Journal: bioRxiv

Article Title: SATB2 and circ3915 RNA chromatin dysregulation drive KRAS -like oncogenic transformation

doi: 10.1101/2024.10.04.616681

Figure Lengend Snippet: Arsenic-transformed cells were generated with the rapid (12 week) transformation model. A , Volcano plot of differentially accessible regions (DARs) by ATAC seq between non-transformed (NT) and iAs-transformed (iAsT) BEAS-2B cells. DARs were defined by a p adj ≤0.1. B , Heatmap of the top 50 DARs between non- transformed (NT) and iAs-transformed (iAsT) cells. Rows are the top genes; columns are the mean centered peak values for each gene in each sample. C , Genomic distribution of all DARs in NT and iAsT cells. DARs regions were defined by p adj <0.01 and Shrunken Log 2 (FC) >1 for opened DARs, and ShrunkenLog 2 (FC) <-1 for closed DARs. D , Proportion of DARs in NT and iAsT cells that are annotated as ENCODE candidate Cis-Regulatory Elements (cCREs). PLS = promoter-like signatures; pELS = proximal enhancer-like signatures; dELS = distal enhancer-like sequences. E , Top enriched de novo transcription factor motifs and best matching transcription factors in iAsT opened and closed DARs. F , Congruence between iAsT DARs and proximal gene expression. ATAC UP = opened chromatin; ATAC DN = closed chromatin; RNA UP = up-regulated transcripts; RNA DN = down-regulated transcripts. Counts are the number of individual genes above the minimum fold-change threshold for mRNA expression (>1 Log 2 (FC)) or open chromatin (>1 ShrunkenLog 2 (FC)). G , MSigDB oncogenic signatures in iAs-transformed cells that were commonly enriched in DARs and DEGs. ATAC UP = opened chromatin; ATAC DN = closed chromatin; RNA UP = up-regulated transcripts; RNA DN = down-regulated transcripts.

Article Snippet: We therefore used the Arraystar Human Circular RNA Array to screen iAs-transformed cells for differentially expressed circRNAs ( Fig S3A,B ), and discovered several dysregulated, cancer-associated circRNAs (e.g., hsa_circ_0002490, hsa_circ_0002874, hsa_circ_0075320, and hsa_circ_0008928) , – .

Techniques: Transformation Assay, Generated, Expressing

Journal: bioRxiv

Article Title: SATB2 and circ3915 RNA chromatin dysregulation drive KRAS -like oncogenic transformation

doi: 10.1101/2024.10.04.616681

Figure Lengend Snippet: Data were generated in the rapid (12 week) iAs transformation model. A , Nanostring-based detection of differential RNA and circRNA expression in iAs-transformed (iAsT) BEAS-2B cells relative to non-transformed (NT) cells (left panel). Up-regulated RNAs (orange dots) were defined by p <0.05, FC >1.2. Down-regulated circRNAs (purple dots) were defined by p <0.05, FC <-1.2. Grey dots represent RNAs with no significant difference in RNA expression or below 1.5 magnitude fold change. Right panel shows SATB2 and circ3915 transcripts and corresponding peptide domains. ULD = ubiquitin-like domain ; CUTL = cut-Like domain; CUT1 = cut domain 1; CUT2 = cut domain 2; HOX = homeobox; INM = inner nuclear membrane; MAR = matrix associated region; NLS = nuclear localization signal. B , Relative SATB2 and circ3915 expression, as measured by RT-qPCR. SATB2 expression increased 18.7-fold and circ3915 expression increased by 15.6-fold. N = 3 replicates; **** p <0.0001. C , Representative Western blot showing SATB2 up-regulation in iAsT BEAS-2B cells. D , SATB2 (purple) and circ3915 (orange) expression in BEAS-2B cells after acute, 48 hr exposure to iAs. Fold-change was measured by RT-qPCR, and calculated relative to non-treated BEAS-2B cells. E , SATB2 and circ3915 expression in BEAS- 2B cells at different time-points in the two-hit iAs transformation model, as measured by Nanostring. N= 3 replicates; * p <0.05. F , SATB2 and circ3915 expression in normal (N) and adjacent tumor (T) samples, as measured by RT-qPCR. All expression data were normalized to the expression of the geometric mean of three common husekeeping genes ( GAPDH, TBP, RPII ).

Article Snippet: We therefore used the Arraystar Human Circular RNA Array to screen iAs-transformed cells for differentially expressed circRNAs ( Fig S3A,B ), and discovered several dysregulated, cancer-associated circRNAs (e.g., hsa_circ_0002490, hsa_circ_0002874, hsa_circ_0075320, and hsa_circ_0008928) , – .

