Journal: Nucleic Acids Research

Article Title: U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP

doi: 10.1093/nar/gkr150

Figure Lengend Snippet: U1 adaptor downregulates multiple mRNAs. A U1 adaptor targeted to RAF1 (U1A-RAF1) was administered to Hela cells at a single-dose of 100 nM. A fully modified 2′MOE ASO (13649) and a chimeric 2′MOE/DNA RNAse H-dependent ASO (194166) complementary to the same target sequence were also administered at the same concentration. After 24 h, cells were harvested, RNA was purified and mRNA reduction assayed by qRT/PCR using primers specific for 3 mRNAs; RAF1, PTEN and SMN2. The bars represent per cent mean untreated control for three replicates and the standard errors. UTC = mock-treated control.

Article Snippet: Cells were rotated at 4°C for 20 min, then centrifuged at 10 000 rpm for 5 min. A 40 µl of cleared cell extract was incubated with 2 µM DNA ASO complimentary to the U1 snRNA 5′-end (5′-CAGGTAAGTAT-3′), 2U Escherichia coli RNAse H (New England Biolabs) for 30 min at 30°C.

Techniques: Modification, Sequencing, Concentration Assay, Purification, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP

doi: 10.1093/nar/gkr150

Figure Lengend Snippet: Potency and specificity of U1 adaptors and RNAse H ASOs. A second U1 adaptor was designed to target SMN2 (U1A-SMN2). HeLa cells were treated at concentrations of 3 nM, 10 nM, 30 nM, or 100 nM with U1A-SMN2, U1A-RAF1, ASO 194166 (RAF1) or ASO 450880 (SMN2). After 24 h, cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. The percent mean mock-treated control (UTC) for three to four replicates and the standard error is shown for each concentration. ( a ) Percent control RAF1 mRNA expression. ( b ) Percent control SMN2 mRNA expression. ( c ) Percent control PTEN mRNA expression.

Article Snippet: Cells were rotated at 4°C for 20 min, then centrifuged at 10 000 rpm for 5 min. A 40 µl of cleared cell extract was incubated with 2 µM DNA ASO complimentary to the U1 snRNA 5′-end (5′-CAGGTAAGTAT-3′), 2U Escherichia coli RNAse H (New England Biolabs) for 30 min at 30°C.

Techniques: Purification, Quantitative RT-PCR, Concentration Assay, Expressing

Journal: Nucleic Acids Research

Article Title: U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP

doi: 10.1093/nar/gkr150

Figure Lengend Snippet: Reduction of U1 snRNA with RNAse H ASOs. ( a ) Chimeric 2′MOE/DNA RNAse H-dependent ASOs were designed to target U1snRNA. The location of 3 RNAse H ASOs is underlined on the U1 snRNA sequence. 469508 (1–20), 469509 (59–78), 469511 (117–138). Northern probe location is highlighted on the U1 snRNA structure. ( b ) HeLa cells were treated with ASOs at the indicated concentrations (nM). The following day total RNA was purified and U1snRNA reduction analyzed by northern blot with the internal U1 probe (U1-1, upper panel), or with a second probe, U1-2, targeted to the U1 5′-end and the non-target control and U6 snRNA probe (lower panel). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA normalized to U6 snRNA signal for each lane. ( c ) Effect of U1 adaptor treatment and U1 snRNA reduction on mRNA expression. HeLa cells seeded in 96-well plates were treated with U1A-RAF1, U1A-SMN2, U1A-PCSK9 or anti-U1 ASO 469508, in triplicate at concentrations ranging from 6 to 200 nM. The following day total RNA was purified and mRNA levels of each targeted gene assessed by qRT/PCR. Results are normalized to the total amount of RNA present in each reaction as determined by Ribogreen assay and plotted as percent control compared to mock-treated cells for three to four replicates with the standard error.

Article Snippet: Cells were rotated at 4°C for 20 min, then centrifuged at 10 000 rpm for 5 min. A 40 µl of cleared cell extract was incubated with 2 µM DNA ASO complimentary to the U1 snRNA 5′-end (5′-CAGGTAAGTAT-3′), 2U Escherichia coli RNAse H (New England Biolabs) for 30 min at 30°C.

