Single-Nucleotide Polymorphisms in the 3' Untranslated Region of CORIN Associated With Cardiovascular Diseases in a Chinese Han Population: A Case–Control Study

Background: Corin is a transmembrane serine protease that activates pro-forms of atrial and brain natriuretic peptides. Numerous studies have indicated that corin played an important role in cardiovascular diseases (CVDs). However, there have been few studies about the correlation between single-nucleotide polymorphisms (SNPs) in the 3' untranslated region (3'UTR) of CORIN and CVDs. The aims of this study were to investigate the associations of three SNPs (rs3749585, rs4695253, and rs12641823) in the 3'UTR of CORIN with CVDs and to find the seed regions of microRNAs (miRNAs) that bind to SNPs of CORIN.Methods and Results: A case–control study (n = 3,537) was performed in a Han population of northeastern China. CVDs included essential hypertension (EH), atrial fibrillation (AF), heart failure (HF), and coronary artery disease (CAD). Genotyping was performed using high-resolution melt analysis. In the EH-control study, rs3749585T was significantly associated with the risk of EH after adjusting for sex and age in allelic (padj = 0.049; OR: 1.113) and dominant (padj = 0.015, OR: 1.233) models. Rs4695253T was significantly associated with the risk of EH in the recessive model after adjusting for sex and age (padj = 0.005, OR: 2.084). Rs3749585T was significantly and negatively associated with AF in the dominant and additive models after adjusting for sex, age, EH, HF, T2DM, and CAD (dominant: padj = 0.009, OR: 0.762; additive: padj = 0.048, OR: 0.873). In the HF-control study and CAD-control study, none of the three SNPs was associated with HF and CAD after adjusting for covariates in any models (padj > 0.05). The levels of high-density lipoprotein (HDL) in rs4695253CC+CT were lower than the levels of HDL in rs4695253TT (42.47 ± 10.30 vs. 48.0 ± 10.24 mg/dl, padj = 0.008). The levels of total cholesterol (TC) in rs4695253CC+CT were lower than the levels of TC in rs4695253TT (164.01 ± 49.15 vs. 180.81 ± 43.92 mg/dl, padj = 0.036). Luciferase assay revealed that the relative luciferase activity of rs3749585CC-transfected cells was significantly decreased by miR-494-3p, in comparison to cells transfected with rs3749585TT (p < 0.001). A significant decrease in the relative luciferase activity of rs3749585TT reporter was observed as compared with rs3749585CC reporter in the presence of miR-1323 or miR-548o-3p (p = 0.017 and 0.012, respectively).Conclusions: We found significant associations between rs3749585T and rs4695253T and EH, between rs4695253T and the levels of TC and HDL, and between rs3749585T and AF. Hsa-miR-494-3p may serve as a potential therapeutic target for EH and AF patients in the future.

Extract of Scutellaria baicalensis induces semaphorin 3A production in human epidermal keratinocytes

In a disease-state-dependent manner, the histamine-resistant itch in dry skin-based skin diseases such as atopic dermatitis (AD) and xerosis is mainly due to hyperinnervation in the epidermis. Semaphorin 3A (Sema3A) is a nerve repulsion factor expressed in keratinocytes and it suppresses nerve fiber elongation in the epidermis. Our previous studies have shown that Sema3A ointment inhibits epidermal hyperinnervation and scratching behavior and improves dermatitis scores in AD model mice. Therefore, we consider Sema3A as a key therapeutic target for improving histamine-resistant itch in AD and xerosis. This study was designed to screen a library of herbal plant extracts to discover compounds with potential to induce Sema3A in normal human epidermal keratinocytes (NHEKs) using a reporter gene assay, so that positive samples were found. Among the positive samples, only the extract of S. baicalensis was found to consistently increase Sema3A levels in cultured NHEKs in assays using quantitative real-time PCR and ELISA. In evaluation of reconstituted human epidermis models, the level of Sema3A protein in culture supernatants significantly increased by application of the extract of S. baicalensis. In addition, we investigated which components in the extract of S. baicalensis contributed to Sema3A induction and found that baicalin and baicalein markedly increased the relative luciferase activity, and that baicalein had higher induction activity than baicalin. Thus, these findings suggest that S. baicalensis extract and its compounds, baicalin and baicalein, may be promising candidates for improving histamine-resistant itch via the induction of Sema3A expression in epidermal keratinocytes.

miR-122-5p regulates the tight junction of the blood-testis barrier of mice via occludin