Techniques: Generated, Transformation Assay, Expressing, RNA Expression, Membrane, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: SATB2 and circ3915 RNA chromatin dysregulation drive KRAS -like oncogenic transformation

doi: 10.1101/2024.10.04.616681

Figure Lengend Snippet: circ3915 is translated into a peptide, and co-localizes with SATB2 in the nucleus . A, Bioluminescence in BEAS-2B cells containing an empty vector (control), or cells that are over-expressing a HiBiT-tagged circ3915 construct (circ3915-OE), as measured with a split luciferase translation assay. RLU = relative light units; N= 3; **** p <0.0001. B , Western blots for 100 µg of total protein or 5 µg of nuclear extract from BEAS-2B cells transfected with an empty vector (control), HiBiT-tagged circ3915 construct (circ3915-OE), or a HiBiT-tagged histone 2BJ (H2BJ) positive control. HiBiT was detected with either LgBiT (top panel) or an anti-HiBiT antibody (middle panel). C , circ3915 is associated with actively translated polysomes. BEAS-2B cells were either transfected with a circ3915 HiBiT overexpression plasmid (top), or transformed with iAs in the rapid (12 week) transformation model (bottom). Line graphs show the distribution of cytoplasmic RNA across sucrose gradient density fractions. Grey lines show the distribution of RNA under normal culture conditions (CHX = cycloheximide), and black lines show the distribution of RNA after adding puromycin (puromycin disrupts translation elongation). An increase in %RNA within the light fractions after treating cells with puromycin indicates that the RNA was being translated under normal conditions. TBP = TATA-box binding protein mRNA. D , Fluorescent microscope images of BEAS2B cells that were transduced with SATB2-GFP and circ3915-HiBiT expression vectors, showing that circ3915-peptide localizes in the nucleus. DAPI-stained DNA (blue) denotes the nucleus.

Article Snippet: We therefore used the Arraystar Human Circular RNA Array to screen iAs-transformed cells for differentially expressed circRNAs ( Fig S3A,B ), and discovered several dysregulated, cancer-associated circRNAs (e.g., hsa_circ_0002490, hsa_circ_0002874, hsa_circ_0075320, and hsa_circ_0008928) , – .

Techniques: Plasmid Preparation, Control, Expressing, Construct, Luciferase, Western Blot, Transfection, Positive Control, Over Expression, Transformation Assay, Binding Assay, Microscopy, Transduction, Staining

Journal: bioRxiv

Article Title: SATB2 and circ3915 RNA chromatin dysregulation drive KRAS -like oncogenic transformation

doi: 10.1101/2024.10.04.616681

Figure Lengend Snippet: A , BEAS-2B cell proliferation after transfection with lentivirus over-expression vectors, as measured with a CellTiter-Glo ATP assay 72 hours after seeding. Control = non-transformed cells containing an empty lentivirus vector. SATB2 + circ3915-OE = cells that contained SATB2 and circ3915 overexpression vectors. RLU = relative light units. Luminescence was normalized to background, and values calculated relative to the control cells. N = 3 independent experiments with 6 technical replicates each; **** p <0.0001. B , BEAS-2B cell migration after transfection with lentivirus over- expression vectors, as measured by a transwell migration assay 24 hr after seeding. Cells were fixed and stained with DAPI, and cell counts obtained with an Olympus cellSens counting module. Counts were normalized to the control cells. N = 3 independent experiments with 3 technical replicates each **** p <0.0001. C , Representative images from the transwell migration assay. D , Differentially expressed genes (DEGs) in BEAS-2B cells with forced SATB2 over-expression. Blue dots represent down-regulated genes with a p adj <0.05, FC <-1.5; orange dots represent up-regulated genes with a p adj <0.05, FC >1.5. Grey dots represent genes with FC<1.5,>-1.5. E , Principal component analysis (left) of RNA-seq expression profiles from non-transformed BEAS-2B cells (NT, brown); non-transformed cells that were transfected with a SATB2 overexpression vector (NT + MCS, red); non- transformed cells containing a SATB2 overexpression vector (SATB2-OE, green); and iAs-transformed BEAS- 2B cells from the rapid (12 week) transformation model (iAsT, pink). The top genes contributing to the variance between PC1 and PC2 are shown in the panel to the right. F , Top enriched MSigDB oncogenic signatures reflected in the DEGs from non-transformed BEAS-2B cells with forced SATB2 over-expression. G , Relative RNA expression of MSigDB oncogenic KRAS signature genes in non-transformed BEAS-2B cells that over-express SATB2 (SATB2-OE) or in iAs-transformed BEAS-2B cells (iAsT, rapid transformation model).

