyroid disease.

Fluorine-doped carbon dots (F-CDs) adsorbing DNA via hydrophobic interaction play dual-role of quenching carrier and signal reference for ratiometric fluorescence strategy to detect microRNA

The expression levels of microRNAs (miRNA) can provide insights into the occurrence, progression, and treatment efficacy in tumors, highlighting the need for precise and easily controllable detection methodologies. The reasonable design of carbon dots enables the precise manipulation of luminescence and surface characteristics. Herein, fluorine doped carbon dots (F-CDs) were designed and prepared to establish a reasonable control of the interaction force between DNA and F-CDs. F-CDs were synthesized through a hydrothermal reaction involving tetrafluoroterephthalic acid and tetraethylene pentaamine, resulting in noteworthy optical and amphiphilic properties. F-CDs proficiently interact with single-stranded DNA (ssDNA) through hydrophobic interaction, leading to the quenching of dye labeled ssDNA, while maintaining self-fluorescence stability. The quenching mechanism was elucidated. The complementary DNA sequence induced nearly 100% desorption of ssDNA from F-CDs. Assisted by the target cycling signal amplification from duplex specific nuclease (DSN) and the self-fluorescence of F-CDs, an effective ratiometric strategy utilizing the adsorption system of F-CDs and probe DNA for let-7a was established for detecting let-7a, a critical miRNA in the assessment of tumor development. Under the optimized conditions, this method demonstrated high sensitivity and reproducibility in detecting let-7a with the linear range from 1.0 fM to 10.0 nM and a low limit of detection (LOD) of 3.0 fM. The recovery of let-7a in plasma and the evaluation of let-7a levels in seven cell types underscored the method's potential for efficient applications in monitoring of miRNA levels in real samples.

Analysis of microRNA expression in rat retina at the early stages of retinopathy development

Age-related macular degeneration (AMD), a neurodegenerative eye disease, is the primary cause of blindness globally, especially affecting the elderly population. MicroRNAs, a type of single-stranded, non-coding RNAs of about 22 nucleotides in length, primarily regulate gene expression negatively. Alterations in the miRNA profile during AMD’s development could potentially aid in early detection and progression monitoring of the disease. However, no data are available on the microRNA expression patterns in the retina during the early stages of AMD. Investigating the molecular mechanisms that underlie age-related retinal degeneration at an early stage, prior to the manifestation of any symptoms, could provide insights into the molecular events that initiate the irreversible stage. In the case of OXYS rats with accelerated aging, dystrophic changes in the RPE, neuroretinal thinning, and impaired choroidal microcirculation — the primary indicators of the dry form of AMD — spontaneously occur by the age of 3 months [1]. A comparative analysis was conducted in this study on microRNA expression in the retinas of OXYS rats at two different stages of retinopathy — 20 days (preclinical) and 3 months (manifestation) — as well as in control Wistar rats. Small RNA-seq sequencing was performed on the DNBSEQ platform. According to differential expression analysis (DESeq2, q-value 0.05), at 20 days of age, 2 microRNAs exhibited altered expression in OXYS rats compared to Wistar rats. Similarly, at 3 months of age, the expression of 16 microRNAs in OXYS rats was altered compared to Wistars, with 7 miRNAs showing increased levels and 9 miRNAs showing decreased levels. Analysis of age-related changes in miRNA expression demonstrated that in the OXYS rat retina between 20 days and 3 months, the levels of 134 miRNAs altered: 59 miRNAs increased, and 75 miRNAs decreased. Over the course of 20 days to 3 months, 94 miRNAs underwent alterations in the retinas of Wistar rats. Among these, the level of 48 miRNAs increased while the level of 46 miRNAs decreased. The RNAhybrid, miRanda, and TargetScan programs were used to search for target genes of differentially presented miRNAs among different groups. Following this, gene ontologies were studied. In comparison to Wistar rats, “adhesion contacts” and “synaptic vesicle cycle” categories are noticeably populated with target genes of miRNA DE at 20 days in OXYS rats. MiRNA target genes with lower levels at 3 months in OXYS rats compared to Wistar rats are significantly enriched in categories such as vascular branching, extracellular matrix organization, adhesion, and protein deubiquitination. Target genes with increased miRNA levels at 3 months in OXYS rats, compared to Wistar rats, are significantly represented in various signaling pathways, such as mTOR, MAPK, VEGF, and thyroid hormone. Target genes, which serve as common targets for at least 10 microRNAs (miRNAs), were chosen for analysis of the targetome subject to regulation by miRNA molecules exhibiting modified expression. Targetomes containing 400 to 600 genes were obtained for miRNAs that increased or decreased levels in both OXYS and Wistar rats between 20 days and 3 months. Notably, these targetomes were enriched in categories such as axonogenesis, retinal layer formation, visual learning, focal adhesion, and endocytosis. Profiling of 84 miRNAs of the retina of OXYS and Wistar rats at the age of 20 days and 3 months was carried out using PCR panels (Qiagen) to verify the results of small RNA-seq. Our analysis revealed highly convergent results between sequencing and PCR panels. Thus, 16 miRNAs showing altered expression in OXYS retina may serve as potential biomarkers and new targets for studying AMD pathogenesis.

