High Throughput Sequencing Following Cross-Linked Immune Precipitation (HITS-CLIP) of Argonaute (AGO) Identifies Mir-193a as a Regulator of Jagged1 In Marrow Stromal Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3847-3847
Author(s):  
Ilango Balakrishnan ◽  
Xiaodong Yang ◽  
Beverly Torok-Storb ◽  
Jay Hesselberth ◽  
Manoj Pillai
Keyword(s):  
Binding Site ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Direct Interaction ◽  
Over Expression ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Immune Precipitation ◽  
Mrna Binding

Abstract Abstract 3847 MicroRNAs (miRNAs) are small non-coding RNAs with important roles in the post-transcriptional regulation of up to 30% of all vertebrate genes. Traditional methods to determine miRNA-mRNA interactions have included transcriptional profiling of miRNAs, bio-informatic prediction of miRNA-mRNA binding, analysis of 3` untranslated region (3`UTR) binding of miRNAs and over-expression of miRNAs in relevant cell types. These studies however fall short of demonstrating direct interaction between a miRNA and its target mRNAs. We applied a recently described biochemical technique of high throughput sequencing following cross-linked immune precipitation (HITS-CLIP) to dissecting the miRNA-mRNA interactions in two functionally distinct human marrow stromal cell lines. HITS-CLIP relies on the ability of ultraviolet (UV) radiation to cross-link RNA to proteins they are bound to, followed by immune-precipitation of the RNA-protein complex to isolate the cross-linked RNA and sequencing by high throughput techniques. As miRNA-mRNA interactions occur in close proximity to the argonaute proteins (AGO), an anti-argonaute monoclonal antibody was used to isolate the Ago-miRNA-mRNA complexes. The two stromal cell lines analyzed by HITS-CLIP (designated HS5 and HS27a) were isolated from a normal marrow primary long term culture (LTC), immortalized and extensively characterized for both function and expression profiles (mRNA and miRNA). HS5 was found to secrete growth factors that stimulate proliferation and differentiation of hematopoietic progenitors (G-CSF, IL-6, IL-1α and IL1β), whereas HS27a expresses activities associated with the stem cell niche (CXCL12, Angiopoietin-1, Jag1 etc). In keeping with this, HS5 conditioned media stimulated proliferation and differentiation of isolated CD34+ cells whereas HS27a supported CD34+ cells in an undifferentiated state. Sequence reads from the HITS-CLIP analysis from each of the cell lines were aligned to the human genome using the UCSC genome browser to identify Ago-mRNA and Ago-miRNA binding sites in both the cell lines. Interestingly, corresponding datasets from HS5 and HS27a were similar for the majority of mRNAs and miRNAs, but distinct for those mRNAs (such as Jag1, CXCL12, IL6 and GCSF) and miRNAs (such as miR-886-3p, miR-221, miR-181a and miR-193a) known to be differentially expressed between the two cell lines. We then validated the use of the HITS-CLIP strategy in stromal cells by analyzing one such Ago-mRNA binding site for Jagged1 (Jag1). Jag1 is a ligand for Notch1 and is expressed in those cells that support the hematopoietic stem cell (HSC) niche. The Notch pathway is a highly conserved signaling system critical in regulating several tissue systems including hematopoietic cells. This binding site, 1749 bp downstream of the transcriptional start-site for Jag1 was significantly more enriched in HS5 compared to HS27a. The site was also a predicted binding site for miR-193a, a miRNA over-expressed in HS5 compared to HS27a cells. Over-expression of miR-193a in HS27a cells resulted in the down-regulation of Jag1 protein (as measured by Western blotting). To confirm the direct interaction between Jag1 and miR-193a, we cloned this purported binding site downstream of the luciferase gene and co-transfected the plasmid with miR-193a. Luciferase activity was down-regulated greater than 50% when compared to control transfections suggesting a direct effect of miR-193a on Jag1 transcript. In summary, our data suggest that HITS-CLIP methodology can be used to define in vivo spatial interactions between miRNA and mRNAs in the marrow microenvironment (ME). It can also be used to define miRNA-based regulation of specific genes such as Jag1, which are critical to defining functional niches in the ME. Disclosures: No relevant conflicts of interest to declare.

