Fig 2: miR-6836 is positively related to stemness signature in CSC and platinum resistance in EOC patients. a Schematic diagram presenting the classification criteria of chemotherapeutic resistance. b Left: Heat map representing the expression levels of miRNAs in EOC tissues from 11 patients sensitive to chemotherapy and 11 resistant patients. Normalized expression levels are represented by color key; higher (yellow) or lower (blue). Right: Table listing miRNAs with significant changes (Fold change = 2.0). c Stemness indices (mRNAsi score) of OV datasets and match TCGA normal data extracted by OCLR machine-learning algorithm derived from TCGA RNA-sequencing data. d Heat map of miRNA profile in SKOV3 attached cells and spheroids. Normalized expression levels are represented by colored bars; higher (red) or lower (blue). e Venn diagram of miRNAs up-regulated in chemotherapy resistant EOC patients compared with sensitive patients and SKOV3 spheroids than attached cells. f RT-qPCR analysis of miR-6836 expression in EOC tissues from chemotherapy sensitive patients (Sensitive, n=57) and resistant patients (Resistant, n=30). g RT-qPCR analysis of miR-6836 in serum samples from EOC patients (EOC, n=65) and healthy individuals or patients with benign ovarian tumor (Non-cancer, n=39). h Kaplan-Meier survival curves analysis of OS in EOC patients with different miR-6836 expression level from Kaplan Meier plotter (KMplot, http://kmplot.com/analysis). i RT-qPCR analysis of miR-6836 levels in EOC cells lines. j RT-qPCR analysis of miR-6836 levels in CAOV3/SKOV3 attached cells, spheroids and reattached cells. k-l A2780/OVCAR3/SKOV3 cells transfected with mimic-miR-6836 (mimic) or mimic-NC (mi-NC) and A2780/CAOV3 cells transfected with inhibitor-miR-6836 (inhibitor) or inhibitor-NC (in-NC). k Tumor spheroids formation assay to present the stemness of SKOV3 treated with mimic/mi-NC or A2780 cells with inhibitor/in-NC. Scale bar, 200 µm. The upper images show representative spheroids, and quantitative analyses are presented as histograms below. l Western blot analysis of Oct4, Nanog, CD44 and Sox2 expression in OVCAR3/A2780 treated with mimic/mi-NC or A2780/CAOV3 with inhibitor/in-NC. Results are presented as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.

Fig 3: Intercellular transfer of miR-6836 by exosomes disseminates cisplatin resistance. a Representative CAOV3 exosome image under electron microscopy. Scale bar, 100 nm. b Nanoparticle tracking analysis measuring the diameter of CAOV3 exosomes. c Western blotting analysis of exosomal protein markers, CD63 and TSG101 expression in exosomes (EXO) and culture medium after exosome purification (CM) gathered from CAOV3 and OVCAR3 culturing. d Exosome internalization: CAOV3 exosomes (Green, PKH67-labeled) containing biotin-miR-6836 (Red, Streptavidin/RBITC-stained) were uptake by OVCAR3/SKOV3 (Phase contrast) cells. Scale bar, 10 μm. e CCK8 assay to evaluate the cell viability of OVCAR3/SKOV3 treated with inhibitor/in-NC and CAOV3 exosomes and further treated with cisplatin at various concentrations. IC50 calculation to evaluate the effect of exosomes on transferring the cisplatin sensitivity of EOC cells by SPSS. f Immunofluorescence assay for γ-H2AX and 53BP1 expression representing DNA damage caused by cisplatin treatment for 24h. Scale bar, 10 μm. g Transwell assay to analyze invasion and migration ability of OVCAR3/SKOV3 treated with inhibitor/in-NC and CAOV3 exosomes. Representative images of transwell assay are shown on the left and quantitative analyses are presented as histograms. h Western blot analysis of DLG2, p-Yap1, Yap1, Bax, Bcl2, Oct4, Nanog, CD44, Sox2 and β-actin expression in OVCAR3/SKOV3 treated with inhibitor/in-NC and CAOV3 exosomes. Results are presented as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.

Fig 4: miR-6836 increases cisplatin resistance by repressing DLG2. a CCK8 assay to evaluate the cell viability of A2780/CAOV3 transfected with in-NC and si-NC, inhibitor and si-NC, or inhibitor and si-DLG2 and further treated with cisplatin at various concentrations. IC50 calculation to evaluate the effect of knocking down DLG2 on the basis of inhibitor on the sensitivity of EOC cells to cisplatin by SPSS. b Immunofluorescence assay for ?-H2AX and 53BP1 expression representing DNA damage caused by cisplatin treatment for 24h. Scale bar, 10 µm. c Western blot analysis of ?-H2AX expression in A2780/CAOV3 treated with inhibitor/in-NC and si-DLG2/si-NC. d Cell growth ability analysis of A2780/CAOV3 transfected with inhibitor/in-NC and si-DLG2/si-NC for 24h using the xCELLigence RTCA-MP system. e Colony formation assay to assess the effect of knocking down DLG2 on the basis of inhibitor on cell growth and colony formation ability of EOC cells. Representative images of colony formation assay are shown as above and quantitative analyses are presented as histograms as below. f Transwell assay to analyze invasion and migration ability of A2780/CAOV3 transfected with inhibitor/in-NC and si-DLG2/si-NC. Representative images of transwell assay are shown on the left and quantitative analyses are presented as histograms. g Western blot analysis of DLG2, p-Yap1, Yap1, Bax, Bcl2, Oct4, Nanog, CD44, Sox2 and ß-actin expression in A2780/CAOV3 treated with inhibitor/in-NC and si-DLG2/si-NC. Results are presented as mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.