LncRNA RUNX1-IT1 affects the differentiation of Th1 cells by regulating NrCAM transcription in Graves' disease.

Graves' disease (GD) is a kind of autoimmune diseases. The development of GD is closely related to the imbalance of Th1/Th2 generated by the differentiation of CD4+ T cells. This study was sought to clarify the role of lncRNA RUNX1-IT1 and explore the mechanism of its function. The expressions of RUNX1-IT1 and Neural cell adhesion molecule (NrCAM) in the peripheral blood of GD patients were detected by qRT-PCR and Western blot. We performed RNA pull down, RIP, and ChIP experiments to verify the correlation between p53 and RUNX1-IT1, p53 and NrCAM. The levels of Th1 cells differentiation markers were detected by Flow cytometry assay and ELISA. The expressions of lncRNA RUNX1-IT1 and NrCAM were most significantly up-regulated in CD4+ T cells of GD patients, and NrCAM expression was significantly positively correlated with RUNX1-IT1 expression. Furthermore, p53 was a potential transcription factor of NrCAM, which could interact with NrCAM. NrCAM level was up-regulated after the overexpression of p53 in CD4+ T cells, while knockdown of RUNX1-IT1 reversed this effect. Down-regulation of NrCAM and RUNX1-IT1 could decrease the mRNA and protein levels of transcriptional regulator T-bet and CXC chemokine ligand 10 (CXCL10) in CD4+ T cells. Our results suggested that RUNX1-IT1 regulated the expressions of the important Th1 factor T-bet, CXCL10, and interferon γ (IFN-γ) by regulating NrCAM transcription, thus participating in the occurrence and development of specific autoimmune disease GD.

Berberine activates the ß-catenin/TCF4 signaling pathway by down-regulating miR-106b to promote GLP-1 production by intestinal L cells.

Berberine facilitates the production of glucagon-like peptide-1 (GLP-1) by intestinal L cells. Here, we aimed to reveal the mechanism of berberine facilitating the production of GLP-1 by intestinal L cells. In this study, we confirmed that the 100 mg/kg berberine daily through diet decreased the miR-106b expression and elevated the expressions of ß-catenin and T-cell factor 4 (TCF4) in colon tissues of high-fat diet mice; berberine decreased the concentrations of triglycerides, total cholesterol and the ratio of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in mouse serum samples; berberine decreased the blood glucose in the mouse tail vein blood and promoted GLP-1 production by intestinal L cells in mouse serum samples and elevated the GLP-1 expression in mouse colon tissues. Meanwhile, the mechanism analysis demonstrated that a dose of 100 µM berberine down-regulated the miR-106b expression by elevating the methylation levels of miR-106b in STC-1 cells and miR-106b bound to TCF4 in 293T cells. Moreover, the 100 mg/kg berberine daily through diet activated the ß-catenin/TCF4 signaling pathway by decreasing miR-106b, thereby facilitating GLP-1 production in intestinal L cells through the in vivo assays. Conclusively, our experimental data illustrated that berberine decreased miR-106b expression by increasing its methylation levels and then activated the ß-catenin/TCF4 signaling pathway, thereby facilitating GLP-1 production by intestinal L cells.

Long noncoding RNA MIR22HG promotes Leydig cell apoptosis by acting as a competing endogenous RNA for microRNA-125a-5p that targets N-Myc downstream-regulated gene 2 in late-onset hypogonadism.

Leydig cells (LCs) apoptosis is responsible for the deficiency of serum testosterone in Late-onset hypogonadism (LOH), while its specific mechanism is still unknown. This study focuses on the role of long noncoding RNA (lncRNA) MIR22HG in LC apoptosis and aims to elaborate its regulatory mechanism. MIR22HG was up-regulated in the testicular tissues of mice with LOH and H2O2-treated TM3 cells (mouse Leydig cell line). Interference of MIR22HG ameliorated cell apoptosis and upregulated miR-125a-5p expression in H2O2-treated TM3 cells. Then, the interaction between MIR22HG and miR-125a-5p was confirmed with RIP and RNA pull-down assay. Further study showed that miR-125a-5p downregulated N-Myc downstream-regulated gene 2 (NDRG2) expression by targeting its 3'-UTR of mRNA. What's more, MIR22HG overexpression aggravated cell apoptosis and reduced testosterone production in TM3 cells via miR-125a-5p/NDRG2 pathway. MIR22HG knockdown elevated testosterone levels in LOH mice. In conclusion, MIR22HG up-regulated NDRG2 expression through targeting miR-125a-5p, thus promoting LC apoptosis in LOH.

MicroRNA-194: a novel regulator of glucagon-like peptide-1 synthesis in intestinal L cells.

