👤 Chunjing Feng

The long non-coding RNAs (lncRNAs) are a type of RNAs with more than 200nt in length and without any long open reading frame, but often have mRNA structural features. They can regulate the expression of target genes in different manners at the transcriptional and post-transcriptional levels. In recent years, various studies demonstrated that lncRNAs play crucial roles in adipogenesis. The long non-coding RNA, lnc-RAP3, located on the mouse chromosome 17, possesses a significantly differential expression pattern during mouse adipocyte differentiation; but its specific biological function (s) remains unclear. To investigate the effect of lnc-RAP3 on adipogenesis in the mouse 3T3-L1 preadipocytes, we first constructed a eukaryotic expression vector pcDNA3.1-RAP3. pcDNA3.1-RAP3 and synthetic RAP3-siRNAs were transfected individually into 3T3-L1 preadipocytes by Lipofectamine Show less

Apolipoprotein B-containing lipoproteins and low-density lipoprotein play a key role in atherosclerotic vascular disease. Modified forms of low-density lipoprotein drive inflammation, an integral aspect of plaque progression. High-density lipoprotein particles are equipped to protect low-density lipoprotein from enzymatic and nonenzymatic modification. Under normal conditions, high-density lipoproteins facilitate cholesterol efflux from tissues, preventing its accumulation with deleterious consequences. However, the high-density lipoprotein particles characteristic of dyslipidemic states associated with premature atherosclerosis are typically dysfunctional as a result of alteration in their metabolism and consequently their structure and composition. Such an effect indirectly enhances low-density lipoprotein atherogenicity. Show less

Mammalian neonates undergo rapid transitions from a sterile uterine environment with a continuous intravenous supply of nutrients to a microbe-rich environment with intermittent ingesting of colostrum/milk via the gut. Currently, little is known about the colostrum-induced alterations of intestinal mucosal proteins in piglets with intra-uterine growth restriction (IUGR). In this study, we sought to investigate the innate differences and effects of colostrum on alterations in small-intestinal proteomes of IUGR piglets. Two IUGR (approximately 0·9 kg) and two normal-birth weight (NBW; approximately 1·3 kg) piglets were obtained from each of six sows at birth. One half (n 12; 6 IUGR v. 6 NBW) of the selected newborn piglets were killed to obtain jejunum samples, and the other half (n 12; 6 IUGR v. 6 NBW) of the newborn piglets were allowed to suckle colostrum from their own mothers for 24 h before jejunum sample collection. On the basis of proteomic analysis, we identified thirty-one differentially expressed proteins in the jejunal mucosa between IUGR and normal neonates before or after colostrum consumption. The intestinal proteins altered by colostrum feeding play important roles in the following: (1) increasing intestinal integrity, transport of nutrients, energy metabolism, protein synthesis, immune response and, therefore, cell proliferation; and (2) decreasing oxidative stress, and therefore cell apoptosis, in IUGR neonates. However, colostrum only partially ameliorated the inferior status of the jejunal mucosa in IUGR neonates. These findings provide the first evidence in intestinal protein alterations of IUGR neonates in response to colostrum ingestion, and thus render new insights into the mechanisms responsible for impaired growth in IUGR neonates and into new nutritional intervention strategies. Show less

This study investigated the effect of angiotensin II (Ang II) on apoptosis and thioredoxin-interacting protein (TXNIP) expression in INS-1 islet cells and the underlying mechanism. INS-1 cells cultured in vitro were treated with different concentration of Ang II for different time, and the viability was measured using cell counting kit-8 (CCK-8). After treatment with 1 × 10 Show less

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent protein deacetylase. Recent studies have demonstrated that enhancing SIRT1 expression or activity may modulate cholesterol and lipid metabolism. However, pharmacological and molecular regulators for SIRT1 are scarce. Here, we aimed to find novel small molecule modulators of SIRT1 to regulate cholesterol and lipid metabolism. A high-throughput screening assay was established to identify SIRT1 activators. Surface plasmon resonance and immunoprecipitation were performed to confirm the interaction of E1231 with SIRT1. Cholesterol assay was performed to demonstrate the in vitro effect of E1231. The in vivo effect of E1231 was evaluated in experimental models. E1231, a piperazine 1,4-diamide compound, was identified as a SIRT1 activator with EC We identified a novel SIRT1 activator E1231 and elucidated its beneficial effects on lipid and cholesterol metabolism. Our study suggests that E1231 might be developed as a novel drug for treating atherosclerosis. Show less