High-throughput sequencing identifies HIV-1-replication- and latency-related miRNAs in CD4+ T cell lines

2017 ◽  
Vol 162 (7) ◽  
pp. 1933-1942 ◽  
Author(s):  
Xiangyun Lu ◽  
Jin Yang ◽  
Haibo Wu ◽  
Zongxing Yang ◽  
Changzhong Jin ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cd4 T Cell ◽  
T Cell Lines ◽  
Hiv 1

scholarly journals High-throughput Sequencing for Species Authentication and Contamination Detection of 63 Cell Lines

2021 ◽  
Author(s):  
Oliver Lung ◽  
Rebecca Candlish ◽  
Michelle Nebroski ◽  
Peter Kruckiewicz ◽  
Cody Buchanan ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Classical Swine Fever ◽  
Virus Genome ◽  
Cox1 Gene ◽  
Species Identity ◽  
Cell Line Authentication ◽  
Dna And Rna

Abstract Cell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency’s National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvβ6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly.

scholarly journals Analysis of Aneuploidy Spectrum From Whole-Genome Sequencing Provides Rapid Assessment of Clonal Variation Within Established Cancer Cell Lines

2021 ◽  
Vol 20 ◽  
pp. 117693512110492
Author(s):  
Ahmed Ibrahim Samir Khalil ◽  
Anupam Chattopadhyay ◽  
Amartya Sanyal
Keyword(s):  
Whole Genome Sequencing ◽  
Cell Lines ◽  
Cancer Cell ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cancer Cell Lines ◽  
Easy Access ◽  
Clonal Variation ◽  
Whole Genome

Background: The revolution in next-generation sequencing (NGS) technology has allowed easy access and sharing of high-throughput sequencing datasets of cancer cell lines and their integrative analyses. However, long-term passaging and culture conditions introduce high levels of genomic and phenotypic diversity in established cell lines resulting in strain differences. Thus, clonal variation in cultured cell lines with respect to the reference standard is a major barrier in systems biology data analyses. Therefore, there is a pressing need for a fast and entry-level assessment of clonal variations within cell lines using their high-throughput sequencing data. Results: We developed a Python-based software, AStra, for de novo estimation of the genome-wide segmental aneuploidy to measure and visually interpret strain-level similarities or differences of cancer cell lines from whole-genome sequencing (WGS). We demonstrated that aneuploidy spectrum can capture the genetic variations in 27 strains of MCF7 breast cancer cell line collected from different laboratories. Performance evaluation of AStra using several cancer sequencing datasets revealed that cancer cell lines exhibit distinct aneuploidy spectra which reflect their previously-reported karyotypic observations. Similarly, AStra successfully identified large-scale DNA copy number variations (CNVs) artificially introduced in simulated WGS datasets. Conclusions: AStra provides an analytical and visualization platform for rapid and easy comparison between different strains or between cell lines based on their aneuploidy spectra solely using the raw BAM files representing mapped reads. We recommend AStra for rapid first-pass quality assessment of cancer cell lines before integrating scientific datasets that employ deep sequencing. AStra is an open-source software and is available at https://github.com/AISKhalil/AStra .

High Throughput Sequencing of Acute Lymphoblastic Leukemia Small RNAs.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3650-3650
Author(s):  
Michael C Wei ◽  
Elizabeth O Osborn ◽  
Michael Cleary
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
High Throughput ◽  
Small Rna ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Rna Expression ◽  
Leukemia Cell Lines

Abstract Abstract 3650 MicroRNAs are small, non-coding RNAs that regulate gene expression and play key roles in cancer by modulating oncogene and tumor suppressor pathways. We are investigating the clinical and prognostic roles of miRNA expression in pediatric leukemias using high-throughput sequencing as a profiling tool. To establish the methodology, we have utilized high-throughput sequencing to quantify small RNA expression from eight acute lymphoblastic leukemia cell lines and one MLL-rearranged infant ALL patient sample. We generated sequencing libraries from these cells, conducted high-throughput sequencing using the Illumina platform, and established a custom bioinformatics pipeline for data analysis. Over 50 million individual sequence reads were analyzed. These sequences were mapped against a database of human miRNAs, and the frequency of miRNA expression among samples was enumerated. Expression of hematopoietic-specific miR-142 and miR-181 cluster miRs was found in these leukemia samples, while the liver-specific miR-122 was not expressed. miR-196b, previously reported to be over-expressed in MLL-rearranged leukemias, was expressed in 3/3 MLL-rearranged leukemia cell lines and 1/5 non-MLL cell lines. Expression of individual miRNAs was validated by quantitative PCR. Additional analysis of MLL-associated miRNAs and novel small RNAs will be presented. Our results demonstrate the feasibility and potential of high-throughput sequencing to profile the expression of small RNAs from leukemia cells, and we plan to apply these methods to additional primary patient samples to examine prognostic and clinical correlations with small RNA expression patterns. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hsa_circ_0008309 May Be a Potential Biomarker for Oral Squamous Cell Carcinoma

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Bowen Li ◽  
Feng Wang ◽  
Xiang Li ◽  
Shuai Sun ◽  
Yuehong Shen ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
High Throughput ◽  
Squamous Cell ◽  
High Throughput Sequencing ◽  
Potential Biomarker ◽  
Bioinformatics Databases

