Targeting LncRNA LLNLR-299G3.1 with antisense oligonucleotide inhibits malignancy of esophageal squamous cell carcinoma cells in vitro and in vivo.

Accumulating evidence has indicated that long non-coding RNAs (lncRNAs) play critical roles in the development and progression of cancers, including esophageal squamous cell carcinoma (ESCC). However, the mechanisms of lncRNAs in ESCC are still incompletely understood and therapeutic attempts for in vivo targeting cancer-associated lncRNA remain a challenge. By RNA-sequencing analysis, we identified that LLNLR-299G3.1 was a novel ESCC-associated lncRNA. LLNLR-299G3.1 was up-regulated in ESCC tissues and cells and promoted ESCC cell proliferation and invasion. Silencing of LLNLR-299G3.1 with ASO (antisense oligonucleotide) resulted in opposite effects. Mechanistically, LLNLR-299G3.1 bound to cancer-associated RNA binding proteins and regulated the expression of cancer-related genes, including OSM, TNFRSF4, HRH3, and SSTR3. ChIRP-seq (chromatin isolation by RNA purification and sequencing) revealed that these genes contained enriched chromatin binding sites for LLNLR-299G3.1. Rescue experiments confirmed that the effects of LLNLR-299G3.1 on ESCC cell proliferation were dependent on interaction with HRH3 and TNFRSF4. Therapeutically, intravenous delivery of placental chondroitin sulfate A binding peptide-coated nanoparticles containing antisense oligonucleotide (pICSA-BP-ANPs) strongly inhibited ESCC tumor growth and significantly improved animal survival in vivo. Overall, our results suggest that LLNLR-299G3.1 promotes ESCC malignancy through regulating gene-chromatin interactions and targeting ESCC by pICSA-BP-ANPs may be an effective strategy for the treatment of lncRNA-associated ESCC.

PM2.5 promotes NSCLC carcinogenesis through translationally and transcriptionally activating DLAT-mediated glycolysis reprograming.

BACKGROUND: Airborne fine particulate matter (PM2.5) has been associated with lung cancer development and progression in never smokers. However, the molecular mechanisms underlying PM2.5-induced lung cancer remain largely unknown. The aim of this study was to explore the mechanisms by which PM2.5 regulated the carcinogenesis of non-small cell lung cancer (NSCLC). METHODS: Paralleled ribosome sequencing (Ribo-seq) and RNA sequencing (RNA-seq) were performed to identify PM2.5-associated genes for further study. Quantitative real time-PCR (qRT-PCR), Western blot, and immunohistochemistry (IHC) were used to determine mRNA and protein expression levels in tissues and cells. The biological roles of PM2.5 and PM2.5-dysregulated gene were assessed by gain- and loss-of-function experiments, biochemical analyses, and Seahorse XF glycolysis stress assays. Human tissue microarray analysis and 18F-FDG PET/CT scans in patients with NSCLC were used to verify the experimental findings. Polysome fractionation experiments, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assay were implemented to explore the molecular mechanisms. RESULTS: We found that PM2.5 induced a translation shift towards glycolysis pathway genes and increased glycolysis metabolism, as evidenced by increased L-lactate and pyruvate concentrations or higher extracellular acidification rate (ECAR) in vitro and in vivo. Particularly, PM2.5 enhanced the expression of glycolytic gene DLAT, which promoted glycolysis but suppressed acetyl-CoA production and enhanced the malignancy of NSCLC cells. Clinically, high expression of DLAT was positively associated with tumor size, poorer prognosis, and SUVmax values of 18F-FDG-PET/CT scans in patients with NSCLC. Mechanistically, PM2.5 activated eIF4E, consequently up-regulating the expression level of DLAT in polysomes. PM2.5 also stimulated transcription factor Sp1, which further augmented transcription activity of DLAT promoter. CONCLUSIONS: This study demonstrated that PM2.5-activated overexpression of DLAT and enhancement in glycolysis metabolism contributed to the tumorigenesis of NSCLC, suggesting that DLAT-associated pathway may be a therapeutic target for NSCLC.

