👤 Shenyou Nie

Hepatitis E virus- (HEV-) mediated hepatitis has become a global public health problem. An important regulatory protein of HEV, ORF3, influences multiple signal pathways in host cells. In this study, to investigate the function of ORF3 from the swine form of HEV (SHEV), high-throughput RNA-Seq-based screening was performed to identify the differentially expressed genes in ORF3-expressing HepG2 cells. The results were validated with quantitative real-time PCR and gene ontology was employed to assign differentially expressed genes to functional categories. The results indicated that, in the established ORF3-expressing HepG2 cells, the mRNA levels of CLDN6, YLPM1, APOC3, NLRP1, SCARA3, FGA, FGG, FGB, and FREM1 were upregulated, whereas the mRNA levels of SLC2A3, DKK1, BPIFB2, and PTGR1 were downregulated. The deregulated expression of CLDN6 and FREM1 might contribute to changes in integral membrane protein and basement membrane protein expression, expression changes for NLRP1 might affect the apoptosis of HepG2 cells, and the altered expression of APOC3, SCARA3, and DKK1 may affect lipid metabolism in HepG2 cells. In conclusion, ORF3 plays a functional role in virus-cell interactions by affecting the expression of integral membrane protein and basement membrane proteins and by altering the process of apoptosis and lipid metabolism in host cells. These findings provide important insight into the pathogenic mechanism of HEV. Show less

BACKGROUND Apoptosis plays an important role in the physiology of platelet function. We aimed to detect the effect of the platelet integrin αIIbβ3 inhibitor, tirofiban, on apoptotic events, including mitochondrial inner-membrane potential (ΔΨm), phosphatidylserine (PS) exposure on platelet surface, and the generation of reactive oxygen species (ROS), when washed platelets were stimulated with thrombin. MATERIAL AND METHODS The study included washed platelets from healthy humans, divided into 4 groups: vehicle, and tirofiban (0.05 μg/ml, 0.25 μg/ml, and 0.5 μg/ml). Platelets were pretreated with vehicle or tirofiban and incubated at 37°C with agitation for 6 h and 24 h. Before thrombin addition, the vehicle group divided into 2 equal groups. Except one vehicle group, the other 4 groups were all stimulated with thrombin (1 U/ml) for 30 min at 37°C. Using flow cytometry, we studied the DYm and PS exposure on platelet surfaces, and the generation of ROS in platelets. RESULTS We observed that at the time of 6 h and 24 h, thrombin-stimulated vehicle platelets induced significant depo-larization of ΔΨm, higher PS exposure, and increased ROS production compared with the vehicle group (P<0.01). However, the tirofiban group had significantly more recovery of DYm, PS exposure, and ROS production compared with the thrombin group (P<0.01). CONCLUSIONS The platelet integrin αIIbβ3 inhibitor, tirofiban, inhibits the depolarization of DYm, PS exposure on platelet surface, and ROS production when stimulated with thrombin. These results suggest that αIIbβ3 inhibitor inhibits the initiation of apoptosis in platelets, showing a potential clinical application of tirofiban as an apoptosis inhibitor. Show less

Perfluorooctane sulfonate (PFOS), one persistent organic pollutant, has been widely detected in the environment, wildlife and human. Currently few studies have documented the effects of chronic PFOS exposure on lipid metabolism, especially in aquatic organisms. The underlying mechanisms of hepatotoxicity induced by chronic PFOS exposure are still largely unknown. The present study defined the effects of chronic exposure to low level of PFOS on lipid metabolism using zebrafish as a model system. Our findings revealed a severe hepatic steatosis in the liver of males treated with 0.5μM PFOS as evidenced by hepatosomatic index, histological assessment and liver lipid profiles. Quantitative PCR assay further indicated that PFOS significantly increase the transcriptional expression of nuclear receptors (nr1h3, rara, rxrgb, nr1l2) and the genes associated with fatty acid oxidation (acox1, acadm, cpt1a). In addition, chronic PFOS exposure significantly decreased liver ATP content and serum level of VLDL/LDL lipoprotein in males. Taken together, these findings suggest that chronic PFOS exposure induces hepatic steatosis in zebrafish via disturbing lipid biosynthesis, fatty acid β-oxidation and excretion of VLDL/LDL lipoprotein, and also demonstrate the validity of using zebrafish as an alternative model for PFOS chronic toxicity screening. Show less

