Differential sensitivity of target genes to translational repression by miR-17~92

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genesCX is a Pew Scholar in Biomedical Sciences. This study is supported by the PEW Charitable Trusts, Cancer Research Institute, National Institute of Health (R01AI087634, R01AI089854, RC1CA146299, R56AI110403, and R01AI121155 to CX), National Natural Science Foundation of China (31570882 to WHL, 31570883 to NX, 31570911 to GF, 91429301 to JH, 31671428 and 31500665 to YZ), 1000 Young Talents Program of China (K08008 to NX), 100 Talents Program of The Chinese Academy of Sciences (YZ), National Program on Key Basic Research Project of China (2016YFA0501900 to YZ), the Fundamental Research Funds for the Central Universities of China (20720150065 to NX and GF), Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A1A01052387 to SGK, NRF-2016R1A4A1010115 to SGK and PHK), and 2016 Research Grant from Kangwon National University (SGK)

Whole genome transcript profiling from fingerstick blood samples: a comparison and feasibility study

<p>Abstract</p> <p>Background</p> <p>Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5 mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small human samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 μl of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit.</p> <p>Results</p> <p>From two sets of fingerstick collections, about 70 uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6 ng total RNA with an average RIN of 9.3. The post-amplification yields for 50 ng of total RNA averaged at 7.0 ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with r²values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r²values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.94 to 0.96 for fingerstick collection 2.</p> <p>Conclusions</p> <p>Our comparisons of RNA quality and gene expression data of the fingerstick method with traditionally processed sample workflows demonstrate excellent RNA quality from the capillary collection as well as very high correlations of gene expression data.</p

Gene Expression in Biopsies of Acute Rejection and Interstitial Fibrosis/Tubular Atrophy Reveals Highly Shared Mechanisms That Correlate With Worse Long‐Term Outcomes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/1/ajt13728.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/2/ajt13728-sup-0005-AppendixS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/3/ajt13728-sup-0003-AppendixS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/4/ajt13728_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/122411/5/ajt13728-sup-0004-AppendixS4.pd

Biomarkers for Early and Late Stage Chronic Allograft Nephropathy by Proteogenomic Profiling of Peripheral Blood

Despite significant improvements in life expectancy of kidney transplant patients due to advances in surgery and immunosuppression, Chronic Allograft Nephropathy (CAN) remains a daunting problem. A complex network of cellular mechanisms in both graft and peripheral immune compartments complicates the non-invasive diagnosis of CAN, which still requires biopsy histology. This is compounded by non-immunological factors contributing to graft injury. There is a pressing need to identify and validate minimally invasive biomarkers for CAN to serve as early predictors of graft loss and as metrics for managing long-term immunosuppression.We used DNA microarrays, tandem mass spectroscopy proteomics and bioinformatics to identify genomic and proteomic markers of mild and moderate/severe CAN in peripheral blood of two distinct cohorts (n = 77 total) of kidney transplant patients with biopsy-documented histology.Gene expression profiles reveal over 2400 genes for mild CAN, and over 700 for moderate/severe CAN. A consensus analysis reveals 393 (mild) and 63 (moderate/severe) final candidates as CAN markers with predictive accuracy of 80% (mild) and 92% (moderate/severe). Proteomic profiles show over 500 candidates each, for both stages of CAN including 302 proteins unique to mild and 509 unique to moderate/severe CAN.This study identifies several unique signatures of transcript and protein biomarkers with high predictive accuracies for mild and moderate/severe CAN, the most common cause of late allograft failure. These biomarkers are the necessary first step to a proteogenomic classification of CAN based on peripheral blood profiling and will be the targets of a prospective clinical validation study

Alu-dependent RNA editing of GLI1 promotes malignant regeneration in multiple myeloma

Despite novel therapies, relapse of multiple myeloma (MM) is virtually inevitable. Amplification of chromosome 1q, which harbors the inflammation-responsive RNA editase adenosine deaminase acting on RNA (ADAR)1 gene, occurs in 30-50% of MM patients and portends a poor prognosis. Since adenosine-to-inosine RNA editing has recently emerged as a driver of cancer progression, genomic amplification combined with inflammatory cytokine activation of ADAR1 could stimulate MM progression and therapeutic resistance. Here, we report that high ADAR1 RNA expression correlates with reduced patient survival rates in the MMRF CoMMpass data set. Expression of wild-type, but not mutant, ADAR1 enhances Alu-dependent editing and transcriptional activity of GLI1, a Hedgehog (Hh) pathway transcriptional activator and self-renewal agonist, and promotes immunomodulatory drug resistance in vitro. Finally, ADAR1 knockdown reduces regeneration of high-risk MM in serially transplantable patient-derived xenografts. These data demonstrate that ADAR1 promotes malignant regeneration of MM and if selectively inhibited may obviate progression and relapse

Deconvoluting Post-Transplant Immunity: Cell Subset-Specific Mapping Reveals Pathways for Activation and Expansion of Memory T, Monocytes and B Cells

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO+CD62L− effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant

Assessing a novel room-temperature RNA storage medium for compatibility in microarray gene expression analysis

RNA integrity is a critical factor in obtaining meaningful gene expression data. Current methodologies rely on maintaining samples in cold environments during collection, transport, processing, and storage procedures, which are also extremely time-sensitive. Several RNA storage products are commercially available to help prevent degradation during the handling and storage steps; however, samples must be kept cold for optimal protection. We have evaluated a novel RNA storage medium based on anhydrobiosis for stabilizing and protecting samples from degradation at room temperature that are intended for use in microarray analysis. Samples were stored dry at room temperature for various time periods to assess any degradation or loss of activity as compared with frozen control samples. Recovered samples were used directly for analysis without further purification and exhibited no interference or inhibition in downstream applications. Comparison of gene expression profiles indicate no significant differences between freezer-stored control samples and those kept at room temperature protected in the RNA storage medium. The quality of recovered RNA was confirmed using spectrophotometry and Bioanalyzer analysis and was identical to control samples. The ability to stabilize RNA samples at ambient temperatures for extended time periods will have tremendous use, particularly for sample shipment to core facilities