Fig. 5.

miR-15b-5p and miR-290a-5p target Ccn2-mediated AKT-mTOR signaling pathway. (A) Venn diagram showing overlapping genes up-regulated in senescent MEFs, down-regulated in ESC-EVs-MEFs and target genes of miR-15b-5p or miR-290a-5p. (B) The table shows 11 target genes of miR-15b-5p (left) and 10 target genes of miR-290a-5p (right) that are up-regulated in senescent MEFs and down-regulated in ESC-EVs treatment. (C) Western blot analysis of Ccn2, AKT, p-AKT, mTOR and p-mTOR levels in P3 MEFs and P7 MEFs exposed to MEF-EVs or ESC-EVs. (D) Grayscale statistical analysis of Ccn2 relative to Tubulin, p-AKT relative to AKT, p-mTOR relative to mTOR. (E) RT-qPCR analysis of Ccn2 transcript levels in P3 MEFs and P7 MEFs overexpressing of miR-15b-5p or miR-290a-5p. (F) Western blot analysis of the level of Ccn2, AKT, p-AKT, mTOR and p-mTOR in P3 MEFs and P7 MEFs overexpression of miR-15b-5p or miR-290a-5p. (G) Grayscale statistical analysis of Ccn2 relative to Tubulin, p-AKT relative to AKT, p-mTOR relative to mTOR. (H) Luciferase reporter constructs. The dual reporter vector pmirGLO expresses Firefly Luciferase (FL) and Renilla Luciferase (RL) under separate promoters. The plasmid pmirGLO-Ccn2 (3′UTR) has the wild-type Ccn2 3′UTR of miR-15b-5p or miR-290a-5p binding site inserted directly after the FL coding region; plasmid pmirGLO-Ccn2 (mut3′UTR) has the Ccn2 3′UTR with a mutation in the miR-15b-5p or miR-290a-5p binding site inserted directly following the FL coding region. (I) Luciferase activity was measured in 293T cells that were co-transfected with miR-15b-5p mimic or miR-15b-5p NC and luciferase reporter plasmids containing wild-type or mutant. (J) Luciferase activity was measured in 293T cells were co-transfected with miR-290a-5p mimic or miR-290a-5p NC and luciferase reporter plasmids containing wild-type or mutant. Data are presented as mean ± SEM. n = 3. Student's t-test: ns, not significance; *p < 0.05 and **p < 0.01.