Journal: Frontiers in Oncology

Article Title: miR-617 interacts with the promoter of DDX27 and positively regulates its expression: implications for cancer therapeutics

doi: 10.3389/fonc.2024.1411539

Figure Lengend Snippet: Identification of DDX27 as a gene target for miR-617 in SCC131 cells. (A) A dose-dependent regulation of DDX27 transcript and protein upon transient transfection of cells with increasing doses of pmiR-617 compared to those transfected with the vector control (B) Upregulation of DDX27 is specific to functional miR-617 level. Transfection of the pmiR-617 construct in SCC131 cells increases the transcript and protein levels of DDX27 compared to those transfected with pmiR-617-M or the vector control. p-values were ≤0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****) and >0.05 (ns).

Article Snippet: The following antibodies were used in this study: anti-DNMT1 antibody (1:5,000 dilution, cat# NB100–56519; Novus Biologicals, Centennial, CO, USA), anti-DDX27 antibody (1:1,000 dilution; cat# ab177950; Abcam, Cambridge, MA, USA), anti-mouse β-actin antibody (1:10,000 dilution; cat# A5441; Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p70 S6 Kinase (Thr421/Ser424) antibody (1:1,000 dilution; cat# 9204; Cell Signaling Technology, Danvers, MA, USA) and anti-p70 S6 Kinase antibody (1: 1,000 dilution; cat# 9202; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Functional Assay, Construct

Journal: Frontiers in Oncology

Article Title: miR-617 interacts with the promoter of DDX27 and positively regulates its expression: implications for cancer therapeutics

doi: 10.3389/fonc.2024.1411539

Figure Lengend Snippet: miR-617 interacts with the DDX27 promoter. (A) Putative miR-617 binding sites in the promoter and 3’UTR of DDX27 . Note, the 11 bp complementarity in the 19 bp putative target site of miR-617 in the DDX27 promoter. (B) Dual-luciferase reporter assay to determine the interaction of miR-617 with 3’UTR and promoter of DDX27 in SCC131 cells. Note, a significant increase in luciferase activity in cells co-transfected with pGL3-PrDDX27 and pmiR-617 compared to those co-transfected with pGL3-PrDDX27 and the vector control, suggesting that miR-617 interacts with the promoter of DDX27 . Further, there was no significant change in luciferase activity in cells co-transfected with p3’UTR-S and pmiR-617 compared to those co-transfected with p3’UTR-S and the vector control, suggesting that miR-617 does not interact with the 3’UTR of DDX27 . (C) miR-617 regulates the promoter activity of DDX27 in a dose-dependent and sequence-specific manner in SCC131 cells. p-values were ≤0.05 (*), <0.01 (**), <0.0001 (****) and >0.05 (ns).

Article Snippet: The following antibodies were used in this study: anti-DNMT1 antibody (1:5,000 dilution, cat# NB100–56519; Novus Biologicals, Centennial, CO, USA), anti-DDX27 antibody (1:1,000 dilution; cat# ab177950; Abcam, Cambridge, MA, USA), anti-mouse β-actin antibody (1:10,000 dilution; cat# A5441; Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p70 S6 Kinase (Thr421/Ser424) antibody (1:1,000 dilution; cat# 9204; Cell Signaling Technology, Danvers, MA, USA) and anti-p70 S6 Kinase antibody (1: 1,000 dilution; cat# 9202; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Binding Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Control, Sequencing

Journal: Frontiers in Oncology

Article Title: miR-617 interacts with the promoter of DDX27 and positively regulates its expression: implications for cancer therapeutics

doi: 10.3389/fonc.2024.1411539

Figure Lengend Snippet: In vitro construct system recapitulates the DDX27 upregulation by miR-617 in SCC131 cells. Cells co-transfected with pPr-DDX27 and miR-617 show a significant induction in the levels of DDX27 transcript and protein compared to those co-transfected with pPr-DDX27 and the vector control. For each experiment, 2 μg of each plasmid construct (total 4 μg) was transfected into the cells. p-values were ≤0.05 (*) and >0.05 (ns).

Article Snippet: The following antibodies were used in this study: anti-DNMT1 antibody (1:5,000 dilution, cat# NB100–56519; Novus Biologicals, Centennial, CO, USA), anti-DDX27 antibody (1:1,000 dilution; cat# ab177950; Abcam, Cambridge, MA, USA), anti-mouse β-actin antibody (1:10,000 dilution; cat# A5441; Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p70 S6 Kinase (Thr421/Ser424) antibody (1:1,000 dilution; cat# 9204; Cell Signaling Technology, Danvers, MA, USA) and anti-p70 S6 Kinase antibody (1: 1,000 dilution; cat# 9202; Cell Signaling Technology, Danvers, MA, USA).

