MicroRNA Analysis of In Vitro Differentiation of Spermatogonial Stem Cells Using a 3D Human Testis Organoid System.

Spermatogenesis produces male gametes from spermatogonial stem cells (SSC), beginning at puberty. Modern-day laboratory techniques allow for the long-term culture of SSC and in vitro spermatogenesis. The specific biochemical processes that occur during spermatogenesis remain poorly understood. One particular element of spermatogenesis that has yet to be characterized is the role of microRNAs (miRNA), short, non-transcribed RNAs that act as post-translational regulators of gene activity. In this study, we seek to describe the presence of miRNA in a two-dimensional (2D) SSC culture and a 3D human testis organoid (HTO) system. Testicular cells were isolated from the frozen tissue of three brain-dead subjects, propagated in cultures for four to five weeks, and used to form 3D HTOs. Following organoid formation, differentiation of testicular cells was induced. RNA was isolated from the whole testis tissue (WT) showing in vivo conditions, HTO Day Zero (2D SSC culture), Day 2 HTOs, and Day 23 differentiated HTOs, then analyzed for changes in miRNA expression using the Nanostring nCounter miRNA panel. One hundred ninety-five miRNAs met the criteria for expression in WT, 186 in 2D culture, 190 in Day 2 HTOs, and 187 in differentiated HTOs. One hundred thirty-three miRNAs were common across all conditions, and 41, 17, 6, and 11 miRNAs were unique for WT, 2D culture, Day 2 HTOs, and differentiated HTOs, respectively. Twenty-two miRNAs were similar between WT and differentiated HTOS. We evaluated the miRNA expression profiles of progressively complex stages of testicular cell culture, culminating in a 3D organoid model capable of meiotic differentiation, and compared these to WT. We identified a great variance between the native tissue and the culture system; however, some miRNAs are preserved. These data may provide avenues for deeper understanding of spermatogenesis and the ability to improve this process in the laboratory. Research on miRNA continues to be an essential avenue for understanding human spermatogenesis.

G2/M Checkpoint Modulation: Insights from miRNA Profiles in FAM and Breast Cancer.

OBJECTIVE: The aim of this research is to understand the role of microRNA in cell cycle regulation especially on G2M Checkpoint from Luminal A samples Indonesian population. The profile results are used as biomarkers and therapeutic targets for breast cancer. For this reason, analysis was carried out on the comparison of miRNA expression between Luminal A and Fibroadenoma  mamae (FAM) using Nanostring nCounter. METHODS: In this study, 5 (Formalin-Fixed Paraffin-Embedded) FFPE Luminal A tissues and 4 FFPE FAM samples were used. RNA was isolated from cancer tissue samples. Differential expression analysis of miRNA was conducted using Nanostring nCounter technology, subsequently followed by the expression analysis between FAM and Luminal A using nSolver softwere. Elevated expression levels of miRNAs were subjected to pathway and gene regulation analysis using KEGG and GSEA MsigDB databases. Data visualization was performed utilizing Cytoscape, NetworkAnalyst, and SRplot tools. RESULT: Based on 792 miRNAs detected on Nanostring nCounter, it was found that 60 miRNAs were upregulated and 6 miRNAs were downregulated. The 15 upregulated miRNAs analyzed show their role in the G2M Checkpoint through several pathways. The five miRNAs that significantly regulate the G2M Checkpoint are hsa-miR-196b-5p, hsa-miR-218-5p, hsa-miR-7-5p, hsa-miR-19a-5p, and hsa-miR-18a-5p Where each of these miRNAs regulates the CDKN1B gene. CONCLUSION: Significant differences in the expression of multiple miRNAs between Luminal A and FAM samples were observed. Furthermore, several of these miRNAs were found to modulate the G2M Checkpoint in Luminal A cancer by suppressing tumor suppressor genes.

miRNA profiling of B16F10 melanoma cell exosomes reveals melanin synthesis-related genes.

