Deep Sequencing Analysis of miRNA Expression in Breast Muscle of Fast-Growing and Slow-Growing Broilers

Growth performance is an important economic trait in chicken. MicroRNAs (miRNAs) have been shown to play important roles in various biological processes, but their functions in chicken growth are not yet clear. To investigate the function of miRNAs in chicken growth, breast muscle tissues of the two-tail samples (highest and lowest body weight) from Recessive White Rock (WRR) and Xinghua Chickens (XH) were performed on high throughput small RNA deep sequencing. In this study, a total of 921 miRNAs were identified, including 733 known mature miRNAs and 188 novel miRNAs. There were 200, 279, 257 and 297 differentially expressed miRNAs in the comparisons of WRRh vs. WRRl, WRRh vs. XHh, WRRl vs. XHl, and XHh vs. XHl group, respectively. A total of 22 highly differentially expressed miRNAs (fold change > 2 or < 0.5; p-value < 0.05; q-value < 0.01), which also have abundant expression (read counts > 1000) were found in our comparisons. As far as two analyses (WRRh vs. WRRl, and XHh vs. XHl) are concerned, we found 80 common differentially expressed miRNAs, while 110 miRNAs were found in WRRh vs. XHh and WRRl vs. XHl. Furthermore, 26 common miRNAs were identified among all four comparisons. Four differentially expressed miRNAs (miR-223, miR-16, miR-205a and miR-222b-5p) were validated by quantitative real-time RT-PCR (qRT-PCR). Regulatory networks of interactions among miRNAs and their targets were constructed using integrative miRNA target-prediction and network-analysis. Growth hormone receptor (GHR) was confirmed as a target of miR-146b-3p by dual-luciferase assay and qPCR, indicating that miR-34c, miR-223, miR-146b-3p, miR-21 and miR-205a are key growth-related target genes in the network. These miRNAs are proposed as candidate miRNAs for future studies concerning miRNA-target function on regulation of chicken growth.     

Global Noncoding microRNA Profiling in Mice Infected with Partial Human Mouth Microbes (PAHMM) Using an Ecological Time-Sequential Polybacterial Periodontal Infection (ETSPPI) Model Reveals Sex-Specific Differential microRNA Expression

Periodontitis (PD) is a polymicrobial dysbiotic immuno-inflammatory disease. It is more prevalent in males and has poorly understood pathogenic molecular mechanisms. Our primary objective was to characterize alterations in sex-specific microRNA (miRNA, miR) after periodontal bacterial infection. Using partial human mouth microbes (PAHMM) (Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) in an ecological time-sequential polybacterial periodontal infection (ETSPPI) mouse model, we evaluated differential mandibular miRNA profiles by using high-throughput Nanostring nCounter^® miRNA expression panels. All PAHMM mice showed bacterial colonization (100%) in the gingival surface, an increase in alveolar bone resorption (p < 0.0001), and the induction of a specific immunoglobin G antibody immune response (p < 0.001). Sex-specific differences in distal organ bacterial dissemination were observed in the heart (82% male vs. 28% female) and lungs (2% male vs. 68% female). Moreover, sex-specific differential expression (DE) of miRNA was identified in PAHMM mice. Out of 378 differentially expressed miRNAs, we identified seven miRNAs (miR-9, miR-148a, miR-669a, miR-199a-3p, miR-1274a, miR-377, and miR-690) in both sexes that may be implicated in the pathogenesis of periodontitis. A strong relationship was found between male-specific miR-377 upregulation and bacterial dissemination to the heart. This study demonstrates sex-specific differences in bacterial dissemination and in miRNA differential expression. A novel PAHMM mouse and ETSPPI model that replicates human pathobiology can be used to identify miRNA biomarkers in periodontitis.     

