Silencing lncRNA NEAT1 reduces nonalcoholic fatty liver fat deposition by regulating the miR-139-5p/c-Jun/SREBP-1c pathway.

INTRODUCTION AND OBJECTIVES: Nonalcoholic fatty liver disease (NAFLD) starts with the abnormal accumulation of lipids in the liver. Long noncoding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) was reported to modulate hepatic metabolic homeostasis in NAFLD. However, little is known about the molecular mechanisms of NAFLD. MATERIALS AND METHODS: To establish a NAFLD cellular model, HepG2 cells and LO2 cells were treated with 1 mM free fatty acids (FFAs) for 24 h. NEAT1, miRNA (miR)-139-5p, c-Jun and sterol-regulatory element binding protein-1c (SREBP-1c) were evaluated using qPCR. The protein levels of c-Jun, SREBP1c, acetyl-CoA carboxylase (ACC) and fatty acid synthetase (FAS) were determined using western blotting. Moreover, Oil Red O staining was employed to assess lipid accumulation. In addition, a kit assay was performed to evaluate TG levels. Finally, the interactions among NEAT1, miR-139-5p, c-Jun and SREBP1c were identified by dual luciferase reporter gene assay. RESULTS: NEAT1, c-Jun and SREBP1c expression was markedly elevated, while miR-139-5p expression was reduced in the NAFLD cellular model. NEAT1 knockdown restrained lipid accumulation in the NAFLD cellular model by directly targeting miR-139-5p. Moreover, miR-139-5p overexpression suppressed lipid accumulation by directly suppressing c-Jun expression. In addition, c-Jun silencing suppressed lipid accumulation by directly targeting SREBP1c. Finally, miR-139-5p inhibition mitigated the inhibitory effect of sh-NEAT1 on lipid accumulation. CONCLUSION: NEAT1 aggravated FFA-induced lipid accumulation in hepatocytes by regulating the c-Jun/SREBP1c axis by sponging miR-139-5p, indicating the potential of NEAT1 as a promising therapeutic target for NAFLD.

Identification of novel hepatitis B virus therapeutic vaccine candidates derived from polymerase protein.

Hepatitis B virus (HBV) infection is a worldwide health problem with high morbidity and mortality rates. The therapeutic vaccine is a promising method of treatment, and HBV polymerase plays a vital role in viral replication. Therefore, a therapeutic vaccine that binds to HBV DNA polymerase may control HBV infection. We predicted and selected epitopes of polymerase using online databases and analysis software. We then performed molecular docking and peptide binding assays to evaluate the binding energies and affinities between polymerase epitopes and the HLA-A0201 molecule. Finally, we induced T cells from the peripheral blood mononuclear cells (PBMCs) of healthy donors using each epitope and quantified the functions of epitope-specific T cells by IFN-γELISPOT assay, T2 cell cytotoxicity assay, HepG2.2.15 cell cytotoxicity assay and HBV gene expression assays. Four epitopes (RVTGGVFLV, GLLGFAAPF, LLDDEAGPL and YMDDVVLGA) had low binding energy and two epitopes (RVTGGVFLV and GLLGFAAPF) had a high binding affinity. The T cells stimulated by two epitopes (GLLGFAAPF and HLYSHPIIL) had a greater ability to induce immune response and suppress HBV. The HBV DNA polymerase epitopes identified in this study are promising targets for designing an epitope-based therapeutic vaccine against HBV.

Hepatitis B virus-specific effector CD8+ T cells are an important determinant of disease prognosis: A meta-analysis.

BACKGROUND: Hepatitis B virus (HBV)-specific effector CD8+ T cells are critical for viral clearance. To determine the effects of HBV-specific effector CD8+ T cells on HBV infection, we performed a meta-analysis of the available literature. METHODS: Electronic database searches identified appropriately designed studies that detected specific CD8+ T cells in HBV-infected patients. Our main endpoints were the course of infection, seroconversion of HBV "e" antigen (HBeAg), the level of HBVDNA, and alanine aminotransferase (ALT) activity. We used a fixed/random model for analysis, according to the results of a heterogeneity test (P value of Q-squared, I2). RESULTS: Our searches found five eligible articles. Pooled estimation of the reported results showed that levels of specific CD8+ T cells were significantly higher in patients with acute hepatitis B than in patients with chronic hepatitis B (odds ratio [OR] = 76.30, 95% confidence interval [CI]: 15.37-378.70). With respect to chronic hepatitis B, patients with <107 copies/ml HBVDNA had higher levels of specific CD8+ T cells relative to patients with >107 copies/ml HBVDNA, but the difference had no statistics significance (OR: 3.89, 95% CI: 0.71-21.33). Patients with negative HBeAg or positive anti-HBeAg antibody (anti-HBe) results had significantly higher levels of specific CD8+ T cells versus patients with positive HBeAg results (OR: 5.82, 95% CI: 1.41-24.13). There were no significant associations between the levels of specific CD8+ T cells and serum ALT activity (OR = 0.86, 95% CI: 0.01-74.15). CONCLUSION: HBV-specific effector CD8+ T cells influence the disease activity in HBV-infected patients in various ways and determine prognosis by eliminating the virus. Therefore, efforts of studying HBV-specific effector CD8+ T cells focused vaccine are potentially needed.

