Background Forkhead box C1 (FOXC1), as a member of the FOX family, is important for promote HCC invasion and metastasis. FOX family protein lays a pivotal role in metabolism. ROS is involved in tumor progression and is associated with the expression of lots of transcription factors. We next explored the mechanism underlying FOXC1 modulating the metabolism and ROS hemostasis in HCC. Methods We used amino acids arrays to verify which metabolism is involved in FOXC1-induced HCC. The kits were used to detect the ROS levels in HCC cells with over-expression or down-expression of FOXC1. After identified the downstream target genes and candidate pathway which regulated by FOXC1 during HCC progression in vitro and in vivo, we used western blot, immunohistochemistry, bisulfite genomic sequencing, methylation-specific PCR, chromatin immunoprecipitation analysis and luciferase reporter assays to explore the relationship of FOXC1 and downstream genes. Moreover, the correlation between FOXC1 and target genes and the correlation between target genes and the recurrence and overall survival were analyzed in two independent human HCC cohorts. Results Here, we reported that FOXC1 could inhibit the cysteine metabolism and increase reactive oxygen species (ROS) levels by regulating cysteine metabolism-related genes, cystathionine γ-lyase (CTH). Overexpression of CTH significantly suppressed FOXC1-induced HCC proliferation, invasion and metastasis, while the reduction in cell proliferation, invasion and metastasis caused by the inhibition of FOXC1 could be reversed by knockdown of CTH. Meanwhile, FOXC1 upregulated de novo DNA methylase 3B (DNMT3B) expression to induce DNA hypermethylation of CTH promoter, which resulted in low expression of CTH in HCC cells. Moreover, low levels of ROS induced by N-acetylcysteine (NAC) which is an antioxidant inhibited the cell proliferation, migration, and invasion abilities mediated by FOXC1 overexpression, whereas high levels of ROS induced by L-Buthionine-sulfoximine (BSO) rescued the suppression results mediated by FOXC1 knockdown. Our study demonstrated that the overexpression of FOXC1 that was induced by the ROS dependent on the extracellular regulated protein kinases 1 and 2 (ERK1/2)- phospho-ETS Transcription Factor 1 (p-ELK1) pathway. In human HCC tissues, FOXC1 expression was positively correlated with oxidative damage marker 8-hydroxy-2′-deoxyguanosine (8-OHdG), p-ELK1 and DNMT3B expression, but negatively correlated with CTH expression. HCC patients with positive co-expression of 8-OHdG/FOXC1 or p-ELK1/FOXC1 or FOXC1/DNMT3B had the worst prognosis, whereas HCC patients who had positive FOXC1 and negative CTH expression exhibited the worst prognosis. Conclusion In a word, we clarify that the positive feedback loop of ROS-FOXC1-cysteine metabolism-ROS is important for promoting liver cancer proliferation and metastasis, and this pathway may provide a prospective clinical treatment approach for HCC.

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Alternative polyadenylation (APA), a post-transcriptional modification, has been implicated in many diseases, but especially in tumor proliferation. CFIm25, the 25 kDa subunit of human cleavage factor Im (CFIm), is a key factor in APA. We show that CFIm25 expression is reduced in human hepatocellular carcinoma (HCC), and its expression correlates with metastasis. Kaplan-Meier analysis indicated that CFIm25 is related to overall survival in HCC. Moreover, CFIm25 expression is negatively related to the metastatic potential of HCC cell lines. CFIm25 knockdown promotes cell invasion and migration in vitro, while overexpression of CFIm25 inhibits cell invasion and migration in vitro and inhibits intrahepatic and lung metastasis in vivo. Additional studies showed that CFIm25 disrupts epithelial-mesenchymal transition by increasing E-cadherin, that it inhibits HCC cell migration and invasion by blocking the p38 and JNK/c-Jun signaling pathways, and that CFIm25 knockdown increases the transcriptional activity of activating protein-1 (AP-1). These findings indicate that therapy directed at increasing CFIm25 expression is a potential HCC treatment.

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The underlying immunopathogenic mechanisms of autoimmune hepatitis (AIH) have not yet been well elucidated. An impairment in regulatory T cells (Tregs) is key to the development of AIH. Krüppel-like factors (KLFs) regulate a broad of cellular processes including immunocyte maturation. KLF14 may regulate Treg differentiation, but the biological functions remain far from elucidated. In this study, we identified the hepatic expression of KLF14 in human and murine liver diseases. Immune-mediated hepatitis was induced by concanavalin A (Con A). A KLF14 recombinant adenoviruses plasmid (Ad-KLF14) was constructed and injected into mice. Tregs were assessed by flow cytometry analysis; inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), were tested by enzyme-linked immunosorbent assay (ELISA). The expression of KLF14 was suppressed in a time-and dose-dependent manner. Changes in cytokine levels were consistent with the degree of hepatic injury. Overexpression of KLF14 protected the liver from immune-mediated damage in vivo. Ad-KLF14 transfection before Con A challenge increased the frequency of Tregs in liver mononuclear cells (MNCs), and suppressed the expression of cytokines. All of these improvements were completely abrogated after Treg deletion in vivo by intraperitoneal injection of a CD25 antibody. In conclusion, these data suggest that KLF14 plays an asyet unrecognized role in immune-mediated hepatitis mainly via induced Treg differentiation. Our findings extend the knowledge of the biological function of KLF14 to the autoimmune disease field, and indicate the possibility of KLF14 as a therapeutic target in AIH patients.