Repression of WT1-Mediated LEF1 Transcription by Mangiferin Governs β-Catenin-Independent Wnt Signalling Inactivation in Hepatocellular Carcinoma

Abstract

Background/Aims: The development of hepatocellular carcinoma (HCC) is a complex process which involves deregulation of multiple signalling pathways. The hyper-activation of Wnt signalling promotes sustained expansion, invasion, and neovascularization of HCC. Mangiferin, a natural small molecule present in Mangifera indica L. has been shown to inactivate β-catenin, which is an indispensable regulator in Wnt pathway. Our study aimed to determine whether mangiferin has any inhibitory effect on HCC and examine how it modulates Wnt signalling. Methods: The tumour inhibitory effect of mangiferin was examined by in vitro cellular models and an in vivo orthotopic HCC implantation model. The genes responsible for mangiferin-mediated anti-HCC were delineated by polymerase chain reaction (PCR) microarray. The expression of target genes was further determined by quantitative PCR and immuno-blotting assays. The binding capacity of Wilms’ tumour 1 (WT1) to the lymphoid enhancer-binding factor 1 (LEF1) promoter was confirmed by chromatin immunoprecipitation-qPCR. Results: Oral administration of mangiferin inhibited orthotopic tumour growth. Cellular investigations confirmed the dose-dependent inhibition of mangiferin on HCC expansion and invasion. PCR array combined with Gene Ontology analysis revealed that the Wnt pathway was the predominant target of mangiferin and LEF1 was the most reduced gene in the Wnt pathway. Overexpression of LEF1 diminished repression of Wnt signalling and reduced proliferation activity in mangiferin-treated HCC cells. The mangiferin-mediated down-regulation of LEF1 was independent of β-catenin but associated with WT1 protein. WT1 knock-in in HCC cells further enhanced LEF1 expression. Chromatin immunoprecipitation assays revealed that the mangiferin induced repression of LEF1 was associated with decreased occupancy of WT1 on the LEF1 promoter. Conclusion: Our study identifies a novel mechanism of hepatocellular carcinoma inhibition through β-catenin-independent Wnt signalling, which is regulated by WT1-associated LEF1 repression. The study also highlights mangiferin as a promising Wnt inhibitor for HCC treatment.

Molecular Mechanisms Involved in Oxidative Stress-Associated Liver Injury Induced by Chinese Herbal Medicine: An Experimental Evidence-Based Literature Review and Network Pharmacology Study

“…Treatment strategies of traditional Chinese medicine (TCM) in alleviating different diseases, such as tumor proliferation, diabetic retinopathy, and liver dysfunctions, have had prolonged utilization and are generally considered as multitarget therapies with minimal adverse actions [ 8 , 9 , 10 , 11 ]. In contrast to the therapeutic effects, side effects of TCM, such as hepatotoxicity, have rarely been reported.…”

Suppression of lncRNA MALAT1 by betulinic acid inhibits hepatocellular carcinoma progression by targeting IAPs via miR‐22‐3p

“…Tissue samples were fixed with 10% formalin and dehydrated by various concentrations of alcohol, according to our previous study 34 . After embedding in paraffin, tissues were cut into sections of approximately 5 μm.…”

“…Cell migration was evaluated as previously described 34 . Briefly, cells were plated onto 6‐well plates (1 × 10 5 /well).…”

“…Tissue samples were fixed with 10% formalin and dehydrated by various concentrations of alcohol, according to our previous study. 34 After embedding in paraffin, tissues were cut into sections of approximately 5 μm. In the process of hematoxylin and eosin (H&E) staining, the slides were incubated in Mayer's hematoxylin and eosin Y solution (Sigma-Aldrich), and then the images were taken using an optical microscope (Olympus, Tokyo, Japan…”

“…Cell migration was evaluated as previously described. 34 Briefly, cells were plated onto 6-well plates (1 × 10 5 /well). After a monolayer-confluent cell layer formed, a sterile micropipette tip (200 μL) was used to create a wound (approximately 0.5 mm), followed by gently rinsing with PBS twice.…”