Techniques: Sequencing, Northern Blot, Purification, Expressing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP

doi: 10.1093/nar/gkr150

Figure Lengend Snippet: U1 adaptors and anti-U1 snRNA ASOs promote reduction of multiple non-targeted mRNAs. HeLa cells were treated with U1A-SMN2, SMN2 ASO 450880, or U1 ASO 469508 at concentrations from 12.5 to 100 nM. The following day cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. Percent mock-treated control expression is shown for the six transcripts identified by Entrez Gene symbol at the bottom of the figure. ( a ) Cells treated with U1A-SMN2. ( b ) Cells treated with anti-U1 RNAse H ASO 469508. ( c ) Cells treated with SMN2 RNAse H ASO 450880.

Article Snippet: Cells were rotated at 4°C for 20 min, then centrifuged at 10 000 rpm for 5 min. A 40 µl of cleared cell extract was incubated with 2 µM DNA ASO complimentary to the U1 snRNA 5′-end (5′-CAGGTAAGTAT-3′), 2U Escherichia coli RNAse H (New England Biolabs) for 30 min at 30°C.

Techniques: Purification, Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP

doi: 10.1093/nar/gkr150

Figure Lengend Snippet: U1 adaptors bind the 5′ sequence of U1 snRNA and inhibit antisense hybridization. ( a ) 293-T cells were transfected with 100 nM U1 adaptor/ASO for 6 h. RNAse H protection assays were performed with whole-cell extracts ± 2U E. coli RNAse H and 2 µM DNA oligonucleotide antisense to the 5′ sequence of U1. Cleavage of U1 snRNA was detected by northern blot using internal U1 probe (U1-1), and normalized using a U3 probe (U3). ( b ) Cellular RNAse protection assay. Cells were treated with U1A_SMN2 or ASO at concentrations ranging from 10 to 200 nM as shown. Cells were then transfected with anti U1 RNAse H ASO 469508 at 50 nM and U1 snRNA cleavage assessed by northern blot using internal U1 probe (U1-1). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA signal normalized to U3 for each lane.

Article Snippet: Cells were rotated at 4°C for 20 min, then centrifuged at 10 000 rpm for 5 min. A 40 µl of cleared cell extract was incubated with 2 µM DNA ASO complimentary to the U1 snRNA 5′-end (5′-CAGGTAAGTAT-3′), 2U Escherichia coli RNAse H (New England Biolabs) for 30 min at 30°C.

Techniques: Sequencing, Hybridization, Transfection, Northern Blot, Rnase Protection Assay

Journal: Molecular cancer

Article Title: Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts.

doi: 10.1186/s12943-023-01824-9

Figure Lengend Snippet: Fig. 4 SOX9 is the downstream gene mediated by circEHD2. A, The cluster heat maps showed the differentially expressed genes regulated by circEHD2 based on next-generation sequencing (NGS) analysis. The red and blue strips indicate up-regulated and down-regulated genes, respectively. B and C, qRT–PCR analysis of the cAMP signal pathway-related genes in OSRC-2 (B) and 786-O (C) cells with circEHD2 knockdown. D and E, qRT–PCR analysis of the expression level of SOX9 when downregulation (D) or upregulation (E) of circEHD2 in OSRC-2 and 786-O cells. F and G, Western blot showed SOX9 expression level in whole protein extracts (F) and nuclear protein extracts (G) of OSRC-2 cells when silencing circEHD2. H, Representative images of IHC and ISH showed the expression of SOX9 and circEHD2 in ccRCC tissues and paired NATs (n = 80). Scale bars: 50 μm. I, The H-score of SOX9 in ccRCC tis sues and paired NATs (n = 80). J, The Pearson correlation analysis showed that circEHD2 expression level was positively correlated with SOX9. Error bars, standard deviation (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Abbreviations ccRCC clear cell renal cell carcinoma NATs normal adjacent tissues circRNA circular RNA circEHD2 circular RNA EHD2 YWHAH yrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta YAP Yes1-associated transcriptional regulator SOX9 SRY-box transcription factor 9 FUS FUS RNA -binding protein hnRNPA2B1 Heterogeneous nuclear ribonucleoprotein A2/B1 ASO antisense oligonucleotides HK2 human renal proximal tubular epithelial cell line ATCC American Type Culture Collection FBS Fetal bovine serum PBS phosphate buffered saline TEM transmission electron microscopy gDNA genomic DNA cDNA complementary DNA qRT-PCR Quantitative real-time PCR WB Western blot MS Mass spectrometry HE Hematoxylin-eosin IHC Immunohistochemistry FISH Fluorescence in situ hybridization FISH-IF Fluorescence in situ hybridization and Immunofluorescence ISH In situ hybridization RIP RNA immunoprecipitation ChIP Chromatin immunoprecipitation ChIRP Chromatin isolation by RNA purification shRNA Short hairpin RNA si-RNA small interfering RNA WT wild type DIG digoxin BCIP/NBT 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium DAPI 4′,6-diamidino-2-phenylindole ROC Receiver operating characteristic PFS progression-free survival EVs Extracellular vesicles CAFs Cancer-associated fibroblasts Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12943-023-01824-9.