The role of microRNAs involved in the disorder of blood-brain barrier in the pathogenesis of multiple sclerosis.

miRNAs are involved in various vital processes, including cell growth, development, apoptosis, cellular differentiation, and pathological cellular activities. Circulating miRNAs can be detected in various body fluids including serum, plasma, saliva, and urine. It is worth mentioning that miRNAs remain stable in the circulation in biological fluids and are released from membrane-bound vesicles called exosomes, which protect them from RNase activity. It has been shown that miRNAs regulate blood-brain barrier integrity by targeting both tight junction and adherens junction molecules and can also influence the expression of inflammatory cytokines. Some recent studies have examined the impact of certain commonly used drugs in Multiple Sclerosis on miRNA levels. In this review, we will focus on the recent findings on the role of miRNAs in multiple sclerosis, including their role in the cause of MS and molecular mechanisms of the disease, utilizing miRNAs as diagnostic and clinical biomarkers, using miRNAs as a therapeutic modality or target for Multiple Sclerosis and drug responses in patients, elucidating their importance as prognosticators of disease progression, and highlighting their potential as a future treatment for MS.

Changes in adiponectin system after ventricular assist device in pediatric heart failure

Ventricular assist device (VAD) implant represents a therapeutic option for pediatric patients with end-stage heart failure (HF). Heart unloading by VAD can modify several molecular pathways underlying cardiac function in HF. Among them, the potential role of microRNA (miRNAs) in response to VAD implant is emerging. This study was aimed at investigating in HF pediatric patients the effect of VAD-modified miRNAs on the adiponectin (ADPN) system, known to exert cardioprotective actions. ADPN was measured in plasma samples obtained from HF children, before and 1month after VAD implant, and from healthy control children. miRNA profile and molecules belonging to ADPN system were determined in cardiac biopsies collected at the time of VAD implantation (pre-VAD) and at the moment of heart transplant (post-VAD). An in vitro study using HL-1 cell line was performed to verify the regulatory role of the VAD-modified miRNA on the ADPN system. VAD implant did not affect circulating and cardiac levels of ADPN, but increased the cardiac mRNA expression of ADPN receptors, including AdipoR1, AdipoR2, and T-cad. AdipoR2 and T-cad were inversely related to the VAD-modified miRNA levels. The in vitro study confirmed the regulatory role of miR-1246 and miR-199b-5p on AdipoR2, and of miR-199b-5p on T-cad. These data suggest that VAD treatment could regulate the expression of the cardioprotective ADPN system by epigenetic mediators, suggesting that miRNAs have a potential role as therapeutic targets to improve cardiac function in HF pediatric patients.

Stability of circulating miRNA in saliva: The influence of sample associated pre-analytical variables

Background and aimsIncreasing evidence supports the practicability of salivary cell-free (cf) miRNA as liquid biopsy markers in cancers. Its successful translation in the clinical setting requires reproducible approaches for saliva manipulation, in order to control for pre-analytical variables influencing miRNA stability. This study aims to define the optimal conditions to maintain the integrity of saliva during collection, transport and processing with respect to cf-miRNA quantification. Materials and methodsSaliva was collected from 20 healthy subjects and 8 oral cancer patients. Two sampling methods were tested and different storage temperatures and times were evaluated. Salivary expression level of target miRNAs was quantified by qPCR. Comparison between group mean values at specific conditions were performed using paired t-tests. Agreement between measurements was evaluated using a Bland-Altman plot. ResultsDifferent collection methods revealed comparable levels of salivary miR-484 and miR-106b-5p in both subject cohorts. MiRNAs were stable for up to 48 h at 4 °C in saliva supernatant, showing significant alteration after 96 h. Mid-term storage of supernatant at −20 °C decreased miRNA stability significantly compared to standard −80 °C. ConclusionsCf-miRNA in saliva were slightly altered by collection methods and storage conditions, both in healthy and in pathological contexts, and remained stable for a period of time compatible with main clinical routine needs.