The development of acquired EGFR-TKI therapeutic resistance is still a serious clinical problem in the management of lung adenocarcinoma. Peroxisome proliferator activated receptor gamma (PPARγ) agonists may exhibit anti-tumor activity by transactivating genes which are closely associated with cell proliferation, apoptosis, and differentiation. However, it remains not clear whether efatutazone has similar roles in lung adenocarcinoma cells of gefitinib resistant such as HCC827-GR and PC9-GR. It has been demonstrated by us that efatutazone prominently increased the mRNA and protein expression of PPARγ, liver X receptor alpha (LXRα),as well as ATP binding cassette subfamily A member 1 (ABCA1). In the presence of GW9662 (a specific antagonist of PPARγ) or GGPP (a specific antagonist of LXRα), efatutazone (40 μmol/L) restored the proliferation of both HCC827-GR and PC9-GR cells and obviously inhibited the increased protein and mRNA expression of PPAR-gamma, LXR-alpha, and ABCA1 induced by efatutazone. LXRα knockdown by siRNA (si-LXRα) significantly promoted the HCC827-GR and PC9-GR cells proliferation, whereas incubation efatutazone with si-LXRα restored the proliferation ability compared with the control group. In addition, combination of efatutazone and LXRα agonist T0901317 showed a synergistic therapeutic effect on lung adenocarcinoma cell proliferation and PPAR gamma, LXR A and ABCA1 protein expression. These results indicate that efatutazone could inhibit the cells proliferation of HCC827-GR and PC9-GR through PPARγ/LXRα/ABCA1 pathway, and synergistic therapeutic effect is achieved when combined with T0901317. Show less

A case-control study. To validate the relationship between POC5 and adolescent idiopathic scoliosis (AIS) in the Chinese patients and to further investigate the functional role of POC5. Three rare functional variants in the POC5 were recently reported to be strongly associated with the disease in a large family with multiple members affected with idiopathic scoliosis. To our knowledge, the association between the mutations of POC5 and AIS remains undetermined in the Chinese population. Single nucleotide variants c.1336G>A, c.1286C>T, and c.1363G>C of POC5 were genotyped in 2432 patients with AIS and 2292 healthy controls using multiple ligase detection reactions. Common variants covering POC5 gene were genotyped in 1446 patients and 2080 controls. The mRNA expression of POC5 was determined in the paraspinal muscles collected from 98 patients and 28 controls. The Student t test was used to compare mRNA expression level between the patients and the controls. In addition, the POC5 expression was compared among different genotypes of the remarkably associated single nucleotide polymorphism (SNP) with analysis of variance test. There was no case of mutation for the three reported variants of POC5. SNP rs6892146 was observed to have significantly different distribution of minor allele frequency in the two group (0.485 vs. 0.446, P = 0.004). The mRNA expression of POC5 was 1.5-fold higher in patients than in the controls (0.00012 ± 0.00009 vs. 0.00008 ± 0.00006, P = 0.02). Patients with genotype GG have a significantly increased expression of POC5 than those with CC (0.00014 ± 0.00007 vs. 0.00009 ± 0.00007, P = 0.03). Common variant rs6892146 of POC5 is associated with the development of AIS in the Chinese population. Targeted regional sequencing of POC5 may help identify novel mutations associated with AIS. 4. Show less

Human amniotic epithelial stem cells (HuAECs) exhibit pluripotent characteristics, which are similar to those of embryonic stem cells, and can differentiate into various adult tissues and cells through directed induction. However, in culture, HuAECs tend to lose their pluripotency, and their directed differentiation capability declines with increasing passage number. The stem cell pluripotency factor octamer‑binding protein 4 (Oct4) is an important transcription factor that promotes stem cell self‑proliferation and maintains their pluripotency. Previous studies have demonstrated that WW domain containing E3 ubiquitin protein ligase 2 (WWP2) negatively regulates Oct4 expression and stem cell pluripotency. Therefore, the present study aimed to investigate the regulation of WWP2 by microRNAs (miRs), and to evaluate the expression of the downstream factor Oct4 and the maintenance of HuAEC pluripotency. Bioinformatics analysis identified a complementary binding site for miR‑32 in the 3'untranslated region of the WWP2 gene, thus suggesting that it may be a target gene of miR‑32. Post‑infection of HuAECs with a vector overexpressing miR‑32, the endogenous expression of WWP2 was significantly decreased, whereas Oct4 expression was significantly increased. Furthermore, miR‑32‑infected cells differentiated into β islet‑like cells by directed induction. The results indicated that after induction, HuAECs overexpressing miR‑32 also overexpressed the biomarkers of β islet‑like cells. In addition, the ability to secrete insulin was markedly enhanced in response to glucose stimulation, in cells overexpressing miR‑32. In conclusion, the present study suggested that miR‑32 may effectively inhibit WWP2 expression in HuAECs and promote Oct4 overexpression to maintain their pluripotency. Show less

