Fig. 6

miR-144/451a upstream CpG island or enhancer deletion modulates HCC progression in vivo by regulating macrophage polarization. a, b The CpG island or enhancer region upstream to miR-144/451a was deleted via CRISPR/cas9 system in luciferase-expressing Hepa1–6 cells, and cells were inoculated intrahepatically into C57BL/6 mice. Tumor development was evaluated by bioluminescence imaging 3 weeks after the injection (n = 5). c The tumor tissues were excised and weighed after mice were sacrificed (n = 5). d The expression of miR-144/miR-451a and their targets in tumor tissues from different groups in (a) was measured via qRT-PCR (n = 5). e Immunofluorescence staining for Ki67 and CD31 was performed to assess tumor cell proliferation and angiogenesis (n = 5). f-i FACS was performed to analyze the subsets and differentiation of TAMs (f, g) and phenotype of lymphocytes (h) from different groups, and the cell percentages were plotted and compared (i) (n = 5). j The tumor tissues were co-stained with F4/80 and CD206 and analyzed for expression of the M2-like macrophage marker, CD206 (n = 5). k The mRNA levels of macrophage polarization markers in sorted TAMs were measured via qRT-PCR (n = 5). Bars, means ± SEMs; *, P < 0.05; **, P < 0.01; ***, P < 0.001