High-altitude polycythemia (HAPC) inducing gastric mucosal lesion (GML) is still out of control and molecular mechanisms remain widely unknown. To address the issues, endoscopy and histopathological analyses were performed. Meanwhile, microarray-based transcriptome profiling was conducted in the gastric mucosa from 3 pairs of healthy subjects and HAPC-induced GML patients. HAPC caused morphological changes and pathological damages of the gastric mucosa of GML patients. A total of 10304 differentially expressed genes (DEGs) were identified, including 4941 up-regulated and 5363 down-regulated DEGs in gastric mucosa of GML patients compared with healthy controls (fold change ≥2, P<0.01 and FDR <0.01). Particularly, apolipoprotein genes APOA4 and APOC3 were 1473-fold and 1468-fold up-regulated in GML patients compared with the controls. In contrast, gastric intrinsic factor (GIF) was 1102-fold down-regulated in GML patients compared with the controls. APOA4 (chr11:116691770-116691711), APOC3 (chr11:116703530-116703589) and GIF (chr11:59603362-59603303) genes are all located on chromosome 11. APOA4 and APOC3 act as an inhibitor of gastric acid secretion while gastric acid promotes ulceration. GIF deficiency activates a program of acute anemia, which may antagonize polycythemia while polycythemia raises the risk of GML. Therefore, the present findings reveal that HAPC-induced GML inspires the protection responses by up-regulating APOA4 and APOC3, and down-regulating GIF. These results may offer the basic information for the treatment of HAPC-induced gastric lesion in the future. Show less

RNA G-quadruplexes (G4s) play important roles in RNA biology. However, the function and regulation of mRNA G-quadruplexes in embryonic development remain elusive. Previously, we identified RHAU (DHX36, G4R1) as an RNA helicase that resolves mRNA G-quadruplexes. Here, we find that cardiac deletion of Rhau leads to heart defects and embryonic lethality in mice. Gene expression profiling identified Nkx2-5 mRNA as a target of RHAU that associates with its 5' and 3' UTRs and modulates its stability and translation. The 5' UTR of Nkx2-5 mRNA contains a G-quadruplex that requires RHAU for protein translation, while the 3' UTR of Nkx2-5 mRNA possesses an AU-rich element (ARE) that facilitates RHAU-mediated mRNA decay. Thus, we uncovered the mechanisms underlying Nkx2-5 post-transcriptional regulation during heart development. Meanwhile, this study demonstrates the function of mRNA 5' UTR G-quadruplex-mediated protein translation in organogenesis. Show less

G-quadruplex (G4) DNA and G4 DNA resolvase are involved in a variety of biological processes. To understand the biological function of G4 DNA structures and their resolvases in spermatogenesis, we investigated the distribution of G4 structures in mouse testis and identified their alterations during spermatogenesis. Meanwhile, we studied the function of RNA helicase associated with AU-rich element (RHAU), a G4 DNA resolvase, in spermatogenesis with a germ-cell-specific knockout mouse model. The results showed that the ablation of RHAU in germ cells caused the increase of G4 structures and thus resulted in the decrease of spermatogonial differentiation. c-kit, a spermatogonia differentiation-related gene, contains two G4 DNA motifs on its promoter. We found its expression was significantly downregulated in RHAU conditional knockout testis. A further analysis demonstrated that RHAU directly bound to the G4 structures to activate c-kit expression. We concluded that RHAU regulates spermatogonia differentiation by promoting c-kit expression via directly binding to the G4 DNA motifs c-kit promoter. Show less

