ABSTRACT: Real-time quantitative miRNA PCR analysis of human plasma samples circulating miRNAs miR-34a and miR-150 associated with colorectal cancer progression
Project description:Background: Screening for the early detection of colorectal cancer is important to improve patient survival. The aim of this study was to investigate the potential of circulating cell-free miRNAs as biomarkers of CRC, and their efficiency at delineating patients with polyps and benign adenomas from normal and cancer patient groups. Methods: The expression of 667 miRNAs was assessed in a discovery set of 48 plasma samples comprising normal, polyp, adenoma, and early and advanced cancer samples. Three miRNAs (miR-34a, miR-150, and miR-923) were further examined in a validation cohort of 97 subjects divided into the same five groups, and in an independent public dataset of 40 CRC samples and paired normal tissues. Results: High levels of circulating miR-34a and low miR-150 levels distinguished groups of patients with polyps from those with advanced cancer (AUC=0.904), and low circulating miR-150 levels separated patients with adenomas from those with advanced cancer (AUC=0.875). In addition, the altered expression of miR-34a and miR-150 in an independent public dataset of forty CRC samples and paired normal tissues was confirmed. Conclusion: We identified two circulating miRNAs capable of distinguishing patient groups with different diseases of the colon from each other, and patients with advanced cancer from benign disease groups.
Project description:The gole of this study was to determine whether circulaitng miRNAs could be used as candidate miRNAs of SLE . In this study a miRNA profile was used to determine aberrant expressed circulating miRNAs in patients with system lupus erythematosus (SLE), compared with rheumatoid arthritis (RA) and healthy control (HCs). To further confirm these microarray data, we identify 8 miRNAs (miR-126, miR-21, miR-451, miR-223, miR-16, miR-125a-3p,miR-155,miR-146a) by real-time quantitative PCR in 20 healthy controls and in 55 cases, of whom 30 were diagnosed with SLE and 25 were diagnosed RA. Using microarray-based expression profiling follwed by real-time quantitative polymerase Cycle Reaction (RT-qPCR)validation, we compared the levels of circulating miRNAs in plasma sample from SLE patients, RA patients, and healthy controls
Project description:Background: Oral squamous cell carcinoma (OSCC) is often diagnosed at a late stage and may be malignantly transformed from oral leukoplakia (OL). This study aimed to identify potential plasma microRNAs (miRNAs) for the early detection of oral cancer. Methods: Plasma from normal, OL, and OSCC patients were evaluated. Small RNA sequencing was used to screen differently expressed miRNAs among the groups. Next, these miRNAs were validated with individual samples by quantitative real-time polymerase chain reaction (qRT-PCR) assays. The possible physiological roles of the identified miRNAs were further investigated using bioinformatics analysis. Results: Three miRNAs (miR-222-3p, miR-150-5p, and miR-423-5p) were identified as differentially expressed among groups; miR-222-3p and miR-423-5p negatively correlated with T stage, lymph node metastasis status, and clinical stage. A high diagnostic accuracy (Area under curve = 0.88) was demonstrated for discriminating OL from OSCC. Bioinformatics analysis reveals that miR-423-5p and miR-222-3p are significantly over-expressed in oral cancer tissues and involved in various cancer pathways. Conclusions: The three plasma miRNA panel may be useful to monitor malignant progression from OL to OSCC and as potential biomarkers for early detection of oral cancer.
Project description:We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics of newly synthesized proteins (pulsed stable isotope labeling with amino acids in cell culture, pSILAC) in combination with mRNA and non-coding RNA expression analyses by next generation sequencing (RNA-, miR-Seq) in the colorectal cancer (CRC) cell line SW480. Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down), lncRNAs (270 up, 123 down) and proteins (542 up, 569 down). Changes in mRNA and protein expression levels showed a positive correlation (r = 0.50, p < 0.0001). More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the downregulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3’-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed upregulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibited proliferation in SW480 cells. Furthermore, HMGB1, KLF12 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of HMGB1, KLF12 and CIT was detected in advanced stages of cancer. This study provides new insights and a comprehensive catalogue of p53-mediated regulations and p53 DNA binding in CRC cells.
Project description:The gole of this study was to determine whether circulaitng miRNAs could be used as candidate miRNAs of SLE . In this study a miRNA profile was used to determine aberrant expressed circulating miRNAs in patients with system lupus erythematosus (SLE), compared with rheumatoid arthritis (RA) and healthy control (HCs). To further confirm these microarray data, we identify 8 miRNAs (miR-126, miR-21, miR-451, miR-223, miR-16, miR-125a-3p,miR-155,miR-146a) by real-time quantitative PCR in 20 healthy controls and in 55 cases, of whom 30 were diagnosed with SLE and 25 were diagnosed RA.
