Journal: International Journal of Biological Sciences

Article Title: MicroRNA-196a promotes renal cancer cell migration and invasion by targeting BRAM1 to regulate SMAD and MAPK signaling pathways

doi: 10.7150/ijbs.60805

Figure Lengend Snippet: Bram1 inhibits cell migration and invasion via MAPK and BMP pathways by binding to EGFR and BMPR1A. (A) HEK293T cells were transfected with Bram1 or shBram1 and the expression level of phosphorylated SMAD1/5/8 and tubulin was examined by Western Blot assay. Data is representative of three independent experiments. (B) HEK293T cells were co-transfected with SMAD luciferase plasmids and Bram1 or shBram1 plasmids, and the lysate was used for luciferase assay. *P<0.05. (C, D) cells were transfected with Bram1 or shBram1, and the expression level of indicated proteins was examined by Western Blot assay. (E) Protein level of phosphate p42/44 and total p42/44 in empty vector (-) and Bram1 overexpression (+) RCC4 cells with or without U0126 treatment (10 µM, 4 hours). (F) Wound healing assay analysis the effect of MAPK inhibitor and Bram1 overexpression on cell migration (10 µM U0126 treatment for 4 hours). (G) The physical interaction between Bram1 and BMPR1A or EGFR was investigated by immunoprecipitation experiments. HEK293T cells were transfected with flag-Bram1, and treated with 50 µM BMP4, 48 hr post-transfection the lysates were collected and incubated with normal beads as a negative control or flag-beads. (H) RCC4 cells were co-transfected with miR-196a and Bram1 plasmids, the expression of Bram1 was detected by RT-PCR. *P<0.05. (I) RCC4 cells were co-transfected with miR-196a and Bram1 plasmids and seeded in the upper chamber of transwell plates. Duplicates were performed in three independent experiments. ***P<0.001. (J) RCC4 cells were co-transfected with miR-196a and Bram1 plasmids, followed by wound healing assay measured at 0 hr and 24 hr. Data is representative of three independent experiments.

Article Snippet: The antibodies used in our experiments included: Anti-Rabbit Bram1( Cell Signaling Technology (CST), Inc Danvers, MA, U.S.A); Anti-Rabbit BMPR1A (Gene tex Inc, San Antonio, Texas, U.S.A); Anti-Rabbit p-ERK (CST, Danvers, MA, U.S.A); Anti-Rabbit total-ERK (CST, Danvers, MA, U.S.A); Anti-Rabbit p-p38 (CST, Danvers, MA, U.S.A); Anti-Rabbit total-p38 (CST, Danvers, MA, U.S.A); Anti-Rabbit p-JNK (CST, Danvers, MA, U.S.A); Anti-Rabbit total-JNK (CST, Danvers, MA, U.S.A); Anti-Rabbit p-SMAD1/5 (CST, Danvers, MA, U.S.A); Anti-Rabbit EGFR (CST, Danvers, MA, U.S.A); Anti-Mouse Tubulin (Sigma-Aldrich Co St. Louis, MO, U.S.A.).

Techniques: Migration, Binding Assay, Transfection, Expressing, Western Blot, Luciferase, Plasmid Preparation, Over Expression, Wound Healing Assay, Immunoprecipitation, Incubation, Negative Control, Reverse Transcription Polymerase Chain Reaction