Growth-suppressor microRNAs mediate synaptic overgrowth and behavioral deficits in Fragile X mental retardation protein deficiency

Abnormal neuronal and synapse growth is a core pathology resulting from deficiency of the Fragile X mental retardation protein (FMRP), but molecular links underlying the excessive synthesis of key synaptic proteins remain incompletely defined. We find that basal brain levels of the growth suppressor let-7 microRNA (miRNA) family are selectively lowered in FMRP-deficient mice and activity-dependent let-7 downregulation is abrogated. Primary let-7 miRNA transcripts are not altered in FMRP-deficiency and posttranscriptional misregulation occurs downstream of MAPK pathway induction and elevation of Lin28a, a let-7 biogenesis inhibitor. Neonatal restoration of brain let-7 miRNAs corrects hallmarks of FMRP-deficiency, including dendritic spine overgrowth and social and cognitive behavioral deficits, in adult mice. Blockade of MAPK hyperactivation normalizes let-7 miRNA levels in both brain and peripheral blood plasma from Fmr1 KO mice. These results implicate dysregulated let-7 miRNA biogenesis in the pathogenesis of FMRP-deficiency, and highlight let-7 miRNA-based strategies for future biomarker and therapeutic development.

Characterization of MicroRNA expression profiles in the ovarian tissue of goats during the sexual maturity period

BackgroundThe ovary is an important reproductive organ in mammals, and its development directly affects the sexual maturity and reproductive capacity of individuals. MicroRNAs (miRNAs) are recognized as regulators of reproductive physiological processes in various animals and have been shown to regulate ovarian development through typical targeting and translational repression. However, little is known about the regulatory role of miRNAs in ovarian tissue development during sexual maturity in goats. To comprehensively profile the different physiological stages of sexual maturation in goats, we performed small-RNA sequencing of ovarian tissue samples collected at four specific time points (1 day after birth (D1), 2 months old (M2), 4 months old (M4), and 6 months old (M6)). In addition, we used ELISAs to measure serum levels of reproductive hormones to study their temporal changes.ResultsThe results showed that serum levels of gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, oestradiol, progesterone, oxytocin, and prolactin were lower in goats at the D1 stage than at the other three developmental stages (P < 0.05). The secretion patterns of these seven hormones show a similar trend, with hormone levels reaching their peaks at 4 months of age. A total of 667 miRNAs were detected in 20 libraries, and 254 differentially expressed miRNAs and 3 groups of miRNA clusters that had unique expression patterns were identified (|log2-fold change|> 1, FDR < 0.05) in the 6 comparison groups. RT‒qPCR was employed to confirm that the expression pattern of the 15 selected miRNAs was consistent with the Illumina sequencing results. Gene ontology analyses revealed significant enrichment of GO terms such as cell proliferation regulation, epithelial cell development, and amino acid transport, as well as important signaling pathways including the MAPK signaling pathway, the PI3K-Akt signaling pathway, and the oestrogen signaling pathway. Further miRNA‒mRNA regulation network analysis revealed that 8 differentially expressed miRNAs (chi-miR-1343, chi-miR-328-3p, chi-miR-877-3p, chi-miR-296-3p, chi-miR-128-5p, chi-miR-331-3p, chi-miR-342-5p and chi-miR-34a) have important regulatory roles in ovarian cell proliferation, hormone secretion and metabolism-related biological processes.ConclusionsOverall, our study investigated the changes in serum hormone and miRNA levels in the ovaries. These data provide a valuable resource for understanding the molecular regulatory mechanisms of miRNAs in ovarian tissue during the sexual maturity period in goats.