Hepatocyte-Specific Deficiency of BAP31 Amplified Acetaminophen-Induced Hepatotoxicity via Attenuating Nrf2 Signaling Activation in Mice

Liver-specific deficiency of B-cell receptor-associated protein 31 knockout mice (BAP31-LKO) and the littermates were injected with acetaminophen (APAP), markers of liver injury, and the potential molecular mechanisms were determined. In response to APAP overdose, serum aspartate aminotransferase and alanine aminotransferase levels were increased in BAP31-LKO mice than in wild-type controls, accompanied by enhanced liver necrosis. APAP-induced apoptosis and mortality were increased. Hepatic glutathione was decreased (1.60 ± 0.31 μmol/g tissue in WT mice vs. 0.85 ± 0.14 μmol/g tissue in BAP31-LKO mice at 6 h, p < 0.05), along with reduced glutathione reductase activity and superoxide dismutase; while malondialdehyde was significantly induced (0.41 ± 0.03 nmol/mg tissue in WT mice vs. 0.50 ± 0.05 nmol/mg tissue in BAP31-LKO mice for 6 h, p < 0.05). JNK signaling activation and APAP-induced hepatic inflammation were increased in BAP31-LKO mice. The mechanism research revealed that BAP31-deficiency decreased Nrf2 mRNA stability (half-life of Nrf2 mRNA decreased from ~1.3 h to ~40 min) and miR-223 expression, led to reduced nuclear factor erythroid 2-related factor 2 (Nrf2) signaling activation and antioxidant genes induction. BAP31-deficiency decreased mitochondrial membrane potentials, reduced mitochondria-related genes expression, and resulted in mitochondrial dysfunction in the liver. Conclusions: BAP31-deficiency reduced the antioxidant response and Nrf2 signaling activation via reducing Nrf2 mRNA stabilization, enhanced JNK signaling activation, hepatic inflammation, and apoptosis, amplified APAP-induced hepatotoxicity in mice.     

Preterm Intraventricular Hemorrhage-Induced Inflammatory Response in Human Choroid Plexus Epithelial Cells

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0–60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41–60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.     

miR-22-3p and miR-30e-5p Are Associated with Prognosis in Cervical Squamous Cell Carcinoma

Alteration in expression of miRNAs can cause various malignant changes and the metastatic process. Our aim was to identify the miRNAs involved in cervical squamous cell carcinoma (SqCC) and metastasis, and to test their utility as indicators of metastasis and survival. Using microarray technology, we performed miRNA expression profiling on primary cervical SqCC tissue (n = 6) compared with normal control (NC) tissue and compared SqCC that had (SqC-M; n = 3) and had not (SqC-NM; n = 3) metastasized. Four miRNAs were selected for validation by qRT-PCR on 29 SqC-NM and 27 SqC-M samples, and nine metastatic lesions (ML-SqC), from a total of 56 patients. Correlation of miRNA expression and clinicopathological parameters was analyzed to evaluate the clinical impact of candidate miRNAs. We found 40 miRNAs differentially altered in cervical SqCC tissue: 21 miRNAs were upregulated and 19 were downregulated (≥2-fold, p < 0.05). Eight were differentially altered in SqC-M compared with SqC-NM samples: four were upregulated (miR-494, miR-92a-3p, miR-205-5p, and miR-221-3p), and four were downregulated (miR-574-3p, miR-4769-3p, miR-1281, and miR-1825) (≥1.5-fold, p < 0.05). MiR-22-3p might be a metastamiR, which was gradually further downregulated in SqC-NM > SqC-M > ML-SqC. Downregulation of miR-30e-5p significantly correlated with high stage, lymph node metastasis, and low survival rate, suggesting an independent poor prognostic factor.     