lncRNA NEAT1 regulates fibrosis and inflammatory response induced by nonalcoholic fatty liver by regulating miR-506/GLI3.

As one of the most common liver disorders worldwide, nonalcoholic fatty liver disease (NAFLD) begins with the abnormal accumulation of triglyceride (TG) in the liver and can lead to inflammation and fibrosis. Long noncoding RNA (lncRNA) NEAT1 was reported to promote NAFLD progress. However, its molecular mechanism in NAFLD was not fully clear. In vitro cellular model of NAFLD was established with BRL3A cell treated by free fatty acid (FFA). Cell Counting Kit-8 (CCK-8) assay was carried out to assess cell proliferation. The expression of mRNA and protein of inflammation and fibrosis in BRL3A cell was detected by qRT-PCR and Western blot. Bioinformatics and dual-luciferase reporter assays were used to predict and validate the interaction between NEAT1 and miR-506 as well as GLI3 and miR-506. NEAT1 was upregulated while miR-506 was downregulated in the progression of NAFLD. Meanwhile, NEAT1 and miR-506 were proved to regulate fibrosis, inflammatory response, and lipid metabolism. Knockdown of NEAT1 inhibited GLI3 expression and promoted miR-506 expression, Overexpression of miR-506 inhibited NEAT1 and GLI3 expression. Moreover, dual-luciferase reporter assays proved that miR-506 could bind to NEAT1 and GLI3, whereas NEAT1 could sponge miR-506 to regulate GLI3 expression. lncRNA NEAT1 could regulate fibrosis, inflammatory response, and lipid metabolism via the miR-506/GLI3 axis as a ceRNA, which is a novel mechanistic role in the regulation of NAFLD. These results provide a new potential treatment target for NAFLD.

miR-1273g silences MAGEA3/6 to inhibit human colorectal cancer cell growth via activation of AMPK signaling.

AMP-activated protein kinase (AMPK) is a metabolic regulator that acts to limit the growth of cancer cells. AMPK is downregulated by melanoma antigens A3/6 (MAGEA3/6), which are cancer-specific proteins that enhance the activity of specific E3 ubiquitin ligases to ubiquitinate and degrade AMP-activated protein kinase α1 (AMPKα1). Here, using a bioinformatic approach, we identified a microRNA, miR-1273g-3p, that is predicted to target the 3' untranslated region (UTR) of MAGEA3/6. Analyzing miR-1273g-3p expression in human colon cancer tissues, we found a reduction in miR-1273g-3p expression correlating with increased MAGEA3/6 expression and AMPKα1 downregulation. Expression of miR-1273g in HT-29 cells and primary human colon cancer cells down-regulated MAGEA3/6, leading to AMPKα1 upregulation, inhibition of proliferation and cell apoptosis. The anti-CRC activity of miR-1273g was blocked by AMPKα1 knockout. MAGEA3/6 shRNA silencing mimicked and abolished miR-1273g-induced actions in HT-29 cells. In vivo, miR-1273g- or MAGEA3/6 shRNA-expressing HT-29 tumors grew significantly slower than control tumors. We propose a novel miRNA-based mechanism, whereby miR-1273g represses MAGEA3/6 expression in human CRC cells and tissues, which may provide a novel cancer-specific therapeutic.

The Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis in Intestinal Epithelial Cells Regulates Intestinal Barrier Function During Intestinal Epithelial Cells-CD4+T-Cell Interactions.

