Figure 5.

MiR-186-5p interacts with the 3'UTR of ROBO1 in HUVECs. (A) The putative binding sequence between miR-186-5p and ROBO1 predicted by the StarBase database is shown. (B) Dual-luciferase reporter assay was performed to confirm if ROBO1 was a target of miR-186-5p in HUVECs. This experiment was performed three times with three technical repetitions each time. An unpaired Student t-test was used to evaluate the differences. (C) RIP assay was applied to confirm the target relation between ROBO1 and miR-186-5p. This experiment was performed three times with three technical repetitions each time. An unpaired Student t-test was used to evaluate the differences. (D) The protein expression of ROBO1 was measured in HUVECs treated with ox-LDL (40 mg/L, 24 h) by Western blot assay. This experiment was performed three times. An unpaired Student t-test was used to evaluate the differences. (E) The overexpression efficiency of ROBO1 plasmid (pc-ROBO1) was analyzed by Western blot assay. This experiment was performed three times. An unpaired Student t-test was used to evaluate the differences. (F) HUVECs were transfected with miR-186-5p mimic alone or together with pc-ROBO1, and the protein level of ROBO1 was measured by Western blot assay. This experiment was performed three times. One-way ANOVA followed by Tukey's post hoc test was used to assess the differences. (G) The protein expression of ROBO1 was determined in HUVECs transfected with si-circ_0068087 alone or together with miR-186-5p inhibitor by Western blot assay. This experiment was performed three times. One-way ANOVA followed by Tukey's post hoc test was used to assess the differences. *P < 0.05.