Cumin Prevents 17β-Estradiol-Associated Breast Cancer in ACI Rats

Breast cancer (BC) is a leading cause of cancer deaths in women in less developed countries and the second leading cause of cancer death in women in the U.S. In this study, we report the inhibition of E2-mediated mammary tumorigenesis by Cuminum cyminum (cumin) administered via the diet as cumin powder, as well as dried ethanolic extract. Groups of female ACI rats were given either an AIN-93M diet or a diet supplemented with cumin powder (5% and 7.5%, w/w) or dried ethanolic cumin extract (1%, w/w), and then challenged with subcutaneous E2 silastic implants (1.2 cm; 9 mg). The first appearance of a palpable mammary tumor was significantly delayed by both the cumin powder and extract. At the end of the study, the tumor incidence was 96% in the control group, whereas only 55% and 45% animals had palpable tumors in the cumin powder and extract groups, respectively. Significant reductions in tumor volume (660 ± 122 vs. 138 ± 49 and 75 ± 46 mm³) and tumor multiplicity (4.21 ± 0.43 vs. 1.16 ± 0.26 and 0.9 ± 0.29 tumors/animal) were also observed by the cumin powder and cumin extract groups, respectively. The cumin powder diet intervention dose- and time-dependently offset E2-related pituitary growth, and reduced the levels of circulating prolactin and the levels of PCNA in the mammary tissues. Mechanistically, the cumin powder diet resulted in a significant reversal of E2-associated modulation in ERα, CYP1A1 and CYP1B1. Further, the cumin powder diet reversed the expression levels of miRNAs (miR-182, miR-375, miR-127 and miR-206) that were highly modulated by E2 treatment. We analyzed the composition of the extract by GC/MS and established cymene and cuminaldehyde as major components, and further detected no signs of gross or systemic toxicity. Thus, cumin bioactives can significantly delay and prevent E2-mediated mammary tumorigenesis in a safe and effective manner, and warrant continued efforts to develop these clinically translatable spice bioactives as chemopreventives and therapeutics against BC.     

miR-196b-5p and miR-107 Expression Differentiates Ocular Sebaceous Carcinoma from Squamous Cell Carcinoma of the Conjunctiva

An Ocular Sebaceous Carcinoma (OSC) is a rare malignant tumor for which initial clinical and pathological diagnosis is often incorrect. OSCs can mimic Squamous Cell Carcinomas of the Conjunctiva (SCCC). The aim of this study was to find microRNA biomarkers to distinguish OSCs and SCCCs from normal tissue and from each other. Clinical OSC and SCCC case files and the corresponding histopathological slides were collected and reviewed. Micro dissected formalin-fixed paraffin-embedded tumor and control tissues were subjected to semi-high throughput microRNA profiling. MicroRNA expression distinguishes OSCs and SCCCs from corresponding control tissues. Selected differentially expressed miRNAs were validated using single RT-PCR assays. No prognostic miRNAs could be identified that reliably predict SCCC metastasis or OSC recurrence. A comparison between OSCs (n = 14) and SCCCs (n = 18) revealed 38 differentially expressed microRNAs (p < 0.05). Differentially expressed miRNAs were selected for validation in the discovery cohort and an independent validation cohort (OSCs, n = 11; SCCCs, n = 12). At least two miRNAs, miR-196b-5p (p ≤ 0.05) and miR-107 (p ≤ 0.001), displayed a statistically significant differential expression between OSCs and SCCCs with miR-196b-5p upregulated in SCCCs and miR-107 upregulated in OSCs. In the validation cohort, microRNA miR-493-3p also showed significant upregulation in SCCCs when compared to OSCs (p ≤ 0.05). ROC analyses indicated that the combined miR-196b-5p and miR-107 expression levels predicted OSCs with 90.0% sensitivity and 83.3% specificity. In conclusion, the combined testing of miR-196b-5p and miR-107, can be of additional use in routine diagnostics to discriminate OSCs from SCCCs.     

Identification and Validation of miR-222-3p and miR-409-3p as Plasma Biomarkers in Gestational Diabetes Mellitus Sharing Validated Target Genes Involved in Metabolic Homeostasis

