Hiroya Yamada

Associations between circulating microRNAs (miR-21, miR-34a, miR-122 and miR-451) and non-alcoholic fatty liver

BACKGROUND In many industrialized countries, non-alcoholic fatty liver disease (NAFLD) is recognized as an important disease that increases the risk of cardiovascular disease, type-2 diabetes, and metabolic syndrome. Most people with NAFLD are asymptomatic, and the disease is discovered incidentally during clinical laboratory tests. Present screening methods for NAFLD use ultrasound, and CT scans that are time-consuming, and a simple screening method for NAFLD is needed. In this study, we investigated whether serum miRNAs are associated with NAFLD and assessed the potential of serum miRNAs as a biomarker for NAFLD. METHODS We assessed intrahepatic fat by ultrasound scan, and the serum levels of five miRNAs (miR-21, miR-34a, miR-122, miR-145, and miR-451), which help regulate cholesterol and fatty acid homeostasis in liver tissue, by real-time PCR in a cross-sectional sample of 403 participants who attended health examinations. RESULTS Serum levels of miRNAs, miR-21, miR-34a, miR-122, and miR-451 were higher in participants with NAFLD. The serum level of miR-122 was correlated with the severity of liver steatosis. CONCLUSION Serum levels of miRNAs, particularly miR-122, may be a useful biomarker for NAFLD.

Associations Between Autoimmune Thyroid Disease Prognosis and Functional Polymorphisms of Susceptibility Genes, CTLA4, PTPN22, CD40, FCRL3, and ZFAT, Previously Revealed in Genome-wide Association Studies

PurposeGenome-wide association studies have revealed several susceptibility genes among patients with autoimmune thyroid disease (AITD), including CTLA4, PTPN22, FCRL3, and ZFAT. However, any possible association between these genes and AITD prognosis remains unknown. The objective of this study was to identify associations between polymorphisms of these genes and AITD prognosis.MethodsWe genotyped functional polymorphisms, including CTLA4 CT60, CTLA4 +49A/G, CTLA4 -1147C/T, CTLA4 -318C/T, PTPN22 -1123C/G, PTPN22 SNP37, CD40 -1C/T, FCRL3 -169C/T, ZFAT Ex9b-SNP10, and ZFAT Ex9b-SNP2, in 197 AITD patients carefully selected from 456 registered AITD patients, and 86 control subjects. The restriction fragment length polymorphism method was used for genotyping.ResultsThe CD40 -1CC genotype and C allele were significantly more frequent in patients with Graves’ disease (GD) in remission than in those with intractable GD (P = 0.041 and P = 0.031, respectively). The FCRL3 -169TT genotype was significantly less frequent in patients with intractable GD than in those with GD in remission (P = 0.0324). For a ZFAT Ex9b-SNP10 polymorphism, the TT genotype and T allele were significantly more frequent in patients with severe Hashimoto’s disease (HD) than in those with mild HD (P = 0.0029 and P = 0.0049, respectively). For a CTLA4 CT60 polymorphism, the antithyrotropin receptor antibody levels at the onset of GD were significantly higher in those with the GG genotype than in those with other genotypes (P = 0.0117).ConclusionsCD40 and FCRL3 gene polymorphisms were associated with GD intractability, and ZFAT polymorphism was associated with HD severity but not its development.

Circulating microRNAs in autoimmune thyroid diseases

Autoimmune thyroid diseases (AITDs), including Graves’ disease (GD) and Hashimotos thyroiditis (HT), are the most common autoimmune diseases. MicroRNAs (miRNAs) are small noncoding RNAs, which can play pivotal roles in immune functions and development of autoimmunity. Recently, it has been recognized that identification of circulating miRNAs can provide important and novel information regarding disease pathogenesis and clinical condition. However, the role circulating miRNAs in AITD has not yet been described.

Longitudinal study of circulating miR-122 in a rat model of non-alcoholic fatty liver disease.

BACKGROUND Circulating microRNAs (miRs) may be promising biomarkers for several diseases. We previously found that miR-122 can function as a biomarker for non-alcoholic fatty liver disease (NAFLD). However, little is known regarding the time course of circulating miR-122 levels during the development of NAFLD. Here, we examined circulating miR-122 levels using a rat model of NAFLD. METHODS To clarify changes in serum levels of miR-122 during development of NAFLD, experimental rats were fed a high-fat diet (HFD) for 2-10 weeks, while control rats received standard chow. Serum and liver tissue was collected from all animals at 2, 6, and 10 weeks of feeding. Clinical laboratory parameters (cholesterol, TG, AST, ALT, NEFA) were determined by biochemistry analyzer. Hepatic lipid accumulation was estimated by Oil red O staining. Circulating miR-122 levels were then measured by real-time polymerase chain reaction. RESULTS Over the 10 weeks of feeding, body weight, total liver lipids, and liver and serum triacylglycerol were increased in the HFD group compared to the control group. However, no significant changes in serum alanine aminotransferase activity were observed, suggesting that NAFLD status was mild. In contrast, we observed drastic up-regulation of circulating miR-122 levels. Our findings suggest that serum miR-122 level is indeed useful for assessing early NAFLD and might be superior to clinical markers traditionally used to monitor hepatic disease.

