Fig. 3

miR-338-3p overexpression suppressed granulosa cell proliferation. (A) The KGN cell line was seeded in 96-well plates and transfected with miR-338-3p mimics or negative-control (NC) mimics, the OD₄₅₀ values were measured at 0, 24, 48, and 72 h using the CCK-8 Kit, and cell-growth curves were plotted. (B) The KGN cells were transfected with miR-338-3p inhibitors or NC inhibitors; the OD₄₅₀ values were measured at 0, 24, 48, and 72 h; and the cell-growth curves were plotted. (C-F) Human primary ovarian GCs were transfected with miR-338-3p mimics/inhibitor or their NCs, in order to manipulate the expression of miR-338-3p. After 48 h of further culture, the cellular proteins were extracted and the western blotting analysis was performed to detect the protein-expression levels of Bcl2 (C) and p53 (E) in each group, and to identify inter-group differences in terms of Bcl-2 protein expression (D) and p53 protein expression (F). The GAPDH protein expression was used as the internal reference control. (G) Primary human ovarian GCs were inoculated on cell slides, and the cells were transfected for 48 h with miR-338-3p mimics/inhibitor or their NCs to manipulate the expression of miR-338-3p. Subsequently, the cells were fixed for immunofluorescence staining to analyse the expression of the proliferation-associated nuclear antigen, Ki-67. The blue fluorescence indicates nuclear staining by DAPI. The green fluorescence represents the proliferation-associated nuclear antigen, Ki-67. Scale bar: 50 μm. The histogram shows the ratio of target antibody/DAPI mean fluorescence intensity (H, I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001