A novel tRNA-derived fragment AS-tDR-007333 promotes the malignancy of NSCLC via the HSPB1/MED29 and ELK4/MED29 axes.

BACKGROUND: Transfer RNA-derived fragments (tRFs) are a new class of small non-coding RNAs. Recent studies suggest that tRFs participate in some pathological processes. However, the biological functions and mechanisms of tRFs in non-small cell lung cancer (NSCLC) are largely unknown. METHODS: Differentially expressed tRFs were identified by tRF and tiRNA sequencing using 9 pairs of pre- and post-operation plasma from patients with NSCLC. Quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) were used to determine the levels of tRF in tissues, plasma, and cells. Gain- and loss-of-function experiments were implemented to investigate the oncogenic effects of tRF on NSCLC cells in vitro and in vivo. Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pulldown, mass spectrum, RNA immunoprecipitation (RIP), Western blot, co-immunoprecipitation (Co-IP) assays, and rescue experiments were performed to explore the regulatory mechanisms of tRF in NSCLC. RESULTS: AS-tDR-007333 was an uncharacterized tRF and significantly up-regulated in NSCLC tissues, plasma, and cells. Clinically, AS-tDR-007333 overexpression could distinguish NSCLC patients from healthy controls and associated with poorer prognosis of NSCLC patients. Functionally, overexpression of AS-tDR-007333 enhanced proliferation and migration of NSCLC cells, whereas knockdown of AS-tDR-007333 resulted in opposite effects. Mechanistically, AS-tDR-007333 promoted the malignancy of NSCLC cells by activating MED29 through two distinct mechanisms. First, AS-tDR-007333 bound to and interacted with HSPB1, which activated MED29 expression by enhancing H3K4me1 and H3K27ac in MED29 promoter. Second, AS-tDR-007333 stimulated the expression of transcription factor ELK4, which bound to MED29 promoter and increased its transcription. Therapeutically, inhibition of AS-tDR-007333 suppressed NSCLC cell growth in vivo. CONCLUSIONS: Our study identifies a new oncogenic tRF and uncovers a novel mechanism that AS-tDR-007333 promotes NSCLC malignancy through the HSPB1-MED29 and ELK4-MED29 axes. AS-tDR-007333 is a potential diagnostic or prognostic marker and therapeutic target for NSCLC.

LncRNA BRCAT54 inhibits the tumorigenesis of non-small cell lung cancer by binding to RPS9 to transcriptionally regulate JAK-STAT and calcium pathway genes.

OBJECTIVES: Increasing evidence suggest that long non-coding RNAs (lncRNAs) play critical roles in cancers. However, the expression pattern and underlying mechanisms of lncRNAs in non-small cell lung cancer (NSCLC) remain incompletely understood. This study aimed to elucidate the functions and molecular mechanisms of a certain lncRNA in NSCLC. METHODS: LncRNA microarray was performed to identify differential expressed lncRNAs between pre- and postoperation plasma in NSCLC patients. The expression level of candidate lncRNA in NSCLC tissues, plasma and cells was determined by quantitative real-time PCR (qRT-PCR) and in situ hybridization. The functional roles of lncRNA were assessed in vitro and in vivo. Furthermore, RNA pull-down, RNA immunoprecipitation, microarray, qRT-PCR and rescue assays were conducted to explore the mechanism action of lncRNA in NSCLC cells. RESULTS: We identified a novel lncRNA (BRCAT54), which was significantly upregulated in preoperative plasma, NSCLC tissues and NSCLC cells, and its higher expression was associated with better prognosis in patients with NSCLC. Overexpression of BRCAT54 inhibited proliferation, migration and activated apoptosis in NSCLC cells. Conversely, knockdown of BRCAT54 reversed the suppressive effects of BRCAT54. Moreover, overexpression of BRCAT54 repressed NSCLC cell growth in vivo. Mechanistically, BRCAT54 directly bound to RPS9. Knockdown of RPS9 substantially reversed the promoting effects of si-BRCAT54 on cell proliferation and enhanced the inhibitive effect of si-BRCAT54 on BRCAT54 expression. In addition, silencing of RPS9 activated JAK-STAT pathway and suppressed calcium signaling pathway gene expressions. CONCLUSION: This study identified BRCAT54 as a tumor suppressor in NSCLC. Targeting the BRCAT54 and RPS9 feedback loop might be a novel therapeutic strategy for NSCLC.

