GSTZ1 Inhibit Hepatocellular Carcinoma Cells Migration And Invasion By Downregulation Of The TGF-? Signalling Pathway

Objective: Hepatocellular carcinoma(HCC)is the sixth most common human malignancy,and its high metastasis rate is an important cause of high mortality in HCC patients.Thus,in-depth exploration of its molecular mechanism is of great significance for the diagnosis and treatment of HCC.Studies have shown that hypertyrosinemia(HT1),obesity,diabetes and hepatic steatosis are prone to progression to HCC,suggesting that metabolic factors are a major cause of the development of HCC.Glutathione s-transferase Zeta 1-1(GSTZ1-1),a member of the GSTs superfamily,is involved in phenylalanine catabolism and detoxification of xenobiotic.Previous studies by our research group found that GSTZ1 deficiency can promote the proliferation of hepatoma cells and inhibit apoptosis.In order to further explore biological role of GSTZ1 in HCC,we found that GSTZ1 can inhibit migration and invasion of hepatoma cells,RNA-seq analysis show that up-regulation of GSTZ1 can significantly downregulate TGF-? signalling,and it is located in the center of pathway interaction networks.TGF-? signalling is one of the core signalling in driving HCC invasion and metastasis.These findings suggest that GSTZ1 may be associated with migration and invasion of HCC.The aim of this study was to clarify the relationship between GSTZ1 and migration and invasion of HCC,and to explore possible molecular mechanisms.Methods: 1.In the Huh7,SK-Hep1 and SNU449 cells,the effects of GSTZ1 overexpression or knockout on the migration and invasion of HCC cells were explored by Wound-Healing and Transwell 2.In the above cell model,q RT-PCR?immunofluorescence staining and Western Blot were used to detect the effects of GSTZ1 on m RNA and protein expression levels of EMT-related markers in hepatoma cells.3.In the above cell model,the regulation effect of GSTZ1 on TGF-? signaling pathway was detected by double luciferase activity assay,cytoplasmic and cytonuclear assay,Western Blot and immunofluorescence staining,respectively.4.In the GSTZ1 knockout cells,the expression of smad2 protein was knocked down using the transfection of shsmad2 plasmid,or the cells were treated with the TGF-? signalling inhibitor SB431542 to observe the changes in the migration and invasion capability and the proteins expression of vimentin and fibronection 5.Western blot was used to observe the correlation between GSTZ1 expression and p-Smad2/3 in paired human HCC and nontumour tissues 6.A nude mouse model of hematogenous metastasis of hepatoma cells was constructed by tail vein injection to observe the effect of GSTZ1 on the lung metastasis ability of hepatoma cells.Results: GSTZ1 could inhibit the migration and invasion of HCC cells.The results of q RT-PCR,Western blot and immunofluorescence staining showed that GSTZ1 could inhibit the epithelial mesenchymal transition of HCC cells.Double luciferase assay,the nuclear localization assay,and Western blot assay showed that GSTZ1 negatively regulated TGF-? signaling pathway.Intervention of the TGF-? signaling pathway with shsmad2,or SB431542,an inhibitor of the TGF-? signalling can reverse the increased migration and invasion abilities of HCC cells caused by GSTZ1 deficiency and enhance the capacity of epithelial-mesenchymal transition.We further analyzed the expression of GSTZ1 in clinical HCC and paracancer tissues,and found that the expression of GSTZ1 in liver cancer and paracancer tissues was negatively correlated with that of psmad2/3.In the lung metastasis model of nude mice,GSTZ1 inhibited the lung metastasis ability of HCC cellsConclusion: We firstly discovered that GSTZ1 inhibits invasion,migration,and epithelial-mesenchymal transition of HCC cells by downregulating the TGF-? signaling pathway in cells,nude mice,and clinical HCC tissue specimens.This study enriched the molecular mechanism of the development of HCC and may shed light on the diagnosis and treatment of HCC.