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Fig. 7 | Journal of Experimental & Clinical Cancer Research

Fig. 7

From: Down-regulation of the tumor suppressor miR-34a contributes to head and neck cancer by up-regulating the MET oncogene and modulating tumor immune evasion

Fig. 7

Administration of miR-34a-5p mimic inhibit tumors growth and PDL1 expressing tumor associated macrophages (TAMs). a 2 × 106 HTB-43 cells were injected to the tongue of nude mice. LNA-control mimic or LNA-miR-34a mimic were once a week (20 mg/kg; intraperitoneal) for two weeks, starting 10 days after tumor cell injections. Mice were euthanized after 20 days and epithelial cell and TAMs were isolated after tissue dissociation using a tumor dissociation kit and isolation of cells with magnetic beads (CD326 (EpCAM) for tumor epithelial cells and CD11b for TAMs). A representative histology of HTB-43 xenograft tumor is included. b Tumor volume was measured by caliper (N = 8 per group). c Level of miR-34a-5p was quantified in tumor lysate using a TaqMan microRNA assay (N = 8 per group). d Levels of MET were assessed in tumor lysates of mice that received the LNA control mimic or the LNA-miR-34a mimic. e, f Levels of miR-34a-5p was quantified in isolated tumor epithelial cells after isolation by Anti-EpCAM magnetic beads (n = 8 per group) and TAMs after isolation by Anti-CD11b magnetic beads (n = 8 per group). g The percentage fold over control of Vimentin expression in mice that received the LNA-control miR or LNA-miR-34a-5p mimic (n = 6 mice per group) measured by qRT-PCR. h Levels of Vimentin protein expression were quantified in tumor cells with rat anti-mouse vimentin APC-conjugated antibody. Filled histogram represents the mice that received LNA-control miR, and open histogram represents the mice that received LNA-miR-34a-mimic. i Percentage of PDL1 expressing TAMs (defined as CD45+CD11b+F4/80+ cells) were quantified by flow cytometry (n = 8 per group). j Luciferase activity in cells transfected with miR-34a-5p mimic or a control miR mimic and a luciferase reporter construct encoding the luciferase gene fused either to the a mutated PDL1 3′ UTR (MUT) or a wild-type PDL1 3′ UTR (WT) in RAW 264.7 cells. Data represent results of at least three independent experiments. * indicates p < 0.05. Data is presented as mean ± SD. * indicates p < 0.05; *** indicates p < 0.001

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