Background Occludin protein is the primary assembling protein of TJs and the structural basis for tight junction formation between Sertoli cells in the spermatogenic epithelium. The expression of miR-122-5p and occludin are negatively correlated. In order to investigate the regulation mechanism of miR-122-5p on occludin and TJ, the present study isolated primary Sertoli cells from C57BL/6 mice, identified a transcription factor of miR-122-5p in testicle, studied the modulating loci of miR-122-5p on occludin using a dual-luciferase reporter assay, analyzed the regulate of miR-122-5p on the expression of occludin with real-time RT-PCR and Western blot, and studied the effect of miR-122-5p on the tight junction using a Millicell Electrical Resistance System. Results The relative luciferase activity in the pcDNA-Sp1 + pGL3-miR-122-5p promoter group was significantly higher than that in the pcDNA-Sp1 + pGL3-basic group, which suggests that transcript factor Sp1 promotes the transcription of miR-122-5p. The relative luciferase activity in the occludin 3′-UTR (wt) + miR-122-5p mimic group was significantly lower than that in the other groups (p < 0.01), which indicates that miR-122-5p modulates the expression of occludin via the ACACTCCA sequence of the occludin-3’UTR. The levels of occludin mRNA and protein in the miR-122-5p mimic group were significantly lower than that in the other groups (p < 0.05), which indicates that miR-122-5p reduces the expression of occludin. The trans-epithelial resistance of the miR-122-5p mimic group was significantly lower than that of the blank control group after day 4 (p < 0.05), which indicates that miR-122-5p inhibited the assembly of the inter-Sertoli TJ permeability barrier in vitro. Conclusion These results displayed that miR-122-5p could regulate tight junctions via the Sp1-miR-122-5p-occludin-TJ axis.

miR-124 Regulates Caveolin-1 Expression and Affects Proliferation and Apoptosis of Osteosarcoma Cells

Osteosarcoma (OS) seriously affects human health. miR-124 expression is closely related to osteosarcoma, but its specific mechanism remains unclear. Our study intends to evaluate miR-124’s effect on osteosarcoma. MG-63 cells were transfected with miR-124 mimics/NC followed by analysis of miR-124 expression by real-time PCR, cell proliferation by CCK8 assay, cell apoptosis by flow cytometry as well as the level of caveolin-1 (CAV1) by Western blot. miR-124 was significantly lower and CAV1 was increased in the four osteosarcoma cells than those in normal osteoblasts (P < 0.05). miR-124 mimics transfection significantly reduced CAV1 level and cell number (P < 0.05) and increased cell apoptosis rate (P < 0.05). Moreover, miR-124 inhibitor significantly promoted the relative luciferase activity in pmirGLO-CAV1-3′UTR-wt-transfected cells (P < 0.05). miR-124 affects osteosarcoma cell proliferation and apoptosis via targeting CAV1.

Acylphloroglucinol Derivatives from Garcinia multiflora with Anti-Inflammatory Effect in LPS-Induced RAW264.7 Macrophages

Two new acylphloroglucinol derivatives, 13,14-didehydroxygarcicowin C (1) and 13,14-didehydroxyisoxanthochymol (2), have been isolated from the stems of Garcinia multiflora, together with seven known compounds (3–9). The structures of new compounds 1 and 2 were elucidated by MS and extensive 1D/2D NMR spectroscopic analyses. Among the isolates, 13,14-didehydroxy-isoxanthochymol (2) and sampsonione B (3) exhibited inhibition against lipopolysaccharide (LPS)-induced NF-κB activation in macrophages at 30 μM with relative luciferase activity values (inhibitory %) of 0.75 ± 0.03 (24 ± 4%) and 0.12 ± 0.03 (88 ± 4%), respectively. Additionally, sampsonione B (3) reduced LPS-induced nitric oxide (NO) production in murine RAW264.7 macrophages and did not induce cytotoxicity against RAW 264.7 cells after 24 h treatment. Compound 3 is worth further investigation and may be expectantly developed as an anti-inflammatory drug candidate.

