Mitochondrial-related gene expression profiles suggest an important role of PGC-1alpha in the compensatory mechanism of endemic dilated cardiomyopathy.

Keshan disease (KD) is an endemic dilated cardiomyopathy with unclear etiology. In this study, we compared mitochondrial-related gene expression profiles of peripheral blood mononuclear cells (PBMCs) derived from 16 KD patients and 16 normal controls in KD areas. Total RNA was isolated, amplified, labeled and hybridized to Agilent human 4 × 44k whole genome microarrays. Mitochondrial-related genes were screened out by the Third-Generation Human Mitochondria-Focused cDNA Microarray (hMitChip3). Quantitative real-time PCR, immunohistochemical and biochemical parameters related mitochondrial metabolism were conducted to validate our microarray results. In KD samples, 34 up-regulated genes (ratios ≥ 2.0) were detected by significance analysis of microarrays and ingenuity systems pathway analysis (IPA). The highest ranked molecular and cellular functions of the differentially regulated genes were closely related to amino acid metabolism, free radical scavenging, carbohydrate metabolism, and energy production. Using IPA, 40 significant pathways and four significant networks, involved mainly in apoptosis, mitochondrion dysfunction, and nuclear receptor signaling were identified. Based on our results, we suggest that PGC-1alpha regulated energy metabolism and anti-apoptosis might play an important role in the compensatory mechanism of KD. Our results may lead to the identification of potential diagnostic biomarkers for KD in PBMCs, and may help to understand the pathogenesis of KD.

[Screening of differentially expressed proteins in serum from subjects with Keshan disease by two-dimensional electrophoresis and mass and mass spectrometry].

OBJECTIVE: To screen differentially expressed proteins in serum in patients with Keshan disease (KD), peripheral blood protein expression spectrum between subjects with Keshan disease and health controls were compared. METHODS: Differentially expressed protein spots were screened by two-dimensional gel electrophoresis (2-DE) between Keshan disease and health control subjects, and constitutive protein were identified by matrix assisted laser adsorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: 9 differentially expressed protein spots were showed in 2-DE images and 8 differentially expressed proteins were identified by MALDI-TOF-MS. In them, 3 up-regulated proteins, mainly relatedd to lipid metabolism, apoptosis resistance, immunological regulation and 3 down-regulated proteins, involved to cellular iron ion homeostasis; 2 up-regulated proteins in serum in patients with KD versus controls from KD areas were detected, mainly associated with protease inhibition. CONCLUSIONS: Haptoglobin, serum albumin, alpha-l-antitrypsin, transferring and alpha2-heremans schmid glycoprotein may be considered as candidate biological indicators used for diagnosis or prognosis of KD.

A study of the differential expression profiles of Keshan disease lncRNA/mRNA genes based on RNA-seq.

BACKGROUND: This study aims to analyze the differential expression profiles of lncRNA in Keshan disease (KSD) and to explore the molecular mechanism of the disease occurrence and development. METHODS: RNA-seq technology was used to construct the lncRNA/mRNA expression library of a KSD group (n=10) and a control group (n=10), and then Cuffdiff software was used to obtain the gene lncRNA/mRNA FPKM value as the expression profile of lncRNA/mRNA. The fold changes between the two sets of samples were calculated to obtain differential lncRNA/mRNA expression profiles, and a bioinformatics analysis of differentially expressed genes was performed. RESULTS: A total of 89,905 lncRNAs and 20,315 mRNAs were detected. Statistical analysis revealed that 921 lncRNAs had obvious differential expression, among which 36 were up-regulated and 885 were down-regulated; 2,771 mRNAs presented with obvious differential expression, among which 253 were up-regulated and 2,518 were down-regulated, and cluster analysis indicated that the gene expression trends among the sample groups were consistent. The differentially expressed lncRNAs were tested for target genes, and 117 genes were found to be regulated by differential lncRNAs, which were concentrated in six signaling pathways, among which the apoptosis FoxO signaling pathway ranked first, so the target genes IGF1R and TGFB2 were screened out. CONCLUSIONS: In this study, RNA-seq technology was used to obtain the differential gene expression profiles of KSD, and bioinformatics analysis was performed to screen out target genes, pointing out the direction for further research into the etiology, pathogenesis and drug treatment targets of KSD.

