Hypoxia-induced exosomes facilitate lung pre-metastatic niche formation in hepatocellular carcinoma through the miR-4508-RFX1-IL17A-p38 MAPK-NF-κB pathway

Abstract

Background: Hypoxia plays an important role in the lung metastasis of hepatocellular carcinoma (HCC). However, the process by which hypoxia promotes the formation of a pre-metastatic niche (PMN) and its underlying mechanism remain unclear. Methods: Exosomes derived from normoxic and hypoxic HCC cells were collected to induce fibroblast activation in vitro and PMN formation in vivo. The micro RNA (miR) profiles of the exosomes were sequenced to identify differentially expressed miRNAs. Gain- and loss-of-function analyses were performed to investigate miR-4508 function. Dual-luciferase, western blotting, and real-time reverse transcription-PCR analyses were used to identify the direct targets of miR-4508 and its downstream signaling pathways. To demonstrate the roles of hypoxic tumor-derived exosomes (H-TDEs) and miR-4508 in the lung metastasis of liver cancer, H22 tumor cells were injected through the tail vein of mice. Blood plasma-derived exosomes from patients with HCC who underwent transarterial chemoembolization (TACE) were applied to determine clinical correlations. Results: We demonstrated that H-TDEs activated lung fibroblasts and facilitated PMN formation, thereby promoting lung metastasis in mice. Screening for upregulated exosomal miRNAs revealed that miR-4508 and its target, regulatory factor X1 (RFX1), were involved in H-TDE-induced lung PMN formation. Moreover, miR-4508 was significantly upregulated in plasma exosomes derived from patients with HCC after TACE. We confirmed that the p38 MAPK-NF-κB signaling pathway is involved in RFX1 knockdown-induced fibroblast activation and PMN formation. In addition, IL17A, a downstream target of RFX1, was identified as a link between RFX1 knockdown and p38 MAPK activation in fibroblasts. Conclusion: Hypoxia enhances the release of TDEs enriched with miR-4508, thereby promoting lung PMN formation by targeting the RFX1-IL17A-p38 MAPK-NF-κB pathway. These findings highlight a novel mechanism underlying hypoxia-induced pulmonary metastasis of HCC.

Keywords: Hypoxia; MicroRNA; Pre-metastatic niche; Tumor-derived exosomes.

Figure 1

H-TDEs promote the activation of fibroblasts in vitro. (A) Transmission electron micrographs of TDEs. (B) Size of TDEs as determined by NTA. (C) Expression of characteristic exosomal markers TSG101, CD9, and CD63 as measured by WB; GAPDH band was present as a negative marker for TDEs. (D) Expression of fibroblast activation markers in MRC5 cells treated with equal quantities of N-TDEs or H-TDEs. (E) and (F) Relative expression of pro-inflammatory factor mRNA in MRC5 cells treated with equal quantities of N-TDEs or H-TDEs from HCCLM3 and MHCC-97H cells. (G) Migration of MRC5 cells treated with equal quantities of exosomes.; (H) Effect of GW4869 (GW) on the expression of fibroblast activation markers induced by CM of HCCLM3 cells. (I) Effect of plasma-derived exosomes from patients with HCC before and after TACE on the expression of fibroblast activation markers in MRC5 cells (NC: MRC5 cells cultured without plasma-derived exosomes; pA, pB, pC: MRC5 cells treated with plasma-derived exosomes from three patients). (J) Quantified gray value of each band presented in Fig. 1I; each bar represents the mean ± S.D.; each experiment was repeated at least 3 times; *P < 0.05, **P < 0.01.

Figure 2

H-TDEs promote PMN formation and lung metastasis. (A) Schematic diagram of the study design. (B) and (C) IF staining of α-SMA and FN in the lungs of the different treatment groups. (D) Modified Masson's staining of the lungs. (E) Percent of CD11b+Gr1+ MDSCs in the lung alveolar perfusion fluid of mice. (F) Double IF staining of CD11b and α-SMA in mouse lungs. (G) IVIS images of mice injected with H22-Luc cells. (H) Quantification of the fluorescence intensity of tumors. (I) and (J) HE staining and Ki67-IHC staining of lungs with metastatic tumors. Each bar represents the mean ± S.D. *P < 0.05, **P < 0.01.

Figure 3

Upregulated miR-4508 in H-TDEs mediates fibroblast activation. (A) High-throughput sequencing of exosomal miRNA from HCCLM3 and MHCC-97H cells. (B) Effect of transferred miR mimics on fibroblast activation. (C) MiR-4508, miR-4488, and miR615-3p levels in plasma-derived exosomes of patients with HCC before and after TACE. (D) MiR-4508 expression in TDEs and tumor cells under hypoxic or normoxic conditions. (E) Intake of HCCLM3-derived exosomal miRNA by MRC5 cells. (F) Effect of miR-4508 mimics on the expression of fibroblast activation markers. (G) Effect of an miR-4508 inhibitor on H-TDE-induced fibroblast activation. (H) Relative expression of inflammation-related mRNA in MRC5 cells transfected with miR-4508 mimics. Each bar represents the mean ± S.D; each experiment was repeated at least 3 times; *P < 0.05, **P < 0.01.