This study investigates the communication between skin cells, specifically melanocytes, keratinocytes, and fibroblasts, which is crucial for the process of melanin production known as melanogenesis. We aimed to understand the role of melanocyte exosomes in regulating melanogenesis and to uncover the microRNAs influencing this process. We isolated exosomes and characterized them using advanced microscopy and protein analysis to achieve this. We conducted experiments on melanoma cells to study melanin production regulation and examined how exosomes influenced gene expression related to melanogenesis. The results revealed that melanocyte exosomes increased certain types of tyrosinases, thereby enhancing melanin production. Furthermore, we acquired the miRNA profile of exosomes and hypothesized that specific siRNAs, such as miR-21a-5p, could potentially facilitate melanin synthesis. Our findings shed light on the importance of exosomes in skin health and provide valuable insights into intercellular communication mechanisms. Understanding these processes can pave the way for innovative therapies to treat melanin-related disorders and maintain healthy skin.

Combined TP53 status in tumor-free resection margins and circulating microRNA profiling predicts the risk of locoregional recurrence in head and neck cancer.

Locoregional recurrences represent a frequently unexpected problem in head and neck squamous cell carcinoma (HNSCC). Relapse often (10-30%) occurs in patients with histologically negative resection margins (RMs), probably due to residual tumor cells or hidden pre-cancerous lesions in normal mucosa, both missed by histopathological examination. Therefore, definition of a 'clean' or tumor-negative RM is controversial, demanding for novel approaches to be accurately explored. Here, we evaluated next generation sequencing (NGS) and digital PCR (dPCR) as tools to profile TP53 mutational status and circulating microRNA expression aiming at scoring the locoregional risk of recurrence by means of molecular analyses. Serial monitoring of these biomarkers allowed identifying patients at high risk, laying the ground for accurate tracking of disease evolution and potential intensification of post-operative treatments. Additionally, our pipeline demonstrated its applicability into the clinical routine, being cost-effective and feasible in terms of patient sampling, holding promise to accurately (re)-stage RMs in the era of precision medicine.

Time-resolved Small-RNA Sequencing Identifies MicroRNAs Critical for Formation of Embryonic Stem Cells from the Inner Cell Mass of Mouse Embryos.

Cells of the inner cell mass (ICM) acquire a unique ability for unlimited self-renewal during transition into embryonic stem cells (ESCs) in vitro, while preserving their natural multi-lineage differentiation potential. Several different pathways have been identified to play roles in ESC formation but the function of non-coding RNAs in this process is poorly understood. Here, we describe several microRNAs (miRNAs) that are crucial for efficient generation of mouse ESCs from ICMs. Using small-RNA sequencing, we characterize dynamic changes in miRNA expression profiles during outgrowth of ICMs in a high-resolution, time-course dependent manner. We report several waves of miRNA transcription during ESC formation, to which miRNAs from the imprinted Dlk1-Dio3 locus contribute extensively. In silico analyses followed by functional investigations reveal that Dlk1-Dio3 locus-embedded miRNAs (miR-541-5p, miR-410-3p, and miR-381-3p), miR-183-5p, and miR-302b-3p promote, while miR-212-5p and let-7d-3p inhibit ESC formation. Collectively, these findings offer new mechanistic insights into the role of miRNAs during ESC derivation.

Small-Extracellular-Vesicle-Derived miRNA Profile Identifies miR-483-3p and miR-326 as Regulators in the Pathogenesis of Antiphospholipid Syndrome (APS).

Primary antiphospholipid syndrome (PAPS) is a systemic autoimmune disease associated with recurrent thrombosis and/or obstetric morbidity with persistent antiphospholipid antibodies (aPL). Although these antibodies drive endothelial injury and thrombophilia, the underlying molecular mechanism is still unclear. Small extracellular vesicles (sEVs) contain miRNAs, key players in intercellular communication. To date, the effects of miRNA-derived sEVs in PAPS are not well understood. We characterised the quantity, cellular origin and miRNA profile of sEVs isolated from thrombotic APS patients (PAPS, n = 50), aPL-carrier patients (aPL, n = 30) and healthy donors (HD, n = 30). We found higher circulating sEVs mainly of activated platelet origin in PAPS and aPL patients compared to HD, that were highly engulfed by HUVECs and monocyte. Through miRNA-sequencing analysis, we identified miR-483-3p to be differentially upregulated in sEVs from patients with PAPS and aPL, and miR-326 to be downregulated only in PAPS sEVs. In vitro studies showed that miR-483-3p overexpression in endothelial cells induced an upregulation of the PI3K-AKT pathway that led to endothelial proliferation/dysfunction. MiR-326 downregulation induced NOTCH pathway activation in monocytes with the upregulation of NFKB1, tissue factor and cytokine production. These results provide evidence that miRNA-derived sEVs contribute to APS pathogenesis by producing endothelial cell proliferation, monocyte activation and adhesion/procoagulant factors.