Smooth muscle 22α (SM22α) is involved in stress fiber formation and enhances contractility in vascular smooth muscle cells (VSMCs). In many cases, SM22α acts as an adapter protein to assemble signaling complexes and regulate signaling, but whether SM22α regulates contractile signaling induced by angiotensin II (AngII) remains unclear. To address this issue, we established a hypertension model of Sm22α(-/-) mice, and demonstrated that hypertension induced by AngII was attenuated in Sm22α(-/-) mice. A decreased vasoconstriction was observed in aortic rings from Sm22α(-/-) mice. Furthermore, loss of SM22α resulted in a reduced contractile response to AngII in VSMCs in vitro. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by AngII was impaired following depletion of SM22α, in parallel with a reduced contractility. The decay of ERK1/2 activity was associated with increased expression of mitogen-activated protein kinase phosphatase 3 (MKP3). Inhibition of MKP3 activity rescued ERK1/2 activity. SM22α depletion caused an enhanced interaction of MKP3 with ERK1/2, and a reduced ubiquitination and degradation of MKP3. Knockdown of SM22α extended the half-life of MKP3. In conclusion, SM22α promotes AngII-induced contraction by maintenance of ERK1/2 signaling cascades through facilitating ubiquitination and degradation of MKP3. The vasoconstriction is attenuated in aortic rings from Sm22α(-/-) mice. MKP3 mediates dephosphorylation of ERK1/2 in AngII-induced VSMC contraction. SM22α inhibits the interaction of ERK1/2 with MKP3. SM22α promotes ubiquitination and degradation of MKP3. SM22α facilitates AngII-induced contraction by maintenance of ERK1/2 signaling. Show less

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator. The present study aimed to investigate the effect of FGF21 on cholesterol efflux and the expression of ATP binding cassette (ABC) A1 and G1 in human THP-1 macrophage-derived foam cells. Furthermore, the present study aimed to investigate the role of the liver X receptor (LXR) α in this process. A model of oxidized low-density lipoprotein-induced foam cells from human THP-1 cells was established. The effect of FGF21 on cholesterol efflux was analyzed using a liquid scintillation counter. The expression of ABCA1 and ABCG1 was determined using quantitative polymerase chain reaction and western blot analyses. FGF21 was found to enhance apolipoprotein A1- and high-density lipoprotein-mediated cholesterol efflux. FGF21 was also observed to increase the mRNA and protein expression of ABCA1 and ABCG1. Furthermore, LXRα-short interfering RNA attenuated the stimulatory effects induced by FGF21. These findings suggest that FGF21 may have a protective effect against atherosclerosis by enhancing cholesterol efflux through the induction of LXRα-dependent ABCA1 and ABCG1 expression. Show less

Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits. Show less

To investigate the effects of neopterin on ABCA1 expression and cholesterol efflux in human THP-1 macrophage-derived foam cells, and to explore the role of the liver X receptor alpha (LXRα) involved. In the present study, THP-1 cells were pre-incubated with ox-LDL to become foam cells. The protein and mRNA expression were examined by Western blot assays and real-time quantitative PCR, respectively. Liquid scintillation counting and high performance liquid chromatography assays were used to test cellular cholesterol efflux and cholesterol content. Neopterin decreased ABCA1 expression and cholesterol efflux in a time- and concentration-dependent manner in THP-1 macrophage-derived foam cells, and the LXRα siRNA can reverse the inhibitory effects induced by neopterin. Neoterin has a negative regulation on ABCA1 expression via the LXRα signaling pathway, which suggests the aggravated effects of neopterin on atherosclerosis. Show less

