The Epigenetic Regulation Of Myeloid Zinc-Finger 1 And Its Association With Metallothionein 2A In Suppression Of Human Gastric Cancer Proliferation

Background and aims:Gastric cancer (GC) is one of the most common cancers, currently ranking second in cancer-related mortality worldwide. The 5-year survival rate of GC patients remains poor mainly due to the development of chemoresistance, or later diagnosis, raising an urgent need to develop more effective strategies for early detection and surveillance. Metallothionein 2A (MT2A) and NF-κB are both involved in carcinogenesis and cancer chemosensitivity. Blocking NF-κB activation in cancer cells has thus shown promisinganti-cancer effects.We previously showed a decreased expression of MT2A and IκB-α in human GC in association with poor prognosis of patients. Our recent studies provide the first evidence for epigenetic upregulation of MT2A in GC by diallyl trisulfide (DATS), a garlic-derived compound, and uncover the molecular mechanisms of the anti-GC activity of DATS as well as its ability to sensitize GC cells to the chemotherapeutic reagent docetaxel (DOC) through enhancing transcription of IκB-α to suppress NF-κB activation in GC cells. However, the precise link between MT2A and IκB-α remains elusive.The DNA sequence analysis of IκB-α gene promoter indicated that, myeloid zinc-finger 1 (MZF1), a member of the Kruppel family of zinc-finger proteins, may involve in the regulatory effect of MT2A on IκB-α in GC cells. We therefore speculated that MZF1, acting as a tumor suppressor in GC cells, might play a critical role in the NF-κB pathway in association with MT2A.In this study, we investigated the expression and epigenetic regulation of MZF1 in human GC cells and primary tumor tissues as well as its potential clinical application. We then accessed the functional role of MZF1 in GC tumorigenesis in relation to MT2A. In addition, we explored the interaction between MZF1 and MT2A in GC cells. Our study would delineate a mechanistic basis of MT2A-NF-κB signaling for anti-GC effect of MT2A and/or DOC, in which MZF1 may act as a key molecule and may be a chemosensitivity predictor in GC patients receiving chemotherpeutic treatment and a promising target for more effective treatment of GC.Methods:Expression and epigenetic modifications of MZF1 in human GC cell lines and primary GC tumors were analyzed by RT-qPCR, methylation-specific PCR, bisulfite sequencing and western blotting. Biofunction of MZF1 in GC was investigated by MTT, colony formation assay and transwell assay. Interplay between MZF1 and MT2A was conducted by co-immunoprecipitation (CoIP), chromatin immunoprecipitation-qPCR assay (ChIP-qPCR) and confocal.Results:1. The expression of MZF1 was decreased or silenced in human GC Cell lines and primary GC tumors associated with its promoter methylation and histone deacetylation. The expression of MZF1 in GC cell lines was restored by treatment with 5-aza-2’-deoxycytidine (5-AZA) and/or trichostatin A (TSA).2. Ectopic expression of MZF1 in GC cell line BGC823 significantly suppressed GC cell proliferation, migration and invasion. In contrast, knockdown of MZF1 provided the growth and metastasis advantage to an immortalized human gastric mucosal epithelial cell line, GES-1, which constitutively expressed MZF1.3. The association of MZF1 with MT2A was shown in BGC823 cells, either upon DATS and/or DOC treatment or ectopic expression of MT2A, or in GES-1 cells. Confocal analysis displayed a colocalization pattern of MZF1 with MT2A in the nuclear of BGC823 cells as well as GES-1 cells. ChIP-qPCR assay further showed the association of MZF1 or MT2A with the promoter region of IκB-α gene.4. MZF1 expression was increased after ectopic expression of MT2A in BGC823 cells, but diminished when knockdown of ectopic MT2A expression in the cells. However, knockdown of MZF1 expression in the ectopic MT2A expressed BGC823 cells showed less effect on the MT2A expression. Similar results were observed in GES-1 cells.5. DATS, in combination with DOC, induced MZF1 expression in GC cells, which was obviously inhibited by knockdown of the ectopic MT2A expression in BGC823 cells.Conclusion:1. MZF1 is epigenetic silenced in human GC Cell lines and primary GC tumors attributable to its promoter methylation & histone deacetylation.2. MZF1 suppresses growth and invasion of human GC cells.3. Tumor-suppressive role of MZF1 in GC is associated with MT2A in regulation of IκB-αgene.4. MT2A is involved in the regulation of MZF1 expression in human GC cells.5. The anti-GC effect of DATS may be attributable to its capacity to epigenetically upregulate MZF1 as well as MT2A.