Fig. 4

CircPLEKHM3 controls cell proliferation and migration through binding miR-9 to regulate BRCA1, DNAJB6 and KLF4 expression. a A schematic illustration of two variants of DNAJB6. The 3’UTR of DNAJB6 variant 2 lacks the miR-9 binding sites. b A schematic illustration of the luciferase report vector of DNAJB6 variant 1 3’UTR wild type (WT) and mutant (Mut) (upper panel), and the relative luciferase activity of LUC–DNAJB6 variant 1 WT and Mut, and LUC–DNAJB6 variant 2 WT in A2780 cells transfected with miR-9 and control mimics (lower panel). The highlighted sequences represent miR-9 seed sequences or sequences that are complementary to miR-9 seed sequences. In the Mut vector, the miR-9 binding sites in the 3’UTR of DNAJB6 variant 1 were mutated on psiCHECK™-2 Vectors. c A schematic illustration of the luciferase report vector of KLF4 3’UTR WT and Mut types (upper panel), and the relative luciferase activity of LUC–KLF4 WT and Mut in A2780 cells transfected with miR-9 and control mimics (lower panel). In the Mut vector, the miR-9 binding sites in the 3’UTR of KLF4 were mutated on psiCHECK™-2 Vectors. d Immunoblotting analyses of DNAJB6a, DNAJB6b, KLF4 and BRCA1 in ovarian cancer cells transfected with circPLEKHM3 overexpression, mock vectors, circPLEKHM3 siRNAs and negative control (NC). e Kaplan–Meier survival analysis of KLF4 protein expression in ovarian cancer patients. KLF4 protein expression was measured by immunohistochemistry analysis. Patient samples were divided into two groups according to their KLF4 immunohistochemistry scores. f Representative images (20× magnification) of immunohistochemical staining for KLF4 in patients with a better prognosis (upper panel) and a worse progno (lower panel). g Representative images (20× magnification) of immunohistochemical staining for KLF4 and BRCA1 in immunodeficient mice injected with A2780 scramble and sh-circPLEKHM3 cells