Glucose-dependent insulinotropic peptide (GIP) exerts multiple biological effects via the G-protein-coupled receptor GIPR, including glucose-stimulated insulin production and secretion, cell proliferation, and antiapoptosis in pancreatic β-cells. In an obese state, the circulating level of GIP is elevated. GIPR-knockout mice are resistant to high-fat-diet-induced obesity. The rising evidence suggests a potential role of GIP in adipocyte biology and lipid metabolism. In our study, we overexpressed GIPR in 3T3-L1 CAR adipocytes and demonstrated that GIP impaired the physiological functions of adipocytes as a consequence of increased production of inflammatory cytokines and chemokines and phosphorylation of IkB kinase (IKK)-β through activation of the cAMP-PKA pathway. Activation of Jun N-terminal kinase (JNK) pathway was also observed during GIP-induced inflammatory responses in adipocytes. The inhibition of JNK blocked GIP-stimulated secretion of inflammatory cytokines and chemokines, as well as phosphorylation of IKKβ. In addition, GIP-induced inflammatory response increased basal glucose uptake but inhibited insulin-stimulated glucose uptake. Moreover, GIP-induced adipocyte inflammation impaired insulin signaling in adipocytes as demonstrated by a reduction of AKT phosphorylation. Our results suggest that GIP might be one of the stimuli attributable to obesity-induced insulin resistance via the induction of adipocyte inflammation. Show less

To investigate the role of liver X receptors (LXRs) on endothelin-1 (ET-1) induced murine HL-1 cardiomyocytes hypertrophy. Cultured murine HL-1 cardiomyocytes were divided into four experiment groups: (1) CONTROL GROUP:treated with DMSO; (2) T0901317 group:treated with LXRs agonist T0901317 (1 µmol/L); (3) ET-1 group:treated with ET-1 (1 nmol/L); (4) T0901317 + ET-1 group:treated with T0901317 (1 µmol/L) for 8 hours, then treated with ET-1 (1 nmol/L). Twenty-four hours later, immunofluorescent staining was performed on HL-1 cells, the surface area of HL-1 cells was analyzed with NIH Image J software, and the synthetic rate of protein in HL-1 cells was detected by (3)H-leucine incorporation. The mRNA level of atrial natriuretic peptide (ANP) and β-myosin heavy chain (β-MyHC) was measured by quantitative realtime PCR. The effect of T0901317 on mRNA expression of ANP was also detected after LXRs gene silencing. The surface area of HL-1 cells, mRNA expression of ANP and β-MyHC, and (3)H-leucine incorporation in ET-1 group were 2.00 ± 0.29, 1.98 ± 0.47, 2.13 ± 0.39 and 1.79 ± 0.17, respectively, which were significantly higher than those of control group (1.00 ± 0.26, 1.00 ± 0.21, 1.00 ± 0.31 and 1.00 ± 0.03, respectively, all P < 0.05). Compared with ET-1 group, the surface area of HL-1 cells, mRNA expression of ANP and β-MyHC, and (3)H-leucine incorporation were significantly decreased in T0901317 + ET-1 group (1.24 ± 0.25, 1.19 ± 0.21, 1.48 ± 0.27 and 1.15 ± 0.11, respectively, all P < 0.05). After inhibition of LXRα/β expression in HL-1 cardiomyocytes using the specific siRNAs, the mRNA expression of ANP in T0901317 + ET-1 group was 1.78 ± 0.05, which was similar as that in ET-1 group (1.94 ± 0.17, P > 0.05). T0901317, an agonist of LXRs, could inhibit ET-1 induced cardiac hypertrophy in vitro, and LXR ligand-mediated inhibition on ANP mRNA expression by T0901317 is receptor dependent. Show less

Insulin resistance results in dysregulated hepatic gluconeogenesis that contributes to obesity-related hyperglycemia and progression of type 2 diabetes mellitus (T2DM). Recent studies show that MAPK phosphatase-3 (MKP-3) promotes gluconeogenic gene transcription in hepatoma cells, but little is known about the physiological role of MKP-3 in vivo. Here, we have shown that expression of MKP-3 is markedly increased in the liver of diet-induced obese mice. Consistent with this, adenovirus-mediated MKP-3 overexpression in lean mice promoted gluconeogenesis and increased fasting blood glucose levels. Conversely, shRNA knockdown of MKP-3 in both lean and obese mice resulted in decreased fasting blood glucose levels. In vitro experiments identified forkhead box O1 (FOXO1) as a substrate for MKP-3. MKP-3-mediated dephosphorylation of FOXO1 at Ser256 promoted its nuclear translocation and subsequent recruitment to the promoters of key gluconeogenic genes. In addition, we showed that PPARγ coactivator-1α (PGC-1α) acted downstream of FOXO1 to mediate MKP-3-induced gluconeogenesis. These data indicate that MKP-3 is an important regulator of hepatic gluconeogenesis in vivo and suggest that inhibition of MKP-3 activity may provide new therapies for T2DM. Show less