Correction: FTSJ1 regulates tRNA 2'-O-methyladenosine modification and suppresses the malignancy of NSCLC via inhibiting DRAM1 expression.

The original version of this article contained an error in the spelling of the author Yuchen Chen, which was incorrectly given as Yuhuan Chen. This has now been corrected in both the PDF and HTML versions of the article.

RNA sequencing of plasma exosomes revealed novel functional long noncoding RNAs in hepatocellular carcinoma.

Exosomal long noncoding RNA (lncRNA) has been found to be associated with the development of cancers. However, the expression characteristics and the biological roles of exosomal lncRNAs in hepatocellular carcinoma (HCC) remain unknown. Here, by RNA sequencing, we found 9440 mRNAs and 8572 lncRNAs were differentially expressed (DE-) in plasma exosomes between HCC patients and healthy controls. Exosomal DE-lncRNAs displayed higher expression levels and tissue specificity, lower expression variability and splicing efficiency than DE-mRNAs. Six candidate DE-lncRNAs (fold change 6 or more, P ≤ .01) were high in HCC cells and cell exosomes. The knockdown of these candidate DE-lncRNAs significantly affected the migration, proliferation, and apoptosis in HCC cells. In particular, a novel DE-lncRNA, RP11-85G21.1 (lnc85), promoted HCC cellular proliferation and migration by targeted binding and regulating of miR-324-5p. More importantly, the level of serum lnc85 was highly expressed in both Alpha-fetoprotein (AFP)-positive and AFP-negative HCC patients and allowed distinguishing AFP-negative HCC from healthy control and liver cirrhosis (area under the receiver operating characteristic curve, 0.869; sensitivity, 80.0%; specificity, 76.5%) with high accuracy. Our finding offers a new insight into the association between the dysregulation of exosomal lncRNA and HCC, suggesting that lnc85 could be a potential biomarker of HCC.

FTSJ1 regulates tRNA 2'-O-methyladenosine modification and suppresses the malignancy of NSCLC via inhibiting DRAM1 expression.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. The mechanisms underlying NSCLC tumorigenesis are incompletely understood. Transfer RNA (tRNA) modification is emerging as a novel regulatory mechanism for carcinogenesis. However, the role of tRNA modification in NSCLC remains obscure. In this study, HPLC/MS assay was used to quantify tRNA modification levels in NSCLC tissues and cells. tRNA-modifying enzyme genes were identified by comparative genomics and validated by qRT-PCR analysis. The biological functions of tRNA-modifying gene in NSCLC were investigated in vitro and in vivo. The mechanisms of tRNA-modifying gene in NSCLC were explored by RNA-seq, qRT-PCR, and rescue assays. The results showed that a total of 18 types of tRNA modifications and up to seven tRNA-modifying genes were significantly downregulated in NSCLC tumor tissues compared with that in normal tissues, with the 2'-O-methyladenosine (Am) modification displaying the lowest level in tumor tissues. Loss- and gain-of-function assays revealed that the amount of Am in tRNAs was significantly associated with expression levels of FTSJ1, which was also downregulated in NSCLC tissues and cells. Upregulation of FTSJ1 inhibited proliferation, migration, and promoted apoptosis of NSCLC cells in vitro. Silencing of FTSJ1 resulted in the opposite effects. In vivo assay confirmed that overexpression of FTSJ1 significantly suppressed the growth of NSCLC cells. Mechanistically, overexpression of FTSJ1 led to a decreased expression of DRAM1. Whereas knockdown of FTSJ1 resulted in an increased expression of DRAM1. Furthermore, silencing of DRAM1 substantially augmented the antitumor effect of FTSJ1 on NSCLC cells. Our findings suggested an important mechanism of tRNA modifications in NSCLC and demonstrated novel roles of FTSJ1 as both tRNA Am modifier and tumor suppressor in NSCLC.