Qingjie Huagong decoction inhibits pancreatic acinar cell pyroptosis by regulating circHipk3/miR-193a-5p/NLRP3 pathway

BackgroundSafer and more effective drugs are needed for the treatment of acute pancreatitis (AP). Qingjie Huagong decoction (QJHGD) has been applied to treat AP for many years and has shown good clinical effects. However, the potential mechanism has not yet been determined. PurposeTo investigate the role and underlying mechanism of the effects of QJHGD on AP both in vitro and in vivo. MethodsQJHGD was characterized by UHPLC-Q-Orbitrap-MS. The protective effect of QJHDG and the underlying mechanism were investigated in MPC-83 cells in vitro. A caerulein-induced AP model was established to evaluate the protective effect of QJHGD in mice. CCK-8 assays were used to detect cell viability. The contents of inflammatory mediators were determined by ELISA. Expression levels of circRNA, miRNA and mRNA were determined by qRT-PCR. Protein expression was determined using Western blot. Pancreatic tissues were assessed by hematoxylin and eosin staining as well as immunohistochemical and immunofluorescence analyses. Pull-down and luciferase activity assays were performed to determine the regulatory relationships of circHipk3, miR-193a-5p and NLRP3. ResultsOur results confirmed that mmu-miR-193a-5p was sponged by mmu-circHipk3, and NLRP3 was a target of miR-193a-5p. In vitro experiments showed that QJHGD enhanced MPC-83 cell viability by regulating circHipk3 sponging mir-193a-5 targeting NLRP3 and inhibiting pyroptosis-related factors. Finally, we showed that QJHGD ameliorated pancreatic tissue injury in AP mice via this pathway. ConclusionThis study demonstrate that QJHDG exerted its anti-AP effects via the circHipk3/miR-193a-5p/NLRP3 pathway, revealing a novel mechanism for the therapeutic effect of QJHDG on AP.

A Glimmer of Hope for Patients with a T3 Transformation Zone: miRNAs Are Potential Biomarkers for Cervical Dysplasia.

This pilot study assesses the potential use of miRNAs in the triage of colposcopy patients with type 3 (nonvisible) cervical transformation zone (TZ). Type 3 TZ is a constitutional finding associated with many problems and controversies in colposcopy patient management. Here, we present miRNAs as a potential biomarker for the detection of CIN3 in these cases. Cervical mucosa samples (CMS) were collected from patients presenting with T3 transformation zone during routine workup using the Cytobrush. Depending on the histological and cytological result, as well as the result of the routinely performed HPV PCR, patients were divided into three groups: patients with a high-grade intraepithelial lesion (CIN3) and a positive high-risk HPV test (CIN3 group), patients without an intraepithelial lesion and a positive high-risk HPV test (HPV group), and healthy controls (N = no intraepithelial lesion and negative HPV test). The cervical mucus samples included in the study were tested for their expression levels of distinct miRNAs using qPCR. All investigated miRNAs were consistently detectable in every sample. The CMSs of histologically graded CIN 3 showed consistently high expression levels of all eight miRNAs, whereas the CMSs from healthy patients (N) show generally lower expression levels. However, CMSs from patients of the HPV group represented a very heterogeneous group. The data presented here can provide a solid basis for future research into a triage test for patients with a T3 transformation zone on the basis of commonly used clinical equipment.

Regulator-carrying dual-responsive integrated AuNP composite fluorescence probe for in situ real time monitoring apoptosis progression

Apoptosis is a typical programmed death mode with complex molecular regulation mechanisms. Developing advanced strategies to monitor apoptosis progression is conducive to disease treatment related with apoptosis. Herein, we developed a regulator-carrying dual-responsive integrated AuNP composite fluorescence probe for in situ real time monitoring apoptosis progression. The nanoprobe is constructed by modifying specially designed double-stranded DNA (dsDNA) and caspase 3-specific cleavable peptides (pep) to the surface of AuNP. After uptake by cells, the nanoprobe recognizes miRNA 21 and triggers fluorescence recovery, enabling silencing and imaging of the upstream signaling molecule miRNA 21. Once miRNA 21 is silenced, the downstream signaling molecule caspase 3 is activated and cleaves the substrate peptides, and fluorescence is restored for in situ imaging of caspase 3. The apoptosis induced by silencing miRNA 21 has been successfully implemented in HeLa and A549 cells. The expression level of miRNA 21 and corresponding changes of caspase 3 have also been effectively monitored. These results suggested this nanoprobe will be a potential tool for apoptosis-related biomedical research and clinical application.