Gestational diabetes mellitus (GDM) causes both maternal and fetal adverse outcomes. The deregulation of microRNAs (miRNAs) in GDM suggests their involvement in GDM pathogenesis and complications. Exosomes are extracellular vesicles (EVs) of endosomal origin, released via exocytosis into the extracellular compartment. Through EVs, miRNAs are delivered in distant target cells and are able to affect gene expression. In this study, miRNA expression was analyzed to find new miRNAs that could improve GDM classification and molecular characterization. MiRNA were profiled in total plasma and EVs in GDM patients and normal glucose tolerance (NGT) women. Samples were collected at third trimester of gestation from two diabetes centers. MiRNA expression was profiled in a discovery cohort using the multiplexed NanoString nCounter Human v3 miRNA. Validation analysis was performed in a second independent cohort using RT-qPCR. A set of miRNAs resulted to be differentially expressed (DE) in total plasma and EVs in GDM. Among them, total plasma miR-222-3p and miR-409-3p were validated in the independent cohort. MiR-222-3p levels correlated with fasting plasma glucose (FPG) (p < 0.001) and birth weight (p = 0.012), whereas miR-409-3p expression correlated with FPG (p < 0.001) and inversely with gestational age (p = 0.001). The major validated target genes of the deregulated miRNAs were consistently linked to type 2 diabetes and GDM pathophysiology. MiR-222-3p and miR-409-3p are two circulating biomarkers that could improve GDM classification power and act in the context of the molecular events leading to the metabolic alterations observed in GDM.     

Plasma miR-9-3p and miR-136-3p as Potential Novel Diagnostic Biomarkers for Experimental and Human Mild Traumatic Brain Injury

Noninvasive, affordable circulating biomarkers for difficult-to-diagnose mild traumatic brain injury (mTBI) are an unmet medical need. Although blood microRNA (miRNA) levels are reportedly altered after traumatic brain injury (TBI), their diagnostic potential for mTBI remains inconclusive. We hypothesized that acutely altered plasma miRNAs could serve as diagnostic biomarkers both in the lateral fluid percussion injury (FPI) model and clinical mTBI. We performed plasma small RNA-sequencing from adult male Sprague–Dawley rats (n = 31) at 2 days post-TBI, followed by polymerase chain reaction (PCR)-based validation of selected candidates. miR-9a-3p, miR-136-3p, and miR-434-3p were identified as the most promising candidates at 2 days after lateral FPI. Digital droplet PCR (ddPCR) revealed 4.2-, 2.8-, and 4.6-fold elevations in miR-9a-3p, miR-136-3p, and miR-434-3p levels (p < 0.01 for all), respectively, distinguishing rats with mTBI from naïve rats with 100% sensitivity and specificity. DdPCR further identified a subpopulation of mTBI patients with plasma miR-9-3p (n = 7/15) and miR-136-3p (n = 5/15) levels higher than one standard deviation above the control mean at <2 days postinjury. In sTBI patients, plasma miR-9-3p levels were 6.5- and 9.2-fold in comparison to the mTBI and control groups, respectively. Thus, plasma miR-9-3p and miR-136-3p were identified as promising biomarker candidates for mTBI requiring further evaluation in a larger patient population.     

Circulating miRNAs as Biomarkers for Mitochondrial Neuro-Gastrointestinal Encephalomyopathy

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disease for which there are currently no validated outcome measures for assessing therapeutic intervention efficacy. The aim of this study was to identify a plasma and/or serum microRNA (miRNA) biomarker panel for MNGIE. Sixty-five patients and 65 age and sex matched healthy controls were recruited and assigned to one of four study phases: (i) discovery for sample size determination; (ii) candidate screening; (iii) candidate validation; and (iv) verifying the performance of the validated miRNA panel in four patients treated with erythrocyte-encapsulated thymidine phosphorylase (EE-TP), an enzyme replacement under development for MNGIE. Quantitative PCR (qPCR) was used to profile miRNAs in serum and/or plasma samples collected for the discovery, validation and performance phases, and next generation sequencing (NGS) analysis was applied to serum samples assigned to the candidate screening phase. Forty-one differentially expressed candidate miRNAs were identified in the sera of patients (p < 0.05, log₂ fold change > 1). The validation cohort revealed that of those, 27 miRNAs were upregulated in plasma and three miRNAs were upregulated in sera (p < 0.05). Through binary logistic regression analyses, five plasma miRNAs (miR-192-5p, miR-193a-5p, miR-194-5p, miR-215-5p and miR-34a-5p) and three serum miRNAs (miR-192-5p, miR-194-5p and miR-34a-5p) were shown to robustly distinguish MNGIE from healthy controls. Reduced longitudinal miRNA expression of miR-34a-5p was observed in all four patients treated with EE-TP and coincided with biochemical and clinical improvements. We recommend the inclusion of the plasma exploratory miRNA biomarker panel in future clinical trials of investigational therapies for MNGIE; it may have prognostic value for assessing clinical status.     

