Fig 1: The direct target of exosomal miR-155 and miR-196a-5p in NSCLC is RASSF4. (A) The underlying targets of miR-155 and miR-196a-5p were predicted by online bioinformatics databases (TargetScan Release 7.0, David’s bioinformatics resources). (B) RT-qPCR assay was carried out to detect the mRNA expression of predicted targets in A549 cells treating M2-exosome. (C) The putative binding sites of RASSF4 for miR-155 and miR-196a-5p by TargetScan Release 7.0 database. (D) The luciferase activities in A549 transfected with wild-type or mutated 3'UTR of RASSF4 were measured. (E,F) The protein expression levels of RASSF4 in A549 cells transfected with negative control (NC), miR-155 mimic (miR-155 OE) or miR-155 inhibitor (miR-155 KD) or miR-196a-5p OE or miR-196a-5p KD were analyzed by western blot. Error bars represent standard deviations and asterisks show significant differences from corresponding control according to Student’s t-test (**P<0.01, ***P<0.001).
Fig 2: MiR-155 and miR-196a-5p promote migration, invasion and EMT of A549 cells through regulating RASSF4 expression. (A) The viability of A549 cells with the corresponding transfection was examined by CCK-8 assay. (B,C,D,E) The migration and invasion abilities were measured by Transwell assay in A549 cells (200× magnification). Scale bar =100 µm. (F,G) The protein expression levels of RASSF4, E-cadherin and vimentin were analyzed by western blot in A549 cells. Error bars represent standard deviations and asterisks show significant differences from corresponding control according to Student’s t-test (*P<0.05, **P<0.01, ***P<0.001).
Fig 3: M2 TAMs secret exosomal miR-155 and miR-196a-5p to promote NSCLC metastasis in vivo. (A) The bioluminescence images in different groups (Control group, M2 exosome group, M2/miR-155 knockdown exosome group, M2/miR-196a-5p knockdown exosome group, and M2/miR-155 knockdown + miR-196a-5p knockdown exosome group) were captured. (B) Bioluminescence signal of both groups at different time points. (C,D) HE staining for lung tissues in different groups and the number of metastases were analyzed (40× magnification). Scale bar =400 µm. (E,F) The protein expression levels of RASSF4 were analyzed in different groups. Error bars represent standard deviations and asterisks show significant differences from corresponding control according to Student’s t-test (*P<0.05, **P<0.01, ***P<0.001).
Supplier Page from Abcam for Anti-RASSF4 antibody