Techniques: Next-Generation Sequencing, Quantitative RT-PCR, Knockdown, Expressing, Western Blot, Standard Deviation

Journal: Molecular cancer

Article Title: Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts.

doi: 10.1186/s12943-023-01824-9

Figure Lengend Snippet: Fig. 5 circEHD2 direct interacts with YWHAH. A, RNA pull-down assay was applied in RCC cells to identify the proteins that interacted with circEHD2. B, The silver staining image of RNA pull-down with circEHD2 probe in OSRC-2 cells. C, Mass spectrometry (MS) analysis of the proteins interacted with circEHD2 via RNA pull-down assay. D, qRT–PCR analysis confirmed that the circEHD2 probe could specifically enrich circEHD2 in OSRC-2 cells by RNA pull-down assay. E, Western blot assay in circEHD2 pull-down proteins confirmed the interaction between circEHD2 and YWHAN. F, The subcellular co- localization of circEHD2 and YWHAH in RCC cells was measured by fluorescence staining assay. Scale bars: 10 μm. G, RIP assay in OSRC-2 cells confirmed that circEHD2 could be enriched by YWHAH. IgG was used as a negative control. H, The three-dimensional structure showed circEHD2 could be bound to YWHAH. I, Serial deletions of circEHD2 were used in RNA pull-down assays to confirm the regions of circEHD2 that were required for YWHAH. J, qRT–PCR analysis of the level of circEHD2 when mutated the binding sites of circEHD2. K, RIP assay was performed in OSRC-2 cells after mutating the 201-252-nt region of circEHD2. L, The knockdown efficiency of YWHAH in RCC cells measured by qRT-PCR. M, qRT–PCR analysis of the level of SOX9 when knockdown of YWHAH in RCC cells. N, Western blot analysis of the expression of SOX9 when downregulation of YWHAH in OSRC-2 cells. O, qRT–PCR analysis of the level of SOX9 in RCC cells after mutating the 201-252-nt region of circEHD2. P, Western blot assay revealed the level of SOX9 in OSRC-2 cells after mutating the 201-252-nt region of circEHD2. Error bars, standard deviation (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Abbreviations ccRCC clear cell renal cell carcinoma NATs normal adjacent tissues circRNA circular RNA circEHD2 circular RNA EHD2 YWHAH yrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta YAP Yes1-associated transcriptional regulator SOX9 SRY-box transcription factor 9 FUS FUS RNA -binding protein hnRNPA2B1 Heterogeneous nuclear ribonucleoprotein A2/B1 ASO antisense oligonucleotides HK2 human renal proximal tubular epithelial cell line ATCC American Type Culture Collection FBS Fetal bovine serum PBS phosphate buffered saline TEM transmission electron microscopy gDNA genomic DNA cDNA complementary DNA qRT-PCR Quantitative real-time PCR WB Western blot MS Mass spectrometry HE Hematoxylin-eosin IHC Immunohistochemistry FISH Fluorescence in situ hybridization FISH-IF Fluorescence in situ hybridization and Immunofluorescence ISH In situ hybridization RIP RNA immunoprecipitation ChIP Chromatin immunoprecipitation ChIRP Chromatin isolation by RNA purification shRNA Short hairpin RNA si-RNA small interfering RNA WT wild type DIG digoxin BCIP/NBT 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium DAPI 4′,6-diamidino-2-phenylindole ROC Receiver operating characteristic PFS progression-free survival EVs Extracellular vesicles CAFs Cancer-associated fibroblasts Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12943-023-01824-9.