A Disease Marker for Aspirin-Induced Chronic Urticaria

There are currently no diagnostic methods in vitro for aspirin-induced chronic urticaria (AICU) except for the provocation test in vivo. To identify disease markers for AICU, we investigated the single nucleotide polymorphism (SNP) of the promoter loci of high-affinity IgE receptor (FcεRIα) and CD203c expression level in Chinese patients with AICU. We studied two genotypic and allelic frequencies of rs2427827 (–344C/T) and rs2251746 (–66T/C) gene polymorphisms of FcεRIα in 20 patients with AICU, 52 subjects with airway hypersensitivity without aspirin intolerance, and 50 controls in a Chinese population. The results showed that the frequencies of two SNPs (–344C>T, –66C>T) were similar to the normal controls. The allele frequency of –344CC was significantly higher in the patients with AICU compared to those with airway sensitivity (p = 0.019). We also studied both histamine release and CD203c expression on KU812 cells to assess aspirin-induced basophil activation. We found that the activity of basophil activation of AICU was significantly higher in the patients with AICU compared to those with airway hypersensitivity without aspirin intolerance. The mean fluorescence intensity of the CD203c expression were 122.5 ± 5.2 vs. 103.3 ± 3.3 respectively, (p < 0.05), and the percentages of histamine release were 31.3% ± 7.4% vs. −24.0% ± 17.5%, (p < 0.05) respectively. Although the mean fluorescence intensity of CD203c expression and the percentage of histamine release were significantly up-regulated by aspirin, they were not affected by anti-IgE antibodies. These results suggest that a single SNP of FcεRIα (–344C>T) is less likely to develop AICU and the basophil activation activity in the sera by measuring CD203c expression can be applicable to confirm the diagnosis of AICU.     

Peripheral Blood miR-328 Expression as a Potential Biomarker for the Early Diagnosis of NSCLC

Lung cancer is often diagnosed at an advanced stage, with subsequently poor prognosis. There are no biomarkers available to facilitate early diagnosis or to discriminate between benign and malignant nodules. MicroRNAs (miRNAs) are stable molecules that can be found and measured in peripheral blood, thus representing potential diagnostic biomarkers. We evaluated 100 individuals comprising 86 patients with predominantly early-stage non-small cell lung cancer (NSCLC) and 24 healthy donors. RNA was extracted from peripheral blood samples and the expression of a panel of miRNAs was analyzed by Real-Time PCR method. Expression levels of miR-328, miR-18a, miR-339 and miR-140 were significantly higher in NSCLC patients than in healthy donors (p < 0.05). In particular, miR-328 showed good diagnostic accuracy in discriminating between patients with early NSCLC and healthy donors (AUC ROC 0.82, 95% CI 0.72–0.92), with 70% sensitivity and 83% specificity at the best relative expression cut-off of 300. Moreover, miR-339 was a good discriminant between healthy donors and late-stage NSCLC patients (AUC ROC 0.79, 95% CI 0.68–0.91). In conclusion, miR-328 represents a potential diagnostic biomarker of NSCLC, especially for the identification of early-stage tumors. Its role in discriminating between benign and malignant nodules detected by spiral CT warrants further investigation.     

