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. 2021 Mar 3;20:46. doi: 10.1186/s12943-021-01343-5

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — miR-144/miR-451a regulate macrophage polarization by targeting HGF and MIF in HCC cells. a, b The abundances of secreted cytokines were determined by cytokine antibody array using sera of HCC patients (a) (n = 36) and supernatant of control HCC cells or those overexpressing miR-144 or miR-451a (b) (n = 3). c The targets of miR-144 or miR-451a were predicted using different online tools and classified according to the protein subcellular localization. The extracellular target proteins were selected. d The production of HGF and MIF was detected in control and miR-144/miR-451a-overexpressing HepG2 cells using ELISA (n = 5). e Bioinformatic prediction identified HGF as a target of miR-144 and MIF as a target of miR-451a. f Reporter assay was performed to validate that miR-144 inhibited luciferase expression from a construct harboring a 3’UTR of HGF or MIF (n = 6). g, h Hepa1–6 cells were subjected to lentivirus-mediated overexpression of miR-144 or seed sequence-mutated miR-144/miR-451a, followed by qRT-PCR (g) and Western blot (h) analyses for the expression of HGF or MIF (n = 6). i The correlation between the levels of HGF mRNA and miR-144 or the levels of MIF and miR-451a in clinical HCC samples was analyzed (n = 125). j Paired clinical para-tumor and HCC tissues were used to determine the levels of HGF and MIF via qRT-PCR (n = 125). HCC patients were grouped according to the levels of HGF or MIF in tumor tissues and were subjected to survival analysis (n = 125). k, l miR-144- or miR-451a-overexpressing Hepa1–6 cells were further modified to express HGF or MIF with WT 3’UTR or the miRNA-binding site-mutated 3’UTR, and the supernatant was prepared and used for culture of BMDMs. The expression of polarization markers (k) (n = 6) and the generation of NO (l) by BMDMs were determined. m BMDMs cultured in conditioned media as described in (k) were further cocultured with CD3⁺ T cells. The activated T cells were then cocultured with Hepa1–6 cells at different ratios of cell numbers. Bars, means ± SEMs; *, P < 0.05; **, P < 0.01; ***, P < 0.001