Exploring the time-dependent regulatory potential of microRNAs in breast cancer cells treated with proteasome inhibitors

PurposeBreast cancer (BrCa) is a predominant type of cancer with a disparate molecular nature. MicroRNAs (miRNAs) have emerged as promising key players in the regulation of pathological processes in BrCa. Proteasome inhibitors (PIs) emerged as promising anticancer agents for several human malignancies, including BrCa, inhibiting the function of the proteasome. Aiming to shed light on the miRNA regulatory effect in BrCa after treatment with PIs, we used two PIs, namely bortezomib and carfilzomib.Materials and methodsFour BrCa cell lines of distinct molecular subtypes were treated with these PIs. Cell viability and IC50 concentrations were determined. Total RNA was extracted, polyadenylated, and reversely transcribed. Next, the levels of specific miRNAs with a significant role in BrCa were determined using relative quantification, and their regulatory effect was assessed.ResultsHigh heterogeneity was discovered in the levels of miRNAs in the four cell lines, after treatment. The miRNA levels fluctuate with distinct patterns, in 24, 48, or 72 hours. Interestingly, miR-1-3p, miR-421-3p, and miR-765-3p appear as key molecules, as they were found deregulated, in almost all combinations of cell lines and PIs. In the SK-BR-3 cell line, the majority of the miRNAs were significantly downregulated in treated compared to untreated cells, with miR-21-5p being the only one upregulated. Finally, various significant biological processes, molecular functions, and pathways were predicted to be affected.ConclusionsThe diversity of pathways predicted to be affected by the diversity in miRNA expression after treatment with PIs paves the way for the recognition of new regulatory axes in BrCa.

Restoration of Altered Oncogenic and Tumor Suppressor microRNA Expression in Breast Cancer and Colorectal Cancer Cell using Epicatechin.

MicroRNAs (miRNA) are small non-coding RNAs that regulate the function of mRNA post-transcriptionally in a tissue-specific manner. miRNA expressions are heavily dysregulated in human cancer cells through various mechanisms, including epigenetic changes, karyotype abnormalities, and miRNA biogenesis defects. miRNAs may act as either oncogenes or tumor suppressors under different conditions. Epicatechin is a natural compound found in green tea which possesses antioxidant and antitumor properties. The objective of this study is to investigate the effect of epicatechin treatment on the expression level of several oncogenic and tumor suppressor miRNAs in breast and colorectal cancer cell lines (MCF7 and HT-29) and identify its mechanism of action. The MCF-7 and HT29 cells were treated with epicatechin for 24 hours and untreated cells were considered control cultures. miRNA was isolated and qRT-PCR was used to measure the expression profile changes of different oncogenic and tumor suppressor miRNAs. Furthermore, the mRNA expression profile was also screened at different concentrations of epicatechin. Our results showed several-fold changes in miRNAs expression level, which is cell line specific. Also, epicatechin at different concentrations induces biphasic changes in mRNA expression levels in both cell lines. Our findings first time demonstrated that epicatechin can reverse the expression of these miRNAs and may trigger the cytostatic effect at a lower concentration.

DDR1 regulates CNS collagen IV expression and vascular fibrosis in AD to improve CNS vascular integrity

AbstractBackgroundBlood‐brain barrier breakdown, a feature associated with AD pathology and treatments, was examined as a potential therapeutic target in both genetic mouse models of AD and clinical trial data from human patients with AD treated with nilotinib.MethodsDDR1 knockout mice crossed with TgAPP mice were probed for levels of CNS collagen to examine the effects of DDR1 in vivo. CSF samples obtained from AD subjects treated with either nilotinib (n = 12) or placebo (n = 11) for 12 months were analyzed using whole genome RNA sequencing; significantly altered miRNA expression was examined for potential downstream effects regulating neurovascular integrity.ResultsCompared to TgAPP mice, staining of CNS sections for collagen IV in TgAPP/DDR1 knockout mice revealed decreased vascular collagen. CSF obtained from AD subjects treated with nilotinib revealed increased levels of miRNAs regulating collagen production, suggesting decreased vascular fibrosis and neuroinflammation, and improved neurovascular integrity.ConclusionDDR1 inhibition with nilotinib may improve neurovascular integrity in AD by reducing CNS collagen IV, vascular fibrosis, and neuroinflammation. Nilotinib should be considered as an adjunctive therapy to approved treatments ‐ including anti‐amyloid antibodies ‐ for individuals with AD.