Techniques: Pull Down Assay, Silver Staining, Mass Spectrometry, Quantitative RT-PCR, Western Blot, Fluorescence, Staining, Negative Control, Binding Assay, Knockdown, Expressing, Standard Deviation

Journal: Molecular cancer

Article Title: Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts.

doi: 10.1186/s12943-023-01824-9

Figure Lengend Snippet: Fig. 6 YWHAH recruits circEHD2 and YWHAH to the promoter of SOX9. A and B, Co-IP assay showed that YAP could be precipitated by anti-YWHAH an tibody (A) and YWHAH could be precipitated by anti-YAP antibody (B) in OSRC-2 cells. C and D, Co-IP assay confirmed that YWHAH could bind to YAP by anti-YWHAH antibody (C) and anti-YAP antibody (D) in the nuclear extracts of OSRC-2 cells. E, Subcellular co-localization of YWHAH and YAP in RCC cells was measured by fluorescence staining assay. Scale bars: 10 μm. F, qRT–PCR analysis of the level of YAP in RCC cells when cocultured with VP at different doses. G and H, qRT–PCR analysis (G) and western blot assay (H) showed the expression level of SOX9 in OSRC-2 cells when cocultured with VP. I, Sche matic illustration of the binding sites of circEHD2 and YAP on SOX9 promoter. J, Schematic illustration of PCR-amplified fragments of SOX9 promoter. K, ChIRP analysis the enrichment of SOX9 promoter fragments in OSRC-2 cells. L, Dual-luciferase reporter assays showed that circEHD2 could bind to the P2 region (-1501-nt to -1001-nt) of the SOX9 promoter. M–P, ChIP-qPCR of YAP (M and O) and YWHAH (N and P) revealed the enrichment of SOX9 promoter in OSRC-2 cells with circEHD2 knockdown (M and N) or overexpression (O and P). Error bars represent the standard deviation (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Abbreviations ccRCC clear cell renal cell carcinoma NATs normal adjacent tissues circRNA circular RNA circEHD2 circular RNA EHD2 YWHAH yrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta YAP Yes1-associated transcriptional regulator SOX9 SRY-box transcription factor 9 FUS FUS RNA -binding protein hnRNPA2B1 Heterogeneous nuclear ribonucleoprotein A2/B1 ASO antisense oligonucleotides HK2 human renal proximal tubular epithelial cell line ATCC American Type Culture Collection FBS Fetal bovine serum PBS phosphate buffered saline TEM transmission electron microscopy gDNA genomic DNA cDNA complementary DNA qRT-PCR Quantitative real-time PCR WB Western blot MS Mass spectrometry HE Hematoxylin-eosin IHC Immunohistochemistry FISH Fluorescence in situ hybridization FISH-IF Fluorescence in situ hybridization and Immunofluorescence ISH In situ hybridization RIP RNA immunoprecipitation ChIP Chromatin immunoprecipitation ChIRP Chromatin isolation by RNA purification shRNA Short hairpin RNA si-RNA small interfering RNA WT wild type DIG digoxin BCIP/NBT 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium DAPI 4′,6-diamidino-2-phenylindole ROC Receiver operating characteristic PFS progression-free survival EVs Extracellular vesicles CAFs Cancer-associated fibroblasts Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12943-023-01824-9.

Techniques: Co-Immunoprecipitation Assay, Fluorescence, Staining, Quantitative RT-PCR, Western Blot, Expressing, Binding Assay, Amplification, Luciferase, ChIP-qPCR, Knockdown, Over Expression, Standard Deviation

Journal: Molecular cancer

Article Title: Extracellular vesicle-circEHD2 promotes the progression of renal cell carcinoma by activating cancer-associated fibroblasts.

doi: 10.1186/s12943-023-01824-9

Figure Lengend Snippet: Fig. 10 circEHD2-ASO inhibits the growth of RCC in vivo. A, Schematic illustration of tumor inoculation and circEHD2-ASO treatment in subcutaneous xenograft model and orthotopic xenograft model. B–E, Representative images of xenograft tumors (B), dissected tumors (C), the volume of tumors (D), and tumor weights (E) in subcutaneous xenograft model treated with ASO-control (n = 6/group) and circEHD2-ASO (n = 6/group), respectively. F-H, Representative images of in vivo bioluminescence imaging (F), the mean photon counts (G), and gross appearance of orthotopic tumor (H) in orthotopic xenograft model treated with ASO-control (n = 4/group) and circEHD2-ASO (n = 4/group), respectively. I, A schematic model showing the mechanism of EVs-circEHD2 mediated the progression of RCC. FUS mediated the biogenesis of circEHD2, then circEHD2 enhanced the growth of RCC through the circEHD2/YWHAH/YAP/SOX9 pathway. While hnRNPA2B1 mediated the packaging of circEHD2 into EVs, and EVs-circEHD2 promote metastasis of RCC by converting fibroblasts to CAFs. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: Abbreviations ccRCC clear cell renal cell carcinoma NATs normal adjacent tissues circRNA circular RNA circEHD2 circular RNA EHD2 YWHAH yrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta YAP Yes1-associated transcriptional regulator SOX9 SRY-box transcription factor 9 FUS FUS RNA -binding protein hnRNPA2B1 Heterogeneous nuclear ribonucleoprotein A2/B1 ASO antisense oligonucleotides HK2 human renal proximal tubular epithelial cell line ATCC American Type Culture Collection FBS Fetal bovine serum PBS phosphate buffered saline TEM transmission electron microscopy gDNA genomic DNA cDNA complementary DNA qRT-PCR Quantitative real-time PCR WB Western blot MS Mass spectrometry HE Hematoxylin-eosin IHC Immunohistochemistry FISH Fluorescence in situ hybridization FISH-IF Fluorescence in situ hybridization and Immunofluorescence ISH In situ hybridization RIP RNA immunoprecipitation ChIP Chromatin immunoprecipitation ChIRP Chromatin isolation by RNA purification shRNA Short hairpin RNA si-RNA small interfering RNA WT wild type DIG digoxin BCIP/NBT 5-Bromo-4-Chloro-3-Indolylphosphate/Nitroblue Tetrazolium DAPI 4′,6-diamidino-2-phenylindole ROC Receiver operating characteristic PFS progression-free survival EVs Extracellular vesicles CAFs Cancer-associated fibroblasts Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12943-023-01824-9.