Techniques: In Vitro, Construct, Transfection, Plasmid Preparation, Control

Journal: Frontiers in Oncology

Article Title: miR-617 interacts with the promoter of DDX27 and positively regulates its expression: implications for cancer therapeutics

doi: 10.3389/fonc.2024.1411539

Figure Lengend Snippet: Biological relevance of the interaction between miR-617 and DDX27 in OSCC patient samples. miR617 levels were downregulated in 11/36 OSCC samples (top panel) and DDX27 levels were downregulated in 19/36 OSCC samples (bottom panel) compared to their matched normal oral tissue samples. Overall, 16/36 OSCC samples showed a positive correlation in the levels of miR-617 and DDX27 . p-values were ≤0.05 (*), <0.01 (**), <0.001 (***) and >0.05 (ns).

Article Snippet: The following antibodies were used in this study: anti-DNMT1 antibody (1:5,000 dilution, cat# NB100–56519; Novus Biologicals, Centennial, CO, USA), anti-DDX27 antibody (1:1,000 dilution; cat# ab177950; Abcam, Cambridge, MA, USA), anti-mouse β-actin antibody (1:10,000 dilution; cat# A5441; Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p70 S6 Kinase (Thr421/Ser424) antibody (1:1,000 dilution; cat# 9204; Cell Signaling Technology, Danvers, MA, USA) and anti-p70 S6 Kinase antibody (1: 1,000 dilution; cat# 9202; Cell Signaling Technology, Danvers, MA, USA).

Techniques:

Journal: Frontiers in Oncology

Article Title: miR-617 interacts with the promoter of DDX27 and positively regulates its expression: implications for cancer therapeutics

doi: 10.3389/fonc.2024.1411539

Figure Lengend Snippet: DDX27 reduces cell viability of SCC131 and SCC084 cells. Transient overexpression of DDX27 significantly decreases viability of both SCC131 and SCC084 cells compared to the vector control transfected cells. For each experiment, 6 μg of plasmid construct was transfected into the cells. p-value was <0.0001 (****).

Article Snippet: The following antibodies were used in this study: anti-DNMT1 antibody (1:5,000 dilution, cat# NB100–56519; Novus Biologicals, Centennial, CO, USA), anti-DDX27 antibody (1:1,000 dilution; cat# ab177950; Abcam, Cambridge, MA, USA), anti-mouse β-actin antibody (1:10,000 dilution; cat# A5441; Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p70 S6 Kinase (Thr421/Ser424) antibody (1:1,000 dilution; cat# 9204; Cell Signaling Technology, Danvers, MA, USA) and anti-p70 S6 Kinase antibody (1: 1,000 dilution; cat# 9202; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Over Expression, Plasmid Preparation, Control, Transfection, Construct

Journal: Frontiers in Oncology

Article Title: miR-617 interacts with the promoter of DDX27 and positively regulates its expression: implications for cancer therapeutics

doi: 10.3389/fonc.2024.1411539

Figure Lengend Snippet: miR-617 downregulates the PI3K/AKT/MTOR pathway, in part, by regulating DDX27 in OSCC cells. Overexpression of miR-617 and DDX27 reduces p-S6K1 (phospho-S6K1) levels in OSCC cells. For each experiment, 6 μg of plasmid construct was transfected into the cells.

Article Snippet: The following antibodies were used in this study: anti-DNMT1 antibody (1:5,000 dilution, cat# NB100–56519; Novus Biologicals, Centennial, CO, USA), anti-DDX27 antibody (1:1,000 dilution; cat# ab177950; Abcam, Cambridge, MA, USA), anti-mouse β-actin antibody (1:10,000 dilution; cat# A5441; Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p70 S6 Kinase (Thr421/Ser424) antibody (1:1,000 dilution; cat# 9204; Cell Signaling Technology, Danvers, MA, USA) and anti-p70 S6 Kinase antibody (1: 1,000 dilution; cat# 9202; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Over Expression, Plasmid Preparation, Construct, Transfection

Journal: Frontiers in Oncology

Article Title: miR-617 interacts with the promoter of DDX27 and positively regulates its expression: implications for cancer therapeutics

doi: 10.3389/fonc.2024.1411539

Figure Lengend Snippet: miR-617 regulates the hallmarks of cancer, in part, by targeting the promoter of DDX27 in SCC131 cells. (A) miR-617 negatively regulates cell viability of cells by targeting the DDX27 promoter. (B) miR-617 negatively regulates anchorage independent growth of cells by targeting the DDX27 promoter. Representative micrographs of soft-agar colonies are shown on the right (scale bar = 500 mm). (C) miR-617 positively regulates cellular apoptosis by targeting the DDX27 promoter. For each experiment, 4 μg of each plasmid construct (total 8 μg) was transfected into the cells. p-values were <0.0001 (****) and >0.05 (ns).