Expression of nestin mRNA is a differentiation marker in thyroid tumors

Nestin is a maker that identifies stem cells in some adult tissues, and its expression is believed to relate to malignancy in cancer cells. In this study, we measured the expression levels of nestin mRNA in various kinds of thyroid tumor by the real-time quantitative reverse transcription-polymerase chain reaction. Unexpectedly, nestin mRNA was detected in almost all differentiated thyroid tumors and normal thyroid tissues, whereas extremely decreased expression was observed in anaplastic carcinomas, which are the most malignant of the thyroid follicular cell-derived tumors. These results suggest that nestin mRNA is a differentiation marker, and its expression does not relate to malignant characteristics in thyroid tumors.

Coffee Consumption and Risk of Colorectal Cancer: The Japan Collaborative Cohort Study

Background Epidemiologic studies have reported coffee consumption to be associated with various health conditions. The purpose of this study was to examine the relationship of coffee consumption with colorectal cancer incidence in a large-scale prospective cohort study in Japan. Methods We used data from the Japan Collaborative Cohort Study for Evaluation of Cancer Risk (JACC Study). Here, we analyzed a total of 58 221 persons (23 607 men, 34 614 women) followed from 1988 to the end of 2009. During 738 669 person-years of follow-up for the analysis of colorectal cancer risk with coffee consumption at baseline, we identified 687 cases of colon cancer (355 males and 332 females) and 314 cases of rectal cancer (202 males and 112 females). We used the Cox proportional-hazard regression model to estimate hazard ratio (HR). Results Compared to those who consumed less than 1 cup of coffee per day, men who consumed 2–3 cups of coffee per day had an HR of 1.26 (95% confidence interval [CI] 0.93–1.70), and men who consumed more than 4 cups of coffee per day had an HR of 1.79 (95% CI 1.01–3.18). A statistically significant increase in the risk of colon cancer was associated with increasing coffee consumption among men (P for trend = 0.03). On the other hand, coffee consumption in women was not associated with incident risk of colon cancer. Coffee consumption was also not associated with rectal cancer incidence in men or women. Conclusions This large-scale population-based cohort study showed that coffee consumption increases the risk of colon cancer among Japanese men.

High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver.

DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status.

Fructose consumption induces hypomethylation of hepatic mitochondrial DNA in rats.

AIMS Fructose may play a crucial role in the pathogenesis of metabolic syndrome (MetS). However, the pathogenic mechanism of the fructose-induced MetS has not yet been investigated fully. Recently, several reports have investigated the association between mitochondrial DNA (mtDNA) and MetS. We examined the effect of fructose-rich diets on mtDNA content, transcription, and epigenetic changes. MAIN METHODS Four-week-old male Sprague-Dawley rats were offered a 20% fructose solution for 14weeks. We quantified mRNAs for hepatic mitochondrial genes and analyzed the mtDNA methylation (5-mC and 5-hmC) levels using ELISA kits. KEY FINDINGS Histological analysis revealed non-alcoholic fatty liver disease (NAFLD) in fructose-fed rats. Hepatic mtDNA content and transcription were higher in fructose-fed rats than in the control group. Global hypomethylation of mtDNA was also observed in fructose-fed rats. SIGNIFICANCE We showed that fructose consumption stimulates hepatic mtDNA-encoded gene expression. This phenomenon might be due to epigenetic changes in mtDNA. Fructose-induced mitochondrial epigenetic changes appear to be a novel mechanism underlying the pathology of MetS and NAFLD.

Gains in disability-free life expectancy from elimination of diseases and injuries in Japan.

Background Although disability-free life expectancy has been investigated in Japan, gains from elimination of diseases and injuries have not been examined. Methods We used data from the 2007 Japanese national health statistics to calculate the number of years with and without activity limitation that could be expected from eliminating 6 selected diseases and injuries. Results At birth, the number of expected years of life without and with activity limitation was 70.8 and 8.4, respectively, in males and 74.2 and 11.8 in females. More than 1.0 expected years without activity limitation were gained from eliminating malignant neoplasms and cerebrovascular diseases; smaller gains were observed after eliminating other diseases and injuries. Elimination of cerebrovascular diseases, dementia, and fracture decreased expected years with activities of daily living (ADL) limitation, and elimination of shoulder lesions/low back pain decreased expected years with non-ADL limitation. Conclusions Elimination of diseases and injuries increased expected years with and without activity limitation among Japanese, which suggests that improved prevention of those diseases and injuries—including cerebrovascular diseases and dementia—would result in longer disability-free life expectancy and fewer years of severe disability.

Messenger RNA quantification after fluorescence activated cell sorting using intracellular antigens

Recent studies using stem cells or cancer stem cells have revealed the importance of detecting minor populations of cells in blood or tissue and analyzing their biological characteristics. The only possible method for carrying out such procedures is fluorescence activated cell sorting (FACS). However, FACS has the following limitations. First, cells without an appropriate cell surface marker cannot be sorted. Second, the cells have to be kept alive during the sorting process in order to analyze their biological characteristics. If an intracellular antigen that was specific to a particular cell type could be stained with a florescent dye and then the cells can be sorted without causing RNA degradation, a more simple and universal method for sorting and analyzing cells with a specific gene expression pattern could be established since the biological characteristics of the sorted cells could then be determined by analyzing their gene expression profile. In this study, we established a basic protocol for messenger RNA quantification after FACS (FACS-mQ) targeting intracellular antigens. This method can be used for the detection and analysis of stem cells or cancer stem cells in various tissues.