Journal: Journal of Experimental & Clinical Cancer Research : CR
Article Title: circGFRA1 and GFRA1 act as ceRNAs in triple negative breast cancer by regulating miR-34a
doi: 10.1186/s13046-017-0614-1
Figure Lengend Snippet: circGFRA1 and GFRA1 act as ceRNAs in TNBC through regulation of miR-34a. a. The expression level of GFRA1 was determined by qRT-PCR (left) and western blot (right) in 11 mammary cell lines. β-actin was used as a control. b. The expression level of GFRA1 in 51 TNBC tissues and their matched normal adjacent tissues was determined by qRT-PCR. c. The predicted binding sites of miR-34a within GFRA1 were shown. d. Luciferase assay of MDA-MB-231 cells cotransfected with miR-34a mimics or miR-34a inhibitor and luciferase reporter containing GFRA1 3′-UTR (GFRA1 wt) or mutant construct (GFRA1 mut). e. Cells were transfected as described, and the expression of GFRA1 was determined by qRT-PCR. f. Cells were transfected as described, and the expression of GFRA1 was determined by western blot analysis. G. Cells were transfected with si-NC or si-GFRA1, and the expression of miR-34a was determined by qRT-PCR. h. Cells were transfected with si-NC, si-circGFRA1 or si-circGFRA1 + miR-34a inhibitor, and the expression of GFRA1 was determined by qRT-PCR. i. Cells were transfected with si-NC, si-GFRA1 or si-GFRA1 + miR-34a inhibitor, and the expression of circGFRA1 was determined by qRT-PCR. j. Cells were transfected with miR-NC, miR-34a or si-circGFRA1 + miR-34a, and CCK8 assay was performed to assess cell proliferation. k. Cells were transfected as described, and apoptosis assay was performed after transfection. All the data are shown as the mean ± s.e.m., * P < 0.05 and ** P < 0.01
Article Snippet: The membrane was then incubated with primary antibody against GFRA1 (1:1000, CST, USA), followed by horseradish peroxidase (HRP)-labeled secondary antibody (Santa Cruz) and detected by chemiluminescence.
Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Binding Assay, Luciferase, Mutagenesis, Construct, Transfection, CCK-8 Assay, Apoptosis Assay
