CCL2 is Upregulated by Decreased miR-122 Expression in Iron-Overload-Induced Hepatic Inflammation

Iron overload (IO) is accompanied by hepatic inflammation. The chemokine (C-C motif) ligand 2 (CCL2) mediates inflammation, and its overexpression is associated with IO. However, whether IO results in CCL2 overexpression in the liver and the underlying mechanisms are unclear.

We subjected mice to IO by administering intraperitoneal injections of dextran-iron or by feeding mice a 3% dextran-iron diet to observe the effects of IO on miR-122/CCL2 expression through real-time qPCR and Western blot analysis. We also used indicators, including the expression of the inflammatory cytokine, the inflammation score based on H&E staining and the serum content of ALT and AST to evaluate the effects of IO on hepatic inflammation. Meanwhile, we observed the effects of vitamin E on IO-induced hepatic inflammation. In cells, we used 100 µΜ FeSO4 or 30 µΜ Holo-Tf to produce IO and observed the roles of miR-122 in regulating CCL2 expression by using miR-122 mimics or inhibitors to overexpress or inhibit miR-122. Then, we used a dual-luciferase reporter assay to prove that miR-122 regulates CCL2 expression through direct binding to its complementary sequence in the CCL2 mRNA 3'UTR.

IO induces the downregulation of miR-122 and the upregulation of CCL2, as well as inflammatory responses both in vitro and in vivo. Although IO-induced oxidative stress is eliminated by the antioxidant vitamin E, IO-induced hepatic inflammation still exists, which probably can be explained by the fact that vitamin E has no effects on the miR-122/CCL2 pathway. In in vitro experiments, the overexpression and inhibition of miR-122 significantly reduced and increased CCL2 expression, respectively. The dual-luciferase reporter assay indicates that miR-122 binds CCL2 mRNA 3'UTR.

We propose the roles of miR-122/CCL2 in IO-induced hepatic inflammation. Our studies should provide a new clue for developing clinical strategies for patients with IO.