Fig 1: Overexpression of miR-146b enhanced xenograft tumor growth but also reduced the invasion of tumor cells.a qPCR analysis of miR-146b in the mouse tumors. b Protein level of FBXL10 in primary cultured tumor cells. c The time and volume of tumors appeared after transplantation. d, e The represented tumors from nude mice after miR-146b overexpression. f Representative H&E-stained images of mouse tumors. 1 represents the internal structure of the human xenograft tumor, 2 represents the control tumor has infiltrated into the adipose tissue of the skin, 3 represents the control tumor has infiltrated into muscle tissue of the skin.Scar bars represent 100 μm. g The representative images of the transwell migration assay in the miR-146b overexpression groups (200×). h During the tumor growth, the tumor sizes in different groups (control, n = 3; control + DDP, n = 4; miR-146b, n = 4; miR-146b + DDP, n = 4). i, j Representative images of ovarian tumor cells with different concentrations of DDP/PTX treatment. k, l Immunoblot analysis of VIM and ZO-1 in mouse models. Scar bars represent 100 μm. m Proposed model for epigenetic regulation of the miR-146b in ovarian cancer. In normal ovary cells, the expression between miR-146b and FBXL10 allows for a mutual balance of each partner, further ensuring the normal function of ovary. In early-phase ovarian cancer cells, high miR-146b expression promotes cell proliferation but inhibits cell migration, following this, an unknown signal reduced the levels of miR-146b, further upregulated the FBXL10 expression, and then reduced Cyclin D1, VIM, and ZO-1 expression, thus resulting in tumor metastasis and chemotherapy failure. *p < 0.05, ** p < 0.01, ***p < 0.001
Fig 2: MiR-146b upregulated the expression of VIM and ZO-1 by targeting FBXL10.a Immunoblot analysis for VIM and ZO-1 in HO8910 and OVCAR-3 with the miR-146b overexpression. b Immunofluorescence staining of VIM in HO8910 and OVCAR-3 cells. Scale bars represent 50 μm. c Immunofluorescence staining of ZO-1 in HO8910 and OVCAR-3 cells. Cell nuclei were stained with DAPI. Scale bars represent 50 μm. d The expression level of VIM and ZO-1 in the FBXL10-knockdown cells and FBXL10-overexpressing cells. e ChIP analysis of FBXL10 binding at the VIM, ZO-1 locus in HO8910-FBXL10 cells. 1, 2 represent different promoter sites of VIM (1:-1183bp of VIM promoter,2: -153bp of VIM promoter), ZO-1 (1: -2453bp of ZO-1 promoter, -1953bp of ZO-1 promoter). f ChIP analysis of H3K4me3 binding at the VIM (VIM-P1: -1183bp of the of VIM promoter, VIM-P5: -153 bpof VIM promoter), ZO-1 (ZO-1-P2: -2453 bp of ZO-1 promoter, ZO-1 P5: -1953bp of ZO-1 promoter) locus in HO8910 cells transduced with miR-146b lentivirus. g H3K4me3 enrichment in sites of ZO-1 promoter with the overexpression of miR-146b. h Representative images of the miR-146b-overexpressing cells with overexpressing FBXL10 in HO8910 and OVCAR-3 cells. i Immunoblot analysis of VIM, ZO-1, and Cyclin D1 of the miR-146b-overexpressing cells with FBXL10 overexpression. j Representative images of immunohistochemical staining for VIM and ZO-1 in normal ovary tissues and ovarian tumor tissues (control samples, n = 8; cancer samples, n = 10). Scale bars represent 100 μm. ***p < 0.001
Fig 3: MiR-146b directly targeted FBXL10.a Schematic representation of the miR-146b and its targeting sites in the 3′-UTR of FBXL10, indicating the binding sites and the corresponding mutations. b miR-146b directly interacted with the FBXL10-3′-UTR. The 293T cells were co-transfected with the luciferase reporter vector, and pcDNA3.1-pri-miR-146b or control plasmids into 293T cells. The luciferase activity was measured after 24 h according to the protocol described in Materials and methods section. c Levels of mature miR-146b were detected using qPCR after transfection with miR-146b mimics or miR-146b inhibitors for 24 h. d, e mRNA (d) and protein (e) levels of FBXL10 in HO8910 and SKOV3 cells transfected with miR-146b mimics or miR-146b inhibitors. f Relative levels of H3K4me3 and H3K36me2 transfected with miR-146b mimics or miR-146b inhibitors. g, h The expression level of FBXL10 in ovarian cancer samples using qPCR (g) and immunohistochemical staining (h) (control samples, n = 21; cancer samples, n = 22). i Regression and correlation analysis between miR-146b and FBXL10 expression in ovarian cancers (n = 37). Scale bars represent 100 μm. *p < 0.05, **p < 0.01, and ***p < 0.001; ns not significant
Fig 4: Functional analysis of FBXL10 in ovarian cancer.a, b shRNA efficiency analysis in ovarian cancer cells by qPCR (a) and western blot (b). c Wild-type FBXL10-Flag overexpression and FBXL10-CxxC(dele)-Flag overexpression in HO8910 cells. Immunostaining was performed with Flag antibody. d Representative images of ovarian cancer cells transduced with FBXL10-shRNA or FBXL10 lentivirus. Scale bars represent 50 μm. e The representative images of the transwell migration assay, respectively (200×). f Quantification of the transmembrane migration abilities of the ovarian cancer cells. g The effects of FBXL10 on cell proliferation using cell count assay. h EdU assay was used to evaluate the proliferation of Ctrl and shRNA of FBXL10 cells. Representative images for EdU-positive ovarian cancer cells (red) and Hoechst-stained nuclei (blue) are shown. Scale bars represent 50 μm. i Quantification of the percentage of EdU-positive cells is shown. j Detection of Cyclin D1 protein expression of ovarian cancer cells after FBXL10 knockdown by western blot analysis. k Histograms showing the percentage of cellular proliferation inhibition rate of ovarian cancer cells with DDP and PTX treatment after FBXL10 knockdown. l The IC50 difference of DDP/PTX between Ctrl and FBXL10-knockdown cells. *p < 0.05, **p < 0.01, and ***p < 0.001; ns not significant
Fig 5: Silencing circ_0000006 ameliorates AD progression in mouse model. BALB/c mice were administrated with 3-aminopropionitrile fumarate salt (BAPN) for 28 days, and with angiotensin II for 16 days to induce AD in the animal model. In the model group, Adeno-associated virus (AAV) carrying scramble shRNA (sh-NC), or circ_0000006 shRNA was injected into the mice on the Day 36 (n = 6 in each group). (A) The measurement of body weight, systolic blood pressure (SBP) and diastolic blood pressure (DBP) in each experimental group. (B) H&E staining of the aorta wall tissue sections in each experimental group. (C) The quantification of aortic wall thickness in each group. (D) Verhoeff-Van Gieson (VVG) staining of the elastic fibers in aortic wall. (E) qRT-PCR analysis of circ_0000006, miR-483-5p and KDM2B expression in the aorta wall tissues. (F) Western blot analysis of contractile markers (α-SMA, SM22α, and MYH11) in the aorta wall tissues. *p < 0.05; **p < 0.01; ***p < 0.001; ****P < 0.0001
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