Figure 3.

circPVT1 negatively regulated miR-145 serving as a molecular sponge. (a) The relative expression levels of circPVT1 and miR-145 in the cytoplasmic and nuclear fractions of Eu_EEC and Eu_ ESC were measured by qRT-PCR, respectively. GAPDH represented the cytoplasmic marker, and U6 was detected as the nuclear reference. (b) Bioinformatics analysis was utilized to predict the putative binding sites between circPVT1 and miR-145. (c) The relative luciferase activity of wide-type circPVT1 (circPVT1-WT) or mutant-type circPVT1 (circPVT1-MT) in the indicated cells cotransfected with miR-145 mimics or miR-145 negative control (miR-145 NC). (d) Cellular lysate of HEK-293T cell was collected and immunoprecipitated. The relative amount of circPVT1 or miR-145 bound to AgO2 antibody was detected using qRT-PCR. IgG was used as a negative control. (e) miR-145 expression levels were decreased in adenomyotic eutopic endometrium (ADS_Euc, n = 45) and ectopic endometrium (ADS_Ec, n = 45). (f) qRT-PCR detection of miR-145 in ADS and normal uterine endometrial cells. Four types of primary cells are as follows: Eu_EEC, adenomyotic eutopic endometrial epithelial cell; Eu_ESC, adenomyotic eutopic endometrial stromal cell; Con_EEC, normal uterine endometrial epithelial cell as control; Con_ESC, normal uterine endometrial stromal cell as control. (g) Relative expression of miR-145 in Eu_EEC and Eu_ESC cells treated with circPVT1 overexpression (GV367-circPVT1) or circPVT1 knockdown (sh-circPVT1), respectively. The CNC group represented cells without any transfection. At least triplicate independent experiments were performed. All data were presented as mean ± SD. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001; NS: no significance.