BACKGROUND/AIMS: Epithelial cells line the intestinal mucosa and form an important barrier for maintaining host health. This study aimed to explore the mechanism of the Sphingosine-1-phosphate (S1P)/Sphingosine-1-phosphate receptor 2 (S1PR2) pathway in intestinal epithelial cells (IECs) that participate in the intestinal barrier function. METHODS: In this study, we constructed a knockout of the S1PR2 gene in mice, and Dextra sulfate sodium (DSS) was used to induce colitis. We isolated IECs from wild type (WT) and S1PR2-/- mice, and the endogenous expression of S1PR2 and Zonula occludens 1 (ZO-1) in IEC were detected by Western blot. Next, the major histocompatibility complex II (MHC-II) expression was analyzed by reverse transcription quantitative real-time (RT-qPCR) and flow cytometry. The in vivo and in vitro intestinal permeability were evaluated by serum fluorescein isothiocyanate (FITC) concentration. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) levels in cell suspension were analyzed by enzyme-linked immuno sorbent assay (ELISA). A carboxyfluorescein diacetate succinimidyl ester (CFSE) assay was used to detect the T-cell proliferation in a co-culture system. RESULTS: The intestinal mucosal barrier damage in S1PR2-/- mice was more severe than in the WT mice, and there were more CD4+T-cells in the colon tissue of DSS-treated S1PR2-/- mice. Either the mouse colon carcinoma cell line (CT26. WT) or the IECs upregulated MHC-II expression, which then promoted CD4+T-cell proliferation. The S1P/S1PR2 pathway controlled MHC-II expression to regulate CD4+T-cell proliferation via the extracellular signal-regulated kinase (ERK) pathway. In addition, the IFN-γ that was secreted by CD4+T-cells increased DSS-induced damage of intestinal epithelial cell barrier function. ZO-1 expression was increased by S1P in CT26.WT cells, while S1PR2 antagonist JTE-013 expression was downregulated. However, in CT26.WTsi-S1PR2 cells, S1P had no effect on ZO-1 expression. CONCLUSIONS: The S1P/S1PR2 axis in IECs mediated CD4+T-cell activation via the ERK pathway and MHC-II expression to regulate intestinal barrier function.

Analysis of epitope-based vaccine candidates against the E antigen of the hepatitis B virus based on the B genotype sequence: An in silico and in vitro approach.

Chronic hepatitis B virus infection is a worldwide health problem with no current effective strategy to achieve a cure. The Hepatitis B virus (HBV) E antigen (HBeAg) has a negative effect on the immune system and a therapeutic vaccine is a promising strategy in order to treat chronic virus infection. In this study, we analyzed and identified the MHC-I, MHC-II and B cell epitopes of the HBeAg based on a B genotype sequence of HBV using a bioinformatic approach and in vitro experiments. The computational approach provided us with four epitopes (LLWFHISCL, YLVSFGVWI, MQLFHLCLI, TVLEYLVSF) of the specific MHC-I allele HLA-A0201 that conformed to all criteria. Molecular docking and a peptide binding assay showed that epitope TVLEYLVSF had the lowest binding energy and epitope LLWFHISCL had the highest binding affinity to the HLA-A0201 molecule. An interferonγenzyme-linked immunospot assay and cytotoxicity assay revealed that epitope LLWFHISCL had the highest ability to induce and stimulate T cells. Furthermore, we determined four core peptides of MHC-II epitopes and a region of the B cell epitope. The epitopes and region identified in this research may be helpful in designing epitope-based vaccines and boosting the mechanism research of HBeAg and its effect on the immune system.

In Silico Analysis of Epitope-Based Vaccine Candidates against Hepatitis B Virus Polymerase Protein.

Hepatitis B virus (HBV) infection has persisted as a major public health problem due to the lack of an effective treatment for those chronically infected. Therapeutic vaccination holds promise, and targeting HBV polymerase is pivotal for viral eradication. In this research, a computational approach was employed to predict suitable HBV polymerase targeting multi-peptides for vaccine candidate selection. We then performed in-depth computational analysis to evaluate the predicted epitopes' immunogenicity, conservation, population coverage, and toxicity. Lastly, molecular docking and MHC-peptide complex stabilization assay were utilized to determine the binding energy and affinity of epitopes to the HLA-A0201 molecule. Criteria-based analysis provided four predicted epitopes, RVTGGVFLV, VSIPWTHKV, YMDDVVLGA and HLYSHPIIL. Assay results indicated the lowest binding energy and high affinity to the HLA-A0201 molecule for epitopes VSIPWTHKV and YMDDVVLGA and epitopes RVTGGVFLV and VSIPWTHKV, respectively. Regions 307 to 320 and 377 to 387 were considered to have the highest probability to be involved in B cell epitopes. The T cell and B cell epitopes identified in this study are promising targets for an epitope-focused, peptide-based HBV vaccine, and provide insight into HBV-induced immune response.