[Surveys on the conditions of Keshan disease and selenium levels of 15 counties in Shandong Province in 2008].

OBJECTIVE: To investigate the conditions of Keshan disease and the selenium nutritional status of residents in Keshan disease endemic area in Shandong Province. METHODS: One site from each of 15 Keshan disease endemic counties in Shandong Province was selected for the survey and 700 subjects in each site were randomly selected for the investigation. Physical examination, electrocardiogram and X-ray radiography of the subjects and the selenium contents of hair, wheat, corn and dried sweet potato were surveyed. RESULTS: A total of 10679 people were investigated in 15 counties and 315 patients were found, including 287 cases of latent type and 28 cases of chronic type, but no acute or subacute type patients were found. There were 1776 cases of abnormal electrocardiogram. Among the 461 cases checked up by radiography, the heart of 195 cases was enlarged. The selenium content of hair, wheat, corn and dried sweet potato were (0.5191 +/- 0.5538), (0.0268 +/- 0.0045), (0.0194 +/- 0.0052) and (0.0193 +/- 0.0039) mg/kg respectively. CONCLUSION: The occurrence of Keshan disease is in a stable status in Shandong Province at present. Hair selenium of residents in Keshan disease endemic area has reached an appropriate level. The selenium nutritional status of residents has improved, and the prevalence of Keshan disease is expected to be decreased in these areas.

Small RNA-sequencing identified the potential roles of neuron differentiation and MAPK signaling pathway in dilated cardiomyopathy.

Dilated cardiomyopathy is a severe disease characterized by ventricular enlargement and subsequent cardiac dysfunction. MiRNAs plays multiple roles in cardiovascular disease. However, diagnosis values and therapeutic effects of miRNAs in dilated cardiomyopathy are yet poorly understood. In the present study, small RNA-sequencing was employed to identify dysregulated circulating miRNAs in DCM patients compared with healthy controls. A total of 48 dysregulated miRNAs were detected, and 7198 mRNAs, the intersection of predicted mRNAs from both Miranda database and RNAhybrid database, were identified as the target mRNAs of these dysregulated miRNAs. Bioinformatics analysis was performed to identify the potential effects of these dysregulated miRNAs in dilated cardiomyopathy. GO analysis and GO-Tree analysis disclosed that neuron differentiation was potentially the core biological process associated with dilated cardiomyopathy. KEGG analysis and Pathway-Act network showed that mitogen-activated protein kinase (MAPK) signaling pathway was the hub pathway in dilated cardiomyopathy. The dysregulated miRNAs and related target mRNAs in neuron differentiation process and MAPK signaling pathway were also presented in the study. In conclusion, forty-eight dysregulated miRNAs were identified by small RNA-sequencing. Bioinformatics analysis suggested these miRNAs might be involved in the pathogenesis of dilated cardiomyopathy via regulating neuron differentiation process and MAPK signaling pathway.

RNA sequencing discloses the genome­wide profile of long noncoding RNAs in dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a common type of non­ischemic cardiomyopathy, of which the underlying mechanisms have not yet been fully elucidated. Long noncoding RNAs (lncRNAs) have been reported to serve crucial physiological roles in various cardiac diseases. However, the genome­wide expression profile of lncRNAs remains to be elucidated in DCM. In the present study, a case­control study was performed to identify expression deviations in circulating lncRNAs between patients with DCM and controls by RNA sequencing. Partial dysregulated lncRNAs were validated by reverse transcription­polymerase chain reaction. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, and lncRNA­messenger RNA (mRNA) co­expression network analyses were employed to probe potential functions of these dysregulated lncRNAs in DCM. Comparison between 8 DCM and 8 control samples demonstrated that there were alterations in the expression levels of 988 lncRNAs and 1,418 mRNAs in total. The dysregulated lncRNAs were found to be mainly associated with system development, organ morphogenesis and metabolic regulation in terms of 'biological processes'. Furthermore, the analysis revealed that the gap junction pathway, phagosome, and dilated and hypertrophic cardiomyopathy pathways may serve crucial roles in the development of DCM. The lncRNA­mRNA co­expression network also suggested that the target genes of the lncRNAs were different in patients with DCM as compared with those in the controls. In conclusion, the present study revealed the genome­wide profile of circulating lncRNAs in DCM by RNA sequencing, and explored the potential functions of these lncRNAs in DCM using bioinformatics analysis. These findings provide a theoretical foundation for future studies of lncRNAs in DCM.