Epigenetic Disruption of ZEB1 Binding Causes Constitutive Activation of IL-15 in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a non-Hodgkin's lymphoma of skin-homing T-cells that represents 70-80% of all cutaneous lymphomas. Studies have shown that interleukin-15 (IL-15) plays a key role in T-cell oncogenesis by increasing T-cell survival, proliferation, migration and invasion in patient-derived CTCL cell lines. Previously, we have demonstrated that IL-15 is overexpressed in lesional biopsies and peripheral blood of CTCL patients (Mishra et al., Blood, 122:1826, 2013). However, the underlying mechanisms of IL-15 deregulation are largely unknown. Since epigenetic modifications such as DNA methylation can alter gene expression, we selected samples highly enriched in neoplastic T-cells from the blood of CTCL patients and performed a pyro-sequencing analysis to determine methylation of the IL-15 gene promoter. We observed significantly higher methylation in the IL-15 promoter of CD4+ T-cells from CTCL patients vs. normal donors (mean ± SEM of relative % methylation= 232.0 ± 49.18, n=9 vs 100.1 ± 7.619, n=6, P =0.03). Furthermore, methylation at the IL-15 promoter was higher in malignant CD4+ T-cells compared to non-malignant neutrophils from the same patient (mean ± SEM of relative % methylation = 232.0 ± 49.18 vs 52.31 ± 6.28%, N=9 each, paired t-test, P =0.0025). Using this approach, we also analyzed methylation of each 'CpG dinucleotide' in the IL-15 promoter. Once again, methylation was higher in CD4+ T-cells of patient versus normal donors and, within each patient, in CD4+ T-cells versus neutrophils. The CpG rich region of the human IL-15 promoter contains three putative binding sites for the known transcriptional repressor Zeb1, which has been reported to be mutated or deleted in CTCL. In order to examine the effect of CpG methylation at the Zeb1 binding region on IL-15 transcription, the CpG rich 5' regulatory region of the IL-15 promoter was amplified and cloned into PGL3 luciferase vector. As measured by relative luciferase activity, the region of the IL-15 promoter containing the 3 ZEB1-binding sites is transcriptionally active in its native form (mean ± SEM of relative luciferase activity of promoter-less vector vs. IL-15 promoter vector = 77.00 ± 3.29 vs. 626.8 ± 25.05 respectively, N=3 each, P<0.0001). Deletion of the putative Zeb1 binding sites (BS#1, BS#2, BS#3 and BS#1-3) or the entire Zeb-binding region (BR) in the IL-15 promoter led to a significant increase in IL-15 transcription as determined by relative luciferase activity (mean ± SEM of relative luciferase activity of pGL3 IL-15 vs. BS#1 vs. BS#2 vs. BS#3 vs. BS#1-3 vs. BR vectors = 99.56 ± 0.33 vs. 7135 ± 108.5 vs. 3940 ± 62.36 vs. 2372 ± 24.65 vs. 1099 ± 9.72 vs. 1525 ± 32.00, n=4 each; P <0.0001 each). Since CpG methylation of DNA can physically prevent transcription factor binding to regulatory regions of DNA, we hypothesized that CD4+ cells from CTCL patients might display reduced binding of Zeb1 to the IL-15 promoter thereby increasing IL-15 transcription. Indeed, using chromatin immunoprecipitation (ChIP)-PCR, we observed a substantial loss of Zeb1 binding at the IL-15 promoter in the CD4+ cells of CTCL patients compared to those of normal donors (mean ± SEM of relative Zeb1 binding to IL-15 promoter in normal donor vs. CTCL patient CD4+ T-cells = 101.3 ± 1.48 vs. 25.31 ± 3.78, n=3 each; P <0.0001). Similarly, silencing Zeb1 in normal donor CD4+ T-cells caused an increase in IL-15 transcription. Finally, constitutive IL-15 overexpression was sufficient to cause a high penetrance lymphoproliferative disease in transgenic mice, with typical dermatologic features of human CTCL, including alopecia, scaly erythematous plaques/patches, and ulcerations, caused by an epidermotropic T-cell infiltrate. Thus, we defined Zeb1 as a novel repressor for IL-15 transcription in normal T-cells and show that epigenetic interference with its repressive function at the IL-15 promoter is associated with T-cell lymphoma in vivo, highlighting the role of epigenetic processes in inflammation-induced cancers and further supporting the role of Zeb1 as a putative tumor suppressor gene in CTCL Disclosures Porcu: Cell Medica: Research Funding; Celgene: Research Funding; Shape: Research Funding; Infinity: Research Funding; Seattle Genetics: Research Funding.

Translational repression of SLC26A3 by miR-494 in intestinal epithelial cells

SLC26A3 [downregulated in adenoma (DRA)] is a Cl−/HCO3− exchanger involved in electroneutral NaCl absorption in the mammalian intestine. Altered DRA expression levels are associated with infectious and inflammatory diarrheal diseases. Therefore, it is critical to understand the regulation of DRA expression. MicroRNAs (miRNAs) are endogenous, small RNAs that regulate protein expression via blocking the translation and/or promoting mRNA degradation. To investigate potential modulation of DRA expression by miRNA, five different in silico algorithms were used to predict the miRNAs that target DRA. Of these miRNAs, miR-494 was shown to have a highly conserved putative binding site in the DRA 3′-untranslated region (3′-UTR) compared with other DRA-targeting miRNAs in vertebrates. Transfection with pmirGLO dual luciferase vector containing DRA 3′-UTR (pmirGLO-3′-UTR DRA) resulted in a significant decrease in relative luciferase activity compared with empty vector. Cotransfection of the DRA 3′-UTR luciferase vector with a miR-494 mimic further decreased luciferase activity compared with cells transfected with negative control. The transfection of a miR-494 mimic into Caco-2 and T-84 cells significantly increased the expression of miR-494 and concomitantly decreased the DRA protein expression. Mutation of the seed sequences for miR-494 in 3′-UTR of DRA abrogated the effect of miR-494 on 3′-UTR. These data demonstrate a novel regulatory mechanism of DRA expression via miR-494 and indicate that targeting this microRNA may serve to be a potential therapeutic strategy for diarrheal diseases.