Techniques: In Vivo, Control, Imaging

Journal:

Article Title: Gene expression profiling in autoimmune non-infectious uveitis disease

doi:

Figure Lengend Snippet: Scatter plot analysis of gene expression profiling on uveitis patients demonstrates differential gene expression. cDNA microarray analysis on 50 RNA samples from uveitis patients and 1 pooled sample from controls was performed. After normalization on house-keeping genes, e.g, GAPDH and beta-2-microglobulin (B2M), the final scores of each of the genes of all arrays were compared to those from the control array. The scatter plot was acquired as described in the Materials and Methods. The Y-axis represents log scores from uveitis patients (group 2) and the X-axis represents log scores from normal controls (group 1). Each symbol represents one gene with red-colored ones represent 2-fold higher expressed in uveitis patients (group 2), green-colored ones represent 2-fold lower expressed in uveitis patients and the black-colored ones represent within 2-fold cut-off threshold.

Article Snippet: Probes were hybridized to a pathway-specific cDNA microarray chip (Inflammatory and autoimmune GEArray; SuperArray Inc., Gaithersburg, MD).

Techniques: Gene Expression, Microarray, Control

Journal:

Article Title: Gene expression profiling in autoimmune non-infectious uveitis disease

doi:

Figure Lengend Snippet: Clustergram analysis of gene expression revealed 4 distinct gene expression profiles. cDNA microarray was performed as described above and clustergram analysis on microarray data was described in the Materials & Methods section. X-axis represents all genes listed in the cDNA microarray chip and Y-axis depicts gene expression profiles from all samples of uveitis patients normalized on normal controls. Each colored band represents expression of a single gene from one patient when compared to that from normal donors with higher expression in red and lower expression in green. Groups I, II, III and IV designate 4 sub-groups of gene expression profiles. Analysis of gene expression profiling among uveitis patients revealed 4 sets of genes designated as molecular signatures A, B, C and D that can be used to define the 4 sub-groups of gene expression profiles.

Article Snippet: Probes were hybridized to a pathway-specific cDNA microarray chip (Inflammatory and autoimmune GEArray; SuperArray Inc., Gaithersburg, MD).

Techniques: Gene Expression, Microarray, Expressing

Journal:

Article Title: Gene expression profiling in autoimmune non-infectious uveitis disease

doi:

Figure Lengend Snippet: Comparison of clustergram analysis of gene expression profiling of 2 male siblings (designated as patient A and patient B) with the same diagnosis of intermediate uveitis with that from normal controls (designated as NL). The cDNA microarray and clustergram analysis were performed as described above and in the Materials and Methods. The X-axis listed all the gene symbols in the cDNA microarray chips. The gene expression profiles from the 2 male siblings with the same diagnosis of intermediate uveitis (designated as patient A and patient B) were aligned with the profile from normal subjects (NL). As described above, each colored band represents expression of a single gene from either patient A or patient B or from normal subjects after normalization on house keeping genes, with higher expression in red and lower expression in green.

Article Snippet: Probes were hybridized to a pathway-specific cDNA microarray chip (Inflammatory and autoimmune GEArray; SuperArray Inc., Gaithersburg, MD).

Techniques: Comparison, Gene Expression, Biomarker Discovery, Microarray, Expressing