TransCRISPR-sgRNA design tool for CRISPR/Cas9 experiments targeting specific sequence motifs.

Eukaryotic genomes contain several types of recurrent sequence motifs, e.g. transcription factor motifs, miRNA binding sites, repetitive elements. CRISPR/Cas9 can facilitate identification and study of crucial motifs. We present transCRISPR, the first online tool dedicated to search for sequence motifs in the user-provided genomic regions and design optimal sgRNAs targeting them. Users can obtain sgRNAs for chosen motifs, for up to tens of thousands of target regions in 30 genomes, either for the Cas9 or dCas9 system. TransCRISPR provides user-friendly tables and visualizations, summarizing features of identified motifs and designed sgRNAs such as genomic localization, quality scores, closest transcription start sites and others. Experimental validation of sgRNAs for MYC binding sites designed with transCRISPR confirmed efficient disruption of the targeted motifs and effect on expression of MYC-regulated genes. TransCRISPR is available from https://transcrispr.igcz.poznan.pl/transcrispr/.

The lncRNA KTN1-AS1 co-regulates a variety of Myc-target genes and enhances proliferation of Burkitt lymphoma cells.

Long non-coding RNAs (lncRNAs) are involved in many normal and oncogenic pathways through a diverse repertoire of transcriptional and posttranscriptional regulatory mechanisms. LncRNAs that are under tight regulation of well-known oncogenic transcription factors such as c-Myc (Myc) are likely to be functionally involved in their disease-promoting mechanisms. Myc is a major driver of many subsets of B cell lymphoma and to date remains an undruggable target. We identified three Myc-induced and four Myc-repressed lncRNAs by use of multiple in vitro models of Myc-driven Burkitt lymphoma and detailed analysis of Myc binding profiles. We show that the top Myc-induced lncRNA KTN1-AS1 is strongly upregulated in different types of B cell lymphoma compared with their normal counterparts. We used CRISPR-mediated genome editing to confirm that the direct induction of KTN1-AS1 by Myc is dependent on the presence of a Myc E-box-binding motif. Knockdown of KTN1-AS1 revealed a strong negative effect on the growth of three BL cell lines. Global gene expression analysis upon KTN1-AS1 depletion shows a strong enrichment of key genes in the cholesterol biosynthesis pathway as well as co-regulation of many Myc-target genes, including a moderate negative effect on the levels of Myc itself. Our study suggests a critical role for KTN1-AS1 in supporting BL cell growth by mediating co-regulation of a variety of Myc-target genes and co-activating key genes involved in cholesterol biosynthesis. Therefore, KTN1-AS1 may represent a putative novel therapeutic target in lymphoma.

Core regions in immunoglobulin heavy chain enhancers essential for survival of non-Hodgkin lymphoma cells are identified by a CRISPR interference screen.

Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eµ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.

Comparison of iSeq and MiSeq as the two platforms for 16S rRNA sequencing in the study of the gut of rat microbiome.

Amplicon-based next-generation sequencing (NGS) of the 16S ribosomal RNA (16S) regions is a culture-free method used to identify and analyze Procaryota occurring within a given sample. The prokaryotic 16S rRNA gene contains conserved regions and nine variable regions (V1-V9) frequently used for phylogenetic classification of genus or species in diverse microbial populations. This work compares the accuracy and efficacy of two platforms, iSeq and MiSeq from Illumina, used in sequencing 16S rRNA. The most important similarities and differences of 16S microbiome sequencing in 20 fecal rat samples were described. Genetic libraries were prepared according to 16S Metagenomic Sequencing Library Preparation (Illumina) for the V3 and V4 regions of the 16S. The species richness obtained using iSeq technology was lower compared to MiSeq. At the second taxonomy level (L2), the abundance of taxa was comparable for both platforms. At the L7, the taxa abundance was significantly different, and the number of taxa was higher for the MiSeq. The alpha diversity was lower for iSeq than for MiSeq, starting from the order to the species level. The beta diversity estimation revealed statistically significant differences in microbiota diversity starting from the class level to the species level in samples sequenced on two investigated platforms. This work disclosed that the iSeq platform could be used to evaluate the bacterial profile of the samples to characterize the overall profile. The MiSeq System seems to be better for a detailed analysis of the differences in the microbiota composition. KEY POINTS: • iSeq platform allows to shorten the sequencing time three times compared to the MiSeq. • iSeq can only be used for an initial and quick microbiome assessment. • MiSeq is better for a detailed analysis of the differences in the microbiota composition.

