The fragment was CYT387 supplier sequenced and inserted into plasmids. Figure 2 Cloning of miR-9 target gene. A, identification of junction fragment of INCB28060 mw norientation. There was a 430 bp fragment, which demonstrated that the fragment was norientation. B, junction fragment digested by XbaI. The 360 bp fragment was destination fragment. Figure 3 Cloning of miR-433 target gene. A, identification of junction fragment of norientation. There was a 580 bp fragment, which demonstrated

that the fragment was norientation. B, junction fragment digested by XbaI. The 360 bp fragment was destination fragment. We measured luciferase activity and the relative light unit (RLU) at 48 h after the transfection. Luciferase activity of cells cotransfected pGL3-miR-9 and hsa-miR-9 decreased Semaxanib ic50 50% compared with pGL3-miR-9 (P < 0.05) (Figure 4A). Luciferase activity of cells cotransfected pGL3-miR-433 and hsa-miR-433 decreased by 54% compared with pGL3-miR-433 (P < 0.05) (Figure 4B). Figure 4 miR-9 and miR-433 down regulated luciferase activity of RAB34 and GRB2. A, miR-9 regulated luciferase activity by integrating the binding site in the 3'-UTR of RAB34. Luciferase activity of SGC7901 cotransfected pGL3-miR-9 and hsa-miR-9 decreased 50% compared with pGL3-miR-9 (P < 0.05). B, miR-433 regulated luciferase activity by integrating the binding site in the 3'-UTR of GRB2. Luciferase activity of SGC7901 cotransfected pGL3-miR-433 and hsa-miR-433

decreased 54% compared with pGL3-miR-433

(P < 0.05). The expression level of RAB34 and GRB2 were measured after miR-9 or miR-433 were transfected into SGC7901. The expression of RAB34 decreased 45% in group 1 and 72% in group 2 compared with control group (P < 0.05) Cobimetinib manufacturer (Figure 5A). The expression of GRB2 decreased 53% in group 1 and 89% in group 2 compared with control group (P < 0.05) (Figure 5B). Meanwhile, we measured the level of miR-9 and miR-433 by qRT-PCR. MiR-9 level increased 1.3-fold and 2.8-fold respectively in group 1 and 2 compared with control group (P < 0.05) (Figure 6A). MiR-433 level increased1.6-fold and 3.0-fold in group 1 and 2 compared with control group (P < 0.05) (Figure 6B). Figure 5 miR-9 and miR-433 down regulated RAB34 and GRB2 expression in SGC7901 cell line. A, RAB34 decreased 45% and 72% compared with control group after 50 pmol (group 1) and 100 pmol (group 2) hsa-miR-9 transfection. Relative gray scale value was compared with β-actin. B, GRB2 decreased 53% and 89% compared with control group after 50 pmol (group 1) and 100 pmo l (group 2) hsa-miR-433 transfection. Relative gray scale value was compared with β-actin. Figure 6 MiR-9 and miR-433 increased after hsa-miR-9 and hsa-miR-433 transfection. A, miR-9 level increased 1.3-fold and 2.8-fold respectively after 50 pmol (group 1) and 100 pmol (group 2) hsa-miR-9 transfection. B, miR-433 level increased 1.6-fold and 3.0-fold respectively after 50 pmol (group1) and 100 pmol (group 2) hsa-miR-433 transfection. (P < 0.05).