The identification of circular RNAs from peripheral blood mononuclear cells in systemic lupus erythematosus.

BACKGROUND: The diagnosis of systemic lupus erythematosus (SLE) is complicated. This study explores the expression of circular RNAs (circRNAs), which are closed non-coding RNAs in which the 5' and 3' ends are covalently linked and which work by sponging microRNAs. CircRNAs were extracted from peripheral blood mononuclear cells (PBMCs) of SLE patients to identify novel circRNA species that might be used for SLE diagnosis. METHODS: Microarray was applied to screening circRNAs changes in PBMCs obtained from SLE patients (n = 10) and healthy participants (n = 10), paired for age and sex. We then verified the selected circRNAs in PBMCs using quantitative reverse transcription-polymerase chain reaction amplification (qRT-PCR) in another cohort, including ten paired SLE patients and healthy participants. The correlation between the differential circRNAs and clinical pathology of SLE were analyzed. RESULTS: 182 up-regulated and 563 significantly down-regulated circRNAs in PBMCs of patients with SLE were identified. Besides, the qRT-PCR results were consistent with the microarray results. The correlation analysis revealed that has_circRNA_100236, has_circRNA_102489, and has_circRNA_101413 were correlated with positive anti-dsDNA, thrombocytopenia, and positive IgG, respectively. Lastly, their miRNAs targets and the binding sites were predicted. CONCLUSION: We identified some dysregulated circRNAs in PBMCs from SLE patients, and these circRNAs may be developed as the novel biomarkers for the diagnosis of SLE.

Circular RNA expression profiles of peripheral blood mononuclear cells in rheumatoid arthritis patients, based on microarray chip technology.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovial inflammation and finally leads to variable degrees of bone and cartilage erosion. The diagnosis of RA is not an accurate indicator, but a series of scores and the mechanisms underlying it remain only partially understood. The present study explored whether circular RNAs (circRNAs) contribute to the RA pathophysiological mechanism. Total RNA from peripheral blood mononuclear cells of 10 RA patients and 10 healthy controls were extracted and circRNA expression profiling was followed by microarray analysis. In addition, circRNA interactions with microRNAs were performed and microRNA response elements were listed to identify differentially expressed binding site targets in RA. Reverse transcription­quantitative polymerase chain reaction amplification (RT­qPCR) was used to verify the differential expression of circRNAs. A total of 584 circRNAs were differentially expressed in RA patients vs. healthy controls, by circRNA microarray, including 255 circRNAs which were significantly upregulated and 329 downregulated among the RA samples. RT­qPCR validation demonstrated that the expression levels of hsa_circRNA_104194, hsa_circRNA_104593, hsa_circRNA_103334, hsa_circRNA_101407 and hsa_circRNA_102594 were consistent with the results from the microarray analysis. The current study presented differentially expressed circRNAs and their corresponding microRNA binding sites in RA. circRNAs may exhibit a role in the regulation of expression of symbol genes that influence the occurrence and development of RA.

Generation of induced pluripotent stem cells from renal tubular cells of a patient with Alport syndrome.

Alport syndrome (AS) is a hereditary disease that leads to kidney failure and is caused by mutations in the COL4A3, COL4A4, and COL4A5 genes that lead to the absence of collagen α3α4α5 (IV) networks in the mature kidney glomerular basement membrane. Approximately 80% of AS is X-linked because of mutations in COL4A5, the gene encoding the alpha 5 chain of type IV collagen. To investigate the pathogenesis of AS at the genetic level, we generated induced pluripotent stem cells (iPSCs) from renal tubular cells of a patient with AS. The successful iPSC generation laid the foundation to master the repair of the COL4A5 gene and to evaluate the differentiation of iPSC into Sertoli cells and the accompanying epigenetic changes at each stage. The generation of iPSCs from AS patients not only confirms that iPSCs could be generated from renal tubular cells, but also provides a novel type of genetic therapy for AS patients. In this study, we generated iPSCs from renal tubular cells via ectopic expression of four transcription factors (Oct4, Sox2, c-myc, and Klf4). According to the human embryonic stem cell (hESC) charter, iPSC formation was confirmed by comparatively analyzing hESC markers via colony morphology, immunohistochemistry, qRT-PCR, flow cytometry, gene expression profiling of the three germ layers, and karyotyping. Our results demonstrated that iPSCs were similar to hESCs with regard to morphology, proliferation, hESC-specific surface marker expression, and differentiation into the cell types of the three germ layers. The efficient generation of iPSCs from the renal tubular cells of an AS patient would provide a novel model to investigate the mechanisms underlying AS and to develop new treatments for AS.