Project description:To reveal the relationship between circulating exosomal miRNAs and the disease severity of psoriasis, we performed next-generation sequencing from plasma exosomes of patients with high psoriasis area and severity index (PASI) score (>10) and low PASI score (<5). We identified 19 differentially expressed exosomal miRNAs that were significantly different between the groups. We validated the top three up-and down-regulated exosomal miRNAs using quantitative real-time PCR.
Project description:We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics of newly synthesized proteins (pulsed stable isotope labeling with amino acids in cell culture, pSILAC) in combination with mRNA and non-coding RNA expression analyses by next generation sequencing (RNA-, miR-Seq) in the colorectal cancer (CRC) cell line SW480. Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down), lncRNAs (270 up, 123 down) and proteins (542 up, 569 down). Changes in mRNA and protein expression levels showed a positive correlation (r = 0.50, p < 0.0001). More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the downregulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3â??-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed upregulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibited proliferation in SW480 cells. Furthermore, HMGB1, KLF12 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of HMGB1, KLF12 and CIT was detected in advanced stages of cancer. This study provides new insights and a comprehensive catalogue of p53-mediated regulations and p53 DNA binding in CRC cells.
Project description:To establish a genomewide miRNA profile of colorectal cancer-derived induced pluripotent cancer cell lines (CRC-iPC). CRC-iPC clones and two parental cell lines were subjected to miRNA microarray analysis using SurePrint Human MiRNA Microarray Release 21.0 (Agilent). In order to identify the differentially-expressed miRNAs after OSKM-reprogramming, the miRNA expression of CRC-iPCs was relatively compared to that of parental colorectal cancer cell lines. Using a stringent selection criteria of log2(fold change) (FC) ≥ 2.0 or < -2.0 and p-value < 0.05, a total of 102 statistically significant differentially-expressed miRNAs were identified. Amongst the 102 miRNAs, 50 miRNAs were down-regulated and 52 miRNAs were up-regulated. Four miRNAs (miR-362-5p, miR-532-3p, miR-125b-5p, and miR199a-3p) were randomly selected for validation by quantitative real-time PCR, the results were consistent with that of the microarray results. To establish a genomewide miRNA profile of colorectal cancer-derived induced pluripotent cancer cell lines (CRC-iPC). CRC-iPC clones and two parental cell lines were subjected to miRNA microarray analysis using SurePrint Human MiRNA Microarray Release 21.0 (Agilent). In order to identify the differentially-expressed miRNAs after OSKM-reprogramming, the miRNA expression of CRC-iPCs was relatively compared to that of parental colorectal cancer cell lines. Using a stringent selection criteria of log2(fold change) (FC) ≥ 2.0 or < -2.0 and p-value < 0.05, a total of 102 statistically significant differentially-expressed miRNAs were identified. Amongst the 102 miRNAs, 50 miRNAs were down-regulated and 52 miRNAs were up-regulated. Four miRNAs (miR-362-5p, miR-532-3p, miR-125b-5p, and miR199a-3p) were randomly selected for validation by quantitative real-time PCR, the results were consistent with that of the microarray results.
Project description:MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of nearly all biological processes. Global miRNA biogenesis is altered in many cancers and RNA-binding proteins (RBPs) have been shown to play a role in this process, presenting a promising avenue for targeting miRNA dysregulation in disease. miR-34a exhibits tumor-suppressive functions by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor Bcl-2, among other regulatory pathways such as Wnt, TGF-, and Notch signaling. Many cancers show downregulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo; however, the post-transcriptional mechanisms by which miR-34a is lost in cancer are not entirely understood. Here, we have used a proteomics-mediated approach to identify Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RBP with no previously reported role in miRNA regulation. We demonstrate that SART3 binds pre-miR-34a with specificity over pre-let-7d and begin to elucidate a new functional role for this protein in non-small lung cancer cells. Overexpression of SART3 led to increased miR-34a levels, downregulation of the miR-34a target genes CDK4 and CDK6, and cell cycle arrest in the G1 phase. In vitro binding studies showed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Collectively, our results present evidence for an influential role of SART3 in miR-34a biogenesis and cell cycle progression.
Project description:To identify the miRNAs associated with endothelial microparticles in plasma, we have employed SurePrint G3 Human v16 miRNA 8x60K Microarray to determine miRNAs in plasma from healthy donors and bicuspid aortic valve patients. Expression of 7 miRNAs (let-7d, let-7g, miR-130a, miR-337, miR-409, miR-494 and miR-718) from this signature was quantified in the same RNA samples by real-time PCR, confirming microarray results.