CircRNAs mediate the effect of chronic lysosomal dysfunction on Alzheimer’s disease pathology

AbstractBackgroundCircular RNAs (circRNAs) and microRNAs (miRNAs) are non‐coding RNAs. CircRNAs modulate miRNA levels by sequestration. MiRNAs regulate amyloidogenic pathways, disrupting transcripts including APP, BACE1, and ADAM10. CircRNAs regulate key endolysosomal transcripts. Chronic lysosomal dysfunction (CLD) is linked to Alzheimer’s Disease (AD). However, the role circRNAs downstream of CLD and AD is unclear. Here, we tested whether CLD affects the amyloidogenic pathway in 5xFAD mice through circRNAs.MethodCortical circRNA expression was obtained of 5xFAD, PPT1+/−, 5xFAD: PPT1+/− (P5X), Naglu+/−, 5xFAD: Naglu+/− (N5X) mice. TruSeq Stranded total RNA was extracted with Ribo‐Zero to deplete rRNA and generate stranded‐RNA‐seq. Libraries were sequenced on a NextSeq 500 platform of Illumina and analysis was done with the DESeq2 package with an FRD &lt;0.05. In silico microRNA binging was done using circAtlas 2.0 browser. The insoluble Aβ‐40 and Aβ‐42 were quantified by ELISA in the hippocampal fraction. The Aβ plaque load was quantified in coronal brain sections using immunohistochemistry.ResultThe P5X and N5X mice exhibit increased amyloid plaque load, hippocampal Aβ‐40, and Aβ‐42 levels, and reduced lifespan compared to 5xFAD mice. We detected 1286 cicRNAs in all the groups. 38 from P5X and N5X groups, 37 from PPT1+/−, and 29 from Naglu+/− passed FRD correction compared with 5xFAD mice. Both linear and circRNAs of 4933406I18Rik, Zfp609, Zfp532, and Nnt changed between PPT1+/− and P5X compared to 5xFAD. The levels of circAdam10, circPan3, circMbtd1, circ4930402H24Rik, circSt6gal2, and circCdc14b were reduced, while circZranb1 and circMyo9a were increased in P5X and N5X compared with 5xFAD. CircNlgn1 levels changed only downstream of PPT1+/−. In silico analyses show amyloidogenic‐related microRNAs exhibit circRNAs binding sites. MiR‐361‐3p has binding sites for circZranb1 and circADAM10, as well as miR‐298‐3p, can bind circNlgn1. Both miR‐361‐3p and miR‐298‐3p trap and inhibit BACE1 function and Aβ production, interfering in the amyloidogenic pathway. CircPan3 regulates the autophagy‐related miR‐421. Experimental evidence shows that overexpression of circPan3 suppresses autophagy through a miR‐421/Pink1 pathway.ConclusionCLD (PPT1+/− and Naglu+/−) boosted AD pathology and induced differential changes in circRNAs levels affecting amyloidogenic and autophagy pathways. There is a positive feedback loop between CLD and circRNAs in AD mouse models.

Diagnostic value of the combination of circulating serum miRNAs and CA125 in endometriosis.

Circulating microRNAs (miRNAs) can serve as noninvasive biomarkers for endometriosis, but their diagnostic and prognostic values require investigation. This study evaluated the potential of 6 miRNAs in diagnosing endometriosis and predicting fertility. The study included patients with endometriosis (stages I-IV) and controls admitted to Sun Yat-Sen Memorial Hospital between May 2013 and March 2014. The serum expression levels of 6 miRNAs (miR-199a, miR-145*, miR-122, miR-9*, miR-141*, and miR-542-3p) were determined using qRT-PCR. Receiver operating characteristics curves were used to determine the diagnostic accuracy. The study included 155 patients with endometriosis and 77 controls. The model combining miR-199a, miR-122, miR-145*, and miR-141* with the carbohydrate antigen 125 (CA125) exhibited 81.8% sensitivity and 92.6% specificity and an area under the curve of 0.939 for diagnosing endometriosis. When combining miR-199a, miR-122, miR-145*, miR-542-3p, and CA125, the receiver operating characteristics curve showed an area under the curve of 0.759 and 79.6% sensitivity and 73.5% specificity for stage I/II versus III/IV endometriosis. Circulating miRNA levels were associated with pelvic adhesions (miR-199a, P < .05), lesion distribution (miR-9*, miR-145*, and miR-141*; all P < .05), and the presence of deep infiltrating endometriosis (miR-199a and miR-122; both P < .001). The expression levels of miR-199a, miR-122, and miR-542-3p decreased with an increasing endometriosis fertility index. The model combining circulating miRNAs (miR-199a, miR-122, miR-145*, and miR-141*) and CA125 is promising for diagnosing endometriosis and its severity. miR-199a, miR-122, and miR-542-3p were associated with the endometriosis fertility index and might be used to predict fertility preoperatively, but these results require confirmation.