In the status of obesity, the glucagon-like peptide-1 (GLP-1) level usually declines and results in metabolic syndrome. This study aimed to investigate the intracellular mechanism of GLP-1 synthesis in L cells from the perspective of microRNA (miRNA). In the present study, we found that GLP-1 level was down-regulated in the plasma and ileum tissues of obese mice, while the ileac miR-194 expression was up-regulated. In vitro experiments indicated that miR-194 overexpression down-regulated GLP-1 level, mRNA levels of proglucagon gene (gcg) and prohormone convertase 1/3 gene (pcsk1), and the nuclear protein level of beta-catenin (ß-catenin). Further investigation confirmed that ß-catenin could promote gcg transcription through binding to transcription factor 7-like 2 (TCF7L2). miR-194 suppressed gcg mRNA level via negatively regulating TCF7L2 expression. What's more, forkhead box a1 (Foxa1) could bind to the promoter of pcsk1 and enhanced its transcription. miR-194 suppressed pcsk1 transcription through targeting Foxa1. Besides, the interference of miR-194 reduced palmitate (PA)-induced cell apoptosis and the anti-apoptosis effect of miR-194 inhibitor was abolished by TCF7L2 knockdown. Finally, in HFD-induced obese mice, the silence of miR-194 significantly elevated GLP-1 level and improved the metabolic symptoms caused by GLP-1 deficiency. To sum up, our study found that miR-194 suppressed GLP-1 synthesis in L cells via inhibiting TCF7L2-mediated gcg transcription and Foxa1-mediated pcsk1 transcription. Meanwhile, miR-194 took part in the PA-induced apoptosis of L cells.

Follicular thyroid carcinoma but not adenoma recruits tumor-associated macrophages by releasing CCL15.

BACKGROUND: The differential diagnosis of follicular thyroid carcinoma (FTC) and follicular adenoma (FA) before surgery is a clinical challenge. Many efforts have been made but most focusing on tumor cells, while the roles of tumor associated macrophages (TAMs) remained unclear in FTC. Here we analyzed the differences between TAMs in FTC and those in FA. METHODS: We first analyzed the density of TAMs by CD68 immunostaining in 59 histologically confirmed FTCs and 47 FAs. Cytokines produced by FTC and FA were profiled using antibody array, and validated by quantitative PCR. Chemotaxis of monocyte THP-1 was induced by condition medium of FTC cell lines (FTC133 and WRO82-1) with and without anti-CCL15 neutralizing antibody. Finally, we analyzed CCL15 protein level in FTC and FA by immunohistochemistry. RESULTS: The average density of CD68(+) cells was 9.5 ± 5.4/field in FTC, significantly higher than that in FA (4.9 ± 3.4/field, p < 0.001). Subsequently profiling showed that CCL15 was the most abundant chemokine in FTC compared with FA. CCL15 mRNA in FTC was 51.4-folds of that in FA. CM of FTC cell lines induced THP-1 cell chemotaxis by 33 ~ 77%, and anti-CCL15 neutralizing antibody reduced THP-1 cell migration in a dose-dependent manner. Moreover, we observed positive CCL15 immunostaining in 67.8% of FTCs compared with 23.4% of FAs. CONCLUSION: Our study suggested FTC might induce TAMs infiltration by producing CCL15. Measurement of TAMs and CCL15 in follicular thyroid lesions may be applied clinically to differentiate FTC from FA pre-operation.

[Determination of three glycosides from herbs of Swertia punicea by RP-HPLC].

OBJECTIVE: To develop a RP-HPLC method for determination of three glycosides in Swertia punicea. METHOD: Chromatographic column: Alltimal C18 (4.6 mm x 250 mm, 5 microm). Mobile phase: methanol-water (including 0.05% H3PO4), and gradient elution. Flow rate: 1 mL x min(-1). Wavelength: 254 nm. Column temperture: 30 degrees C. RESULT: The calibration curves of gentiopicroside, mangiferin and swertrianolin were in good linearity over the range of 31.3-281.7, 0.31-2.78, 0.55-4.91 microg, (r = 0.9996, 0.9993, 0.9995). The average recoveries were 103.36%, 101.42% and 97.39%, with RSD less then 3% (n = 5). CONCLUSION: It is a simple and sensitive meathod in controlling the quality of S. punicea.

An experiment on standardized cell culture assay in assessing the activities of Composite Artemisia Capillaris Tablets against hepatitis B virus replication in vitro.

OBJECTIVE: To explore the activities of Composite Artemisia Capillaris Tablet (CACT) against hepatitis B virus replication in vitro. METHODS: By means of radioimmunoassay (RIA), Dot blot and Southern blot, the surface and e antigen production of 2.2.15 cells, HBV DNA in 2.2.15 cell culture medium and that in 2.2.15 cells were examined respectively. RESULTS: HBsAg, HBeAg values of 2.2.15 cells treated by CACT were lower than those of the control, the HBV DNA quantities in culture medium and in 2.2.15 cells decreased as compared with those cells with no treatment by CACT given to them. CONCLUSION: CACT could inhibit HBV DNA replication, showing its potential antiviral activity in hepatitis B treatment.