MicroRNAs and Drinking: Association between the Pre-miR-27a rs895819 Polymorphism and Alcohol Consumption in a Mediterranean Population

Recently, microRNAs (miRNA) have been proposed as regulators in the different processes involved in alcohol intake, and differences have been found in the miRNA expression profile in alcoholics. However, no study has focused on analyzing polymorphisms in genes encoding miRNAs and daily alcohol consumption at the population level. Our aim was to investigate the association between a functional polymorphism in the pre-miR-27a (rs895819 A>G) gene and alcohol consumption in an elderly population. We undertook a cross-sectional study of PREvención con DIeta MEDiterránea (PREDIMED)-Valencia participants (n = 1007, including men and women aged 67 ± 7 years) and measured their alcohol consumption (total and alcoholic beverages) through a validated questionnaire. We found a strong association between the pre-miR-27a polymorphism and total alcohol intake, this being higher in GG subjects (5.2 ± 0.4 in AA, 5.9 ± 0.5 in AG and 9.1 ± 1.8 g/day in GG; p_adjusted = 0.019). We also found a statistically-significant association of the pre-miR-27a polymorphism with the risk of having a high alcohol intake (>2 drinks/day in men and >1 in women): 5.9% in AA versus 17.5% in GG; p_adjusted < 0.001. In the sensitivity analysis, this association was homogeneous for sex, obesity and Mediterranean diet adherence. In conclusion, we report for the first time a significant association between a miRNA polymorphism (rs895819) and daily alcohol consumption.     

miR-16-5p Is a Stably-Expressed Housekeeping MicroRNA in Breast Cancer Tissues from Primary Tumors and from Metastatic Sites

For quantitative microRNA analyses in formalin-fixed paraffin-embedded (FFPE) tissue, expression levels have to be normalized to endogenous controls. To investigate the most stably-expressed microRNAs in breast cancer and its surrounding tissue, we used tumor samples from primary tumors and from metastatic sites. MiRNA profiling using TaqMan^® Array Human MicroRNA Cards, enabling quantification of 754 unique human miRNAs, was performed in FFPE specimens from 58 patients with metastatic breast cancer. Forty-two (72%) samples were collected from primary tumors and 16 (28%) from metastases. In a cross-platform analysis of a validation cohort of 32 FFPE samples from patients with early breast cancer genome-wide microRNA expression analysis using SurePrintG3 miRNA (8 × 60 K)^® microarrays from Agilent^® was performed. Eleven microRNAs could be detected in all samples analyzed. Based on NormFinder and geNorm stability values and the high correlation (rho ≥ 0.8) with the median of all measured microRNAs, miR-16-5p, miR-29a-3p, miR-126-3p, and miR-222-3p are suitable single gene housekeeper candidates. In the cross-platform validation, 29 human microRNAs were strongly expressed (mean log2-intensity > 10) and 21 of these microRNAs including miR-16-5p and miR-29a-3p were also stably expressed (CV < 5%). Thus, miR-16-5p and miR-29a-3p are both strong housekeeper candidates. Their Normfinder stability values calculated across the primary tumor and metastases subgroup indicate that miR-29a-3p can be considered as the strongest housekeeper in a cohort with mainly samples from primary tumors, whereas miR-16-5p might perform better in a metastatic sample enriched cohort.     

miR-9-5p,miR-675-5p and miR-138-5p Damages the Strontium and LRP5-Mediated Skeletal Cell Proliferation,Differentiation, and Adhesion

This study was designed to evaluate the effects of strontium on the expression levels of microRNAs (miRNAs) and to explore their effects on skeletal cell proliferation, differentiation, adhesion, and apoptosis. The targets of these miRNAs were also studied. Molecular cloning, cell proliferation assay, cell apoptosis assay, quantitative real-time PCR, and luciferase reporter assay were used. Strontium altered the expression levels of miRNAs in vitro and in vivo. miR-9-5p, miR-675-5p, and miR-138-5p impaired skeletal cell proliferation, cell differentiation and cell adhesion. miR-9-5p and miR-675-5p induced MC3T3-E1 cell apoptosis more specifically than miR-138-5p. miR-9-5p, miR-675-5p, and miR-138-5p targeted glycogen synthase kinase 3 β (GSK3β), ATPase Aminophospholipid Transporter Class I Type 8A Member 2 (ATP8A2), and Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1), respectively. Low-density lipoprotein receptor-related protein 5 (LRP5) played a positive role in skeletal development. miR-9-5p, miR-675-5p, and miR-138-5p damage strontium and LRP5-mediated skeletal cell proliferation, differentiation, and adhesion, and induce cell apoptosis by targeting GSK3β, ATP8A2, and EIF4EBP1, respectively.     