Article Snippet: We therefore used the Arraystar Human Circular RNA Array to screen iAs-transformed cells for differentially expressed circRNAs ( Fig S3A,B ), and discovered several dysregulated, cancer-associated circRNAs (e.g., hsa_circ_0002490, hsa_circ_0002874, hsa_circ_0075320, and hsa_circ_0008928) , – .

Techniques: Transfection, Over Expression, ATP Assay, Control, Transformation Assay, Plasmid Preparation, Migration, Transwell Migration Assay, Staining, RNA Sequencing Assay, Expressing, RNA Expression

Journal: bioRxiv

Article Title: SATB2 and circ3915 RNA chromatin dysregulation drive KRAS -like oncogenic transformation

doi: 10.1101/2024.10.04.616681

Figure Lengend Snippet: A , Differentially accessible regions (DARs) between non-treated BEAS-2B cells containing an empty expression vector, and BEAS-2B cells containing a SATB2 over-expression construct. DARs were defined by an FDR ( p adj ) ≤0.1. B , Heatmap of the top 50 DARs between non-transformed BEAS-2B cells containing an empty expression vector (control) or over-expressing SATB2 (SATB2-OE). Columns are the mean centered peak values for each gene (row) in each sample. The genomic location of each peak is shown to the right of the heatmap, with most peaks occurring in promoters, introns, and distal intergenic regions. C , Principal component analysis of DARs from non-transformed BEAS-2B cells (NT, green); non-transformed cells that were transfected with a SATB2 overexpression vector (NT + MCS, red); non-transformed cells over-expressing SATB2 (SATB2-OE, blue); and iAs-transformed BEAS-2B cells (rapid transformation model; iAsT, purple). D , Overlapping transcription factor motifs between BEAS-2B cells over-expressing SATB2 (SATB2-OE) and iAs-transformed BEAS-2B cells (rapid transformation model). E , Top enriched de novo transcription factor motifs and best matching transcription factors in opened and closed DARs from BEAS-2B cells over-expressing SATB2 . F , Congruence between SATB2-OE DARs and proximal gene expression. RNA UP = up-regulated transcripts; RNA DN = down-regulated transcripts. Counts are the number of individual genes above the minimum fold-change threshold for mRNA expression (>1 Log 2 (FC)) or open chromatin (>1 ShrunkenLog 2 (FC)). G , Top enriched C6 oncogenic signatures in non- transformed BEAS-2B cells that over-express SATB2 . H, GSEA plot shows that genes upregulated in the KRAS pathway in iAsT cells were also enriched in SATB2OE cells, while downregulated genes were similarly downregulated. I , In SATB2-KD cells, this enrichment is reversed: previously upregulated genes in the KRAS pathway are downregulated, and previously downregulated genes are upregulated.

Article Snippet: We therefore used the Arraystar Human Circular RNA Array to screen iAs-transformed cells for differentially expressed circRNAs ( Fig S3A,B ), and discovered several dysregulated, cancer-associated circRNAs (e.g., hsa_circ_0002490, hsa_circ_0002874, hsa_circ_0075320, and hsa_circ_0008928) , – .

Techniques: Expressing, Plasmid Preparation, Over Expression, Construct, Transformation Assay, Control, Transfection

Journal: bioRxiv

Article Title: SATB2 and circ3915 RNA chromatin dysregulation drive KRAS -like oncogenic transformation