RNA sequencing of exosomes revealed differentially expressed long noncoding RNAs in early-stage esophageal squamous cell carcinoma and benign esophagitis.

Aim: To explore the roles of exosomal long noncoding RNAs (lncRNAs) in early-stage esophageal squamous cell carcinoma (ESCC) and benign esophagitis. Materials & methods: Exosomal lncRNAs were analyzed using RNA-seq and validated by quantitative real-time PCR, loss-of-function, co-culture and RNA pulldown assays. Results: Exosomal lncRNAs displayed tighter tissue-specificity, higher expression level and lower splicing efficiency than that of mRNAs. A total of 152 exosomal lncRNAs were differentially expressed between ESCC and controls. A total of 124 exosomal lncRNAs were dysregulated between ESCC and esophagitis. Knockdown of 13 ESCC-associated lncRNAs modified proliferation, migration, and apoptosis of ESCC cells. A novel lncRNA RP5-1092A11.2 was highly expressed in ESCC-derived exosomes, ESCC cells and tumor tissues. Exosomes released from RP5-1092A11.2-knockdown cells inhibited ESCC cell proliferation. Conclusion: Dysregulated exosomal lncRNAs were functionally associated with different disease status in esophagus.

Long-term outcomes of macrovascular diseases and metabolic indicators of bariatric surgery for severe obesity type 2 diabetes patients with a meta-analysis.

There is currently no detailed evidence for the long-term effects of bariatric surgery on severely obese with type 2 diabetes, such as the risk of myocardial infarction and stroke. In order to provide evidence on the risks of macrovascular diseases and metabolic indicators of bariatric surgery follow-up for more than five years, we searched in the Cochrane library, Pubmed, and EMBASE databases from the earliest studies to January 31, 2019. Randomized clinical trials or cohort studies compared bariatric surgery and conventional medical therapy for long-term incidence of macrovascular events and metabolic outcomes in severely obese patients with T2DM. Fixed-effects and random-effects meta-analyses were performed to pool the relative risks (RRs), hazard ratios (HRs) and weighted mean difference (WMD). Publication bias and heterogeneity were examined. Four RCTs and six cohort studies were finally involved in this review. Patients in the bariatric surgery group as compared to the conventional treatment group had lower incidence of macrovascular complications (RR = 0.43, 95%CI = 0.27~0.70), cardiovascular events (CVEs) (HR = 0.52, 95%CI = 0.39~0.71), and myocardial infarction (MI) (RR = 0.40, 95%CI = 0.26~0.61). At the same time, the results demonstrate that bariatric surgery is associated with better weight and better glycemic control over the long-term than non-surgical therapies, and reveal that different surgical methods have different effects on various metabolic indicators. Bariatric surgery significantly decreases macrovascular complications over the long term and is associated with greater weight loss and better intermediate glucose outcomes among T2DM patients with severe obesity as compared to patients receiving only conservative medical measures.

Interactions between genetic polymorphisms of glucose metabolizing genes and smoking and alcohol consumption in the risk of type 2 diabetes mellitus.

