Research Paper Volume 8, Issue 8 pp 1608—1624

Figure 3. Senescent oral fibroblasts differentially express SASP-associated miRNAs. The volcano plot illustrates the differentially expressed miRNA signature in cisplatin induced senescent oral fibroblasts. cDNA synthesized from RNA isolated from proliferating fibroblasts and fibroblasts induced to senesce using cisplatin (RNA isolated 15 days post-senescence) was analyzed using TaqMan miRNA tiling low density array (TLDA) to determine miRNA expression profile in senescent fibroblasts (n=3) (A). qRT-PCR showed that miR-335 (B) levels gradually increase in senescent fibroblasts with a time course corresponding to the increase in the levels of SASP components IL-6 (C) and MCP-1 (D), (n=3). Over-expression of miR-335 in young fibroblasts increased synthesis and secretion of MCP-1 (E-F) and IL-6 (G-H) (n=3), and stimulated chemotaxis and invasion of H357 cells in 2D-assay (n=3) (I-J). Blockade of MCP-1 in conditioned media of senescent fibroblasts significantly reduced migration of H357 cells compared to negative isotype IgG treated control (n=3) (K). Blockade of MCP-1 in conditioned media derived from miR-335 over-expressing fibroblasts also reduced H357 cell migration than those fibroblasts transfected with negative miRNA control (n=3) (L). All experiments were performed independently as indicated by n and with technical repeats. The bars represent mean ± STDEV (B-E, G) or mean ± SEM (F, H-L). *p<0.05 was determined by paired student's t-test (A, E, I-L), two-way repeated measure ANOVA for time course studies with post-hoc corrections by Holm-Sidak method (B-D), and Mann-Whitney U-test (F,H). (See Supplementary Figure S3 and Supplementary Table S1).