Journal: Biomarker Research

Article Title: Circular RNA circEMB promotes osteosarcoma progression and metastasis by sponging miR-3184-5p and regulating EGFR expression

doi: 10.1186/s40364-022-00442-9

Figure Lengend Snippet: Identification and characterisation of circEMB in OSA cells. a Cluster heatmap demonstrating the expression levels of circRNA based on sequencing results. b qRT-PCR analysis of candidate genes correlated with tumour invasion and metastasis. c Sanger sequencing showing the circEMB loop formation characteristics. The circEMB sequence in the circBASE database was verified to contain seven exons (954) from the EMB gene. d Relative expression levels of circEMB in OSA cell lines and normal osteoblasts were determined via qRT-PCR. e After treatment with or without RNase R in 143B and U2OS cells, resistance exonuclease digestion of circEMB and EMB were determined by qRT-PCR. f The presence of circEMB in 143B and U2OS cells was determined via agarose gel electrophoresis. circEMB was amplified in cDNA instead of gDNA. GAPDH was used as the negative control. g-h The expression of circEMB and reference genes (GAPDH and U6) in the cytoplasm and nucleus was verified via nuclear plasma separation assay. circEMB was highly abundant in the cytoplasm. i circEMB localisation was analysed via FISH using a Cy3-labelled oligonucleotide. Scale bar, 50 μm. Data are expressed as mean ± SD ( n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: The human normal osteoblast cell line hFOB1.19 and human OSA cell lines 143B, U2OS, MNNG, MG63 and Saos-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Sequencing, Quantitative RT-PCR, Agarose Gel Electrophoresis, Amplification, Negative Control, Clinical Proteomics

Journal: Biomarker Research

Article Title: Circular RNA circEMB promotes osteosarcoma progression and metastasis by sponging miR-3184-5p and regulating EGFR expression

doi: 10.1186/s40364-022-00442-9

Figure Lengend Snippet: CircEMB promotes OSA cell proliferation, migration, invasion, apoptosis and cell cycle distribution. a Two shRNAs were designed to silence circEMB expression without affecting the mRNA expression of EMB. sh-circEMB #1 had the highest silencing efficiency. b CCK-8 assay revealed that circEMB knockdown inhibited the proliferation of 143B and U2OS cells. c Colony formation assay revealed that circEMB knockdown inhibited the proliferation of 143B and U2OS cells. d Transwell assay was performed to assess the influence of circEMB knockdown on the invasion and migration capabilities of 143B and U2OS cells. Scale bar, 100 μm. e Wound-healing assay was used to assess the effects of circEMB knockdown on 143B and U2OS cell migration. Scale bar, 200 μm. f Effects of circEMB silencing on apoptosis were examined via APC annexin V/PI staining as well as flow cytometry. g Flow cytometry was performed to assess the effects of circEMB on the cell cycle of 143B and U2OS cells. Data are expressed as mean ± SD ( n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: The human normal osteoblast cell line hFOB1.19 and human OSA cell lines 143B, U2OS, MNNG, MG63 and Saos-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Migration, Expressing, CCK-8 Assay, Knockdown, Colony Assay, Transwell Assay, Wound Healing Assay, Staining, Flow Cytometry

Journal: Biomarker Research

Article Title: Circular RNA circEMB promotes osteosarcoma progression and metastasis by sponging miR-3184-5p and regulating EGFR expression

doi: 10.1186/s40364-022-00442-9

Figure Lengend Snippet: CircEMB sponges miR-3184-5p. a-b RIP assay was performed to verify the binding between circEMB and the Ago2 protein. c Schematic representation of the overlap among target miRNAs of circEMB predicted using the TargetScan, miRanda, RNAhybrid, ENCORI and RNA22 databases. d-e RNA pull-down assay verified the binding between seven candidate miRNAs and circEMB. f-h RIP assay of miRNA showed that in contrast to the NC group, the miR-3184-5p mimics group effectively suppressed circEMB levels in 143B and U2OS cells. i-j HEK293T cells were co-transfected with miR-3184-5p mimics and wild-type or mutant circEMB luciferase reporter vector, and luciferase reporter activity was assessed. k Subcellular colocalization between circEMB (red) and miR-3184-5p (green) was observed in in 143B and U2OS by confocal FISH. Scale bar, 50 μm. Data are expressed as mean ± SD ( n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: The human normal osteoblast cell line hFOB1.19 and human OSA cell lines 143B, U2OS, MNNG, MG63 and Saos-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Binding Assay, Pull Down Assay, Transfection, Mutagenesis, Luciferase, Plasmid Preparation, Activity Assay