The impact of gene-environment interaction on diabetes remains largely unknown. We aimed to investigate if interaction between glucose metabolizing genes and lifestyle factors is associated with type 2 diabetes mellitus (T2DM). Interactions between genotypes of 4 glucose metabolizing genes (MTNR1B, KCNQ1, KLF14, and GCKR) and lifestyle factors were estimated in 722 T2DM patients and 759 controls, using multiple logistic regression. No significant associations with T2DM were detected for the single nucleotide polymorphisms of MTNR1B, KLF14 and GCKR. However, rs151290 (KCNQ1) polymorphisms were found to be associated with risk of T2DM. Compared with AA, the odds ratios (ORs) of AC or CC genotypes for developing T2DM were 1.545 (P = 0.0489) and 1.603 (P = 0.0383), respectively. In stratified analyses, the associations were stronger in smokers with CC than smokers with AA (OR = 3.668, P = 0.013); drinkers with AC (OR = 5.518, P = 0.036), CC (OR = 8.691, P = 0.0095), and AC+CC (OR = 6.764, P = 0.016) than drinkers with AA. Compared with nondrinkers with AA, drinkers who carry AC and CC had 12.072-fold (P = 0.0007) and 8.147-fold (P = 0.0052) higher risk of developing T2DM. In conclusions, rs151290 (KCNQ1) polymorphisms are associated with increased risk of T2DM, alone and especially in interaction with smoking and alcohol.

Genetic effects on hypertriglyceridemic waist phenotype: rs780094, rs10830963, rs151290, and rs972283 polymorphisms and the interactions between them and behavior risk factors.

BACKGROUNDS: We aimed to evaluate the association of SNPs in GCKR, MTNR1B, KCNQ1, and KLF14 genes confirmed in previous studies and hapertriglyceridemic waist (HTGW) in Han Chinese, and assess the interactions between genes and behavior risk factors. METHODS: We genotyped the single nucleotide polymorphisms (SNPs) for GCKR, MTNR1B, KCNQ1, and KLF14 gene were genotyped in 373 patients with HTGW and 466 normal healthy subjects. We used logistic regression to investigate the gene-gene, gene-behavior interactions for the risk of HTGW. RESULTS: Among the 4 SNPs, the AG genotype of rs780094 was protective factor, whereas the recessive model of rs151290 was risk factor after adjusting for confounders. Stratified by sex, only for women, the recessive model of rs151290 was still significance. The significant synergies interactions between SNPs were found between rs780094 in GCKR and rs972283 in KLF14 and rs10830963 in MTNR1B, respectively; meanwhile, the antagonistic interaction was revealed for rs151290 and rs780094 only for women. For male, there were significant synergies interactions between rs780094, smoking and alcohol drinking; and antagonistic interaction was revealed between rs780094 and severe activity both for men and women. CONCLUSIONS: GCKR and KLF14 genes play a significant role in risk of HTGW in a Han Chinese population.

Polymorphisms in Four Genes (KCNQ1 rs151290, KLF14 rs972283, GCKR rs780094 and MTNR1B rs10830963) and Their Correlation with Type 2 Diabetes Mellitus in Han Chinese in Henan Province, China.

Genetic variants at KCNQ1 rs151290, KLF14 rs972283, GCKR rs780094 and MTNR1B rs10830963 have been associated with type 2 diabetes mellitus (T2DM), but the results are contradictory in Chinese populations. The aim of the present study was to investigate the association of these four SNPs with T2DM in a large population of Han Chinese at Henan province, China. Seven-hundred-thirty-six patients with T2DM (cases) and Seven-hundred-sixty-eight healthy glucose-tolerant controls were genotyped for KCNQ1 rs151290, KLF14 rs972283, GCKR rs780094 and MTNR1B rs10830963. The association of genetic variants in these four genes with T2DM was analyzed using multivariate logistic regression. Genotypes and allele distributions of KCNQ1 rs151290 were significantly different between the cases and controls (p < 0.05). The AC and CC genotypes and the combined AC + CC genotype of rs151290 in KCNQ1 were associated with increases risk of T2DM before (OR = 1.482, 95% CI = 1.062-2.069; p = 0.021; OR = 1.544, 95% CI = 1.097-2.172, p = 0.013; and OR = 1.509, 95% CI = 1.097-2.077, p = 0.011, respectively) and after (OR = 1.539, 95% CI = 1.015-2.332, p = 0.042; OR = 1.641, 95% CI = 1.070-2.516, p = 0.023; and OR = 1.582, 95% CI = 1.061-2.358, p = 0.024; respectively) adjustment for sex, age, anthropometric measurements, biochemical indexes, smoking and alcohol consumption. Consistent with results of genotype analysis, the C allele of rs151290 in KCNQ1 was also associated with increased risk of T2DM (OR = 1.166, 95% CI = 1.004-1.355, p = 0.045). No associations between genetic variants of KLF14 rs972283, GCKR rs780094 or MTNR1B rs10830963 and T2DM were detected. The AC and CC genotypes and the C allele of rs151290 in KCNQ1 may be risk factors for T2DM in Han Chinese in Henan province.

