Figure 2.

Expression of miR‐34c is regulated by ROS‐JNK‐p53 pathway in neurons. (a) ROS generation was induced by H₂O₂ in HT‐22 cells. HT‐22 cells treated with 60 μmol/l H₂O₂ for 24 hr, and ROS generation was detected using flow cytometry (FCM). (b) Western blot analysis of p‐JNK, JNK, and P53 protein in the HT‐22 cells exposed to different concentrations of H₂O₂ (0, 20, 40, 60, 100, and 200 μmol/l) for 24 hr. Densitometry analysis was performed and normalized to β‐actin. The values are the mean ± SD (n = 3). *p < .05,**p < .01 versus 0 μmol/l H₂O₂ group. (c) Effects of JNK inhibitor SP600125 on p‐JNK, JNK, and P53 protein (top and middle panel) and miR‐34c expression (bottom panel) in HT‐22 cells. HT‐22 cells were treated with SP600125 (50 μmol/l) for 3 hr and then incubated with H₂O₂ (60 μmol/l) for 24 hr. The values are the mean ± SD (n = 3). *p < .05 ** p < .01, n = 3. (d–g) Changes of P53 protein (top and middle panel) and miR‐34c expression (bottom panel) in the indicated groups. HT‐22 cells were treated with actinomycin D (Act D, 10 nmol/l) (d) or pifithrin‐α (PFT‐α, 10 nmol/l) (e) for 3 hr, or transfected with pEZ‐p53 (f) or shP53 (g) for 24 hr, and then treated with (or without) H₂O₂ (60 μmol/l) for 24 hr. All data of P53 levels were normalized to β‐actin, and the relative expression of miR‐34c was normalized to U6‐snRNA expression levels. *p < .05, **p < .01. n = 3. (h) The predicted P53 binding sites in the promoter region of miR‐34c and the designed mutant sequences were indicated. (i) Relative luciferase activities of the miR‐34c promoter were measured in the 293A cells cotransfected pEZ‐p53 with wild‐type luciferase vector or mutant luciferase vector. *p < .05 versus Con, n = 3