Antiarrhythmic ionic mechanism of Guanfu base A--Selective inhibition of late sodium current in isolated ventricular myocytes from guinea pigs.

The present study was designed to determine the effects of Guanfu base A (GFA) on the late sodium current (INa.L), transient sodium current (INa.T), HERG current (IHERG), and Kv1.5 current (IKv1.5). The values of INa.L, INa.T, IHERG and IKv1.5 were recorded using the whole-cell patch clamp technique. Compared with other channels, GFA showed selective blocking activity in late sodium channel. It inhibited INa.L in a concentration-dependent manner with an IC50 of (1.57 ± 0.14) µmol · L(-1), which was significantly lower than its IC50 values of (21.17 ± 4.51) µmol · L(-1) for the INa.T. The inhibitory effect of GFA on INa,L was not affected by 200 µmol · L(-1) H2O2. It inhibited IHERG with an IC50 of (273 ± 34) µmol · L(-1) and has slight blocking effect on IKv1.5, decreasing IKv1.5 by only 20.6% at 200 µmol · L(-1). In summary, GFA inhibited INa.L selectively and remained similar inhibition in presence of reactive oxygen species. These findings may suggest a novel molecular mechanism for the potential clinical application of GFA in the treatment of cardiovascular disorders.

Cell-specific expression of the analgesic-antitumor peptide coding sequence under the control of the human α-fetoprotein gene promoter and enhancer.

The aim of the present study was to construct a gene-modified hepatocellular carcinoma (HCC)-specific analgesic-antitumor peptide (AGAP) expression vector regulated by the α-fetoprotein (AFP) promoter and enhancer, in order to evaluate its effect. The AFP promoter is generally used in HCC-specific gene therapy strategies. However, this approach is limited by the weak activity of the AFP promoter. Linking the AFP enhancer and promoter has been shown to generate a stronger and more HCC-selective promoter. The AGAP DNA fragment was amplified from the total RNA of the Chinese scorpion, Buthus martensii Karsch. The fragment was subsequently cloned into the pAFP plasmid with the minimal essential DNA fragment, which included the AFP gene promoter and enhancer, to construct the recombinant plasmid, pAFP-AGAP. The plasmid was transfected into HepG2 cells and the mRNA expression levels of AGAP were detected by reverse transcription polymerase chain reaction (RT-PCR). In addition, Cell Counting Kit 8 (CCK-8) was used to analyze the cytotoxicity of plasmid transfection. The length, position and orientation of the inserted AGAP gene were all confirmed to be correct; thus, the recombinant vector was successfully constructed. Using RT-PCR and CCK-8 analysis, the mRNA expression levels of AGAP and the cytotoxicity in AFP-producing human HCC cells were determined. The AFP promoter and enhancer were found to specifically accelerate the expression of the target genes within the cells that were positive for AFP. Therefore, the method used in the present study was demonstrated to be a novel integration of traditional Chinese medicine with western medicine.

Antiarrhythmic efficacy of CPUY102122, a multiple ion channel blocker, on rabbits with ischemia/reperfusion injury.

BACKGROUND: The antiarrhythmic potential of a novel multichannel blocker CPUY102122 (CY22) was investigated in the present study. METHODS: The effect of CY22 on rapid delayed rectifier potassium channel current (IKr) was studied using whole-cell patch clamp techniques in Chinese Hamster Ovary cells stably expressing human Ether-à-go-go-Related Gene. We further evaluated the antioxidant effects of CY22 and demonstrated the reversal of connexin down-regulation in the development of cardiac ventricular arrhythmias, which was produced using coronary ligation/reperfusion in rabbits. CY22 and Amiodarone were administered 30min prior to the procedure. Next, electrocardiograms were recorded, protein expression of left ventricular Connexin43 (Cx43), non-phosphorylation-Cx43 (np-Cx43), Rac-1 and gp-91[phox] were assayed using Western blot analysis, microstructural changes in the myocardium were observed and redox system activity was assayed. RESULTS: CY22 inhibited IKr in a concentration-dependent manner with IC50 value of 2.8±0.8µmol/L. CY22 treatment significantly decreased T-wave amplitude and QTc arrhythmic scores and ameliorated the shape of the infarcted myocardium compared to the model group. CY22 decreased the serum levels of creatine kinase, lactate dehydrogenase, and myocardial levels of malondialdehyde, as well as increased superoxide dismutase activity. Cx43 expression in the left ventricle was significantly increased by CY22 treatment, which significantly decreased np-43 expression, Rac-1 activity and gp-91[phox] protein expression. CONCLUSIONS: These results indicated that CY22 has both antiarrhythmic and cardiovascular protective effects partly by blocking IKr, the production of antioxidants and protection of Cx43.

