LOXL2 Promotes Vasculogenic Mimicry By Upregulating IQGAP1 In Hepatocellular Carcinoma

Objective:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors that its incidence is increasing year by year and it has become the third most common cause of cancer death worldwide.Angiogenesis is an important step for tumor growth,invasion and metastasis.Therefore,antiangiogenic therapy has become a hot spot in the prevention and treatment of cancer.Vasculogenic mimicry refers to the unique capability to provide blood support and promote the growth of tumors which is different from endothelium-dependent blood vessels.VM is related to the poor prognosis but the mechanism is not clear.Immumohistochemical and CD31/PAS double staining was done to validate the expression of LOXL2? IQGAP1 and VM in 201 cases of HCC tissues.The relationship between LOXL2,IQGAP1,VM and clinical data was analyzed,and the correlation between LOXL2 and IQGAP1 was observed.The effects of LOXL2 and IQGAP1 on migration,invasion,proliferation and VM formation of HCC cells were studied by cell transfection,Western blotting and other cell experiments.Our study aims to study the mechanisms that how LOXL2 and IQGAP1 promote the development of HCC which could provide a new perspect for the treatment of HCC.Methods:1.A total of 201 HCC tissue specimens were collected from patients undergoing hepatectomy at the tumor Tissue Bank of Tianjin Cancer Hospital and all the diagnosis was confirmed by trained pathologists.Immumohistochemical was used to validate the expression of LOXL2? IQGAP1 and CD31/PAS double staining was used to analyze the presence of VM.Further,the relationship between LOXL2,IQGAP1 and clinical data ? VM ? survival was analyzed,and the correlation between LOXL2 and IQGAP1 was observed.2.Western blot was used to screen HCC cell lines with low expression and high expression of LOXL2.Using the plasmid transfection to control LOXL2 expression,and cultured the stable transfected cell lines.The effects of LOXL2 on the migration,invasion and VM formation of HCC cells were detected by scratch,migration,invasion and three-dimensional culture.The transfection efficiency and VM related molecules VE-vadherin,MMP2 and MMP9 were detected by western blot and immunofluorescence was used to identify the results.3.Plasmid transfection was used to transfer the IQGAP1 down regulated plasmid into the above cell which was LOXL2 up-regulating.While the IQGAP1 up plasmid was transferred into the cells which was LOXL2 downregulating.The effects of IQGAP1 on the functional recovery which was caused by the decreased LOXL2 were detected by scratch,migration,invasion,three-dimensional culture and Western blot.Co-IP was used to detect the relationship between LOXL2 and IQGAP1.4.Western blot was used to screen HCC cell lines with low expression and high expression of IQGAP1.Transfected with IQGAP1 plasmid,and cultured the cells.We used scratch,migration,invasion,three-dimensional culture,plate cloning,soft agar cloning and MTT proliferation assay to detect the effects of IQGAP1 on migration,invasion,VM formation and proliferation of HCC cells.Western blot was used to detect VM related molecules VE-vadherin,MMP2 and MMP9.Results:1.IHC results showed that LOXL2 was predominantly localized to the cytoplasm and/or nucleus and the location of IQGAP1 staining was mainly in the cytoplasm and/or cell membrane.LOXL2 and IQGAP1 were both high expression in 201 HCC tissues,and related with tumor grade,metastasis and VM.And besides,LOXL2 was positively correlated with the IQGAP1.Kaplan-Meier survival analysis showed HCC patients with LOXL2 or IQGAP1 overexpression have a poor prognosis and survival rate.2.Bel7402 and Hep G2 were screened as LOXL2 high expression and LOXL2 low expression through western blot.After transfected with LOXL2 interference plasmid in Bel7402(Bel7402 sh LOXL2),the expression levels of IQGAP1,VE-cadherin,MMP2 and MMP9 were declined while the ability of migration,invasion and VM formation were suppressed.After transfected with LOXL2 plasmid in Hep G2(Hep G2 LOXL2),the expression of IQGAP1,VE-cadherin,MMP2 and MMP9 were increased while the ability of migration,invasion and VM formation were promoted..3.Transfected with IQGAP1 plasmid in Bel7402 sh LOXL2,the expression of IQGAP1,VE-cadherin,MMP2 and MMP9 were increased while the ability of migration,invasion and VM formation were promoted.Transfected with IQGAP1 interference plasmid in Hep G2 LOXL2,the expression levels of IQGAP1,VE-cadherin,MMP2 and MMP9 were declined while the ability of migration,invasion and VM formation were suppressed.Co-IP results showed that LOXL2 could combined with IQGAP1.In summary,LOXL2 may affect the invasion,migration and VM by regulating the expression of IQGAP1.4.SMMC7721 and Hep G2 were selected as IQGAP1 high expression and IQGAP1 low expression.The expression of IQGAP1,VE-cadherin,MMP2 and MMP9 were increased while the ability of migration,invasion and VM formation were promoted after IQGAP1 pasmid were transfected with Hep G2.Plate cloning and soft agar assay and MTT proliferation showed that the up-regulation of IQGAP1 promoted the proliferation of Hep G2 cells.There is a reverse result after transfected with IQGAP1 interference plasmid in SMMC7721.5.The above results show that IQGAP1 plays an important role in the development of HCC.The up regulation of IQGAP1 can significantly promote the malignant biological behavior of HCC cells.Conclusion:1.LOXL2 and IQGAP1 are both high expression in HCC,and they are associated with tumor grade?metastasis and VM.HCC patients with LOXL2 and/or IQGAP1 overexpression have a poor prognosis and survival rate.LOXL2 expression is related to IQGAP1 expression.2.LOXL2 promotes migration ? invasion and VM formation by upregulating IQGAP1 in HCC cells.3.IQGAP1 can enhance the ability of migration,invasion,proliferation,VM formation to promote the progress of malignant biological behavior.