The Potential Role of Electronegative High-Density Lipoprotein H5 Subfraction in RA-Related Atherosclerosis

Although the heterogeneity of high-density lipoprotein-cholesterol (HDL-c) composition is associated with atherosclerotic cardiovascular risk, the link between electronegative subfractions of HDL-c and atherosclerosis in rheumatoid arthritis (RA) remains unknown. We examined the association of the percentage of the most electronegative subfraction of HDL-c (H5%) and RA-related atherosclerosis. Using anion-exchange purification/fast-protein liquid chromatography, we demonstrated significantly higher H5% in patients (median, 7.2%) than HC (2.8%, p < 0.005). Multivariable regression analysis revealed H5% as a significant predictor for subclinical atherosclerosis. We subsequently explored atherogenic role of H5 using cell-based assay. The results showed significantly higher levels of IL-1β and IL-8 mRNA in H5-treated (mean ± SD, 4.45 ± 1.22 folds, 6.02 ± 1.43-folds, respectively) than H1-treated monocytes (0.89 ± 0.18-folds, 1.03 ± 0.26-folds, respectively, both p < 0.001). In macrophages, H5 upregulated the mRNA and protein expression of IL-1β and IL-8 in a dose-dependent manner, and their expression levels were significantly higher than H1-treated macrophages (all p < 0.001). H5 induced more foam cell formation compared with H1-treated macrophages (p < 0.005). In addition, H5 has significantly lower cholesterol efflux capacity than H1 (p < 0.005). The results of nanoLC-MS/MS approach reveal that the best discriminator between high-H5% and normal-H5% is Apo(a), the main constituent of Lp(a). Moreover, Lp(a) level is a significant predictor for high-H5%. These observations suggest that H5 is involved in RA-related atherosclerosis.     

Does Melatonin Homeostasis Play a Role in Continuous Epigastric Pain Syndrome?

Two clinical forms of functional dyspepsia (FD) are listed in the Rome III criteria: postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS), differing in the recurrence of ailments depending on the diet. Continuous EPS (CEPS) is observed in some EPS patients, also at night, but its cause is still unknown. We showed previously that melatonin (MEL) homeostasis may be associated with FD. In the present work we evaluated selected components of melatonin homeostasis in patients with CEPS. The study included 30 patients with CEPS, 21 women and nine men, aged 21–49 years and 30 control subjects (EPS excluded); organic and mental diseases, as well as Helicobacter pylori infection, were excluded in both groups. The average severity of abdominal pain in the last three months was estimated in a 10-point scale (Visual Analog Scale). The levels of mRNA expression of arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT), the main components of MEL homeostasis, were determined in gastric mucosa with real time PCR. The fasting serum level of MEL (at 09:00 a.m.) and circadian urine excretion of 6-sulfatoxymelatonin (6-HMS) were determined with ELISA. AANAT expression in antral mucosa of control subjects was 1.76 ± 0.41, in the gastric body 1.35 ± 0.38, and in the dyspeptic group 1.42 ± 0.38 (p < 0.05) and 0.92 ± 0.55 (p < 0.05), respectively. HIOMT expression in the control was 2.05 ± 0.70 in the antrum and 1.57 ± 0.69 in the body and in the CEPS group, it was: 1.51 ± 0.57 (p < 0.05) and 0.74 ± 0.31 (p < 0.001), respectively. MEL concentration (pg/mL) was 9.41 ± 3.09 in the control group and 5.62 ± 1.34 (p < 0.01) in the CEPS group. Urinary 6-HMS excretion (μg/24 h) was 11.40 ± 4.46 in the controls and 7.68 ± 2.88 (p < 0.05) in the CEPS. Moreover, a negative correlation was found between the tested parameters and severity of epigastric pain. These results indicate that patients with CEPS may display low level of AANAT and HIOMT expression in gastric mucosa, resulting in decreased MEL synthesis.