Zinc finger protein 668 (ZNF668) is a recently discovered protein and its expression levels, as well as its involvement in the invasion and metastasis of non-small cell lung cancer (NSCLC), are largely unknown. In the present study, immunohistochemical analysis demonstrated that ZNF668 protein expression was decreased in lung tumors (51/167, 30.5%) compared with adjacent normal lung tissues (43/62, 69.4%; P<0.001). Subsequent statistical analysis revealed that ZNF668 expression was negatively associated with increased tumor-node-metastasis stage (P=0.019) and lymph node metastasis (P=0.002). Following ZNF668 downregulation by transfection of a Show less

To investigate the influence of acute blood stasis on nitric oxide (NO), angiotensin Ⅱ(AngⅡ), angiopoietin-like protein 4 (ANGPTL4) mRNA, neuregulin 1 (NRG-1) mRNA, and platelet endothelial cell adhesion molecule-1 (PECAM-1) in rats with stasis induced by high-molecular-weight dextran (HMWD). Seventy-five Sprague Dawley rats were divided randomly into five groups (n = 15 in each group): control group, immediate group, 1 h group, 3 h group, and 6 h group. A model of acute blood stasis was established via injection of HMWD into the tail vein. After performing electrocardiogram at the predetermined times according to the grouping, we collected blood and cardiac samples for hematoxylin-eosin (HE) staining and histopathological examination via transmission electron microscopy. Enzyme-linked immunosorbent assay was used to detect plasma levels of NO, AngⅡ, and fibrinogen. Real-time polymerase chain reaction was used to detect the expression of ANGPTL4 mRNA and NRG-1 mRNA. Immunohistochemical methods were used to detect PECAM-1 protein expression. The rat model of blood stasis showed blood retention in the capillary lumens. The ST segment showed gradual elevation, and was still elevated at 3 and 6 h after induction of blood stasis. HE staining showed myocardial cell necrosis and dissolution after modeling, along with basement membrane rupture and mitochondrial structural damage. Transmission electron microscopy showed endothelial cell swelling and an increase in absorption vesicles immediately after modeling. Endothelial cell apoptosis was increased at 3 and 6 h after modeling. Cardiac muscle fibers were disordered and intercalated discs were blurred immediately after modeling. There were massive numbers of dissolved cardiac muscle fibers, ruptured basement membranes, and mitochondrial structural damage at 3 and 6 h after modeling. NO plasma concentration was significantly reduced immediately and 1 h after modeling, while it was increased at 3 and 6 h. Ang¢ò plasma concentration was decreased immediately after modeling, but increased at 1, 3, and 6 h. Fibrinogen plasma concentration was significantly increased at immediate, 1, 3, and 6 h after modeling. PECAM-1 protein expression was obviously increased immediately after modeling, at 1, 6 h was found mild augment. Expression of AngPTL4 mRNA was increased at immediate, 1, 3, and 6 h after modeling, and was found further augment at 3, and 6 h. Expression of NRG-1 mRNA was increased at immediate, 1, 3, and 6 h after modeling, and the strongest expression was at 1 h. The pathological manifestation of acute blood stasis is characterized by microvascular blood retention. Prolonged blood stasis leads to worsening endothelial cell and cardiomyocyte damage, along with imbalances in the expression of vasomotor factors and increased vascular tone. The pathological damage caused by blood stasis also promotes the expression of cell protection factors. Show less

This study aimed to investigate the effects of benazepril hydrochloride (BH) on proteinuria and ANGPTL-4 expression in a diabetic nephropathy (DN) rat model. A total of 72 Wistar male rats were randomly divided into three groups: normal control (NC), DN group and BH treatment (BH) groups. The DN model was induced by streptozotocin (STZ). Weight, glucose, proteinuria, biochemical indicators and the kidney weight index were examined at 8, 12 and 16 weeks. In addition, ANGPTL-4 protein and mRNA expressions were assessed by immunohistochemistry and qRT-PCR, respectively. Relationships between ANGPTL-4 and biochemical indicators were investigated using Spearman analysis. Weight was significantly lower but glucose levels were significantly higher in both the DN and BH groups than in the NC group (P < 0.05). Compared with the DN group, proteinuria, urea, creatinine, triglycerides and total cholesterol levels were decreased, whereas the albumin level was increased after BH treatment (all P < 0.05). Furthermore, BH diminished kidney volume and ameliorated the pathological changes associated with DN. ANGPTL-4 expression was significantly decreased after BH treatment, and ANGPTL-4 expression was highly correlated with biochemical indicators of DN (P < 0.05). Benazepril hydrochloride improves DN and decreases proteinuria by decreasing ANGPTL-4 expression. Show less