[Observation on conditions and related factors of Keshan disease in Shandong province, 1990-2007].

OBJECTIVE: To observe the serial changes of condition and related factors of Keshan disease (KSD) and provide the scientific basis for disease control and further research. METHODS: The villages in KSD epidemic area of Juxian, Yishui, Pingyi, Zoucheng counties were selected. Since 1990, every five-year was counted as a stage. For the first to third stage, 3-14 year-old residents and over 14 year-old suspicious patients were selected as surveillance subjects. And in the fourth stage, all residents were selected as surveillance subjects. The same group of surveillance subjects in each stage were observed consecutively for 5 years. Surveillance contents included physical checkup, electrocardiogram (ECG) and Xray. At the same time, the selenium (Se) concentration in hair, wheat, corn and sweet potato was measured. The economic income and grain availability were also investigated. RESULTS: Totally, 14,510 cases were visited during 18 years. The incidence of KSD was 3.02% in the first stage, 2.31% in the second stage, 3.57% in the third stages and 3.65% in the fourth stage. Totally, 14,510 cases were examined by ECG, 809 cases showed the abnormal ECG and the total incidence of abnormal ECG was 5.49%. The incidence of abnormal ECG was 3.52% -5.24% from 1990 to 2004 but was 10.97%-10.91% from 2005 to 2007. 732 of hair samples, 701 of wheat samples, 615 of corn samples and 643 of sweet potato samples were collected and the Se concentration was determined by the fluorescent method. Se levels in hair samples had increased (P < 0.05) year by year but Se levels in food have not changed significantly. The economic income and grain availability had increased gradually from 535.8 yuan and 254.6 kg per person in 1990 to 2968.0 yuan and 602.0 kg per person in 2007. CONCLUSION: The condition of KSD was in a stable situation in Shandong Province. Related factors improvement should be an important environmental condition.

[Effects of high level Zn intake on metabolism in man].

OBJECTIVE: To observe the effects of high level Zn intake on zinc (Zn) copper (Cu), lipids metabolism and antioxidation function in man so as to provide scientific basis for Zn supplementation. METHODS: 40 rural healthy men were chosen and tablets of 50 mg Zn (as 0.2 g of zinc glucose) per day was taken by them for eight weeks. All subjects were checked up for five times: at week 0, 2, 4, 8 during the period of Zn supplementation and at week 12 and their serum, RBC, hair, and 24h-urine were collected for assays at the same time. RESULTS: (1) Zn contents in serum, RBC and hair increased significantly after 2 to 4 weeks supplementation and decreased at 4 weeks after stopping supplementation, but they were still higher than those before supplementation. Zn contents in 24h-urine increased significantly after supplementation and decreased at 4 weeks after stopping supplementation to the same level as before supplementation. (2) Cu contents in serum, RBC and hair increased significantly after 4 weeks supplementation and decreased at 4 weeks after stopping supplementation. Cu contents in 24h-urine did not change significantly during experiment. (3) RBC superoxide dismutase (SOD) activities were decreased persistently after 2 weeks supplementation and were not recovered at 4 weeks after stopping supplementation. RBC glutathione peroxidase (GPX) activities increased significantly after 4 weeks supplementation. The content of lipid peroxide (LPO) increased significantly after 2 weeks supplementation. (4) Total cholesterol (TC), triglyceride (TG), Low density lipoprotein cholesterol (LDL-C) and apolipoprotein B100 (ApoB100) increased significantly, high density lipoprotein cholesterol (HLD-C) and apolipoprotein A1 (ApoA1) decreased after supplementation. CONCLUSION: When healthy men received 50 mg Zn supplement per day, it might interfere Zn, Cu and lipids metabolism and inhibit antioxidation process.