Hypoxia-Induced FAM13A Regulates the Proliferation and Metastasis of Non-Small Cell Lung Cancer Cells.

Hypoxia in non-small cell lung cancer (NSCLC) affects cancer progression, metastasis and metabolism. We previously showed that FAM13A was induced by hypoxia in NSCLC but the biological function of this gene has not been fully elucidated. This study aimed to investigate the role of hypoxia-induced FAM13A in NSCLC progression and metastasis. Lentiviral shRNAs were used for FAM13A gene silencing in NSCLC cell lines (A549, CORL-105). MTS assay, cell tracking VPD540 dye, wound healing assay, invasion assay, BrdU assay and APC Annexin V staining assays were performed to examine cell proliferation ability, migration, invasion and apoptosis rate in NSCLC cells. The results of VPD540 dye and MTS assays showed a significant reduction in cell proliferation after FAM13A knockdown in A549 cells cultured under normal and hypoxia (1% O2) conditions (p < 0.05), while the effect of FAM13A downregulation on CORL-105 cells was observed after 96 h exposition to hypoxia. Moreover, FAM13A inhibition induced S phase cell cycle arrest in A549 cells under hypoxia conditions. Silencing of FAM13A significantly suppressed migration of A549 and CORL-105 cells in both oxygen conditions, especially after 72 and 96 h (p < 0.001 in normoxia, p < 0.01 after hypoxia). It was showed that FAM13A reduction resulted in disruption of the F-actin cytoskeleton altering A549 cell migration. Cell invasion rates were significantly decreased in A549 FAM13A depleted cells compared to controls (p < 0.05), mostly under hypoxia. FAM13A silencing had no effect on apoptosis induction in NSCLC cells. In the present study, we found that FAM13A silencing has a negative effect on proliferation, migration and invasion activity in NSCLC cells in normal and hypoxic conditions. Our data demonstrated that FAM13A depleted post-hypoxic cells have a decreased cell proliferation ability and metastatic potential, which indicates FAM13A as a potential therapeutic target in lung cancer.

Utility of fragility fracture prediction tools in a group of postmenopausal women.

OBJECTIVES: Fractures are a common complication of osteoporosis. The main aim of our study was to assess the relation between fractures identified as low energy fractures (fragility), bone mineral density (BMD), trabecular bone score (TBS), and handgrip in a group of postmenopausal women. An additional aim was to determine the relation between fragility fractures and age, height loss, and falls (reported in the last 12 months and 5 years). MATERIAL AND METHODS: The study was conducted in a group of 120 (mean age 69 years; 59-81, SD 5.3) postmenopausal patients who were referred to the Medical Centre for an osteoporosis screening appointment by their general practitioner. All patients were interviewed (with a questionnaire containing questions on fracture risk factors and highest height), had their anthropometric measures taken (current height and weight) as well as TBS analysis following their DXA (dual-energy X-ray absorptiometry) scan and handgrip measure. RESULTS: Sixty patients from the study group had a history of fractures (with a total of 92 fractures), of whom 39 women (76 fractures) were identified as those with a low-energy fracture. Fragility fractures were more likely to be reported in older patients (Me 71 vs. 68 years, p < 0.05). Differences observed between TBS, handgrip and BMD in reference to fragility fractures were not statistically significant. Analysis showed significant correlations between BMD (neck and L1-L4) and TBS fracture risk categories. Falls reported in the last 5 years and height loss were factors which correlated with fragility fractures (p < 0.05). CONCLUSIONS: Risk of fragility fractures increases with age. Bone mineral density is insufficient as a fracture risk assessment tool. Information on falls and height loss may provide additional data on fracture risk assessment.

Hypomethylation of the promoter region drives ectopic expression of TMEM244 in Sézary cells.