ANGPTL4 (angiopoietin-like protein 4) is a LPL (lipoprotein lipase) inhibitor and is present in high-density lipoprotein (HDL). However, it is not defined whether ANGPTL4 in HDLs could affect HDL metabolism and function in type 2 diabetes mellitus (T2DM). ANGPTL4 levels in the circulation and HDLs were quantified in nondiabetic participants (n=201, 68.7% females) and T2DM patients (n=185, 66.5% females). HDLs were isolated from nondiabetic controls and T2DM patients to assess cholesterol efflux Physically, ANGPTL4 in HDLs protected HDLs from hydrolysis. Resulting from increased circulating ANGPTL4 levels in T2DM, ANGPTL4 levels in HDLs were elevated but with compromised inhibitory effect on EL, leading to increased HDL hydrolysis and dysfunction. Show less

Axis inhibition protein 1 (AXIN1) is characterized as a tumor suppressor in numerous types of cancer. However, the functional role of AXIN1 in the testicular germ cell tumors (TGCTs) remains unclear. The human embryonal carcinoma-derived cell line NTera2 was transfected with a recombinant AXIN1 expression vector (pcDNA3.1-AXIN1) and/or a small interfering RNA (siRNA) directed against AXIN1 (siAXIN). Following transfection, the mRNA and protein levels of AXIN1 were determined via reverse transcription-quantitative polymerase chain reaction analysis and western blotting, respectively. In addition, cell viability, apoptosis and the expression of apoptosis-associated proteins [apoptosis regulator Bax (Bax) and B-cell lymphoma (Bcl)-2] and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway proteins [phosphorylated (p)-mTOR, mTOR, p-AKT, AKT, P-70S ribosomal protein S6 (S6) and S6] were assessed. AXIN1 mRNA and protein levels were increased following transfection with pcDNA3.1-AXIN1 and decreased following transfection with siAXIN1 compared with their respective control groups. After overexpression of AXIN1, NTera2 cell viability and expression of Bcl-2, p-mTOR p-AKT and p-S6 protein was decreased, while apoptosis and Bax protein levels were increased, compared with the control group. However, there was no significant difference in AXIN1 mRNA expression, apoptosis or Bax/Bcl-2 protein expression when NTera2 cells were simultaneously transfected with pcDNA3.1-AXIN1+siAXIN1. In conclusion, the results of the present study indicate that overexpression of AXIN1 protects against TGCTs via inhibiting the PI3K/AKT/mTOR signaling pathway, suggesting that AXIN1 may be a potential target for gene therapy in TGCTs. Show less

Circulating lipid concentrations are among the strongest modifiable risk factors for coronary artery disease (CAD). Most genetic studies have focused on Caucasian populations with little information available for populations of African ancestry. Using a cohort of ~2800 African-Americans (AAs) from a biobank at Vanderbilt University (BioVU), we sought to trans-ethnically replicate genetic variants reported by the Global Lipids Genetics Consortium to be associated with lipid traits in Caucasians, followed by fine-mapping those loci using all available variants on the MetaboChip. In AAs, we replicated one of 56 SNPs for total cholesterol (TC) (rs6511720 in LDLR, P=2.15 × 10 Show less

Hypertrophic cardiomyopathy (HCM), one of the most common forms of myocardial diseases, is the major cause of sudden cardiac death in young adults and competitive athletes. Analyses of gene mutations associated with HCM are valuable for its molecular diagnosis, genetic counseling, and management of familial HCM. To dissect the relationship between the clinical presentation and gene mutations of HCM, the genetic characterizations of 19 HCM-related genes in 18 patients (8 cases from 6 pedigrees with familial HCM and 10 cases without familial HCM) were detected using next-generation sequencing (NGS). As a result, 12 disease-related mutations were identified in the 18 subjects, including 6 single mutations and 3 double mutations [MYBPC3 (p.Gln998Glu) plus TNNI3 (p.Arg145Gly), PRKAG2 (p.Gly100Ser) plus MYBPC3 (p.Lys1209Serfs*28) and TNNI3 (p.Glu124Gln) plus GLA (p.Trp47*)]. The 3 heterozygous double mutations were discovered for the first time in the malignant familial HCM patients. Of the 6 single mutations, a novel mutation was found in tafazzin (TAZ, p.Ile208Val), and a mutation in β-myosin heavy chain gene (MYH7, p.Arg54Gln), which was reported as rare in the general population, was firstly found in one HCM patient. Identification of novel and rare mutations in HCM patients have added new data to the spectrum of gene mutations associated with this disease. These findings provide an essential basis for the molecular diagnosis and better management of family members at risk of familial HCM. Show less

