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

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

Comprehensive Identification and Profiling of miRNAs Involved in Terpenoid Synthesis of Gleditsia sinensis Lam.

Gleditsia sinensis Lam. is a tree with worldwide distribution and important economic and medicinal values; its pods contain terpenoids including gleditsioside, thiamine, and brassinosteroids. However, thus far, there are few studies on the terpenoid regulation of G. sinensis at the molecular level. microRNA (miRNA) is a class of small RNAs with conserved and crucial roles in the regulation of diverse biological processes during plant growth and development. To identify the miRNAs of G. sinensis and evaluate their involvement in terpenoid synthesis, this investigation quantified the content changes in saponins in pods at three developmental stages: May (pod-setting stage), July (elongation stage), and September (browning stage), and then we performed genome-wide miRNA profiles during the three development stages of the G. sinensis pods. A total of 351 conserved miRNAs belonging to 216 families were identified, among which 36 conserved miRNAs exist specifically in legumes. Through target analysis, 708 unigenes were predicted to be candidate targets of 37 differentially expressed miRNAs. The targets of miR838-3p and miR2093-5p were involved in the derived branches of monoterpenes and gleditsioside, in brassinosteroid biosynthesis (BRB), and in indole alkaloid biosynthesis (IAB). Intriguingly, the targets of miR829-3p.1 were predicted to take part in thiamine biosynthesis, and the targets of miR4414b and miR5037a were involved in the main process of cytokinin synthesis. The corresponding targets participated in BRB, IAB, and terpenoid backbone biosynthesis, which were enriched significantly, suggesting that miR2093-5p, miR4414b, miR5037a, miR829-3p.1, and miR838-3p play indispensable roles in the regulation of triterpenoid saponin and monoterpenoid biosynthesis. To date, this is the first report of miRNA identification in G. sinensis and miRNA expression profiles at different developmental stages of G. sinensis pods, which provides a basis for further uncovering the molecular regulation of terpenoid synthesis in G. sinensis and new insights into the role of miRNAs in legumes.

Mechanistic Basis of Arsenic Induced Carcinogenesis: Differential miRNA Expression

Arsenic, a toxic metalloid, provokes many detrimental consequences to human health. It is prevalent in earth's crust and poses a major threat to humans globally. Inorganic arsenic exposure occurs mainly via drinking water or food and is metabolized in mammals to form organic metabolites/ end products. Chronic exposure to arsenic causes lung, skin and urinary bladder cancers and increases the risks of liver, kidney and prostate cancers. Arsenic-induced ROS generation, disturbances in several signaling pathways, DNA repair inhibition, chromosomal aberrations, and epigenetic changes including alterations in DNA methylation, histone modifications and differential miRNA expression profiles are involved in cancer progression, and malignant transformation. However, details of arsenic-induced carcinogenesis and molecular mechanisms involved are still remaining obscure. MicroRNAs are post-transcriptional gene expression regulators and themselves may act as oncogenes and tumor suppressor genes. Differential miRNA expression is implicated in several human cancers. This review covers general mechanistic basis of arsenic-induced carcinogenesis, explores recent in-vitro, in-vivo and cohort studies on differential miRNA expression profiles and shares associated molecular mechanistic data on miRNA dysregulation and their functional consequences leading to arsenic induced tumorigenesis, metastasis and cancer, also discusses the future directions.

Renal Tissue miRNA Expression Profiles in ANCA-Associated Vasculitis—A Comparative Analysis

Anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) comprises autoimmune disease entities that cause target organ damage due to relapsing-remitting small vessel necrotizing vasculitis, and which affects various vascular beds. The pathogenesis of AAV is incompletely understood, which translates to considerable disease- and treatment-related morbidity and mortality. Recent advances have implicated microRNAs (miRNAs) in AAV; however, their accurate characterization in renal tissue is lacking. The goal of this study was to identify the intrarenal miRNA expression profile in AAV relative to healthy, non-inflammatory and inflammatory controls to identify candidate-specific miRNAs. Formalin-fixed, paraffin-embedded renal biopsy tissue samples from 85 patients were obtained. Comprehensive miRNA expression profiles were performed using panels with 752 miRNAs and revealed 17 miRNA that differentiated AAV from both controls. Identified miRNAs were annotated to characterize their involvement in pathways and to define their targets. A considerable subset of differentially expressed miRNAs was related to macrophage and lymphocyte polarization and cytokines previously deemed important in AAV pathogenesis, lending credence to the obtained results. Interestingly, several members of the miR-30 family were detected. However, a validation study of these differentially expressed miRNAs in an independent, larger sample cohort is needed to establish their potential diagnostic utility.