Comprehensive analysis of miRNA-mRNA regulatory pairs associated with colorectal cancer and the role in tumor immunity

BackgroundMicroRNA (miRNA) which can act as post-transcriptional regulators of mRNAs via base-pairing with complementary sequences within mRNAs is involved in processes of the complex interaction between immune system and tumors.In this research, we elucidated the profiles of miRNAs and target mRNAs expression and their associations with the phenotypic hallmarks of colorectal cancers (CRC) by integrating transcriptomic, immunophenotype, methylation, mutation and survival data.ResultsWe conducted the analysis of differential miRNA/mRNA expression profile by GEO, TCGA and GTEx databases and the correlation between miRNA and targeted mRNA by miRTarBase and TarBase. Then we detected using qRT-PCR and validated the diagnostic value of miRNA-mRNA regulator pairs by the ROC, calibration curve and DCA. Phenotypic hallmarks of regulatory pairs including tumor-infiltrating lymphocytes, tumor microenvironment, tumor mutation burden, global methylation and gene mutation were also described. The expression levels of miRNAs and target mRNAs were detected in 80 paired colon tissue samples. Ultimately, we picked up two pivotal regulatory pairs (miR-139-5p/ STC1 and miR-20a-5p/ FGL2) and verified the diagnostic value of the complex model which is the combination of 4 signatures above-mentioned in 3 testing GEO datasets and an external validation cohort.ConclusionsWe found that 2 miRNAs by targeting 2 metastasis-related mRNAs were correlated with tumor-infiltrating macrophages, HRAS, and BRAF gene mutation status. Our results established the diagnostic model containing 2 miRNAs and their respective targeted mRNAs to distinguish CRCs and normal controls and displayed their complex roles in CRC pathogenesis especially tumor immunity.

Multiplex miRNA reporting platform for real-time profiling of living cells

Accurately characterizing cell types within complex cell structures provides invaluable information for comprehending the cellular status during biological processes. In this study, we have developed an miRNA-switch cocktail platform capable of reporting and tracking the activities of multiple miRNAs (microRNAs) at the single-cell level, while minimizing disruption to the cell culture. Drawing on the principles of traditional miRNA-sensing mRNA switches, our platform incorporates subcellular tags and employs intelligent engineering to segment three subcellular regions using two fluorescent proteins. These designs enable the quantification of multiple miRNAs within the same cell. Through our experiments, we have demonstrated the platform's ability to track marker miRNA levels during cell differentiation and provide spatial information of heterogeneity on outlier cells exhibiting extreme miRNA levels. Importantly, this platform offers real-time and in situ miRNA reporting, allowing for multidimensional evaluation of cell profile and paving the way for a comprehensive understanding of cellular events during biological processes.

Investigation of the miRNA levels changes to acceptable daily intake dose pesticide mixture exposure on rat mesentery and pancreas

Consumers are constantly exposed to a variety of chemical mixtures as part of their everyday activities and lifestyle. Food, water and commercial products are only some examples of the possible ways people get exposed to these mixtures. However, following federal and local guidelines for risk assessment related to chemical exposure, risk analysis focuses on a single substance exposure scenario and not on a mixture, as in real life. Realizing the pronounced gap of this methodology, the real-life risk simulation scenario approach tries to address this problem by investigating the possible effect of long-term exposure to chemical mixtures closely resembling the actual circumstances of modern life. As part of this effort, this study aimed to identify the cumulative effects of pesticides belonging to different classes and commonly used commercial products on long-term exposure with realistic doses. Sprague Dawley rats were given a pesticide mix of active ingredients and formulation chemicals in a daily acceptable dose (ADI) and 10xADI for 90 days. Following thorough everyday documentation of possible side-effects, after 90 days all animals were sacrificed and their organs were examined. Exposure to pesticides particularly affects the miRNA levels at that point will provide us with more information about whether they can be potential biomarkers.

