Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-137 and miR-34a directly target Snail and inhibit EMT, invasion and sphere-forming ability of ovarian cancer cells

doi: 10.1186/s13046-016-0415-y

Figure Lengend Snippet: MiR-137 and miR-34a are downregulated in OC tissues and decreased expressions of miR-137 and miR-34a are associated with poor survival in OC patients. a Venn diagram showing the overlap of miRNAs that were predicted to bind to the Snail 3′-UTR by alternative algorithms (TargetScan, miRSystem and DIANA-MicroT-CDS). The 6 predicted miRNAs were common to these three algorithms. b , c qPCR analysis of miR-137 ( b ) and miR-34a ( c ) levels in 50 paired cancerous and normal tissue samples from OC patients. d , e Kaplan-Meier analysis of overall survival in 50 OC patients with high median ( n = 25) or low median ( n = 25) expression levels of miR-137 ( d ) or miR-34a ( e )

Article Snippet: The Snail 3′-UTR luciferase vector was purchased from OriGene.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-137 and miR-34a directly target Snail and inhibit EMT, invasion and sphere-forming ability of ovarian cancer cells

doi: 10.1186/s13046-016-0415-y

Figure Lengend Snippet: Snail is a direct target of miR-137 and miR-34a in OC cells. a Relative miR-34a expression in OC cell lines (SKOV-3 and ES-2) and normal ovarian epithelial NOEC cells. b , c ES-2 cells were transfected with reporter constructs containing either wild-type (WT) Snail , or Snail 3′-UTR with mutation (MUT), along with miR-137 mimic ( b ), miR-34a mimic ( c ), or negative control mimic (Neg mimic), respectively. Relative luciferase activity was measured. d , e qPCR analysis of Snail expression in OC cells after overexpression ( d ) or knockdown ( e ) of miR-137 and miR-34a. f Western blotting analysis of Snail expression in OC cells after overexpression or knockdown of miR-137 and miR-34a. g , h qPCR analysis of indicated mRNAs in OC cells after transient overexpression or knockdown of miR-137 ( g ) and miR-34a ( h ). i Relative mRNA expression of Snail in OC tissues and matched normal tissues. j Analysis of Snail mRNA expression using microarray (Oncomine) on normal ovary versus OC tissue. ** P < 0.01

Article Snippet: The Snail 3′-UTR luciferase vector was purchased from OriGene.

Techniques: Expressing, Transfection, Construct, Mutagenesis, Negative Control, Luciferase, Activity Assay, Over Expression, Western Blot, Microarray

Journal: Cancer Research Communications

Article Title: MARCH2, a Novel Oncogene-regulated SNAIL E3 Ligase, Suppresses Triple-negative Breast Cancer Metastases

doi: 10.1158/2767-9764.CRC-23-0090

Figure Lengend Snippet: MARCH2 promotes ubiquitination and downregulation of SNAIL. A, HA-tagged WT, but not RING domain–mutant (W97A), MARCH2 downregulates SNAIL levels in MDA-MB-231 cells. B, MARCH2 coprecipitates with SNAIL in MDA-MB-231 cells coexpressing FLAG-SNAIL and HA-MARCH2. C, WT, but not RING domain– or transmembrane domain–mutant, MARCH2 ubiquitinates SNAIL. FLAG-Snail was immunoprecipitated from MDA-MB-231 cells coexpressing FLAG-Snail, HA-Ubiquitin and GST-tagged MARCH2 constructs. Immunoprecipitates were blotted with antibodies to HA to assess ubiquitination. D, MARCH2 ubiquitinates SNAIL in in vitro ubiquitination assay. Reactions were performed with recombinant SNAIL (Myc-tagged) in the presence of ubiquitin, recombinant E1 (UBE1), recombinant E2 (UBE2D3) and/or recombinant MARCH2.

Article Snippet: The in vitro ubiquitination reactions were performed using 2 µmol/L recombinant human SNAIL protein (Origene TP304581), 3 µmol/L recombinant human March2 E3 ligase (Origene TP307517), 100 µmol/L Ubiquitin (R&D Systems U-100H-10M), 100 nmol/L recombinant E1(UBE1, Biotechne E-305), 500 nmol/L recombinant E2 (UBE2D3, Biotechne E2-627), 1X ATP Energy Regeneration buffer (Enzo BML-EW9810-0100) in a 25 µL reaction buffer containing 50 mmol/L Hepes, pH 8.0, 50 mmol/L NaCl, and 1 mmol/L TCEP.

Techniques: Mutagenesis, Immunoprecipitation, Construct, In Vitro, Ubiquitin Assay, Recombinant