Integrated Analysis and Identification of mRNAs, Circular RNAs, and Long Noncoding RNAs in Immunoglobulin A Nephropathy.

Circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) have a role in monitoring the appearance and progression of a great many diseases. They are useful markers for the prognosis and diagnosis of some diseases. In previous studies, the expression patterns of mRNAs, circRNAs, and lncRNAs related to immunoglobulin A (IgA) nephropathy have not been sufficiently discussed. Active prevention methods and treatment for IgA nephropathy (IgAN) are still not used. Integrated analyses and identification of the circRNAs and lncRNAs in IgAN have not been executed. We carried out a deep RNA sequencing analysis between controls and subjects with IgAN. In total, 125 antisense lncRNAs were identified to be greatly differentially expressed between the control and experimental groups. In addition, 606 mRNAs and 1275 circRNAs with differential expression levels were found between the groups. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways were used as bioinformatic methods in this study. Our study showed the expression patterns of mRNAs, circRNAs, and lncRNAs in IgAN. We revealed the key roles of circRNAs and lncRNAs in the molecular mechanism of IgAN.

Differential proteomics analysis of liver failure in peripheral blood mononuclear cells using isobaric tags for relative and absolute quantitation.

The aim of the present study was to examine differentially expressed proteome profiles for candidate biomarkers in peripheral blood mononuclear cells (PBMCs) of liver failure (LF) patients. Ten patients were diagnosed as LF and 10 age- and gender-matched subjects were recruited as healthy controls. Isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic technology is efficiently applicable for identification and relative quantitation of the proteomes of PBMCs. Eight-plex iTRAQ coupled with strong cation exchange chromatography, and liquid chromatography coupled with tandem mass spectrometry were used to analyze total proteins in LF patients and healthy control subjects. Molecular variations were detected using the iTRAQ method, and western blotting was used to verify the results. LF is a complex type of medical emergency that evolves following a catastrophic insult to the liver, and its outcome remains the most ominous of all gastroenterologic diseases. Serious complications tend to occur during the course of the disease and further exacerbate the problems. Using the iTRAQ method, differentially expressed proteome profiles of LF patients were determined. In the present study, 627 proteins with different expression levels were identified in LF patients compared with the control subjects; with 409 proteins upregulated and 218 proteins downregulated. Among them, four proteins were significantly differentially expressed; acylaminoacyl-peptide hydrolase and WW domain binding protein 2 were upregulated, and resistin and tubulin ß 2A class IIa were downregulated. These proteins demonstrated differences in their expression levels compared with other proteins with normal expression levels and the significant positive correlation with LF. The western blot results were consistent with the results from iTRAQ. Thus, investigation of the molecular mechanism of the proteins involved in LF may facilitate an improved understanding of the pathogenesis of LF and elucidation of novel biomarker candidates.

Integrated microRNA and protein expression analysis reveals novel microRNA regulation of targets in fetal down syndrome.

Down syndrome (DS) is caused by trisomy of human chromosome 21 and is associated with a number of deleterious phenotypes. To investigate the role of microRNA (miRNA) in the regulation of DS, high­throughput Illumina sequencing technology and isobaric tagging for relative and absolute protein quantification analysis were utilized for simultaneous expression profiling of miRNA and protein in fetuses with DS and normal fetuses. A total of 344 miRNAs were associated with DS. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to investigate the proteins found to be differentially expressed. Functionally important miRNAs were determined by identifying enriched or depleted targets in the transcript and the protein expression levels were consistent with miRNA regulation. The results indicated that GRB2, TMSB10, RUVBL2, the hsa­miR­329 and hsa­miR­27b, hsa­miR­27a targets, and MAPK1, PTPN11, ACTA2 and PTK2 or other differentially expressed proteins were connected with each other directly or indirectly. Integrative analysis of miRNAs and proteins provided an expansive view of the molecular signaling pathways in DS.

Integrated analyses of a major histocompatibility complex, methylation and transcribed ultra-conserved regions in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease which affects different organs and systems that, has a complex genetic inheritance, and is affected by both epigenetic and environmental risk factors. Previous studies on SLE have lacked the statistical power and genetic resolution to fully determine the influence of major histocompatibility complex (MHC) on SLE. In this study, in order to determine this influence, a total of 15 patients with SLE and 15 healthy controls were enrolled. MHC region capture technology, hMeDIP-chip, transcribed ultra-conserved region (T-UCR) microarray and bioinformatics analysis were utilized for both groups. The results revealed methylated CpG enrichment at 6 loci in the MHC segment of SLE. We found 4 single-nucleotide polymorphisms (SNPs) in the CpG promoter of human leukocyte antigen-B (HLA-B) and 2 SNPs in chr6:29521110­29521833. No significant GO term or KEGG pathway enrichment was noted for an immune-correlated process in the SLE patients for the corresponding CpG-methylated genes. In this study, T-UCR was not discovered in the MHC segment. The analysis of SNPs (rs1050683, rs12697943, rs17881210, rs1065378, rs17184255 and rs16895070) and gene expression in peripheral blood lymphocytes indicated that these SNPs were associated with the occurrence of SLE. Further studies are warranted to examine the roles of these SNPs in the pathogenesis of SLE. Integrative analysis technology provided a view of the molecular signaling pathways in SLE.