Figure 4

MiR-4508 stimulates fibroblast activation by targeting RFX1. (A) Target gene prediction of miR-4508 using bioinformatics tools. (B) GO analysis of the predicted genes. (C) Wild-type and mutated form of the binding site between miR-4508 and RFX (left); relative luciferase activity of RFX1-wt and RFX1-mut plasmid-transfected cells (right). (D) Expression of RFX1 and α-SMA in MRC5 cells transfected with miR-4508 mimics. (E) IF staining of FAP and RFX1 in MRC5 cells. (F) Effect of an miR-4508 inhibitor on H-TDE-induced downregulation of RFX1 in MRC5 cells. (G) Effect of RFX1 shRNAs on the expression of RFX1, FAP, and α-SMA in MRC5 cells. (H) Effect of an RFX1 overexpression plasmid on miR-4508 mimic-induced downregulation of RFX1 and upregulation of POSTN, FAP, and α-SMA in MRC5 cells. (I) Relative expression of inflammation-related mRNA in MRC5 cells transfected with RFX1 shRNA. (J) IF staining of RFX1 in the lungs of mice treated with TDEs. Each bar represents the mean ± S.D.; each experiment was repeated at least 3 times; *P < 0.05, **P < 0.01.

Figure 5

MiR-4508 facilitates the formation of PMN and lung metastasis of HCC. (A) Effect of miR-4508 on mouse lung fibroblasts. (B) Schematic diagram of miR-4508-rich TDE-mediated induction and analysis of PMN formation and tumor metastasis in mice. (C)-(E) Expression of RFX1 (C), FN (D), and α-SMA (E) in the lungs of mice treated by miR-4508-rich TDEs as determined by IF staining. (F) IF staining of CD11b+Ly6G+ cells in the pre-metastatic lungs of mice. (G) In vivo live imaging of the metastatic tumors in mice preconditioned with miR-4508-rich exosomes (n=4). (H) Quantification of fluorescence in mice at indicated time points. (I) Ex vivo fluorescence images of the lungs with metastatic tumors. (J) and (K) HE (J) and IHC (K) staining of the metastatic tumors in lungs. *P < 0.05, **P < 0.01; each experiment was repeated at least 3 times; *P < 0.05, **P < 0.01.

Figure 6

Downregulated RFX1 activates p38 MAPK-NF-κB in fibroblasts. (A) Effect of RFX1 knockdown on p38 MAPK activation and IκB degradation. (B) Levels of cytoplasmic and nuclear p65 in MRC5 cells transfected with RFX1 shRNA. (C) Effect of the NF-κB inhibitor DHMEQ on RFX1 knockdown-induced activation of fibroblasts and NF-κB. (D) Effect of the p38 inhibitor adezmapimod on RFX1 knockdown-induced activation of fibroblasts and p38 MAPK. (E) Effect of adezmapimod on RFX1 knockdown-induced upregulation of IL6 and IL8 mRNA. (F) IF staining of FAP and RFX1 in MRC5 cells treated with RFX1 shRNA with or without adezmapimod. (G) Effect of adezmapimod treatment on miR-4508-enriched exosome-induced metastatic tumor formation in mice. Each bar represents the mean ± S.D; each experiment was repeated at least 3 times; *P < 0.05, **P < 0.01.

Figure 7

RFX1 activates p38 MAPK by upregulating IL17A. (A) Relative expression of IL8 and IL17A mRNA in MRC5 cells treated with RFX1 shRNA with or without the p38 inhibitor adezmapimod. (B) Relative expression of IL8 and IL17A mRNA in MRC5 cells transfected with miR-4508 mimics and/or an RFX1 overexpression plasmid. (C) Effect of IL17A on p38 MAPK-NFκB activation in MRC5 cells. (D) Effect of an IL17A neutralizing antibody on RFX1 knockdown-induced activation of fibroblasts and p38 MAPK. (E) IF staining of IL17A in the lungs of mice treated with miR-4508-rich exosomes. Each bar represents the mean ± S.D.; each experiment was repeated at least 3 times; *P < 0.05, **P < 0.01.

Figure 8

RFX1 negatively correlates with metastatic lung cancer. (A) Kaplan-Meier survival analysis based on data from Surveillance, Epidemiology, and End Results (SEER) database to investigate median survival time of patients with HCC with or without lung metastasis. (B) Data analysis of RFX1 expression in patients with and without metastatic lung cancer from TCGE database. (C) and (D) PCA analysis of the correlation between RFX1 (C) and ACTA2 (D) expression and tumor signature score. (E) Proposed mechanism of H-TDE-induced lung PMN formation.