To investigate the potential association between FADS1 rs174537 polymorphism and serum proteins in patients with aggressive periodontitis, which may provide benefits for diagnosis and treatment of aggressive periodontitis. A total of 353 patients with aggressive periodontitis (group AgP) and 125 matched controls (group HP) were recruited in the study. Genotyping of FADS1 rs174537 and serum biochemical indexes were tested at the study's start. The relationships between the levels of TP, GLB, ALB, A/G and genotyping were analyzed. (1) The detection rate of allele G in group AgP was higher than that in group HP(68.1% vs. 61.2%, P=0.046,OR=1.35,95% CI 1.00-1.83); the detection rate of genotype GG in group AgP was higher than in group HP(45.5% vs. 34.4%,P=0.029, OR=1.60, 95% CI 1.05-2.44). (2) In group AgP, the patients with GG genotype exhibited significantly lower TP, GLB than the patients with GT+TT genotype [(77.08 ± 7.88) g/L vs. (79.00 ± 4.66) g/L, P=0.007; (28.17 ± 7.63) g/L vs.(29.88 ± 3.49) g/L,P=0.007) and the higher A/G(1.72 ± 0.22 vs.1.67 ± 0.22, P=0.040), but there was no significant difference in ALB between the patients with GG genotype and the patients with GT+TT genotype. In group HP, there were no significant differences in TP, GLB, A/G and ALB between individuals with genotype GT+TT and with genotype GG. (3)Compared with individuals with genotype GT+TT in group HP, the AgP patients with genotype GT+TT exhibited significantly higher TP, GLB [(79.00 ± 4.66) g/L vs. (75.20 ± 4.53) g/L, P<0.01; (29.88 ± 3.49) g/L vs.(26.55 ± 2.94) g/L, P<0.01) and the lower A/G(1.67 ± 0.22 vs. 1.88 ± 0.30, P<0.01), but there was no significant difference in ALB. There were no significant differences in TP, GLB, A/G and ALB the between the AgP patients with genotype GG and the healthy subjects with the same genotype either. FADS1 rs174537 polymorphism is associated with aggressive periodontitis. The patients with genotype GG in group AgP had relatively lower TP,GLB and higher A/G. Genotype GG might be a risk indicator for aggressive periodontitis by reducing host defense capability and contributing to inflammatory response in the occurrence and development of aggressive periodontitis. Show less

The Notch and transforming growth factor (TGF)-β signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-β signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-β signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-β signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-β and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs. Show less

Transcription factor carbohydrate responsive element binding protein (ChREBP) promotes glycolysis and lipogenesis in metabolic tissues and cancer cells. ChREBP-α and ChREBP-β, two isoforms of ChREBP transcribed from different promoters, are both transcriptionally induced by glucose. However, the mechanism by which glucose increases ChREBP mRNA levels remains unclear. Here we report that hepatocyte nuclear factor 4 alpha (HNF-4α) is a key transcription factor for glucose-induced ChREBP-α and ChREBP-β expression. Ectopic HNF-4α expression increased ChREBP transcription while knockdown of HNF-4α greatly reduced ChREBP mRNA levels in liver cancer cells and mouse primary hepatocytes. HNF-4α not only directly bound to an E-box-containing region in intron 12 of the ChREBP gene, but also promoted ChREBP-β transcription by directly binding to two DR1 sites and one E-box-containing site of the ChREBP-β promoter. Moreover, HNF-4α interacted with ChREBP-α and synergistically promoted ChREBP-β transcription. Functionally, HNF-4α suppression reduced glucose-dependent ChREBP induction. Increased nuclear abundance of HNF-4α and its binding to cis-elements of ChREBP gene in response to glucose contributed to glucose-responsive ChREBP transcription. Taken together, our results not only revealed the novel mechanism by which HNF-4α promoted ChREBP transcription in response to glucose, but also demonstrated that ChREBP-α and HNF-4α synergistically increased ChREBP-β transcription. Show less

