Research Paper Volume 10, Issue 9 pp 2511—2534

Figure 6. The circDDX10-miR-1301-3p/miR-4660-SIRT3 interaction axis is associated with ovarian aging. (A) The predicted circRNA-miRNA-mRNA interaction network was predicted using bioinformatics online programs (starBase, circBase, TargetScan, and miRBase). The red triangle indicates circRNA, the green diamond indicates miRNA, and the blue circle indicates the targeted gene. (B) The predicted binding sites between circDDX10-miR-1301-3p/miR-4660 and miR-1301-3p/miR-4660-SIRT3 were exhibited. The matching types (in blue) and matching positions (in black and brown) were presented. The 7mer-m8, was an exact match to positions 2-8 of the mature miRNA (the seed + position 8), and the 8mer was an exact match to positions 2-8 of the mature miRNA (the seed + position 8) followed by an 'A'. (C) The expression of circDDX10 and SIRT3 in the ovarian cortex and granulosa cells (GCs) was examined by qPCR. (D) Co-localization of the SIRT3 protein to mitochondria in GCs. Patients (n = 5) per group and all samples were run in duplicate. Blue indicates 4′, 6-diamidino-2-phenylindole (DAPI) or nuclear staining. Red indicates cyto-chrome C (Cyt-c) and was used as a mitochondrial marker. Green indicates the SIRT3 protein. The scale bar is shown in the image of the bottom right corner (a) and equals 100 μm. (E) The expression of SIRT3 protein was determined using western blot. Proteins were isolated from GCs as described. Semi-quantitative analyses of protein levels were performed. Data indicate the mean ± SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.