Research Paper Volume 8, Issue 8 pp 1608—1624

Figure 4. miR-335 represses PTEN function in senescent oral fibroblasts. The 3′UTR of PTEN bears a conserved seed sequence complementary to miR-335 (A). Over-expression of miR-335 in oral fibroblasts negatively regulated PTEN protein level in dose-response manner (n=3) (B). Co-transfection of miR-335 with pmiR-reporter vector bearing a 1.45 kb insert of PTEN 3′UTR showed diminished luminescence compared to controls (n=3) (C). Western blot showed PTEN expression is reduced in senescent fibroblasts, compared to presenescence (proliferating) controls (n=9) (D) and this is associated with an increased phosphorylation of Akt (E). Transient knockdown of PTEN in oral fibroblasts stimulated transmigration of H357 cells in vitro (n=3) and increased MMP2 transcript levels (F-H). All experiments were performed independently as indicated by n and with technical repeats. The bars represent mean ± SEM or mean ± STDEV (H). *p<0.05 and ***p<0.01, were determined by paired student's t-test (C, F-H), Mann-Whitney U-test (D). The arrows indicate the respective molecular weight of detectable proteins in western blot using EZ-Run prestained rec protein ladder. (See Supplementary Figure S4 and Supplementary Table S2-S3).