Objective. Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region. The circular RNA (circRNA) is known to serve an important role in the carcinogenesis of different types of cancer. However, the circRNA role of OSCC remains unclear. Methods. 8 pairs of OSCC tissues and adjacent normal tissues were obtained to detect circRNAs expression by high-throughput sequencing, and 45 pairs of OSCC tissues were selected to verify the differentially significant circRNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To further investigate the role of hsa_circ_0008309, the circRNA-microRNA (miR)-mRNA network was predicted using bioinformatics databases. The expression levels of hsa_circ_0008309, miR-1290, miR-136-5P, and miR-382-5P in SCC-15 and CAL27 cell lines were detected by RT-qPCR. Western blotting was performed to detect the protein level of Ataxin 1 (ATXN1). Results. The high-throughput sequencing results demonstrated that circRNAs were abundantly expressed in OSCC, and 16 circRNAs were significantly differentially expressed. Hsa_circ_0008309 was significantly downregulated in 45 pairs of OSCC tissue samples and was statistically correlated with pathological differentiation. The bioinformatics databases suggested that hsa_circ_0008309 could combine with miR-1290, miR-136-5P, and miR-382-5P, respectively, to regulate the expression of ATXN1. It was subsequently identified that hsa_circ_0008309 may inhibit miR-136-5P and miR-382-5P expression and increase ATXN1 expression in the OSCC cell lines. Conclusion. In summary, the results of the present study revealed that OSCC tissues have abundant circRNAs and, to the best of our knowledge, we firstly explore the regulatory role of the hsa_circ_0008309-miR-136-5P/hsa-miR-382-5P-ATXN1 network in OSCC. The results indicated that hsa_circ_0008309 may be a potential biomarker for OSCC.

scholarly journals High-throughput sequencing reveals circular RNA hsa_circ_0000592 as a novel player in the carcinogenesis of gastric carcinoma

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Min Liang ◽  
Zhaoyu Liu ◽  
Hai Lin ◽  
Boyun Shi ◽  
Ming Li ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
Epithelial Cells ◽  
Gastric Carcinoma ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Gastric Epithelial Cells ◽  
Environmental Carcinogen ◽  
Functional Roles

Abstract Background/Aim: Gastric cancer is one of the most common malignant tumors, and its complex pathogenesis has not been fully elucidated. Circular RNAs (circRNAs) are involved in various biological processes and human diseases. However, their exact functional roles and mechanisms of action remain largely unclear. We previously discovered the differential expression of non-coding RNAs (ncRNAs) during the malignant transformation of human gastric epithelial cells. In this study, we investigated the functional roles of a significantly up-regulated circRNA (hsa_circ_0000592) in gastric cancer. Methods:N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced malignant-transformed gastric epithelial cells (GES-1-T) and normal gastric epithelial cells (GES-1-N) were analyzed by high-throughput circRNA sequencing. The top 15 up-regulated circRNAs in high-throughput sequencing results were further confirmed by qRT-PCR in different gastric epithelial cell lines. The function of the most significant circRNA (hsa_circ_0000592) was investigated by using RNA interference (RNAi) assays, fluorescence in situ hybridization analysis (FISH), and bioinformatics prediction methods. Results: A total of 1509 genes were up-regulated and 3142 genes were down-regulated in GES-1-T cells when compared with GES-1-N cells. When compared with GES-1-N cells, hsa_circ_0000592 was obviously up-regulated in GES-1-T cells, as well as in other gastric cancer cell lines. The silencing of hsa_circ_0000592 mRNA led to a decrease in cell proliferation, cell cycle arrest at the G0/G1 phase, an increased rate of apoptosis, and a reduction in cell migration. Furthermore, FISH showed that hsa_circ_0000592 was mainly located in the cytoplasm, and a bioinformatics analysis suggested that hsa_circ_0000592 might function by sponging multiple miRNAs, and most notably four conserved miRNAs, including miR-139-3p, miR-200, miR-367-3p, and miR-33a-3p. Conclusion: This study is the first to identify hsa_circ_0000592 as a novel circRNA with a critical role in MNNG-induced gastric cancer. Due to the essential role of hsa_circ_0000592 in gastric carcinoma cells, it may be considered as a potential biomarker for use in diagnosing gastric carcinoma. Our findings provide a new insight into the function of circRNAs in environmental carcinogen-induced gastric cancer.

scholarly journals Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines

2015 ◽  
Vol 43 (11) ◽  
pp. 5601-5616 ◽  
Author(s):  
J. P. Tosar ◽  
F. Gambaro ◽  
J. Sanguinetti ◽  
B. Bonilla ◽  
K. W. Witwer ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Small Rna ◽  
High Throughput Sequencing ◽  
Breast Cell Lines