Identification of microRNAs and their target genes in Alport syndrome using deep sequencing of iPSCs samples.

MicroRNAs (miRNAs) are a class of small RNA molecules that are implicated in post-transcriptional regulation of gene expression during development. The discovery and understanding of miRNAs has revolutionized the traditional view of gene expression. Alport syndrome (AS) is an inherited disorder of type IV collagen, which most commonly leads to glomerulonephritis and kidney failure. Patients with AS inevitably reach end-stage renal disease and require renal replacement therapy, starting in young adulthood. In this study, Solexa sequencing was used to identify and quantitatively profile small RNAs from an AS family. We identified 30 known miRNAs that showed a significant change in expression between two individuals. Nineteen miRNAs were up-regulated and eleven were down-regulated. Forty-nine novel miRNAs showed significantly different levels of expression between two individuals. Gene target predictions for the miRNAs revealed that high ranking target genes were implicated in cell, cell part and cellular process categories. The purine metabolism pathway and mitogen-activated protein kinase (MAPK) signaling pathway were enriched by the largest number of target genes. These results strengthen the notion that miRNAs and their target genes are involved in AS and the data advance our understanding of miRNA function in the pathogenesis of AS.

Microarray analysis of long non-coding RNA expression in human acute rejection biopsy samples following renal transplantation.

Rejection is still a major obstacle in long-term allograft survival of renal transplant recipients. Long non­coding RNAs (lncRNAs) are an important class of pervasive RNAs involved in a variety of biological functions, and which are often found to be differentially expressed between healthy and pathological conditions. The aim of this study was to compare the expression profiles of lncRNAs between samples from acute rejection following kidney transplantation and control samples. Three patients were enrolled, diagnosed by renal biopsy with acute rejection upon kidney transplantation. We used lncRNA microarrays to study the lncRNA expression profiles in the kidney biopsies of these patients and in kidneys from healthy donors. Reverse transcription­quantitative polymerase chain reaction (RT-qPCR) was used to validate the microarray results. In addition, potential functions of the identified lncRNAs were further explored by searching the UCSC, RNAdb, RefSeq and NRED databases. Five candidate lncRNAs displaying differential expression in acute rejection samples were validated by RT-qPCR. The results were in agreement with the microarray data. Among the identified lncRNAs, certain have been previously identified in relevant conditions, thereby supporting previous evidence, but certain may constitute novel biomarker candidates. This is the first report to date using lncRNA microarrays to identify unique expression signatures of acute rejection in transplant biopsies. Our data indicate that lncRNAs are potentially involved in the pathogenesis of acute rejection. Our results may have important implications in the identification of diagnostic biomarkers, as well as in the understanding and treatment of acute rejection following renal transplantation.

Analysis of microRNAs in patients with systemic lupus erythematosus, using Solexa deep sequencing.

OBJECTIVES: Our aim in this study was to identify and examine the differential expression of microRNAs in patients with systemic lupus erythematosus (SLE). METHODS: We employed high-quality, high-throughput Solexa sequencing to clone and identify microRNAs in SLE patients and a control group. RESULTS: From the sequencing data, we identified numerous microRNAs displaying significantly different levels of expression in patients with SLE and in healthy controls. The 212 and 199 microRNAs were upregulated and downregulated, respectively. Only 61 novel microRNAs exhibited significantly different levels of expression in the two groups. The target genes of the novel microRNAs identified in the SLE group were found to have cell metabolism functions. We also analyzed the chromosomal locations of the microRNAs with high level of expression between the two groups. A profile comparison revealed that the majority of transcripts were expressed at a similar level. The functional classes of the most abundant microRNAs were equally represented on each chromosome. CONCLUSION: We identified novel and known microRNAs significantly enhancing our understanding of the microRNA expression profiles of SLE patients. These data also provide insight into the function of microRNAs in SLE and provide new strategies for future therapies.