Identifying Biomarkers for Osteogenic Potency Assay Development.

There has been extensive exploration of how cells may serve as advanced therapy medicinal products to treat skeletal pathologies. Osteoblast progenitors responsible for production of extracellular matrix that is subsequently mineralized during bone formation have been characterised as a rare bone marrow subpopulation of cell culture plastic adherent cells. Conveniently, they proliferate to form single-cell derived colonies of fibroblastoid cells, termed colony forming unit fibroblasts that can subsequently differentiate to aggregates resembling small areas of cartilage or bone. However, donor heterogeneity and loss of osteogenic differentiation capacity during extended cell culture have made the discovery of reliable potency assay biomarkers difficult. Nonetheless, functional osteoblast models derived from telomerised human bone marrow stromal cells have allowed extensive comparative analysis of gene expression, microRNA, morphological phenotypes and secreted proteins. This chapter highlights numerous insights into the molecular mechanisms underpinning osteogenic differentiation of multipotent stromal cells and bone formation, discussing aspects involved in the choice of useful biomarkers for functional attributes that can be quantitively measured in osteogenic potency assays.

Exploring conserved and novel MicroRNA-like small RNAs from stress tolerant Trichoderma fusants and parental strains during interaction with fungal phytopathogen Sclerotium rolfsii Sacc.

The study investigated potential microRNA-like small RNAs (milRNAs) from multi-stress-tolerant Tricho-fusants and parental strains (P1- Trichoderma virens NBAIITvs12 and P2- Trichoderma koningii MTCC796) for antagonistic activity during interaction with phytopathogen Sclerotium rolfsii. The Trichoderma was cultured in-vitro, with and without antagonism, against the pathogen and total RNA was extracted followed by small RNA library construction and sequencing. The milRNAs were identified by mapping high-quality unique reads against a reference genome. The milRNAs were recognized higher in antagonist Trichoderma during interaction with test pathogen compared to normal growth. The novel milRNAs candidates were found to vary during interaction with the pathogen and normal growth. The gene ontology and functional analysis illustrated that a total of 5828 potential targeted genes were recognized for 93 milRNAs of potent Fu21_IB and 3053 genes for 62 milRNAs of least fusant Fu28_IL. Functional annotation of milRNA-predicted genes integrating KEGG pathways indicates new insights into regulatory mechanisms, by interfering with milRNAs, associated with signal transduction, amino sugar metabolism, benzoate degradation, amino acid metabolism, and steroid and alkaloid metabolism for potential biocontrol of stress-tolerant Tricho-fusant FU21 during interaction with S. rolfsii. The present investigation is the first report of conserved and novel milRNAs from Tricho-fusants and parental strains interacting with S. rolfsii.

A Major Downregulation of Circulating microRNAs in Zika Acutely Infected Patients: Potential Implications in Innate and Adaptive Immune Response Signaling Pathways.

Zika virus (ZIKV) is an arbovirus mainly transmitted by mosquitos of the genus Aedes. The first cases of ZIKV infection in South America occurred in Brazil in 2015. The infection in humans causes diverse symptoms from asymptomatic to a syndrome-like dengue infection with fever, arthralgia, and myalgia. Furthermore, ZIKV infection during pregnancy is associated with fetal microcephaly and neurological disorders. The identification of host molecular mechanisms responsible for the modulation of different signaling pathways in response to ZIKV is the first step to finding potential biomarkers and therapeutic targets and understanding disease outcomes. In the last decade, it has been shown that microRNAs (miRNAs) are important post-transcriptional regulators involved in virtually all cellular processes. miRNAs present in body fluids can not only serve as key biomarkers for diagnostics and prognosis of human disorders but also contribute to cellular signaling offering new insights into pathological mechanisms. Here, we describe for the first time ZIKV-induced changes in miRNA plasma levels in patients during the acute and recovery phases of infection. We observed that during ZIKV acute infection, among the dysregulated miRNAs (DMs), the majority is with decreased levels when compared to convalescent and control patients. We used systems biology tools to build and highlight biological interactions between miRNAs and their multiple direct and indirect target molecules. Among the 24 DMs identified in ZIKV + patients, miR-146, miR-125a-5p, miR-30-5p, and miR-142-3p were related to signaling pathways modulated during infection and immune response. The results presented here are an effort to open new vistas for the key roles of miRNAs during ZIKV infection.