Genome-wide study reveals an important role of spontaneous autoimmunity, cardiomyocyte differentiation defect and anti-angiogenic activities in gender-specific gene expression in Keshan disease.

BACKGROUND: Keshan disease (KD) is an endemic cardiomyopathy in China. The etiology of KD is still under debate and there is no effective approach to preventing and curing this disease. Young women of child-bearing age are the most frequent victims in rural areas. The aim of this study was to determine the differences between molecular pathogenic mechanisms in male and female KD sufferers. METHODS: We extracted RNA from the peripheral blood mononuclear cells of KD patients (12 women and 4 men) and controls (12 women and 4 men). Then the isolated RNA was amplified, labeled and hybridized to Agilent human 4×44k whole genome microarrays. Gene expression was examined using oligonucleotide microarray analysis. A quantitative polymerase chain reaction assay was also performed to validate our microarray results. RESULTS: Among the genes differentially expressed in female KD patients we identified: HLA-DOA, HLA-DRA, and HLA-DQA1 associated with spontaneous autoimmunity; BMP5 and BMP7, involved in cardiomyocyte differentiation defect; and ADAMTS 8, CCL23, and TNFSF15, implicated in anti-angiogenic activities. These genes are involved in the canonical pathways and networks recognized for the female KD sufferers and might be related to the pathogenic mechanism of KD. CONCLUSION: Our results might help to explain the higher susceptibility of women to this disease.

Serum biomarkers of Keshan disease assessed using a protein profiling approach based on ClinProt technique.

The etiology of Keshan disease (KD), an endemic myocardiopathy in regions of China, is largely unknown. To show the protein changes in serum from KD patients versus controls and idiopathic dilated cardiomyopathy (IDCM) and to search specific biological markers for differential diagnosis for KD. Serum of 65 patients with KD was compared with 29 patients with IDCM, 62 controls from KD areas and 28 controls from non-KD areas by ClinProt/MALDI-ToF technique. The genetic algorithm, quick classifier algorithm and supervised neural network algorithm methods were used to screen marker proteins and establish diagnostic model. Thirty-four differential peaks were identified in KD patients compared with the healthy controls from non-KD areas. Thirty-eight differentially peaks were identified in KD patients and controls from KD areas; and sixty-seven differentially peaks were identified in patients with KD and patients with IDCM. We believe that marker protein peaks screened in KD patients, healthy controls and IDCM patients may provide clues for the differential diagnosis and treatment of KD.

Comparing gene expression profiles of Kashin-Beck and Keshan diseases occurring within the same endemic areas of China.

In this study, differentially expressed genes in peripheral blood from patients with Kashin-Beck disease and Keshan disease were compared to further investigate the etiology and pathogenesis of both diseases, which occur in a common endemic area of China. Twenty Kashin-Beck disease patients and 12 healthy controls, and 16 Keshan disease patients and 16 healthy controls, were grouped into four pairs. Patients and controls were selected from common endemic areas for the two diseases. Total RNA was isolated from peripheral blood mononuclear cells from all patients and controls, and gene expression profiles analyzed by oligonucleotide microarrays. Sixteen genes differentially expressed in both Kashin-Beck disease and Keshan disease (versus controls) were identified, and comprised nine genes showing synchronous and seven asynchronous expression. The Comparative Toxicogenomics Database shows that expression and biological function of these genes can be affected by multiple environmental factors, including mycotoxin and selenium content, potential environmental risk factors for the two diseases. Thus, these shared differentially expressed genes may contribute to the distinct organ lesions, caused by common environmental risk factors of Kashin-Beck disease and Keshan disease.