HMGA1 Promotes Macrophage Recruitment via Activation of NF-κB-CCL2 Signaling in Hepatocellular Carcinoma

Figure 3

CCL2 is required for HMGA1-induced macrophage recruitment. (a) A gene signature consists of 33 genes (AIF1, CCL1, CCL14, CCL23, CCL26, CD300LB, CNR1, CNR2, EIF1, EIF4A1, FPR1, FPR2, FRAT2, GPR27, GPR77, RNASE2, MS4A2, BASP1, IGSF6, HK3, VNN1, FES, NPL, FZD2, FAM198B, HNMT, SLC15A3, CD4, TXNDC3, FRMD4A, CRYBB1, HRH1, and WNT5B) was used to define infiltrating macrophages, and the correlation between CCL2 and macrophage was investigated by Spearman’s analysis. (b) HCC-LM3 and SMC-7721 cells were transfected with two specific siRNAs against HMGA1 or negative control (NC) siRNAs, and Western blotting analysis showed the protein level of HMGA1 in the HCC-LM3 and SMC-7721 cells. (c) Real-time qPCR analysis showed the mRNA level of CCL2 in the HCC-LM3 and SMC-7721 cells transfected with NC-siRNA or HMGA1-siRNA1/2. (d) Enzyme-linked immunosorbent assay (ELISA) showed the secreted level of CCL2 in the conditioned medium (CM) of HCC-LM3 and SMC-7721 cells transfected with NC-siRNA or HMGA1-siRNA1/2. (e and f) HCC-LM3 and SMC-7721 cells were treated with NC-siRNA or HMGA1-siRNA1/2 for 48 h, and CM was acquired and subjected for Transwell assay; the migratory ability of human monocytes isolated from PBMCs was evaluated after stimulation with indicated CM and recombinant human CCL2 protein for 12 h. The ANOVA followed by post hoc Tukey’s multiple comparison test was used for group comparisons. ; ; .

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