Proteomic analysis and microRNA expression profiling of plasma-derived exosomes in primary immune thrombocytopenia.

Exosomes are released into extracellular fluids and have emerged as vital biological mediators in autoimmune diseases. Plasma-derived exosomes have been reported to take part in the pathogenesis of primary immune thrombocytopenia (ITP), but the protein and miRNA cargoes have not been entirely elucidated. Via proteomic analysis and RNA sequencing on plasma-derived exosomes from ITP patients and healthy controls, we found one upregulated exosomal protein (apolipoprotein E, ApoE), six downregulated exosomal miRNAs (miR-584-5p, miR-4433a-5p, miR-4433b-3p, miR-6842-3p, miR-130b-5p and miR-222-3p), and 10 upregulated exosomal miRNAs (miR-29a-3p, miR-142-5p, miR-16-2-3p, miR-29b-3p, miR-501-3p, miR-144-5p, miR-192-5p, miR-182-5p, miR-363-3p and miR-96-5p) in ITP patients. The elevated exosomal protein candidate ApoE in the ITP cohort was further validated using western blot. Via quantitative real-time polymerase chain reaction assays, three differentially expressed miRNAs (miR-584-5p, miR-142-5p and miR-29b-3p) were identified. This study provides direct evidence for a restricted signature of exosomal protein and miRNAs which distinguishes ITP from healthy controls. The results require further validation in larger independent ITP cohorts, which will provide insights into the potential pathophysiological significance of circulating exosomes in ITP.

Silver birch pollen-derived microRNAs promote NF-κB-mediated inflammation in human lung cells.

The pollen of Betula pendula Roth (silver birch) is considered to be the main cause of allergy-related rhinitis in Europe and its protein-based allergens such as Bet v 1 are well characterized. However, little is known about non-protein components of birch pollen, e.g., small RNAs and their proinflammatory activity. In the present study, next-generation sequencing (NGS) and bioinformatic approaches were used for silver birch pollen (SBP)-derived microRNA profiling and evaluation of microRNA target genes and pathways in human. Human lung cells, namely WI-38 fibroblasts and A549 alveolar epithelial cells were then stimulated with SBP microRNA in vitro and imaging cytometry-based analysis of the levels of proinflammatory cytokines, autophagy parameters and small RNA processing regulators was conducted. Bioinformatic analysis revealed that SBP microRNA may interfere with autophagy, inflammation and allergy pathways in human. SBP and SBP-derived microRNA induced NF-κB-mediated proinflammatory response in human lung cells as judged by increased levels of NF-κB p65, IL-8 and TNFα. NSUN2 and NSUN5 were involved in pollen-derived microRNA processing. Pollen-derived microRNA also modulated autophagic pathway by changes in the pools of LC3B and p62 that may affect autophagy-based adaptive responses during allergic lung inflammation. We postulate that SBP-derived microRNAs can be considered as novel proinflammatory environmental agents.

MiR-451a enhances the phagocytosis and affects both M1 and M2 polarization in macrophages.

Leukemia associated macrophages (LAMs), which are different from tumor-associated macrophages as well as classical M1 and M2 macrophages, are specifically activated by leukemic microenvironment. We have reported the heterogeneity of gene expression profiles in LAMs. However, the expression profiles of microRNA (miRNA) in LAMs and their regulatory mechanisms have not been established. Here, the expression profiles of miRNA in LAMs from bone marrow and spleen of acute myeloid leukemia mice were analyzed. Then, the effects of miR-451a, which was upregulated in LAMs, on macrophages were studied by transfecting miRNA mimic to peritoneal macrophages. The results showed that overexpression of miR-451a altered the morphology, enhanced the phagocytic ability of macrophages, and promotes the expression of differentiation marker CD11b in macrophages. Furthermore, miR-451a increased the proliferation capacity of both M1- and M2-polarized macrophages, but not M0 macrophages. Moreover, miR-451a further enhanced the expression of iNOS upon M1 activation. Therefore, our results reveal the miRNA expression profiles in LAMs, and broaden the knowledge about miRNA regulation in macrophages.