Role of microRNAs in the Pathophysiology of Ulcerative Colitis

Ulcerative colitis (UC) is an intractable disorder characterized by a chronic inflammation of the colon. Studies have identified UC as a multifactorial disorder affected by both genetic and environmental factors; however, the precise mechanism remains unclear. Recent advances in the field of microRNA (miRNA) research have identified an association between this small non-coding RNA in the pathophysiology of UC and altered miRNA expression profiles in patients with UC. Nevertheless, the roles of individual miRNAs are uncertain due to heterogeneity in both research samples and clinical backgrounds. In this review, we focus on miRNA expression in colonic mucosa where inflammation occurs in UC and discuss the potential roles of individual miRNAs in disease development, outlining the pathophysiology of UC.

Integrated microRNA Analysis Identifies miR-512-3p as a Potential Biomarker of Poor Outcome in Pediatric Medulloblastoma

Background: Medulloblastoma, a genetically heterogeneous tumor, is the most frequent malignant brain tumor in children. Although several studies have been carried out, the molecular mechanism underlying medulloblastoma tumorigenesis is not completely known. microRNA (miRNA) expression profiles have been associated with development, progression, and prognosis of human cancers, including medulloblastoma. However, the role of miRNAs in pediatric medulloblastoma has been poorly explored.Methods: Global miRNA expression in 24 microdissected medulloblastoma specimens (19 pediatric and 5 adult specimens) was evaluated by microarray assay. miR-512-3p, the most differentially expressed miRNA in these two groups, was analyzed by qRT-PCR in a cohort of 51 consecutive pediatric medulloblastoma samples and 7 pediatric non-neoplastic cerebellum control samples, and its clinical significance was assessed. Further in silico miRNA prediction of target genes was performed with bioinformatics tools.Results: Compared to the controls, miR-512-3p was significantly downregulated in the pediatric medulloblastoma samples. Higher miR-512-3p was associated with incomplete degree of resection, high risk group classification, and poor overall survival. In silico analysis in an independent cohort of medulloblastoma identified that some of the miR-512-3p target genes (SMAD9, SSX2IP, MAPK10, PTCH1, CCDC6, and BMPR2) were statistically correlated with overall survival, metastasis, and death.Conclusions: For the first time, our results have shown that miR-512-3p is significantly associated with poor clinical outcome in pediatric medulloblastoma, suggesting that miR-512-3p is a potential biomarker of prognosis.

miR-4454 Promotes Hepatic Carcinoma Progression by Targeting Vps4A and Rab27A

Hepatocellular carcinoma (HCC) has high morbidity and mortality. MicroRNAs (miRNAs), which could be regulated by cancer-derived exosomes, play critical regulatory roles in the initiation and development of cancer. However, the expressions, effects, and mechanisms of abundant miRNAs regulated by HCC cancer-derived exosomes in HCC remain largely unclear. Exosomes of HepG2 cells under heat shock, TGF-β1, doxorubicin, acid and hypoxia/reoxygenation (H/R) conditions, and exosomes were successfully identified by transmission electron microscopy and Western blot analysis. The identified exosomes were then applied to evaluate the miRNA expression profiles by RNA sequencing. Mechanically, we discovered that doxorubicin was upregulated, TGF-β1 downregulated the expressions of Vps4A, Rab27A, Alix, and Hrs in HepG2 cells and exosomes, and Vps4A and Rab27A, as target genes for miR-4454, could also be downregulated by miR-4454. Functionally, we revealed that miR-4454 inhibitor and miR-4454 inhibitor-mediated exosomes could markedly suppress proliferation, migration, invasion, and vascularization and accelerate cycle arrest, apoptosis, and ROS of HepG2 cells. This study provided many potential HCC cancer-derived exosome-mediated miRNAs in HCC under 5 different stimulus conditions. Meanwhile, we certified that miR-4454 in exosomes could provide a novel and effective mechanism for HCC function.

miR-19b-3p is associated with a diametric response to resistance exercise in older adults and regulates skeletal muscle anabolism via PTEN inhibition.