Inherited cardiomyopathy is the major cause of sudden cardiac death (SCD) and heart failure (HF). The disease is associated with extensive genetic heterogeneity; pathogenic mutations in cardiac sarcomere protein genes, cytoskeletal protein genes and nuclear envelope protein genes have been linked to its etiology. Early diagnosis is conducive to clinical monitoring and allows for presymptomatic interventions as needed. In the present study, the entire coding sequences and flanking regions of 12 major disease (cardiomyopathy)-related genes [namely myosin, heavy chain 7, cardiac muscle, β (MYH7); myosin binding protein C, cardiac (MYBPC3); lamin A/C (LMNA); troponin I type 3 (cardiac) (TNNI3); troponin T type 2 (cardiac) (TNNT2); actin, α, cardiac muscle 1 (ACTC1); tropomyosin 1 (α) (TPM1); sodium channel, voltage gated, type V alpha subunit (SCN5A); myosin, light chain 2, regulatory, cardiac, slow (MYL2); myosin, heavy chain 6, cardiac muscle, α (MYH6); myosin, light chain 3, alkali, ventricular, skeletal, slow (MYL3); and protein kinase, AMP-activated, gamma 2 non-catalytic subunit  (PRKAG2)] in 8 patients with dilated cardiomyopathy (DCM) and in 8 patients with hypertrophic cardiomyopathy (HCM) were amplified and then sequenced using the Ion Torrent Personal Genome Machine (PGM) system. As a result, a novel heterozygous mutation (MYH7, p.Asn885Thr) and a variant of uncertain significance (TNNT2, p.Arg296His) were identified in 2 patients with HCM. These 2 missense mutations, which were absent in the samples obtained from the 200 healthy control subjects, altered the amino acid that was evolutionarily conserved among a number of vertebrate species; this illustrates that these 2 non-synonymous mutations play a role in the pathogenesis of HCM. Moreover, a double heterozygous mutation (PRKAG2, p.Gly100Ser plus MYH7, p.Arg719Trp) was identified in a patient with severe familial HCM, for the first time to the best of our knowledge. This patient provided us with more information regarding the genotype-phenotype correlation between mutations of MYH7 and PRKAG2. Taken together, these findings provide insight into the molecular mechanisms underlying inherited cardiomyopathy. The mutations identified in this study may be further investigated in the future in order to improve the diagnosis and treatment of patients with inherited cardiomyopathy. Furthermore, our findings indicated that sequencing using the Ion Torrent PGM system is a useful approach for the identification of pathogenic mutations associated with inherited cardiomyopathy, and it may be used for the risk evaluation of individuals with a possible susceptibility to inherited cardiomyopathy. Show less

The molecular mechanism of macroautophagy/autophagy induction has been intensively studied, but little is known about downregulation of autophagy and how this process is restricted. In particular, how is autophagy maintained at an appropriate homeostatic level when cells are subjected to prolonged stress? In this study (see the related punctum in Autophagy 12-5), Liu et al. report a function of the CUL3-KLHL20 ubiquitin ligase in feedback regulation, leading to the downregulation of autophagy through the degradation of the ULK1 and PIK3C3/VPS34 complexes. Show less

The SNP of rs964184 in ZPR1 has recently been associated with type 2 diabetes mellitus (T2DM) in Japanese individuals. To comprehensively investigate the association of common variants in ZPR1 with T2DM in Han Chinese individuals, we designed a two-stage case-control study of 3,505 T2DM patients and 6,911 unrelated healthy Han Chinese individuals. A total of 24 single nucleotide polymorphisms (SNPs) were genotyped, and single-SNP association, imputation and gender-specific association analyses were performed. To increase the coverage of genetic markers, we implemented imputation techniques to extend the number of tested makers to 280. A novel SNP, rs2075290, and the previously reported SNP, rs964184, were significantly associated with T2DM in the two independent datasets, and individuals harboring the CC genotype of rs2075290 and GG genotype of rs964184 exhibited higher levels of fasting plasma glucose (FPG) and blood hemoglobin A1c (HbA1c) than individuals of other genotypes. Additionally, haplotype analyses indicated that two haplotype blocks containing rs2075290 or rs964184 were also significantly associated with T2DM. In summary, these results suggest that ZPR1 plays an important role in the etiology of T2DM, and this gene might be involved in abnormal glucose metabolism. Show less