Comparative Small RNA Profiling and Functional Exploration on Wheat With High- and Low-Cadmium Accumulation.

Cadmium is a toxic metal widely found in workplaces and plant soil because of extensive industrialization. Wheat is an important source of food generated from plant soil. The different responses of wheat against different omic levels of cadmium have been observed and widely studied worldwide. With the development of high-throughput sequencing, micro-level biological research has extended to the microRNA level. In this study, high-cadmium-accumulating wheat cultivars (Annong9267) and low-cadmium-accumulating wheat cultivars (Qian 102032) were used as experimental models. The two cultivars were treated by Cd for 2 h to explore the microRNA profiles in root and leaf tissues through small RNA sequencing. Important small RNAs, such as tae-miR9663-5p and tae-miR6201, and potential small RNA-mediated mechanisms associated with cadmium accumulation were identified by summarizing specific microRNA profiling patterns and their respective target genes. At the wheat roots and leaves, differentially expressed small RNAs related to cadmium accumulation in different plant tissues (roots or leaves) were identified, and functional enrichment analyses on target genes of differentially expressed miRNAs in low- and high-cadmium-accumulating wheat cultivars in different plant tissues (roots or leaves) obtained some known mature miRNAs and new miRNAs. The identified miRNA will be regarded as a potential screening biomarker for low-cadmium-accumulating wheat.

Sedentary and Trained Older Men Have Distinct Circulating Exosomal microRNA Profiles at Baseline and in Response to Acute Exercise.

Exercise has multi-systemic benefits and attenuates the physiological impairments associated with aging. Emerging evidence suggests that circulating exosomes mediate some of the beneficial effects of exercise via the transfer of microRNAs between tissues. However, the impact of regular exercise and acute exercise on circulating exosomal microRNAs (exomiRs) in older populations remains unknown. In the present study, we analyzed circulating exomiR expression in endurance-trained elderly men (n = 5) and age-matched sedentary males (n = 5) at baseline (Pre), immediately after a forty minute bout of aerobic exercise on a cycle ergometer (Post), and three hours after this acute exercise (3hPost). Following the isolation and enrichment of exosomes from plasma, exosome-enriched preparations were characterized and exomiR levels were determined by sequencing. The effect of regular exercise on circulating exomiRs was assessed by comparing the baseline expression levels in the trained and sedentary groups. The effect of acute exercise was determined by comparing baseline and post-training expression levels in each group. Regular exercise resulted in significantly increased baseline expression of three exomiRs (miR-486-5p, miR-215-5p, miR-941) and decreased expression of one exomiR (miR-151b). Acute exercise altered circulating exomiR expression in both groups. However, exomiRs regulated by acute exercise in the trained group (7 miRNAs at Post and 8 at 3hPost) were distinct from those in the sedentary group (9 at Post and 4 at 3hPost). Pathway analysis prediction and reported target validation experiments revealed that the majority of exercise-regulated exomiRs are targeting genes that are related to IGF-1 signaling, a pathway involved in exercise-induced muscle and cardiac hypertrophy. The immediately post-acute exercise exomiR signature in the trained group correlates with activation of IGF-1 signaling, whereas in the sedentary group it is associated with inhibition of IGF-1 signaling. While further validation is needed, including measurements of IGF-1/IGF-1 signaling in blood or skeletal muscle, our results suggest that training status may counteract age-related anabolic resistance by modulating circulating exomiR profiles both at baseline and in response to acute exercise.

miR-582-5p serves as an antioncogenic biomarker in intermediate risk AML with normal cytogenetics and could inhibit proliferation and induce apoptosis of leukemia cells.