Article Snippet: The following antibodies were used in this study: anti-DNMT1 antibody (1:5,000 dilution, cat# NB100–56519; Novus Biologicals, Centennial, CO, USA), anti-DDX27 antibody (1:1,000 dilution; cat# ab177950; Abcam, Cambridge, MA, USA), anti-mouse β-actin antibody (1:10,000 dilution; cat# A5441; Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p70 S6 Kinase (Thr421/Ser424) antibody (1:1,000 dilution; cat# 9204; Cell Signaling Technology, Danvers, MA, USA) and anti-p70 S6 Kinase antibody (1: 1,000 dilution; cat# 9202; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Plasmid Preparation, Construct, Transfection

Journal: Frontiers in Oncology

Article Title: miR-617 interacts with the promoter of DDX27 and positively regulates its expression: implications for cancer therapeutics

doi: 10.3389/fonc.2024.1411539

Figure Lengend Snippet: The effect of a synthetic miR-617 mimic on SCC131 cells-derived xenografts in nude mice. (A) Top panel: photographs of nude mice showing tumor growth on Day 27 of injection of cells pre-treated with miR-617 mimic and mimic control. Bottom panel: Excised xenografts from cells-pretreated with miR-617 mimic and mimic control on Day 27. The dotted red circle denotes the absence of tumor development. (B) Effect of miR-617 mimic on the volume of xenografts during a time course of 13–27 days. For mimic control group: n=5, Day 13–27 and, for miR-617 mimic group: n=4, Day 13; n=3, Day 15–21; n=4; Day 23; n=6, Day 25–27 (n=number of tumors measured). (C) Effect of miR-617 mimic on the weight of xenografts on Day 27. For miR-617 mimic and the mimic control groups, 3 animals were injected per group. (D) The levels of miR-617 and DDX27 in OSCC xenografts. L, left posterior flank; R, right posterior flank; MC, mimic control; and, M, mimic-617. p-values were ≤0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****) and >0.05 (ns).

Article Snippet: The following antibodies were used in this study: anti-DNMT1 antibody (1:5,000 dilution, cat# NB100–56519; Novus Biologicals, Centennial, CO, USA), anti-DDX27 antibody (1:1,000 dilution; cat# ab177950; Abcam, Cambridge, MA, USA), anti-mouse β-actin antibody (1:10,000 dilution; cat# A5441; Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p70 S6 Kinase (Thr421/Ser424) antibody (1:1,000 dilution; cat# 9204; Cell Signaling Technology, Danvers, MA, USA) and anti-p70 S6 Kinase antibody (1: 1,000 dilution; cat# 9202; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Derivative Assay, Injection, Control

Journal: Molecular Cell

Article Title: Circular RNA ZNF609/ CKAP5 mRNA interaction regulates microtubule dynamics and tumorigenicity

doi: 10.1016/j.molcel.2021.11.032

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Ddx27 , Proteintech , Cat#17087-1-AP; RRID: AB_2092569.

Techniques: Virus, Subcloning, Recombinant, Transfection, Western Blot, cDNA Synthesis, SYBR Green Assay, Magnetic Beads, Cloning, Expressing, Plasmid Preparation, Construct, Software, Single Cell Gel Electrophoresis

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Characterization of R-Loop–Interacting Proteins in Embryonic Stem Cells Reveals Roles in rRNA Processing and Gene Expression

doi: 10.1016/j.mcpro.2021.100142

Figure Lengend Snippet: Many R-loop–binding proteins are enriched within the nucleolus. A , immunofluorescence staining of three R-loop–associated DEAD-box proteins, DDX18, DDX24, and DDX27, costained with FBRL to mark the nucleolus. DNA was stained by DAPI. The scale bar represents 10 μm. B , comparison of rRNA-related and rRNA-unrelated proteins enriched by S9.6. R-loop–associated proteins were classified as rRNA related or unrelated by GO term analysis, using the biological process term “ribosomebiogensis,” “rRNAmetabolicprocess,” and “rRNAprocessing.” Boxes of the box plot represent the first and third quartiles, the band represents the median, and the whiskers depict 1.5 times the interquartile range. C , Venn diagram of the overlap between the R-loop interactomes (with and without filtering) and the nucleolar proteome from Kar et al. . The upper diagram includes the 335 proteins enriched in S9.6 co-IP samples with the 76 most enriched (corresponding to the orange dots in Fig. 1 B ) depicted in the lower diagram . p Values were calculated using hypergeometric tests. DAPI, 4′,6-diamidino-2-phenylindole; FBRL, fibrillarin; GO, Gene Ontology; IP, immunoprecipitation.

Article Snippet: Antibodies used included DDX18 (Bethyl Laboratories; A300-535A), DDX24 (Abcam; ab70463), DDX27 (Bethyl Laboratories; A302-216A), DDX54 (MilliporeSigma; AV36498-100UL), DHX9 (Abcam; ab26271), RPB1 (Santa Cruz Biotechnology; sc-899x), fibrillarin (FBRL; Novus Biologicals; NB300-269), CEBPZ (Proteintech; 25612-1-AP), SFPQ (Abcam; ab38148), CTCF (MilliporeSigma; 07-729), and mouse IgG2a (Abcam; ab18413).

Techniques: Binding Assay, Immunofluorescence, Staining, Comparison, Co-Immunoprecipitation Assay, Immunoprecipitation