Manipulation of KCNE2 expression modulates action potential duration and Ito and IK in rat and mouse ventricular myocytes.

In heterologous expression systems, KCNE2 has been demonstrated to interact with multiple α-subunits of voltage-dependent cation channels and modulate their functions. However, the physiological and pathological roles of KCNE2 in cardiomyocytes are poorly understood. The present study aimed to investigate the effects of bidirectional modulation of KCNE2 expression on action potential (AP) duration (APD) and voltage-dependent K(+) channels in cardiomyocytes. Adenoviral gene delivery and RNA interference were used to either increase or decrease KCNE2 expression in cultured neonatal and adult rat or neonatal mouse ventricular myocytes. Knockdown of KCNE2 prolonged APD in both neonatal and adult myocytes, whereas overexpression of KCNE2 shortened APD in neonatal but not adult myocytes. Consistent with the alterations in APD, KCNE2 knockdown decreased transient outward K(+) current (Ito) densities in neonatal and adult myocytes, whereas KCNE2 overexpression increased Ito densities in neonatal but not adult myocytes. Furthermore, KCNE2 knockdown accelerated the rates of Ito activation and inactivation, whereas KCNE2 overexpression slowed Ito gating kinetics in neonatal but not adult myocytes. Delayed rectifier K(+) current densities were remarkably affected by manipulation of KCNE2 expression in mouse but not rat cardiomyocytes. Simulation of the AP of a rat ventricular myocyte with a mathematical model showed that alterations in Ito densities and gating properties can result in similar APD alterations in KCNE2 overexpression and knockdown cells. In conclusion, endogenous KCNE2 in cardiomyocytes is important in maintaining cardiac electrical stability mainly by regulating Ito and APD. Perturbation of KCNE2 expression may predispose the heart to ventricular arrhythmia by prolonging APD.

Association of Canonical Wnt/ß-Catenin Pathway and Type 2 Diabetes: Genetic Epidemiological Study in Han Chinese.

We aimed to investigate the associations of polymorphisms in Canonical Wnt/ß-catenin pathway (WNT) signaling genes (including low-density lipoprotein-related protein 5 [LRP5] and transcription factor 7-like 2 [TCF7L2] gene) and the downstream gene glucagon (GCG) and risk of type 2 diabetes mellitus (T2DM) in a Han Chinese population. We genotyped the single nucleotide polymorphisms (SNPs) for LRP5, TCF7L2 and GCG gene were genotyped in 1842 patients with T2DM and 7777 normal glucose-tolerant healthy subjects. We used multifactor dimensionality reduction (MDR) and multiplicative logistic regression adjusting for sex, age, anthropometric measurements and lipid levels to investigate the gene-gene interactions for the risk of T2DM. Among the five SNPs in LRP5, the recessive model of rs7102273 and the haplotype GCTCC were associated with T2DM risk; the haplotype GCTTC was associated with decreased risk. For TCF7L2, the rs11196218 genotype GA and the haplotype CCG, TTG, TTA were associated with T2DM risk; whereas, the haplotype CTG and TCG were associated with decreased risk. Both MDR and multiplicative logistic regression revealed potential gene-gene interactions among LRP5, TCF7L2, and GCG associated with T2DM. The WNT signaling pathway may play a significant role in risk of T2DM in Han Chinese people.

Association of LRP5, TCF7L2, and GCG variants and type 2 diabetes mellitus as well as fasting plasma glucose and lipid metabolism indexes.