Numerous studies confirmed that aberrant microRNA (miRNA) expression contributes to cancer development and progression. We carried out this study to explore the expression profile of miRNAs in intermediate risk acute myeloid leukemia (AML) and locate certain miRNAs as biomarkers. We profiled differentially expressed miRNAs by performing miRNA sequencing analysis in the patients' samples. Bioinformatic analysis showed the most significantly expressed genes mostly involved in cellular component organization, cell differentiation, and cell development. Reverse-transcription polymerase chain reaction validated the expression of miR-582-5p in different groups of AML samples. It was confirmed that miR-582-5p was downregulated in newly diagnosed AML and relapse/refractory AML compared with CR AML or controls. Among intermediate risk AML patients with normal cytogenetics, a lower level of miR-582-5p is correlated with an unfavorable outcome, and a shorter overall survival. Gain- and loss-of-function experiments revealed that miR-582-5p could inhibit proliferation, suppress migration, and invasion ability and induce apoptosis of leukemia cells. Furthermore, overexpression of miR-582-5p can increase sensitivity of cells to Ara-C. In conclusion, miR-582-5p can serve as an antioncogenic biomarker in intermediate risk AML with normal cytogenetics for risk classification and outcome prediction. These results showed a novel role for miR-582-5p in predicting the prognosis and promoting the tumor growth of AML.

Identification of Cerebrospinal Fluid MicroRNAs Associated With Leptomeningeal Metastasis From Lung Adenocarcinoma.

Background: Leptomeningeal metastasis (LM) has frequently been observed in patients with lung adenocarcinoma. So far, its diagnosis and disease course monitoring are still extremely difficult. Moreover, there is no effective treatment regimen for LM due to a lack knowledge on the molecular mechanism of LM. This study aimed to identify LM-related cerebrospinal fluid (CSF) miRNAs, which have potential value for diagnosing and monitoring LM and exploring the molecular mechanism. Methods: CSF miRNAs were screened and verified by microarray analysis and quantitative real-time PCR (qRT-PCR) in LM patients with lung adenocarcinoma and non-LM controls, and the diagnostic performance of candidate miRNAs was evaluated. Then, candidate miRNAs in matched CSF samples from LM patients at diagnosis, after initial therapy, at relapse, and after salvage therapy, were analyzed to assess the relationship between CSF miRNAs and LM disease course. The effect of candidate miRNAs on proliferation, invasion, and migration of lung adenocarcinoma cell lines was assessed. The targeted genes of the candidate miRNA were predicted by TargetScan, miRDB, and miRTarbase online analysis tools. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the functional categories of predicted target genes. Results: CSF miR-7975, miR-7977, and miR-7641 were screened and verified to be statistically significantly up-regulated in LM patients compared to non-LM controls. The three miRNAs, when combined, exhibited optimal diagnostic performance. Longitudinal data of CSF miR-7975 and miR-7977 correlated well with clinical courses of LM. Overexpression of miR-7977 promoted proliferation, migration, and invasion of lung adenocarcinoma cells. Moreover, 385 targeted genes of miR-7977 were predicted and were involved in various pathways related to cancer metastasis. Conclusions: This study offers insights for future research of CSF miRNAs as robust tools for diagnosing and monitoring LM. It also reveals a novel pathway for exploration of underlying mechanisms of LM.

Identification of differentially expressed miRNAs associated with thermal injury in epidermal stem cells based on RNA-sequencing.

Current research indicates that epidermal stem cells (EpSCs) play an important role in promoting wound healing, but the mechanism of action of these cells during wound repair following thermal damage remains unclear. In the present study, the trypsin digestion method was used to isolate human EpSCs and the cells were incubated in a 51.5°C water tank for 35 sec to construct a thermal injury model. The differentially expressed miRNAs were identified using high-throughput sequencing technology, and bioinformatic methods were used to predict their target genes and signaling pathways that may be involved in wound repair. A total of 33 miRNAs including, hsa-miR-1973, hsa-miR-4485-3p, hsa-miR-548-5p, hsa-miR-212-3p and hsa-miR-4461 were upregulated, whereas 21 miRNAs including, hsa-miR-4520-5p, hsa-miR-4661-5p, hsa-miR-191-3p, hsa-miR-129-5p, hsa-miR-147b and hsa-miR-6868-3p were downregulated following thermal injury of the human EpSCs. The bioinformatic analysis indicated that the differentially expressed miRNAs are involved in biological processes such as cell proliferation and differentiation, cell growth apoptosis, cell adhesion and migration. The results showed that there is a differential expression pattern of miRNAs after thermal injury of human EpSCs and these differences are involved in the regulation of the wound healing process. These findings provide new clues for further study of the wound healing mechanism and targeted therapy.

MicroRNA expression profiling of peripheral blood mononuclear cells associated with syphilis.