ARHGAP4 mutated in a Chinese intellectually challenged family.

OBJECTIVE: Mental retardation is characterized by lower intelligence compared to the average intelligence of persons the same age. These patients have low adaptive capacity acquired by society. The genetic factors of causing MR include monogenic disease, chromosome structural aberration, and chromosome number aberration and so on. We explored the cause of a Chinese family suffering from mental retardation. METHODS: We used karyotyping technology to determine the karyotype of the proband, and we used FISH to verify the result of the karyotyping. We used whole-exome sequencing to identify the disease-causing gene and used Sanger sequencing to verify the result of whole-exome sequencing to assess the family's gene expression. RESULTS: The G-banding of the karyotype revealed that the patient's karyotype is 46, XY. FISH revealed that the patient does not have a trisomy syndrome. The karyotype of the proband is normal. Using whole-exome sequencing, we identified 108,767 variants in the exome gene of the patient, including 101,787 SNPs and 6980 InDels. Combining clinical information and bioinformatics analysis, including databases filtering and SIFT analysis, we found ARHGAP4 in X chromosome was candidate MR disease-causing gene. PCR and Sanger sequencing results were consistent with whole-exome sequencing. ARHGAP4 (T491M) mutation was present in the genome of the proband and his mother is a carrier, while his father, sister, and brother do not carry this mutation. CONCLUSION: According to clinical information, whole-exome sequencing results and Sanger verification results, ARHGAP4 (T491M) mutation may be disease-causing gene of the MR patient. The relation between ARHGAP4 mutation and MR clinical characteristic is needed to be illuminated with participation of more MR patients.

Genome-wide analysis of 5-hmC in the peripheral blood of systemic lupus erythematosus patients using an hMeDIP-chip.

Systemic lupus erythematosus (SLE) is a chronic, potentially fatal systemic autoimmune disease characterized by the production of autoantibodies against a wide range of self-antigens. To investigate the role of the 5-hmC DNA modification with regard to the onset of SLE, we compared the levels 5-hmC between SLE patients and normal controls. Whole blood was obtained from patients, and genomic DNA was extracted. Using the hMeDIP-chip analysis and validation by quantitative RT-PCR (RT-qPCR), we identified the differentially hydroxymethylated regions that are associated with SLE. There were 1,701 genes with significantly different 5-hmC levels at the promoter region in the SLE patients compared with the normal controls. The CpG islands of 3,826 genes showed significantly different 5-hmC levels in the SLE patients compared with the normal controls. Out of the differentially hydroxymethylated genes, three were selected for validation, including TREX1, CDKN1A and CDKN1B. The hydroxymethylation levels of the three genes were confirmed by RT-qPCR. The results suggested that there were significant alterations of 5-hmC in SLE patients. Thus, these differentially hydroxymethylated genes may contribute to the pathogenesis of SLE. These findings show the significance of 5-hmC as a potential biomarker or promising target for epigenetic-based SLE therapies.

Genome-wide analysis of DNA 5-hmC in peripheral blood of uremia by hMeDIP-chip.

BACKGROUND: Treatment of uremia is now dominated by dialysis; in some cases, patients are treated with dialysis for decades, but overall outcomes are disappointing. A number of studies have confirmed the relevance of several experimental insights to the pathogenesis of uremia, but the specific biomarkers of uremia have not been fully elucidated. To date, our knowledge about the alterations in DNA 5-hydroxymethylcytosine (5-hmC) in uremia is unclear, to investigate the role of DNA 5-hmC in the onset of uremia, we performed hMeDIP-chip between the uremia patients and the normal controls from the experiment to identify differentially expressed 5-hmC in uremia-associated samples. METHODS: Extract genomic DNA, using hMeDIP-chip technology of Active Motif companies for the analysis of genome-wide DNA 5-hmC, and quantitative real-time PCR confirmation to identify differentially expressed 5-hmC level in uremia-associated samples. RESULTS: There were 1875 genes in gene Promoter, which displayed significant 5-hmC differences in uremia patients compared with normal controls. Among these genes, 960 genes displayed increased 5-hmC and 915 genes decreased 5-hmC. 4063 genes in CpG Islands displayed significant 5-hmC differences in uremia patients compared with normal controls. Among these genes, 1780 genes displayed increased 5-hmC and 2283 genes decreased 5-hmC. Three positive genes, HMGCR, THBD, and STAT3 were confirmed by quantitative real-time PCR. CONCLUSION: Our studies indicate the significant alterations of 5-hmC. There is a correlation of gene modification 5-hmC in uremia patients. Such novel findings show the significance of 5-hmC as a potential biomarker or promising target for epigenetic-based uremia therapies.