doi: 10.1101/2024.10.04.616681

Figure Lengend Snippet: A , Western blot showing lentiviral SATB2 knockdown in iAs-transformed BEAS-2B cells. EV = empty vector control. B , Relative SATB2 and circ3915 expression in iAs-transformed BEAS-2B cells after SATB2 knockdown with shSATB2, as measured by RT-qPCR. EV = empty vector control. N = 3 replicates; * p <0.05. C , Same as panel B , except after circ3915 knockdown with shcirc3915. N = 3 replicates; * p <0.05, ns = not statistically significant. D , Principal component analysis (left) of RNA-seq expression profiles from non- transformed BEAS-2B cells (NT; brown); non-transformed cells containing an empty lentiviral expression vector (NT + MCS; red); non-transformed cells over-expressing SATB2 (SATB2-OE, green); iAs-transformed cells (iAsT; pink); iAs-transformed cells containing an empty lentiviral vector (iAsT + EmV; turqoise); iAsT cells with stable SATB2 knockdown (shSATB2; purple); and iAsT cells with stable circ3915 knockdown (shC3915; light blue). iAs-transformed cells were generated with the rapid (12 week) transformation model. Arrows indicate how RNA expression profiles shifted from control samples to the matching experimental group. E , Bar plot of normalized enrichment scores for significantly enriched ( p adj <0.05) MSigDB oncogenic signatures in iAs- transformed BEAS-2B cells (iAsT, rapid transformation model), non-transformed cells overexpressing SATB2 (SATB2-OE), BEAS-2B cells with stable SATB2 knockdown (SATB2-KD), and BEAS-2B cells with stable circ3915 knockdown (circ3915-KD). F , iAs-transformed BEAS-2B cell proliferation after shRNA knockdown, as measured with a CellTiter-Glo ATP assay 72 hours after seeding. Control = iAs-transformed cells containing an empty lentivirus vector. RLU = relative light units. Luminescence was normalized to background, and values calculated relative to the control cells. N = 3 independent experiments with 6 technical replicates each; **** p <0.0001. G , iAs-transformed BEAS-2B cell migration after after shRNA knockdown, as measured by a transwell migration assay 24 hr after seeding. Cells were fixed and stained with DAPI, and cell counts obtained with an Olympus cellSens counting module. Cell counts were normalized to the control cells. N = 3 independent experiments with 3 technical replicates each **** p <0.0001. H , Representative images from the transwell migration assay of panel I, J . Kaplan-Meier plots of overall survival relative to SATB2 expression in patients that either did not have a KRAS mutation ( I , n=342) or harbored a KRAS mutation ( J , n=150). Patient data were controlled for age at diagnosis, sex, and pack years.

Article Snippet: We therefore used the Arraystar Human Circular RNA Array to screen iAs-transformed cells for differentially expressed circRNAs ( Fig S3A,B ), and discovered several dysregulated, cancer-associated circRNAs (e.g., hsa_circ_0002490, hsa_circ_0002874, hsa_circ_0075320, and hsa_circ_0008928) , – .

Techniques: Western Blot, Knockdown, Transformation Assay, Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, RNA Sequencing Assay, Generated, RNA Expression, shRNA, ATP Assay, Migration, Transwell Migration Assay, Staining, Mutagenesis

Journal: Physiological Genomics

Article Title: Circular RNAs in rat models of cardiovascular and renal diseases

doi: 10.1152/physiolgenomics.00064.2017

Figure Lengend Snippet: Box plots show the distribution of expression levels for all samples in the circular (circ)RNA microarray data set. R-1, R-2, and R-3 are 3 individual Dahl salt-resistant rats (R). S-1, S-2, and S-3 are 3 individual Dahl salt-sensitive rats (S). WKY-1, WKY-2, and WKY-3 are 3 individual Wistar Kyoto rats (WKY). SHR-1, SHR-2, and SHR-3 are 3 individual spontaneously hypertensive rats (SHR).

Article Snippet: Arraystar Rat Circular RNA Microarray ( www.arraystar.com ) was used for microarray experiments.

Techniques: Expressing, Microarray

Journal: Physiological Genomics

Article Title: Circular RNAs in rat models of cardiovascular and renal diseases

doi: 10.1152/physiolgenomics.00064.2017

Figure Lengend Snippet: Differentially expressed circRNAs between normotensive and hypertensive rats. The figure represents the number of circRNAs differentially expressed according to the microarray analysis. Based on the location and direction on the genome, circRNAs are subcategorized into exonic, intronic, antisense, sense overlapping, and intergenic. S vs. R up indicates circRNAs were upregulated in Dahl salt-sensitive rats (S) compared with Dahl salt-resistant rats (R). S vs. R down indicates circRNAs were downregulated in S compared with R. SHR vs. WKY up indicates circRNAs were upregulated in spontaneously hypertensive rats (SHR) compared with Wistar Kyoto rats (WKY). SHR vs. WKY down indicates circRNAs were downregulated in SHR compared with WKY.

Article Snippet: Arraystar Rat Circular RNA Microarray ( www.arraystar.com ) was used for microarray experiments.

Techniques: Microarray

Journal: Physiological Genomics

Article Title: Circular RNAs in rat models of cardiovascular and renal diseases

doi: 10.1152/physiolgenomics.00064.2017

Figure Lengend Snippet: Validation of differentially expressed circRNAs in the microarray study by quantitative RT-PCR. R, Dahl salt-resistant rat; S, Dahl salt-sensitive rat; WKY, Wistar Kyoto rat; SHR, spontaneously hypertensive rat. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Arraystar Rat Circular RNA Microarray ( www.arraystar.com ) was used for microarray experiments.

Techniques: Biomarker Discovery, Microarray, Quantitative RT-PCR