BACKGROUND: Treponema pallidum (T. pallidum) infection evokes significant immune responses, resulting in tissue damage. The immune mechanism underlying T. pallidum infection is still unclear, although microRNAs (miRNAs) have been shown to influence immune cell function and, consequently, the generation of antibody responses during other microbe infections. However, these mechanisms are unknown for T. pallidum. METHODS: In this study, we performed a comprehensive analysis of differentially expressed miRNAs in healthy individuals, untreated patients with syphilis, patients in the serofast state, and serologically cured patients. miRNAs were profiled from the peripheral blood of patients obtained at the time of serological diagnosis. Then, both the target sequence analysis of these different miRNAs and pathway analysis were performed to identify important immune and cell signaling pathways. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed for microRNA analysis. RESULTS: A total of 74 differentially regulated miRNAs were identified. Following RT-qPCR confirmation, three miRNAs (hsa-miR-195-5p, hsa-miR-223-3p, hsa-miR-589-3p) showed significant differences in the serofast and serologically cured states (P < 0.05). One miRNA (hsa-miR-195-5p) showed significant differences between untreated patients and healthy individuals. CONCLUSIONS: This is the first study of miRNA expression differences in peripheral blood mononuclear cells (PBMCs) in different stages of T. pallium infection. Our study suggests that the combination of three miRNAs has great potential to serve as a non-invasive biomarker of T. pallium infections, which will facilitate better diagnosis and treatment of T. pallium infections.

Regulatory roles of differentially expressed MicroRNAs in metabolic processes in negative Lens-induced myopia Guinea pigs.

BACKGROUND: Myopia is one of the most common vision defects worldwide. microRNAs can regulate the target gene expression, influencing the development of diseases. RESULTS: To investigate the alterations of microRNA profiling in negative lens-induced myopia (NLIM) guinea pigs and to explore the regulatory role of microRNAs in the occurrence and the development of myopia, we first established the NLIM guinea pig model after induction for 2 weeks. Further, we isolated sclera to purify total messenger RNA (mRNA) in both NLIM and NLIM fellow sclera. Using next generation sequencing technique and bioinformatics analysis, we identified the differentially expressed microRNAs in NLIM guinea pigs, performed the bioinformatics annotation for the differentially expressed microRNAs, and validated the expression of differentially expressed microRNAs. As a result, we successfully established an NLIM model in guinea pigs, identified 27 differentially expressed microRNAs in NLIM guinea pig sclera, including 10 upregulated and 17 downregulated microRNAs. The KEGG annotation showed the main signaling pathways were closely associated with PPAR signaling, pyruvate and propanoate metabolisms, and TGF-beta signaling pathways. CONCLUSIONS: Our findings indicate that the development of myopia is mainly involved in the disorder of metabolic processes in NLIM guinea pigs. The PPAR signaling, pyruvate and propanoate metabolism pathways may play roles in the development of myopia.

Bovid microRNAs involved in the process of spermatogonia differentiation into spermatocytes.

The male infertility of cattleyak resulted from spermatogenic arrest has greatly restricted the effective utilization of the heterosis from crossbreeding of cattle and yak. Based on our previous studies, the significant divergences of the transcriptomic and proteomic sequencing between yak and cattleyak prompt us to investigate the critical roles of microRNAs in post-transcriptional regulation of gene expression during spermatogenesis. TUNEL-POD analysis presented sharply decreased spermatogenic cell types and the increased apoptotic spermatogonia in cattleyak. The STA-PUT velocity sedimentation was employed to obtain spermatogonia and spermatocytes from cattle, yak and cattleyak and these spermatogenic cells were verified by the morphological and phenotypic identification. MicroRNA microarray showed that 27 differentially expressed miRNAs were simultaneously identified both in cattleyak vs cattle and in cattleyak vs yak comparisons. Further analysis revealed that the down-regulation of bta-let-7 families, bta-miR-125 and bta-miR-23a might impair the RA-induced differentiation of spermatogonia. Target gene analysis for differentially expressed miRNAs revealed that miRNAs targeted major players involved in vesicle-mediated transport, regulation of protein kinase activity and Pathways in cancer. In addition, spermatogonia transfection analysis revealed that the down-regulation of bta-miR-449a in the cattleyak might block the transition of male germ cells from the mitotic cycle to the meiotic program. The present study provided valuable information for future elucidating the regulatory roles of miRNAs involved in spermatogenic arrest of cattleyak.