New diterpenoid alkaloids from Aconitum coreanum and their anti-arrhythmic effects on cardiac sodium current.

Two new diterpenoid alkaloids, Guan-Fu base J (GFJ, 1) and Guan-Fu base N (GFN, 2) along with nineteen known alkaloids (3-21) were isolated from the roots of Aconitum coreanum (Lèvl.) Rapaics, which is the raw material of a new approval anti-arrhythmia drug "Acehytisine Hydrochloride". The structures of isolated compounds were established by means of 1D, 2D NMR spectroscopic and chemical methods. All isolates obtained in the present study were evaluated for their inhibitory effects on blocking the ventricular specific sodium current using a whole-cell patch voltage-clamp technique. Among these 21 compounds, Guan-Fu base S (GFS, 3) showed the strongest inhibitory effect with an IC50 value of 3.48 µM, and only hetisine-type C20 diterpenoid alkaloids showed promising IC50 values for further development.

[Strontium ranelate promotes osteogenic differentiation of rat bone mesenchymal stem cells through bone morphogenetic protein-2/Smad signaling pathway].

OBJECTIVE: To explore whether strontium ranelate (Sr) promotes osteoblast lineage differentiation of rat bone mesenchymal stem cells (BMSCs) through the bone morphogenetic protein-2 (BMP-2)/Smad signaling pathway. METHODS: Cultured rat BMSCs were exposed to different concentrations of Sr, noggin (an inhibitor of BMP-2) or Smad1 siRNA. The activity of alkaline phosphatase (ALP) in the exposed cells was detected by colorimetry, and the formation of mineralized nodules was observed with alizarin red staining. The expressions of phosphorylated (p) Smad1/5/8 and Runt-related transcription factor 2 (Runx2) in the cells were detected by Western blotting. RESULTS: Exposure to Sr at 0.1 to 10 mmol/L for 1 h markedly increased the expression of p-Smad1/5/8 in the BMSCs, and the increment was the most obvious following 1 mmol/L Sr exposure. Preconditioning with 100 ng/ml noggin for 2 h inhibited Sr-induced up-regulation of p-Smad1/5/8 expressions. Exposure of the cells to 0.1 to 5 mmol/L Sr for 6 h significantly enhanced Runx2 expression, and the peak enhancement occurred following 1 mmol/L Sr exposure. Transfection of the BMSCs with Smad1 siRNA decreased the basal level of Smad1/5/8 protein expression, and also inhibited Sr-induced up-regulation of p-Smad1/5/8 and Runx2 expressions as well as Sr-induced enhancement of ALP activity and formation of mineralized nodules. CONCLUSION: The BMP-2/Smad pathway is involved in Sr-induced osteoblast differentiation of rat BMSCs.

Transforming growth factor beta-1 C-509T polymorphism and cancer risk: a meta-analysis of 55 case-control studies.

AIM: To investigate the association of transforming growth factor-beta 1 (TGF-ß1) C-509T polymorphism and susceptibility to cancer by means of meta-analysis. METHODS: An extensive search was performed to identify eligible case-control studies investigating such a link. The strength of the association between TGF-ß1 C-509T polymorphism and cancer risk was assessed by pooled odds ratios (ORs) and 95%confidence intervals (95%CIs) in fixed or random effects models. RESULTS: 55 published case-control studies with a total number of 21,639 cases and 28,460 controls were included. Overall, there was no association between TGF-ß1 C-509T and cancer risk in all genetic comparison models (TT vs. CC: OR=1.01, 95%CI=0.89-1.15; T vs. C: OR=1.01, 95%CI=0.94-1.07). However, a stratified analysis by cancer type indicated -509 T allele was significantly associated with decreased risk of colorectal cancer (CRC) (TT vs. CT/CC: OR=0.85, 95%CI=0.76-0.95), especially for Caucasians (TT vs. CT/CC: OR=0.83, 95%CI=0.71-0.98) and for population-based studies (TT vs. CT/CC: OR=0.78, 95%CI=0.68- 0.89). CONCLUSION: This meta-analysis suggested that TGF-ß1 C-509T polymorphism might contribute to a decreased risk on colorectal cancer susceptibility, especially for Caucasians.