Sézary syndrome (SS) is an aggressive form of cutaneous T-cell lymphoma (CTCL) characterized by the presence of circulating malignant CD4+ T cells (Sézary cells) with many complex changes in the genome, transcriptome and epigenome. Epigenetic dysregulation seems to have an important role in the development and progression of SS as it was shown that SS cells are characterized by widespread changes in DNA methylation. In this study, we show that the transmembrane protein coding gene TMEM244 is ectopically expressed in all SS patients and SS-derived cell lines and, to a lower extent, in mycosis fungoides and in a fraction of T-cell lymphomas, but not in B-cell malignancies and mononuclear cells of healthy individuals. We show that in patient samples and in the T-cell lines TMEM244 expression is negatively correlated with the methylation level of its promoter. Furthermore, we demonstrate that TMEM244 expression can be activated in vitro by the CRISPR-dCas9-induced specific demethylation of TMEM244 promoter region. Since both, TMEM244 expression and its promoter demethylation, are not detected in normal lymphoid cells, they can be potentially used as markers in Sézary syndrome and some other T-cell lymphomas.

Argonaute 2 RNA Immunoprecipitation Reveals Distinct miRNA Targetomes of Primary Burkitt Lymphoma Tumors and Normal B Cells.

miRNAs are small noncoding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been linked to Burkitt lymphoma (BL) pathogenesis. To date, the number of known pathogenesis-related miRNA-target gene interactions is limited. Here, we determined for the first time the miRNA targetomes of primary BL tumors and normal B cells. AGO2-RNA immunoprecipitation of two frozen diagnostic BL tissue samples and three CD19+ B-cell samples isolated from routinely removed tonsils showed distinct miRNA targetomes of BL and normal B cells. In contrast to normal B cells, miRNA target genes in BL were enriched for targets of the oncogenic miR-17 to 92 cluster, and were involved mainly in cell cycle and cell death. Immunohistochemistry on BL and tonsil tissues confirmed altered protein levels for two of six selected miRNA targets, in line with the differential AGO2-IP enrichment between BL and normal B cells. A comparison of AGO2-IP-enriched genes in primary BL cases with BL cell lines indicated that despite a considerable overlap, the miRNA targetomes of BL cell lines show substantial differences with the targetomes of primary BL tumors. In summary, we identified distinct miRNA targetomes of BL and normal B cells, and showed both the necessity and feasibility of studying miRNA-target gene interactions in primary tumors.

Relative homogeneity of oral bacterial oral in Crohn's disease compared to ulcerative colitis and its connections with antioxidant defense - preliminary report.

INTRODUCTION: Interactions between oral microbiota and systemic diseases have been suggested. We aimed to examine the composition of oral microbiota with reference to antioxidative defense and its correlation with clinical state in Crohn's disease (CD) in comparison to ulcerative colitis (UC). MATERIALS AND METHODS: Smears were taken from the buccal and tongue mucosa of patients with CD, UC and controls, and cultured with classical microbiology methods. Bacterial colonies were identified using matrix-assisted laser desorption/ionization (MALDI) with a time-of-flight analyzer (TOF). Blood morphology and C-reactive protein (CRP) were analyzed in the hospital laboratory. Antioxidative defense potential (FRAP) was determined using spectrophotometry in saliva and serum. RESULTS: Oral microbiota in CD patients were characterized by lower diversity in terms of the isolated bacteria species compared to UC and this correlated with reduced FRAP in the oral cavity and intensified systemic inflammation. Oral microbiota composition in CD did not depend on the applied treatment. In CD patients, a negative correlation was observed between the FRAP value in saliva and serum and the CRP value in serum. Individual differences in the composition of oral microbiota suggest that different bacteria species may be involved in the induction of oxidative stress associated with a weakening of antioxidative defense in the oral cavity, manifested by ongoing systemic inflammation. CONCLUSIONS: Analysis of both the state of the microbiota and antioxidative defense of the oral cavity, as well as their referencing to systemic inflammation may potentially prove helpful in routine diagnostic applications and in aiding a better understanding of CD and UC pathogenesis associated with oral microbiota.

MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma.

BACKGROUND/AIMS: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. METHODS: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth / apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. RESULTS: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth- and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. CONCLUSION: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1.