The Role Of Nrf2 In The Hypoxia Induced Migration And Invasion And Vasculogenic Mimicry In Hepatocellular Carcinoma

ObjectiveHepatocellular carcinoma(HCC)is one of the most common malignant tumors of digestive system in China.The clinical prognosis of HCC is generally poor as a result of its malignance and metastasis.Hypoxic microenvironment is an important driving force for tumor development,which the molecular mechanism involved is not clear As we all known,Nrf2 is a nuclear transcription factor that mainly regulates the redox homeostasis of the cells.It has been reported that Nrf2 is highly expressed in a variety of malignant tumors,involving in the proliferation and metastasis in tumor cells However,the molecular mechanism of Nrf2 in the metastasis and invasion in HCC is still unknown.The formation of vasculogenic mimicry(VM)which is a kind of vascular pattern independent of endothelial cells has been found that can promote the metastasis in HCC.In this study,we aimed to reveal the relationship between Nrf2 and VM formation under hypoxia.The mechanism studied thoroughly both Nrf2 and the metastasis in HCC may provide a novel idea for HCC disease in diagnosis,treatment and prognosis.Methods1.HCC-LM3 and Bel-7402 were selected and CoCl2 added for the hypoxia model Wound-healing assay,invasion assay and Matrigel three-dimensional culture were used to detect the ability change in migration,invasion and tube formation in condition of hypoxia in HCC.The expression of Nrf2 and VM related factors HIF-1α,MMP2,MMP9 and VEGF was detected by Western blot and Immunofluorescence in the condition of hypoxia in HCC2.We selected four HCC cell lines containing HCC-LM3,Huh7,MHCC-97H,and MHCC-97L.The constitutive expression of Nrf2 was detected by Western blot in four HCC cells and two of them chose for experiment.The objective cell lines were transfected with Nrf2 plasmids packaged by lentivirus.We established the stable HCC cell lines transfected with Nrf2 upregulation and downregulation expression plasmids via adding puromycin,which were verified by Western blot3.Through wound-healing assay,invasion assay and Matrigel three-dimensional culture,we explored the role of Nrf2 in the migration and invasion ability,tube formation ability in HCC under hypoxia.The VM related factors MMP2,MMP9,VEGF,HIF-1α,Twist-1 and VE-cadherin were tested by Western blot after transfecting Nrf2 plasmids in the condition of hypoxia4.We selected Huh7 cell line stably transfected with sh-Nrf2 plasmid and control plasmid and SUMO inhibitor 2-D08 was added under hypoxia condition.Through wound-healing assay,invasion assay and Matrigel three-dimensional culture,we explored the role of SUMO inhibitor 2-D08 in the migration and invasion and tube formation ability in HCC under hypoxia.The expression of Nrf2 was tested by Western blot after adding SUMO inhibitor 2-D08 in the condition of hypoxiaResults1.HCC-LM3 and Bel-7402 were both treated in normoxic and hypoxic environments.In hypoxic condition,the results of migration and invasion experiments and Matrigel three-dimensional culture showed enhanced ability of migration and invasion and tube formation in hepatocellular carcinoma cells compared with normoxic(P<0.05).With treatment of CoCl2 in HCC,compared with normoxic condition,the results of Western blot and Immunofluorescence assay showed that the expression of Nrf2 was enhanced under hypoxia condition(P<0.005).The expression of VM related factors HIF-1α,MMP2,MMP9 and VEGF in hepatocellular carcinoma cells increased under hypoxic condition detected by Western blot(P<0.05)2.The expression of Nrf2 in four hepatocellular carcinoma cell lines(HCC-LM3,Huh7,MHCC-97H,MHCC-97L)was detected by Western blot,and the result showed the expression of Nrf2 was the highest in the Huh7 cell line,whereas the expression of Nrf2 was the lowest in the HCC-LM3 cell line.Nrf2 overexpression plasmid was transfected into HCC-LM3 cell line,while sh-Nrf2 plasmid was transfected into Huh7 cell line.Both HCC cell lines added puromycin for screening the stable transfected cells examined by Western blot.Western blot showed that the expression of Nrf2 in HCC-LM3 cell line transfected with the Nrf2 plasmid increased compared with the control(P<0.05),while reduced expression of Nrf2 in the Huh7 cell line with sh-Nrf2 plasmid(P<0.05).3.The results of wound-healing assay,invasion assay and Matrigel three-dimensional culture showed that HCC-LM3 cell line with overexpressing Nrf2 had a higher ability in the migration and invasion and tube formation than those of the control under hypoxia(P<0.05).However,compared with the control,Huh7 cell line transfected with sh-Nrf2 plasmid had a weaker ability in the migration and invasion and tube formation under hypoxia(P<0.05).Western blot showed that the expression of VM-related factors MMP2,MMP9 and VEGF in HCC-LM3 cell line transfected with Nrf2 overexpression plasmid was higher compared with the control under hypoxia(P<0.05).In contrast,the expression of MMP2,MMP9 and VEGF in Huh7 cell line with knocking down Nrf2 was lower than those of the control in the condition of hypoxia(P<0.05).4.After adding SUMO inhibitor 2-D08,the wound-healing assay result showed that 2-D08 decreased the migration rate under hypoxia condition in HCC(P<0.05),and the migration rate was negatively correlated with the concentration of 2-D08.While knocking out the expression of Nrf2 in the Huh7 cell line and adding inhibitor 2-D08,the migration rate did not change significantly at the same time point under hypoxia(P>0.05).The invasion assay and Matrigel three-dimensional culture showed that SUMO inhibitor 2-D08 weakened the ability in the invasion and tube formation in HCC under hypoxia(P<0.05),While knocking out the expression of Nrf2 in the Huh7 cell line and adding inhibitor 2-D08,the numbers of cells penetrating and tube formation in HCC did not change significantly under hypoxia(P>0.05).The result of Western blot showed that SUMO inhibitor 2-D08 did not affect the expression of Nrf2 protein(P>0.05).Conclusions1.Hypoxia increases the ability of migration and invasion and tube formation in HCC.2.Hypoxia can promote the expression of the VM related factors HIF-1α,MMP2,MMP9 and VEGF in HCC.3.In condition of hypoxia,Nrf2 can promote the migration and invasion and tube formation in HCC.4.Nrf2 can promote the expression of the VM related protein MMP2,MMP9 and VEGF in HCC under hypoxia.5.Inhibition of SUMO modification of Nrf2 reduces the migration,invasion and tube formation in HCC under hypoxia.6.SUMO inhibitor 2-D08 do not affect the protein level of Nrf2 in HCC under hypoxia.