Apolipoprotein A5 (apoA5) is a potent regulator of triglyceride (TG) metabolism and therefore may contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD), a disease characterised by excessive TG-rich lipid droplets in hepatocytes. To test this hypothesis, we examined the mRNA expression of apoA5 in paediatric NAFLD livers in comparison to healthy controls. According to microarray and quantitative real-time PCR, human NAFLD livers exhibited elevated apoA5 expression compared to healthy controls. The apoA5 expression levels were positively correlated with hepatic TG storage and a marker for lipid droplets (perilipin), but were not correlated with plasma TG levels. These observations were confirmed with a NAFLD rat model. Interestingly, apoA5 expression was not altered in cultured fat-laden HepG2 cells, demonstrating that fat storage does not induce apoA5 in NAFLD livers. Therefore, the correlation between apoA5 and intracellular fat storage is likely explained by the potent effect of apoA5 in promoting intracellular fat storage. Our NAFLD patients and rats had elevated insulin resistance, which may have a role in elevating apoA5 expression in NAFLD livers. Our data support the hypothesis that apoA5 promotes hepatic TG storage and therefore contributes to the pathogenesis of NAFLD, and may represent a potential target for therapeutic intervention. Show less

Advances in exome sequencing and the development of exome genotyping arrays are enabling explorations of association between rare coding variants and complex traits. To ensure power for these rare variant analyses, a variety of association tests that group variants by gene or functional unit have been proposed. Here, we extend these tests to family-based studies. We develop family-based burden tests, variable frequency threshold tests and sequence kernel association tests. Through simulations, we compare the performance of different tests. We describe situations where family-based studies provide greater power than studies of unrelated individuals to detect rare variants associated with moderate to large changes in trait values. Broadly speaking, we find that when sample sizes are limited and only a modest fraction of all trait-associated variants can be identified, family samples are more powerful. Finally, we illustrate our approach by analyzing the relationship between coding variants and levels of high-density lipoprotein (HDL) cholesterol in 11,556 individuals from the HUNT and SardiNIA studies, demonstrating association for coding variants in the APOC3, CETP, LIPC, LIPG, and LPL genes and illustrating the value of family samples, meta-analysis, and gene-level tests. Our methods are implemented in freely available C++ code. Show less

Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a β-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/β-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism. Show less

Non-obstructive azoospermia (NOA), a severe form of male infertility, is often suspected to be linked to currently undefined genetic abnormalities. To explore the genetic basis of this condition, we successfully sequenced ~650 infertility-related genes in 757 NOA patients and 709 fertile males. We evaluated the contributions of rare variants to the etiology of NOA by identifying individual genes showing nominal associations and testing the genetic burden of a given biological process as a whole. We found a significant excess of rare, non-silent variants in genes that are key epigenetic regulators of spermatogenesis, such as BRWD1, DNMT1, DNMT3B, RNF17, UBR2, USP1 and USP26, in NOA patients (P = 5.5 × 10(-7)), corresponding to a carrier frequency of 22.5% of patients and 13.7% of controls (P = 1.4 × 10(-5)). An accumulation of low-frequency variants was also identified in additional epigenetic genes (BRDT and MTHFR). Our study suggested the potential associations of genetic defects in genes that are epigenetic regulators with spermatogenic failure in human. Show less

Thiazide-sensitive sodium chloride cotransporter (NCC) plays an important role in maintaining blood pressure. Aldosterone is known to modulate NCC abundance. Previous studies reported that dietary salts modulated NCC abundance through either WNK4 [with no lysine (k) kinase 4]-SPAK (Ste20-related proline alanine-rich kinase) or WNK4-extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling pathways. To exclude the influence of SPAK signaling pathway on the role of the aldosterone-mediated ERK1/2 pathway in NCC regulation, we investigated the effects of dietary salt changes and aldosterone on NCC abundance in SPAK knockout (KO) mice. We found that in SPAK KO mice low-salt diet significantly increased total NCC abundance while reducing ERK1/2 phosphorylation, whereas high-salt diet decreased total NCC while increasing ERK1/2 phosphorylation. Importantly, exogenous aldosterone administration increased total NCC abundance in SPAK KO mice while increasing DUSP6 expression, an ERK1/2-specific phosphatase, and led to decreasing ERK1/2 phosphorylation without changing the ratio of phospho-T53-NCC/total NCC. In mouse distal convoluted tubule (mDCT) cells, aldosterone increased DUSP6 expression while reducing ERK1/2 phosphorylation. DUSP6 Knockdown increased ERK1/2 phosphorylation while reducing total NCC expression. Inhibition of DUSP6 by (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased ERK1/2 phosphorylation and reversed the aldosterone-mediated increments of NCC partly by increasing NCC ubiquitination. Therefore, these data suggest that aldosterone modulates NCC abundance via altering NCC ubiquitination through a DUSP6-dependent ERK1/2 signal pathway in SPAK KO mice and part of the effects of dietary salt changes may be mediated by aldosterone in the DCTs. Show less

Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death and heart failure, and it is characterized by genetic and clinical heterogeneity, even for some patients with a very poor clinical prognosis; in the majority of cases, DCM necessitates a heart transplant. Genetic mutations have long been considered to be associated with this disease. At present, mutations in over 50 genes related to DCM have been documented. This study was carried out to elucidate the characteristics of gene mutations in patients with DCM. The candidate genes that may cause DCM include MYBPC3, MYH6, MYH7, LMNA, TNNT2, TNNI3, MYPN, MYL3, TPM1, SCN5A, DES, ACTC1 and RBM20. Using next-generation sequencing (NGS) and subsequent mutation confirmation with traditional capillary Sanger sequencing analysis, possible causative non-synonymous mutations were identified in ~57% (12/21) of patients with DCM. As a result, 7 novel mutations (MYPN, p.E630K; TNNT2, p.G180A; MYH6, p.R1047C; TNNC1, p.D3V; DES, p.R386H; MYBPC3, p.C1124F; and MYL3, p.D126G), 3 variants of uncertain significance (RBM20, p.R1182H; MYH6, p.T1253M; and VCL, p.M209L), and 2 known mutations (MYH7, p.A26V and MYBPC3, p.R160W) were revealed to be associated with DCM. The mutations were most frequently found in the sarcomere (MYH6, MYBPC3, MYH7, TNNC1, TNNT2 and MYL3) and cytoskeletal (MYPN, DES and VCL) genes. As genetic testing is a useful tool in the clinical management of disease, testing for pathogenic mutations is beneficial to the treatment of patients with DCM and may assist in predicting disease risk for their family members before the onset of symptoms. Show less

Curcumin, a traditional Chinese derivative from the rhizomes of Curcuma longa, is beneficial to health by modulating lipid metabolism and suppressing atherogenesis. A key part of atherosclerosis is the failure of macrophages to restore their cellular cholesterol homeostasis and the formation of foam cells. In this study, results showed that curcumin dramatically increased the expression of ATP-binding cassette transporter 1 (ABCA1), promoted cholesterol efflux from THP-1 macrophage-derived foam cells, and reduced cellular cholesterol levels. Curcumin activated AMP-activated protein kinase (AMPK) and SIRT1, and then activated LXRα in THP-1 macrophage-derived foam cells. Inhibiting AMPK/SIRT1 activity by its specific inhibitor or by small interfering RNA could inhibit LXRα activation and abolish curcumin-induced ABCA1 expression and cholesterol efflux. Thus, curcumin enhanced cholesterol efflux by upregulating ABCA1 expression through activating AMPK-SIRT1-LXRα signaling in THP-1 macrophage-derived foam cells. This study describes a possible mechanism for understanding the antiatherogenic effects of curcumin on attenuating the progression of atherosclerosis. Show less

Liver X receptor (LXR) isoforms, LXRα and LXRβ, have similar protein structures and ligands, but diverse tissue distribution. We used two synthetic, non-steroidal LXR agonists, T0901317 and GW3965, to investigate the effects of LXR agonist modulation on prostate specific antigen (PSA) via the expressions of androgen receptors (AR), LXRα, or LXRβ, in prostate carcinoma cells. LXRα- or LXRβ-knockdown cells were transduced with specific shRNA lentiviral particles. LXRα and LXRβ expressions were assessed by immunoblotting and RT-qPCR assays. Cell proliferation was determined by (3) H-thymidine incorporation assays. The effects of LXR agonists and epigallocatechin gallate (EGCG) on PSA expression were determined by ELISA, immunoblotting, or transient gene expression assays. Treatment with either T0901317 or GW3965 significantly attenuated cell proliferation of LNCaP cells. T0901317 treatment suppressed PSA expression while GW3965 treatment enhanced PSA expression. The increase of PSA promoter activity by GW3965 was dependent on the expression of AR. Either LXRα- or LXRβ-knockdown did not affect the activation of androgen on PSA gene expression. However, as compared with mock knockdown-LNCaP cells, the LXRα-knockdown but not the LXRβ-knockdown attenuated the effects of T0901317 and GW3965 on PSA expressions. The effect of GW3965 on PSA expression was blocked by the addition of EGCG. Our results indicate that T0901317 and GW3965 have divergent effects on PSA expressions. The effects of LXR agonists on PSA expression are LXRα-dependent and AR-dependent. EGCG blocks the inducing effect of GW3965 on PSA expression. Show less