Fig. 2
MIR181A1HG in CRC cell-derived EVs promotes CRLM by inducing liver premetastatic niche formation. (a) The expression levels of MIR181A1HG in 8 CRC cell lines and 1 normal colorectal mucosa cell line (FHC) were assessed by qPCR. (b) qPCR assays were used to evaluate MIR181A1HG expression in EVs derived from 4 CRC cell lines, HT29 and SW480, which possess relatively weak metastatic potential, and RKO and SW620, which possess relatively high metastatic potential. (c) HT29 and RKO CRC cells were transfected with MIR181A1HG overexpression or knockdown constructs, respectively; luciferase-labeled HCT8 CRC cells were then injected into the spleens of nude mice to construct mouse models of CRLM, followed by injection of EVs into the tail vein. The impact of EVs derived from CRC cells on CRLM in vivo was determined via spleen injection to construct mouse models of CRLM. c. Representative live images of LMs in nude mice. (d) Number of metastatic colonies in the livers of the nude mice from different groups determined by live imaging and HE staining. (e) Overall survival of each group of mice (n = 10 for each group). (f) Representative flow cytometry analysis of MDSCs. (g) Quantification of MDSCs in each group of mice. (h) The expression levels of Ly6G+, α-SMA and fibronectin in the LM tissues of each group were assessed by western blotting. (i) qPCR assays were used to determine the expression levels of MIR181A1HG in LX2 cells, which were incubated with conditioned medium (CM) from RKO and SW620 cells with or without EV depletion via an inhibitor of EV secretion (GW4869). (j) Representative immunofluorescence staining of LX2 cells that were cocultured with EVs derived from HT29, RKO, SW480, and SW620 cells pretransfected with Cy3-tagged MIR181A1HG (red). (nsP>0.05, *P < 0.05, **P < 0.01, and ***P < 0.001)