ObjectiveAutophagy participates in the progression of hepatocellular carcinoma (HCC) and the resistance of HCC cells to sorafenib. We investigated the feasibility of sensitising HCC cells to sorafenib by modulating miR-541-initiated microRNA-autophagy axis.DesignGain- and loss-of-function assays were performed to evaluate the effects of miR-541 on the malignant properties and autophagy of human HCC cells. Autophagy was quantified by western blotting of LC3, transmission electron microscopy analyses and confocal microscopy scanning of mRFP-GFP-LC3 reporter construct. Luciferase reporter assays were conducted to confirm the targets of miR-541. HCC xenograft tumours were established to analyse the role of miR-541 in sorafenib-induced lethality.ResultsThe expression of miR-541 was downregulated in human HCC tissues and was associated with malignant clinicopathologic phenotypes, recurrence and survival of patients with HCC. miR-541 inhibited the growth, metastasis and autophagy of HCC cells both in vitro and in vivo. Prediction software and luciferase reporter assays identified autophagy-related gene 2A (ATG2A) and Ras-related protein Rab-1B (RAB1B) as the direct targets of miR-541. Consistent with the effects of the miR-541 mimic, inhibition of ATG2A or RAB1B suppressed the malignant phenotypes and autophagy of HCC cells. Furthermore, siATG2A and siRAB1B partially reversed the enhancement of the malignant properties and autophagy in HCC cells mediated by the miR-541 inhibitor. More interestingly, higher miR-541 expression predicted a better response to sorafenib treatment, and the combination of miR-541 and sorafenib further suppressed the growth of HCC cells in vivo compared with the single treatment.ConclusionsDysregulation of miR-541-ATG2A/RAB1B axis plays a critical role in patients’ responses to sorafenib treatment. Manipulation of this axis might benefit survival of patients with HCC, especially in the context of the highly pursued strategies to eliminate drug resistance.
The forkhead box transcription factor A2 (FOXA2) is a member of the hepatocyte nuclear factor family and plays an important role in liver development and metabolic homeostasis, but its role in the metastasis of hepatocellular carcinoma (HCC) has not been evaluated. In this study, we found that the expression of FOXA2 was decreased in 68.1% (49/72) of human HCC tissues compared with their paired non-cancerous adjacent tissues. Clinicopathological analysis revealed that reduced FOXA2 expression was correlated with aggressive characteristics (venous invasion, poor differentiation, high tumor node metastasis grade). FOXA2 level was even lower in portal vein tumor thrombus compared with primary tumor tissues and correlated with epithelial-mesenchymal transition in HCC cells. Overexpression of FOXA2 inhibited migration and invasion of Focus cells, whereas knockdown of FOXA2 in HepG2 showed the opposite effect. Moreover, upregulation of FOXA2 suppressed HCC metastasis to bone, brain and lung in two distinct mouse models. Finally, we proved that FOXA2 repressed the transcription of matrix metalloproteinase (MMP)-9 and exerted its antimetastasis effect partially through downregulation of MMP-9. In conclusion, our findings indicate that FOXA2 plays a critical role in HCC metastasis and may serve as a novel therapeutic target for HCC.
Background
: Liver cancer stem cells (LCSCs) are responsible for the initiation, progression and chemoresistance of liver cancer. However, no agent targeting LCSC is available in the clinic to date. Here, we investigated the effects of targeting protein arginine methyltransferase 5 (PRMT5), an epigenetic regulator, on LCSCs and HCC using a novel PRMT5 inhibitor DW14800.
Methods
: Tumor spheroid formation culture was used to enrich LCSCs and assess their self-renewal capability. Human alpha-1-antitrypsin (A1AT) ELISA, acetylated low-density lipoprotein (ac-LDL) uptake, periodic acid-Schiff (PAS) reactions and senescence associated β-galactosidase (SA-β-gal) activity assays were performed to examine the differentiation status of HCC cells. The effects of DW14800 on HCC malignancy were assessed in HCC cell lines and on an HCC xenograft model in mice. Chromatin immunoprecipitation was applied to clarify the transcriptional regulation of HNF4α by PRMT5-mediated Histone H4 arginine-3 symmetrical dimethylation (H4R3me2s).
Results
: Quantitative real-time PCR revealed that the expression of PRMT5 was upregulated in LCSCs. DW14800 specifically decreased the symmetrical dimethylation of arginine residues in HCC cells. Treatment of DW14800 suppressed the self-renewal capacity of LCSCs while re-establishing hepatocyte-specific characteristics in HCC cells. DW14800 displayed antitumor effects in HCC cells
in vitro
and in xenograft HCC
in vivo
. Importantly, ChIP assay showed that PRMT5 and H4R3me2s bound to the promoter region of HNF4α gene, and DW14800 increased the expression of HNF4α via reducing the H4R3me2s levels and enhancing the transcription of HNF4α.
Conclusions
: Our data revealed the significance of targeting PRMT5 activity in LCSC elimination and HCC differentiation, and proposed that DW14800 may represent a promising therapeutic agent for HCC in the clinic.
These results suggest that FOXA2 induces the differentiation of gastric cancer and highlight FOXA2 as a novel therapeutic target and prognostic marker for human gastric cancer.
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