Recent data puts WNT signaling pathway in a pivotal role in regulating pancreas development as well as islet function, insulin production and secretion. The key effectors in the WNT signaling pathway are low-density lipoprotein receptor-related protein 5 (LRP5), transcription factor 7-like 2 (TCF7L2), and downstream-regulated glucagon (GCG). Our previous studies suggest that the WNT signaling pathway plays a significant role in risk of type 2 diabetes mellitus (T2DM) in Chinese population. The main purpose of the present study was to investigate the associations of single nucleotide polymorphisms (SNPs) in LRP5, TCF7L2 and glucagon (GCG) and quantitative traits in a healthy population. We used tag SNP to screen candidate SNPs for LRP5 and GCG; for TCF7L2, used the confirmed SNP rs11196218. A total of 1842 patients with T2DM and 7777 healthy controls underwent genotyping for the SNPs. We found a significant association of rs3758644 in LRP5 and fasting plasma glucose (p=0.006), and rs11196218 in TCF7L2 and triglycerides level (p=0.004). Among the SNPs in LRP5, TCF7L2, and GCG analyzed, only rs3758644 of LRP5 and rs11196218 of TCF7L2 were significantly associated with fasting plasma glucose and triglycerides index, respectively, in a healthy population.

Effects of advanced glycation end products on calcium handling in cardiomyocytes.

BACKGROUND AND AIMS: Advanced glycation end products (AGEs) accumulate in diabetes and the engagement of receptor for AGE (RAGE) by AGEs contributes to the pathogenesis of diabetic cardiomyopathy. This study aims to investigate the effects of AGE/RAGE on ryanodine receptor (RyR) activity and Ca(2+) handling in cardiomyocytes to elucidate the possible mechanism underlying cardiac dysfunction in diabetic cardiomypathy. METHODS AND RESULTS: Confocal imaging Ca(2+) spark, the elementary Ca(2+) release event reflecting RyR activity in intact cell, as well as SR Ca(2+) content and systolic Ca(2+) transient were performed in cultured neonatal rat ventricular myocytes. The results show that 50 mg/ml AGE increased the frequency of Ca(2+) sparks by 160%, while 150 mg/ml AGE increased it by 53%. AGE decreased the amplitude, width and duration of Ca(2+) sparks. Blocking RAGE with anti-RAGE IgG completely abolished the alteration of Ca(2+) sparks. The SR Ca(2+) content indicated by the amplitude (ΔF/F0) of 20 mM caffeine-elicited Ca(2+) transient was significantly decreased by 150 mg/ml AGE. In parallel, the amplitude of systolic Ca(2+) transient evoked by 1 Hz-field stimulation was remarkably decreased by 150 mg/ml AGE. The anti-RAGE antibody completely restored the impaired SR load and systolic Ca(2+) transient. CONCLUSION: AGE/RAGE signal enhanced Ca(2+) spark-mediated SR Ca(2+) leak, causing partial depletion of SR Ca(2+) content and consequently decreasing systolic Ca(2+) transient, which may contribute to contractile dysfunction in diabetic cardiomyopathy.

Glucagon gene polymorphism modifies the effects of smoking and physical activity on risk of type 2 diabetes mellitus in Han Chinese.

Few genome-wide association studies have considered interactions between multiple genetic variants and environmental factors associated with disease. The interaction was examined between a glucagon gene (GCG) polymorphism and smoking, alcohol consumption and physical activity and the association with risk of type 2 diabetes mellitus (T2DM) in a case-control study of Chinese Han subjects. The rs12104705 polymorphism of GCG and interactions with environmental variables were analyzed for 9619 participants by binary multiple logistic regression. Smoking with the C-C haplotype of rs12104705 was associated with increased risk of T2DM (OR=1.174, 95% CI=1.013-1.361). Moderate and high physical activity with the C-C genotype was associated with decreased risk of T2DM as compared with low physical activity with the genotype (OR=0.251, 95% CI=0.206-0.306 and OR=0.190, 95% CI=0.164-0.220). However, the interaction of drinking and genotype was not associated with risk of T2DM. Genetic polymorphism in rs12104705 of GCG may interact with smoking and physical activity to modify the risk of T2DM.