Journal: Biomarker Research

Article Title: Circular RNA circEMB promotes osteosarcoma progression and metastasis by sponging miR-3184-5p and regulating EGFR expression

doi: 10.1186/s40364-022-00442-9

Figure Lengend Snippet: CircEMB affects the malignant behaviors of OSA by sponging miR-3184-5p. a-c CCK-8 and colony formation assays showed that miR-3184-5p knockdown partly reversed circEMB knockdown-induced decrease in cell proliferation. d-e Transwell and wound healing assays revealed the mechanisms through which miR-3184-5p knockdown partly reversed circEMB-mediated invasion and migration abilities of 143B and U2OS cells. Scale bar, 100 μm Scale (Transwell); Scale bar, 200 μm(wound-healing assays). f Flow cytometry revealed that silencing miR-3184-5p partly reversed circEMB-mediated apoptosis of 143B and U2OS cells. g Flow cytometry revealed that silencing miR-3184-5p partly reversed circEMB-mediated G1/S phase arrest of 143B and U2OS cells. Data are expressed as mean ± SD ( n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: The human normal osteoblast cell line hFOB1.19 and human OSA cell lines 143B, U2OS, MNNG, MG63 and Saos-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: CCK-8 Assay, Knockdown, Migration, Flow Cytometry

Journal: Biomarker Research

Article Title: Circular RNA circEMB promotes osteosarcoma progression and metastasis by sponging miR-3184-5p and regulating EGFR expression

doi: 10.1186/s40364-022-00442-9

Figure Lengend Snippet: The circEMB/miR-3184-5p/EGFR axis was activated in OSA cells. a 143B cells transfected with sh-circEMB and scramble shRNA were subjected to RNA-seq. b Schematic representation of the overlap among target mRNAs of miR-3184-5p predicted using the TargetScan, miRanda, RNAhybrid, and miRDB databases. c Schematic representation of the overlap among target mRNAs of miR-3184-5p and mRNAs downregulated via circEMB knockdown. d Pathway enrichment analysis and GSEA revealed that the overlap genes were markedly enriched in the Notch signalling pathway. e-f After transfection of sh-circEMB into 143B and U2OS cells, the relative expression of four candidate mRNAs was determined via qRT-PCR. g-h Verification of the binding between miR-3184-5p and EGFR in HEK293T cells by dual-luciferase reporter gene assay. i-j Correlation between the mRNA expression of circEMB and EGFR and between that of miR-3184-5p and EGFR in 53 OSA samples. k Expression of EGFR in paired OSA and adjacent normal tissues was examined via western blotting. Data are expressed as mean ± SD ( n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: The human normal osteoblast cell line hFOB1.19 and human OSA cell lines 143B, U2OS, MNNG, MG63 and Saos-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Transfection, shRNA, RNA Sequencing, Knockdown, Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Gene Assay, Western Blot

Journal: Biomarker Research

Article Title: Circular RNA circEMB promotes osteosarcoma progression and metastasis by sponging miR-3184-5p and regulating EGFR expression

doi: 10.1186/s40364-022-00442-9

Figure Lengend Snippet: circEMB affects the malignant behaviour of OSA via the circEMB/miR-3184-5p/EGFR axis. a-c CCK-8 and colony formation assays revealed the changes in proliferation abilities of 143B and U2OS after simultaneous cell transfection with sh-circ-EMB, miR-3184-5p inhibitors, and sh-EGFR. d Transwell assay was performed to examine the effects of the circEMB/miR-3184-5p/EGFR axis on the migration and invasion abilities of cells. Scale bar, 100 μm. e-f Effects of the circEMB/miR-3184-5p/EGFR axis on cell cycle arrest and apoptosis were evaluated by flow cytometry. h Western blotting was used to assess the effects of the circEMB/miR-3184-5p/EGFR axis on the expression of E-cadherin, N-cadherin, Vimentin, PCNA, CDK4, CCND1, BAX, BCL2, Jagged1, and EGFR. i An in vivo imaging system was used to examine the effects of the circEMB/miR-3184-5p/EGFR axis on cell proliferation. j IHC staining for Ki-67, PCNA, Vimentin, N-cadherin and E-cadherin in each group. Scale bar, 100 μm. k An in vivo imaging system was used to examine the effects of the circEMB/miR-3184-5p/EGFR axis on metastasis in nude mice. Scale bar, 1000 μm. l Microscopic images of HE staining of metastatic nodules in the lungs of nude mice. Data are expressed as mean ± SD ( n = 3) (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Article Snippet: The human normal osteoblast cell line hFOB1.19 and human OSA cell lines 143B, U2OS, MNNG, MG63 and Saos-2 were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: CCK-8 Assay, Transfection, Transwell Assay, Migration, Flow Cytometry, Western Blot, Expressing, In Vivo Imaging, Immunohistochemistry, Staining