Activin-a Increases B-Cell Acute Lymphoblastic Leukemia (B-ALL) Vesiculation and Modifies the Micro-RNA Cargo: Special Focus on MiR-491-5p

We recently demonstrated that ActivinA (ActA) is abundant in the leukemic bone marrow microenvironment where it exerts a pro-leukemic action by increasing the migratory and invasive properties of B-ALL cells. Extracellular vesicles (EV) are a new way of intercellular communication. They can be categorized in small-EV (sEV) and large-EV (lEV) based on size and origin. Similarly to migration, EV release is dependent upon cytoskeleton activation and membrane remodeling. EV play a prominent role in carcinogenesis, depending on the molecules they enclose, including miRNA that can downmodulate the expression of target mRNAs in recipient cells. The aim of the project was to study the impact of ActA on B-ALL vesiculation: quantity and miRNA cargo. Moreover, we investigated whether deregulated miRNAs could contribute to ActA-mediated leukemic progression. To investigate the role of ActA on B-ALL vesiculation, we used 697 and Nalm-6 cells lines. By means of nanoparticle tracking analysis, we demonstrated that after 24h, ActA significantly increased of 2.5 and 1.4 folds the number of sEV (p&amp;lt;0.0001) and lEV (p&amp;lt;0.0026, n=9) produced by 697 cells, compared to not stimulated cells (NS). A significant increase of both EV was also observed after 48h of ActA stimulation. The ability of ActA to promote B-ALL cell vesiculation was also confirmed on Nalm-6. Furthermore, we investigated the miRNA cargo of EV derived from 697 cells stimulated or not with ActA by OpenArray. After 24h, ActA significantly upregulated miR491-5p, let-7i-3p, and downregulated miR-639, miR135b-3p and miR139-5p. On the other hand, after 48h, ActA significantly upregulated miR15b-3p, let-7-e-5p, miR23b-3p and downregulated miR1236-3p, miR-18a-3p. We focused on miR491-5p, the most upregulated in ActA EV (3 folds at 24h), compared to NS (p&amp;lt;0.0001). ActA-mediated increase of miR491-5p was observed also in 697 cells (FC=1.5, p&amp;lt;0.0001, n=14). To investigate miR491-5p modulation also in Nalm-6 cells, they were stimulated or not for 24h and miRNA levels were evaluated in cells and their EV, isolated by ultracentrifugation. We preliminary observed that ActA increased miRNA expression of 479 folds in Nalm-6 EV (p=0.09, n=3), but only slightly in cells. Interestingly, absolute miRNA levels were higher of 4 folds in NS EV compared to NS cells (p=0.05), and showed an increasing trend in stimulated condition (FC=1552, p=0.09, n=3), suggesting that upon ActA stimulation, miR491-5p is upregulated and mostly encapsulated in EV. It has been described that miR491-5p promotes cell survival and proliferation in solid tumors. Indeed, we investigated whether ActA could impact on B-ALL cell viability through miR491-5p modulation. Indeed, 697 cells were stimulated every 3 days with ActA. Cell counts showed no differences between cells stimulated or not in the exponential growth phase. Interestingly, at plateau (day 7), ActA-stimulated cells were more viable (76%, range: 69%-80%) compared to NS cells (56%, range: 50%-66%, p=0.05, n=3). Alongside, we preliminary observed higher miR491-5p in ActA-stimulated cells (FC=3, n=1). It has also been reported that miR491-5p regulates chemoresistance in several cancers. Interestingly, by using Annexin-V/7-AAD assay, we demonstrated that ActA-stimulated 697 cells had a viability advantage of 11.3% (p=0.006, n=3), compared to NS cells, after 72h of ASNase-based chemotherapy, suggesting a chemoprotective role of ActA in B-ALL. Moreover, we observed that the combination of ActA and ASNase treatments induced the overexpression of miR491-5p compared to NS (FC=2.5, p=0.001) and to cells stimulated with ActA alone (FC=2.3, p=0.04) or ASNase alone (FC=1.4, p=0.004, n=8). To investigate the possible role of miR491-5p, we downregulated it into 697 cells by transfecting a miR491-5p inhibitor. Interestingly, under ASNase treatment, ActA still induced a viability advantage in cell transfected with a random inhibitor (negative control, 9.8%, p=0.006), comparable to untransfected cells. On the contrary, in miR491-5p inhibitor transfected cells, the viability advantage mediated by ActA decreased to 5.9% (p=0.03). Therefore, miR491-5p inhibition reduced of 30% the chemoprotective ability of ActA (p=0.04, n=3). In conclusion, these results suggest that ActA not only increases EV production, but also modifies their miRNA cargo and that miR491-5p could be one of the key players involved in ActA-mediated pro-leukemic action.