Association of the rs11196218 polymorphism in TCF7L2 with type 2 diabetes mellitus in Asian population.

OBJECTIVE: To clarify the association of rs11196218 polymorphism in transcription factor 7-like 2 (TCF7L2) and type 2 diabetes mellitus (T2DM) in Asian population by a case-control study and meta-analysis. METHODS: In the case-control study, 1842 patients with T2DM and 7777 normal glucose-tolerant controls in the Henan province of China were genotyped for rs11196218 in TCF7L2 by PCR-ligase detection reaction. We used allele, co-dominant, dominant and recessive models to evaluate the risk association and performed a meta-analysis of the results of different genetic models in previous studies and the current study. RESULTS: The AG genotype of rs11196218 was associated with risk of T2DM in the Henan population (odds ratio 1.37, 95% confidence interval 1.06-1.78), and dominant model showed marginal significant association (1.28, 0.99-1.67). Meta-analysis of 10 studies revealed the dominant model associated with T2DM in the overall population (1.20, 1.05-1.36). When stratified by region (southern and northern China and Japan), both the AG genotype and the dominant model were associated with risk of T2DM in southern Chinese (1.31, 1.03-1.66; 1.27, 1.01-1.60, respectively). CONCLUSION: The rs11196218 polymorphism in TCF7L2 is associated with risk of T2DM in Asian population.

Human branch point consensus sequence is yUnAy.

Yeast carries a strictly conserved branch point sequence (BPS) of UACUAAC, whereas the human BPS is degenerative and is less well characterized. The human consensus BPS has never been extensively explored in vitro to date. Here, we sequenced 367 clones of lariat RT-PCR products arising from 52 introns of 20 human housekeeping genes. Among the 367 clones, a misincorporated nucleotide at the branch point was observed in 181 clones, for which we can precisely pinpoint the branch point. The branch points were comprised of 92.3% A, 3.3% C, 1.7% G and 2.8% U. Our analysis revealed that the human consensus BPS is simply yUnAy, where the underlined is the branch point at position zero and the lowercase pyrimidines ('y') are not as well conserved as the uppercase U and A. We found that the branch points are located 21-34 nucleotides upstream of the 3' end of an intron in 83% clones. We also found that the polypyrimidine tract spans 4-24 nucleotides downstream of the branch point. Our analysis demonstrates that the human BPSs are more degenerative than we have expected and that the human BPSs are likely to be recognized in combination with the polypyrimidine tract and/or the other splicing cis-elements.

Effect of lactase preparations in asymptomatic individuals with lactase deficiency--gastric digestion of lactose and breath hydrogen analysis.

We compared two lactase preparations derived from Aspergillus orizae (AOL) and Penicillinase multicolor (PML) for stability in the stomach and overall enzymatic activity in 10 asymptomatic subjects with lactase deficiency. The subjects were given 10,000 FCC units of either AOL or PML 30 min prior to or simultaneously with 300 ml of milk. Gastric juice was withdrawn through a nasogastric tube immediately after and every 15 min for 60 min, and breath was sampled before and every 15 min for 6 h after the milk ingestion. When lactase was given simultaneously with the milk, gastric juice lactase activity and galactose concentration were significantly higher than the control levels. When lactase preparations were given 30 min prior to the milk, neither lactase activity nor galactose was detected in the gastric juice. The pH of the gastric juice was about 6.0 after the milk ingestion. Breath hydrogen did not increase when milk was ingested simultaneously with enzymes, but did increase if enzymes were given 30 min prior to milk ingestion. There were no significant differences in lactase activity, galactose concentration in gastric juice, or breath hydrogen when AOL and PML were compared. In conclusion, with exogenous lactase, digestion of lactose begins in the stomach when pH is raised to 6.0 by the buffering action of milk. Lactase preparations are effective assessed by breath hydrogen analysis in asymptomatic individuals with lactase deficiency if the enzymes are given simultaneously with milk.