To screen the MYBPC3 gene mutations in Han Chinese patients with hypertrophic cardiomyopathy (HCM). Sixty-six patients with HCM were enrolled for the study. The exons in the functional regions of MYBPC3 were amplified with PCR and the products were sequenced. Four novel mutations and four common polymorphisms were identified in this patient cohort. A Lys301fs mutation in exon10 was evidenced in a H30, and when he was 47 years old, he had the chest tightness, shortness of breath with septal hypertrophy of 18.7mm; a Asp463stop mutation in exon17 was detected in a H48, he was 24 years old 24-year-old when a medical examination showed ventricular septal hypertrophy of 15.4 mm; both Gly523Arg mutation in exon18 and Tyr847His mutation in exon26 were found in a H53 with onset age 36 years old, feeling chest tightness after excise and his ventricular septal hypertrophy was 27 mm that time. MYBPC3 mutations occurred in 4.5% patients in this cohort. These mutations were not found in 100 non-HCM control patients. MYBPC3 mutation is presented in a small portion of Han Chinese patients with HCM. Show less

Liver X receptors (LXRs) are important transcriptional regulators of lipid homeostasis and proliferation in several cell types. However, the roles of LXRs in pancreatic beta cells have not been fully established. The aim of this study was to investigate the effects of LXRs on pancreatic beta cell proliferation. Gene expression was analysed using real-time RT-PCR. Transient transfection and reporter gene assays were used to determine the transcriptional activity of LXRs in pancreatic beta cells. Cell viability and proliferation were analysed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DNA fluorometric, BrdU labelling and [(3)H]thymidine incorporation assays. Cell cycle distribution was investigated by flow cytometry analysis. Adenovirus-based RNA interference was used to knockdown LXRalpha, LXRbeta and p27 in MIN6 cells and mouse islets. We found that both Lxralpha (also known as Nr1h3) and Lxrbeta (also known as Nr1h2) were expressed and transactivated the LXR response element in HIT-T15 and MIN6 cells. Activation of LXRs dose-dependently inhibited pancreatic beta cell viability and proliferation. This was accompanied by beta cell cycle arrest at the G1 phase. Furthermore, LXR activation increased levels of the p27 protein by inhibiting its degradation. Knockdown of p27 reversed these effects of LXR activation on growth inhibition and cell cycle arrest. Our observations indicate that LXR activation inhibits pancreatic beta cell proliferation through cell cycle arrest. A well-known regulator of pancreatic beta cell cycle progression, p27, is upregulated and mediates the effects of LXRs on growth inhibition in beta cells. These observations suggest the involvement of aberrant activation of LXR in beta cell mass inadequacy, which is an important step in the development of type 2 diabetes. Show less

Intermittent administration of PTH stimulates bone formation, but the precise mechanisms responsible for PTH responses in osteoblasts are only incompletely understood. Here we show that binding of PTH to its receptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternary complex containing PTH, PTH1R, and LRP6 promoted rapid phosphorylation of LRP6, which resulted in the recruitment of axin to LRP6, and stabilization of beta-catenin. Activation of PKA is essential for PTH-induced beta-catenin stabilization, but not for Wnt signaling. In vivo studies confirmed that PTH treatment led to phosphorylation of LRP6 and an increase in amount of beta-catenin in osteoblasts with a concurrent increase in bone formation in rat. Thus, LRP6 coreceptor is a key element of the PTH signaling that regulates osteoblast activity. Show less