Fig. 5.

miR-550a-3-5p, down-regulated by E6, inhibited YAP/CCL2 to suppress M2 macrophages polarization and M2 macrophages-induced EMT. a, b Relative expressions of miR-550a-3-5p in E6/7-knockdown HPV-positive OSCC cells or E6-overexpressed HPV-negative OSCC cells by qRT-PCR. Western blot showed the corresponding protein levels of E6 or E7. c Levels of CD163 in PMA-THP-1 cells by qRT-PCR after exposure to CM from differentially transfected UPCI:SCC090 and UM-SCC-47 cells for 24 h. d mRNA and protein levels of YAP and TAZ in miR-550a-3-5p-overexpressed HPV-positive OSCC cells using qRT-PCR and Western blot. e Schematic representation of the Vector containing 3’UTR region. Three predicted binding sites for miR-550a-3-5p within YAP 3’UTR wild type (WT) and the mutated sequence (MUT) were indicated in red. Relative activity of luciferase reporters was significantly suppressed in cells co-transfected with WT binding site vectors of YAP 3’UTR in the presence of miR-550a-3-5p mimic. f mRNA levels of CCL2, TLR9, CXCL5 and CSF1 in YAP-inhibited and miR-550a-3-5p-overexpressed HPV-positive OSCC cells using qRT-PCR. Western blot showed protein levels of YAP after siRNA-mediated knockdown. g mRNA and protein levels of CCL2 in HPV-positive OSCC cells after treatment with verteporfin at a dose of 10 μM for 24 h (DMSO was used as a negative control). h mRNA and protein levels of CCL2 in UPCI:SCC090 and UM-SCC-47 cells co-transfected with miR-550a-3-5p and YAP-5SA. i Levels of CD163 in PMA-THP-1 cells by qRT-PCR after exposure to CM from different UPCI:SCC090 and UM-SCC-47 cells for 24 h. Recombinant human CCL2 was added to CM at 500 ng/mL before inducing PMA-THP-1 cells. j Levels of E-cadherin and Vimentin in differentially transfected HPV-positive OSCC cells after co-culturing with their corresponding TAMs for 48 h by qRT-PCR and Western blot. All assays were carried out in triplicate. Results were presented as means ± SD. *P<0.05, **P<0.01, ***P<0.001