Fig. 7
miR-744 transferred by cancer cell-derived EVs represses the development of NSCLC by regulating the SUV39H1/Smad9/BMP4 axis in A549 cells. a Overexpression efficiency of Smad9 determined by RT-qPCR in A549 cells. b Western blot analysis validating the overexpression efficiency of Smad9 in A549 cells (* p < 0.05, vs. OE-NC-treated A549 cells). c Expression of miR-744, SUV39H1, Smad9, and BMP4 determined by RT-qPCR in OE-Smad9-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells. d Representative Western blots of SUV39H1, Smad9, and BMP4 and p-Smad9 and their quantitation in OE-Smad9-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells. e Viability of OE-Smad9-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells measured using CCK-8 assay. f Colony formation of OE-Smad9-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells measured using colony formation assay (* p < 0.05, vs. si-NC-transfected A549 cells treated with EVs from BEAS-2B cells; # p < 0.05, vs. si-NC-transfected A549 cells treated with EVs from H1299 cells). g–j Results of in vivo tumor growth. A549 cells with or without Smad9 overexpression were injected into mice to establish the xenograft model and the EVs were injected into the tumors 7 days after transplantation. Tumor volume (g) was measured every 3 days; and final tumor volume (h) and weight (i) were measured, averaged, and compared after 4 weeks. The expression of proliferation marker, Ki-67 (j), was measured by IHC staining images were taken (* p < 0.05, vs. mice injected with A549 cells treated with BEAS-2B-EVs; # p < 0.05, vs. mice injected with A549 cells treated with H1299-EVs). k Knockdown efficiencies of 3 siRNAs targeting BMP4 determined by RT-qPCR in A549 cells. l Western blot analysis validating the knockdown efficiencies of 3 siRNAs targeting BMP4 in A549 cells (* p < 0.05, vs. A549 cells transfected with si-NC). m Expression of SUV39H1, Smad9, and BMP4 in si-BMP4-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells. n Western blots of SUV39H1, Smad9, BMP4 and p-Smad9 and their quantitation in si-BMP4-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells. o Viability of si-BMP4-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells measured using CCK-8 assay. p Colony formation of si-BMP4-transfected A549 cells treated with EVs from BEAS-2B and H1299 cells measured using colony formation assay (* p < 0.05, vs. si-NC-transfected A549 cells treated with EVs from BEAS-2B cells; # p < 0.05, vs. si-NC-transfected A549 cells treated with EVs from H1299 cells). Data are summarized as mean ± standard deviation. Data from two groups were compared using unpaired t-test and data from multiple groups were assessed by one-way ANOVA with Tukey's tests. Data obtained at different time points were assessed by Bonferroni-corrected repeated measures ANOVA. Mice: n = 8/group. Each experiment was repeated three times