Molecular Mechanism Of Kidney-type Glutaminase Regulating Epithelial-mesenchymal Transition Of Hepatocellular Carcinoma

Objective: To research the effects and mechanism of kidney-type glutaminase(GLS1)on epithelial-mesenchymal transition(EMT)of hepatocellular carcinoma(HCC)in vitro and in vivo as well as clinical verification.Methods: HCC cell line HCCLM3 overexpressing GLS1,a key glycolytic enzyme of glutamine,was constructed by lentiviral vector PCDH-CMV-MCS-EF1-Puro GLS1.HCCLM3 cells with GLS1 knockdown were constructed by lentiviral vector LV-GLS-sg RNA.Cell migration and invasion were detected by the Transwell assay.Cell fusion speeds were measured by the scratch assay.Glycolysis of glutamine and expressions of epithelial and mesenchymal phenotype-related proteins were detected by Western blot.The expressions of matrix metalloproteinases MMP2 and MMP9 were detected by ELISA.Nude mice were subcutaneously implanted with HCCLM3-LV-GLS1 cells(Group A,GLS1 overexpression group),HCCLM3-sg RNA-GLS1 cells(Group B,GLS1 knockdown group)and HCCLM3 cells(Group C,control group)respectively.The effects of GLS1 expression on HCCLM3 cell tumorigenesis,distal metastasis and tumor growth were evaluated.The mice were sacrificed 5 weeks after tumor formation to compare the tumor size and metastasis in the liver and lungs.The expression of GLS1 and EMT related proteins in hepatocellular carcinoma tissues and adjacent tissues were detected by paraffin section immunohistochemistry.The correlation between these proteins was evaluated.The relationship between GLS1 expression,microvascular invasion and the prognosis of HCC were clinically verified.Results: HCCLM3-LV-GLS1 and HCCLM3-sg RNA-GLS1 cells were successfully constructed.Transwell assay showed that the migration and invasion capacities of HCCLM3-LV-GLS1 cells increased,but those of HCCLM3-sg RNA-GLS1 cells decreased.Scratch assay showed that HCCLM3-LV-GLS1 cells fused more rapidly.Western blot exhibited that the expression of epithelial marker E-cadherin decreased in HCCLM3-LV-GLS1 cells,but those of mesenchymal marker vimentin and transcription factor ZEB1 negatively regulating E-cadherin increased,indicating that the epithelial-mesenchymal transition of HCCLM3 cells was promoted.ELISA revealed that the expressions of MMP2 and MMP9 proteins in the GLS1 overexpression group increased with extended time.In vivo experiments showed that Group B had significantly lower tumor formation rate and smaller tumor volume.Tumor formation was unaffected in Group A which also had a similar tumor volume to that of Group C.With decreasing GLS1 expression,the expression of E-cadherin was up-regulated but that of vimentin was down-regulated.The expressions of key factors(Slug and ZEB1)in EMT-related signaling pathways were also up-regulated.Group A had a higher MMP2 expression level than that of Group B.GLS1 knockout group liver orthotopic xenograft experiments,suggesting that low metastasis rate of lung,and was significantly different with over expression group and control group.The clinical specimens of paraffin section immunohistochemical experiments showed that different GLS1 expression level,and EMT related proteins such as Vimentin,Snail,ZEB1 and ZO1 have a certain correlation between the expression of GLS1.The expression of Gl S1 was associated with tumor microvascular invasion and the prognosis of HCC patients.Conclusion: Inhibition of glutamine metabolism can be used as an effective strategy for HCC treatment,and GLS1 is a potentially new target for treating human malignancies.