EBV Microrna Mir-BHRF1-2 Targets PRDM1/Blimp1: Potential Role in EBV Lymphomagenesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3547-3547
Author(s):  
Jiao Ma ◽  
Kui Nie ◽  
David Redmond ◽  
Yifang Liu ◽  
Daniel M Knowles ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Binding Site ◽  
Cell Line ◽  
Cell Lines ◽  
Target Genes ◽  
Rna Seq ◽  
B Cell Lymphomas ◽  
Down Regulation ◽  
Over Expression

Abstract PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in the pathogenesis of diffuse large B-cell lymphoma (DLBCL). In DLBCL, PRDM1 is inactivated by mutations and deletions; however, there is also evidence that PRDM1 is down-regulated by microRNAs (miRNAs) in DLBCL and Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma (cHL). A decrease in PRDM1 activity contributes to the pathogenesis of DLBCL and cHL by inhibiting plasma cell differentiation triggered by signal transduction pathways such as the NF-kB pathway. Since malignant EBV-positive B-cell lymphoproliferations are often associated with increased NF-kB activity, it is conceivable that abnormal PRDM1 down-regulation may play a role in their pathogenesis. EBV-positive B-cell lymphomas are postulated to originate from EBV-infected B-cells with latency III growth program of EBV gene expression. Thus, EBV-immmortalized lymphoblastoid cell lines (LCLs), which are of latency III type, serve as a good model to study EBV lymphomagenesis. We observed discordance in PRDM1 mRNA and protein levels in LCLs. By quantitative real-time reverse transcriptase PCR, PRDM1 mRNA levels in LCLs varied from 14.6% to 1259.7% relative to the multiple myeloma cell line U266, which expresses high levels of PRDM1. However, PRDM1 protein was discordantly low in LCLs compared to U266 based on immunohistochemistry and Western blotting assays, consistent with post-transcriptional regulation. EBV encodes 25 viral miRNAs, and we postulate that one of more of them may function to dampen PRDM1 expression. Indeed, a miRNA binding site containing seed match to bases 2-7 of EBV miR-BHRF1-2 was identified in positions 1565 to 1589 of PRDM1 3’ untranslated region. MiR-BHRF1-2 functionally targeted this specific binding site and repressed luciferase reporter activity. Mutation in the seed region of this site relieved the repression in comparison to the wild type control. MiR-BHRF1-2 was highly expressed in LCLs, while it was barely detectable in the EBV-positive Burkitt lymphoma cell line MUTU I, which has latency type I. Importantly, immunoblotting assay demonstrated an up-regulation of PRDM1 protein level in CCL156 and CCL159 LCL cells transfected with miR-BHRF1-2 inhibitor relative to those transfected with miRNA Inhibitor negative control, supporting a role of miR-BHRF1-2 in PRDM1 down-regulation in vivo. To examine the biological consequences of increased PRDM1 expression in LCL cells, PRDM1 was over-expressed in JY25 and CCL159 LCL cell lines. Enforced expression of PRDM1 induced apoptosis in both cell lines. Furthermore, bromodeoxyuridine (Brdu) incorporation study demonstrated that overexpression of PRDM1 reduced the percentage of S phase from 43.4% to 27.6% in CCL159 cells, and 39.5% to 27.9% in JY25 cells, respectively. Whole transcriptome sequencing (RNA-seq) identified a set of potential PRDM1 direct target genes whose expressions decreased in both LCL cell lines upon PRDM1 over-expression. These genes have broad functions including cell proliferation and survival, transcription and translation, mitochondrial functions, and cytoskeleton. Although no significant changes in cell cycle and apoptosis were observed upon transfection of miR-BHRF1-2 inhibitor, RNA-seq analysis of CLL159 cells transfected with miR-BHRF1-2 inhibitor revealed a small subset of repressed genes which overlapped with those identified by PRDM1 over-expression. This finding suggests that the increase in PRDM1 expression upon miR-BHRF1-2 inhibition, albeit small, is capable of repressing a subset of PRDM1 target genes with potential biological effects. In summary, our findings demonstrate that PRDM1 is a target of EBV miR-BHRF1-2. MiR-BHRF1-2 mediated PRDM1 down-regulation may contribute to the pathogenesis of EBV-associated B-cell lymphomas by inhibiting the transcription repression program of PRDM1 and limiting PRDM1-mediated cellular changes detrimental to tumor growth, including cell cycle arrest and apoptosis. Further characterization of the target genes whose expression is up-regulated by miR-BHRF1-2-mediated PRDM1 down-regulation may provide important clues to the pathogenetic function of miR-BHRF1-2 and EBV oncogenesis in general. Disclosures No relevant conflicts of interest to declare.

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