Project description:We undertook an integrative technological approach to compare miRNA detection capability of three high-throughput commercial platforms. We artificially introduced human precursor, 2’-O-methyl modified and mature spiked-in miRNAs in a controlled fashion into native human placenta total RNA.
Project description:We undertook an integrative technological approach to compare miRNA detection capability of three high-throughput commercial platforms. We artificially introduced human precursor, 2M-bM-^@M-^Y-O-methyl modified and mature spiked-in miRNAs in a controlled fashion into native human placenta total RNA.
Project description:We undertook an integrative technological approach to compare miRNA detection capability of three high-throughput commercial platforms. We artificially introduced human precursor, 2M-bM-^@M-^Y-O-methyl modified and mature spiked-in miRNAs in a controlled fashion into native human placenta total RNA.
Project description:Defects in stress responses are important contributors in many chronic conditions including cancer, cardiovascular disease, diabetes, and obesity-driven pathologies like non-alcoholic steatohepatitis (NASH). Specifically, endoplasmic reticulum (ER) stress is linked with these pathologies and control of ER stress can ameliorate tissue damage. MicroRNAs have a critical role in regulating diverse stress responses including ER stress. Here we show that miR-494-3p plays a functional role during ER stress. ER stress inducers (tunicamycin and thapsigargin) robustly increase the expression of miR-494 in vitro in an ATF6 dependent manner. Surprisingly, miR-494 pretreatment dampens the induction and magnitude of ER stress in response to tunicamycin in endothelial cells. Conversely, inhibition of miR-494 increases ER stress de novo and amplifies the effects of ER stress inducers. Using Mass Spectrometry (TMT-MS) we identified many proteins that are downregulated by both tunicamycin and miR-494 in cultured human umbilical vein endothelial cells (HUVECs). Among these, we found 6 transcripts which harbor a putative miR-494 binding site. Our data indicates that ER stress driven miR-494 may act in a feedback inhibitory loop to dampen downstream ER stress signaling. We propose that RNA-based approaches targeting miR-494 or its targets may be attractive candidates for inhibiting ER stress dependent pathologies in human disease.
Project description:It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function. This series includes microRNA sequencing data for human cell lines 293, C8166, A549, SH-SY5Y, and an EBV-transformed LCL line. For each cell line, total small RNA isolated by TRIzol was sequenced for comparison to RISC-associated microRNAs as determined by sequencing small RNAs from an Argonaute immunoprecipitation.
Project description:We present “centered sites,” a class of microRNA target sites that lacks both perfect seed pairing and 3'-compensatory pairing and instead has 11–12 contiguous Watson–Crick pairs to the center of the microRNA. In elevated Mg2+, centered sites impart mRNA cleavage, but in cells, centered sites repress protein output without consequential Agronaute-catalyzed cleavage. Our study also identified novel extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many microRNAs are evolutionarily conserved. To study centered sites and identify miRNA cleavage targets, mRNA degradomes were sequenced from human brain and HeLa cells, and smallRNAs were sequenced from human brain and zebrafish embryo at 24 hours post fertilization (hpf). Replicates were combined before the analysis. Fastq files are not available for GSM548638 and GSM548639.
Project description:Transmissible gastroenteritis virus (TGEV) is a member of Coronaviridae family. Our previous research showed that TGEV infection could induce mitochondrial dysfunction and up-regulat miR-222 level. Therefore, we presumed that miR-222 might be implicated in regulating mitochondrial dysfunction induced by TGEV infection. To verify the hypothesis, the effect of miR-222 on mitochondrial dysfunction was detected and showed that miR-222 attenuated TGEV-induced mitochondrial dysfunction. To investigate the underlying molecular mechanism of miR-222 in TGEV-induced mitochondrial dysfunction, a quantitative proteomic analysis of PK-15 cells that were transfected with miR-222 mimics and infected with TGEV was performed. In total, 4151 proteins were quantified and 100 differentially expressed proteins were obtained (57 up-regulated, 43 down-regulated), among which thrombospondin-1 (THBS1) and cluster of differentiation 47 (CD47) were down-regulated. THBS1 was identified as the target of miR-222. Knockdown of THBS1 and CD47 increased mitochondrial Ca2+ level and decreased mitochondrial membrane potential (MMP) level. Together, our data establish a significant role of miR-222 in regulating mitochondrial dysfunction in response to TGEV infection.
Project description:The goal of the current study was to identify miRNAs regulated by TGF-b in human CD8+ T cells and analyze the function of these miRNAs in shaping the immunomodulatory effect of TGF-b in these cells. We identified the miR-23a cluster to be upregulated and found that this cluster could target key molecules (IFN-g and LAMP1) involved in immune response by CD8+ T cells. CD8+ T cells were isolated and purified from healthy human peripheral blood of 5 donors and were activated using beads coupled to anti-CD2, anti-CD3 and anti-CD24. They were then treated with 5ng/ml TGF-b, with 1µM SD-208 or left untreated. RNA from these cells were then isolated and used for deep sequencing.
Project description:we combined their genome-wide profiles of tumors and normal adjacent tissues in 10 ccRCC patients. The results showed that 283 miRNAs were down-regulated and 187 up-regulated, meanwhile 7473 mRNAs were up-regulated and 3439 down-regulated in ccRCC. Expressions of 12 miRNAs and genes were validated in 10 patients we studied by RT-qPCR (validation rate from 60% to 100%). Differentially expressed gene analysis showed the collective change of miR-200 and SLC22A gene family, and pathway analysis revealed down-regulation of multiple metabolic pathways and up-regulation of focal adhesion, ECM-receptor interaction, etc. Moreover, A miRNA cluster located in chromosome Xq27.3 were significantly down-regulated in ccRCC, which were verified in 53 ccRCC patients by RT-qPCR (validation rate from 63.8% to 88.6%).56M-BM- and 586 potentially novel miRNAs andM-BM- mRNA we detected according toM-BM- statistical analyses. In addition,M-BM- 14M-BM- miRNA candidates were randomly selected to validateM-BM- usingM-BM- qRT-PCR and Sanger sequcencing technology, 10 of out 14 could be successfully amplified,.Plus, the sequences of all 5 randomly selected amplified products were confirmed by cloned Sanger sequencing.Our data demonstrated a combined profiling of miRNAs and mRNAs in ccRCC, which showed the decreased metabolism and the perturbation of renal cell function. Moreover, this study identified the expression alteration of a miRNA cluster on Xq27.3, which suggested its potential function in carcinogenesis of ccRCC Examination of 10 pairs of kidney normal cells and cancer cells .
Project description:miRNA sequencing of mammary tumor RNA from 18 [AKXD subline(n) x PyMT]F1. The PyMT strain was FVB/N-TgN(MMTV-PyVT)634Mul. Mammary tumor total small RNA from mice representing each of the 18 AKXD RI strains was pooled to represent each strain and sequenced using the Illumina Genome Analyzer IIx sequencer.