Our laboratory has discovered that dysregulation in microRNA (miRNA) that target anabolic signaling between younger and older adults is a potential molecular mechanism resulting in age-associated decreases in skeletal muscle mass and function (sarcopenia). Whether differences in miRNA expression profiles account for inter-individual variability in exercise adaptation in older adults is unclear. Understanding paradoxical responses to anabolic stimulation and identifying the mechanisms for this inconsistency in mobility-limited older adults may provide new targets for the treatment of sarcopenia. The objective of the current study was to assess circulating miRNA expression profiles in diametric response of leg lean mass in mobility-limited older individuals after a 6 month progressive resistance exercise training intervention (PRET). Participants were dichotomized by gain (Gainers; n = 33) or loss (Losers; n = 40) of leg lean mass after PRET. Gainers signifcantly increased fat-free mass. Six miRNA (miR-1-3p, miR-19b-3p, miR-92a, miR-126, miR-133a-3p, and miR-133b) were identified to be differentially expressed between Gainers and Losers, with miR-19b-3p being the miRNA most highly associated with increases in fat-free mass. We then used a novel integrative approach to determine if differences in circulating miR-19b-3p potentially translate to augmented anabolic response in human skeletal muscle cells in vitro. Results from this analysis identified that overexpression of miR-19b-3p targeted and downregulated PTEN to facilitate increases in muscle protein synthetic rate. Together these data identify miR-19b-3p as a potent regulator of muscle anabolism that may contribute to an inter-individual response to PRET in mobility-limited older adults.

miR-218-5p mediates myocardial fibrosis after myocardial infarction by targeting CX43

Background: Myocardial fibrosis after myocardial infarction (MI) has been considered a core factor in the deterioration of cardiac function. Previous studies have shown that miRNA plays an important role in various pathophysiological processes of the heart. However, the role of miRNA in myocardial fibrosis regulation after MI remains unclear. In the present study, we documented that miR-218-5p was significantly decreased in myocardial fibroblasts after MI. Methods: The miRNA expression profiles of MI were downloaded from GEO Datasets. The expression of a fibrosis-related gene in vivo and in vitro was analyzed by RT-PCR, western blotting, and immunohistochemical staining. Result: Total 7 up- and 9 downregulated common miRNAs were found in the two profiles. Among these common genes, miR-218-5p was downregulated in the MI mice. MiR-218-5p mediated the myocardial fibrosis in vivo and in vitro. Mechanistically, we found that GJA1 (CX43) may be the target of miR218-5p, and overexpressed CX43 can partly block the function of miR-218-5p in fibrosis inhibition. Conclusion: Our results suggested that miR-218-5p plays an important role in myocardial fibrosis after MI by targeting CX43. Thus, miR-218-5p promises to be a potential diagnosis and treatment of myocardial fibrosis after MI.

Vector competence of Aedes aegypti and screening for differentially expressed microRNAs exposed to Zika virus

Background Zika virus (ZIKV) is transmitted to humans primarily by Aedes aegypti. Previous studies on Ae. aegypti from Jiegao (JG) and Mengding (MD) in Yunnan province, China have shown that these mosquitoes are able to transmit ZIKV to their offspring through vertical transmission, indicating that these two Ae. aegypti strains pose a potential risk for ZIKV transmission. However, the vector competence of these two Ae. aegypti strains to ZIKV has not been evaluated and the molecular mechanisms influencing vector competence are still unclear. Methods Aedes aegypti mosquitoes from JG and MD were orally infected with ZIKV, and the infection rate (IR), dissemination rate (DR), transmission rate (TR) and transmission efficiency (TE) of these two mosquito strains were explored to evaluate their vector competence to ZIKV. On 2, 4 and 6 days post-infection (dpi), the small RNA profiles between ZIKV-infected and non-infected Ae. aegypti midgut and salivary gland tissues were compared to gain insights into the molecular interactions between ZIKV and Ae. aegypti. Results There were no significant differences in the IR, DR, TR and TE between the two Ae. aegypti strains (P > 0.05). However, ZIKV RNA appeared 2 days earlier in saliva of the JG strain, which indicated a higher competence of the JG strain to transmit ZIKV. Significant differences in the microRNA (miRNA) expression profiles between ZIKV-infected and non-infected Ae. aegypti were found in the 2-dpi libraries of both the midgut and salivary gland tissues from the two strains. In addition, 27 and 74 miRNAs (|log2 fold change| > 2) were selected from the miRNA expression profiles of ZIKV-infected and non-infected midgut and salivary gland tissues from the JG and MD strains, respectively. Conclusions Our results provide novel insights into the ZIKV–mosquito interactions and build a foundation for future research on how miRNAs regulate the vector competence of mosquitoes to this arbovirus. Graphical abstract