The Study Of HIF-1? Affected Vasculogenic Mimicry Formation In Hepatocellular Carcinoma Through LOXL2 Regulation In Hypoxic Tumor Microenvironment

Objective The incidence and mortality rates of hepatocellular carcinoma(HCC)have steadily increased in recent years.A hypoxic microenvironment is one of the most important characteristics of solid tumors which has been shown to promote tumor metastasis,epithelial-mesenchymal transition(EMT)and angiogenesis.EMT and vasculogenic mimicry have been regarded as crucial contributing factors to cancer progression.HIF-1? functions as a master transcriptional regulator in the adaptive response to hypoxia.Lysyl oxidases like 2(LOXL2)is a member of the lysyl oxidase family,which main function is to catalyze the covalent cross linkages of collagen and elastin in the extracellular matrix.Recent work has demonstrated that HIF-1? promotes the expression of LOXL2,which is believed to amplify tumor aggressiveness.LOXL2 has shown to promote metastasis and is correlated with poor prognosis in hepatocellular carcinoma.The purpose of our study is to explore the role of HIF-1? in progression and metastasis of hepatocellular carcinoma by promoting the expression of LOXL2 as well as the potential regulatory mechanism.Methods 1.To access HIF-1? and LOXL2 expression level in HCC samples,we performed immunohistochemistry staining on 201 HCC tissue sections,which were obtained from the Tumor Tissue Bank of the Tianjin Cancer Hospital and Tianjin General Hospital.The VM formation was examined by CD31/periodic acid-Schiff double staining.Then we analyzed correlations between the expression level of HIF-1?,LOXL2 and VM formation,sex,age,tumor size,stage,metastasis by two-tailed ?2 and Spearman tests.Lastly,prognostic values of HIF-1?,LOXL2 and VM were assessed by survival analysis.2.HIF-1? promoted invasion,metastasis,EMT and VM formation by promoting LOXL2 expression in vitro.1)Hep G2 and Bel7402 cells were treated with Co Cl2 and tranfected with sh HIF-1? plasmids to decrease the expression of HIF-1?.Western blot,q RT-PCR and IF were used to observe the regulation of HIF-1? on LOXL2.2)Invasion and migration assays were used to observe that HIF-1? promoted invasion and migration in HCC cells by regulating LOXL2 expression.And western blot and q RT-PCR assays were used to detect the EMT-related proteins.3)The ability of VM formation was tested using 3D culture assay.3.Then we performed Gene Chip? Human Transcriptome Array(HTA)2.0 in Hep G2 cells,Hep G2 cells overexpressed LOXL2 and Hep G2 cells treated with Co Cl2? Then quantitative real-time PCR was carried out to validate the changes in expression levels of the 12 genesResults 1.Correlation of HIF-1? and LOXL2 expression as well as VM with the clinicopathological parameters and prognosis of patients with hepatocellular carcinoma.Immunohistochmeical analysis showed that HIF-1? protein was highly expressed in 94 of 201 HCC sample tissues(46.77%).LOXL2 protein was highly expressed in 102 of 201 HCC sample tissues(50.75%).Elevated HIF-1? and LOXL2 expression was associated with an increase in tumor grade and VM(<0.05).Finally,Kaplan–Meier survival analysis indicated that the HIF-1? and LOXL2 high expression groups have poor overall survival compared with the low expression group(P<0.05).In 48 specimens(23.88%),VM was observed.Notably,our results demonstrated that the presence of VM correlates with the tumor grade,metastasis,and poor prognosis(P<0.05).To understand the clinical relevance of HIF-1? and LOXL2,we also explored the relationship between HIF-1? and LOXL2 by conducting a correlation analysis of HIF-1? and LOXL2 in 201 HCC patients.The data showed that HIF-1? expression was positively correlated with LOXL2 in the HCC samples(r=0.1990,P =0.004).2.HIF-1? significantly regulates LOXL2 expression and changes in LOXL2 expression counteract the aggressive phenotype and VM formation induced by HIF-1? in HCC cells.1)HIF-1? significantly regulates LOXL2 expression in HCC cells.The expression of HIF-1? was down-regulated under hypoxia condition,and the ability of invasion,migration and VM formation of HCC cells were decreased.The expression of E-cadherin was increased and the expression of vimentin was decreased.2)In hypoxic conditions,down-regulation of HIF-1? expression,LOXL2 the expression of LOXL2 decreased.After co-transfection of HIF-1? down-regulated plasmid and LOXL2 uptake plasmid,it was found that the ability of invasion,migration and VM formation of hepatocellular carcinoma cells were enhanced,and EMT was promoted.3)In hypoxia,the expression of HIF-1? increased,LOXL2 expression also increased.The down-regulation of LOXL2 attenuated the ability of invasion,migration and VM formation of hepatocarcinoma cells,and inhibited EMT.3.The molecules related to the role of LOXL2 in tumor hypoxia microenvironment were analyzed by Affymetrix HTA2.0 gene expression profile analysis.The gene expression profiles of Hep G2-LOXL2,Hep G2-hypoxia,Hep G2-control were analyzed,and the molecules related to the action of LOXL2 in tumor hypoxia microenvironment were found.Conclusions 1.In HCC sample tissues,HIF-1? protein and LOXL2 protein were highly expressed.HIF-1? and LOXL2 expression in HCC were positively associated with TNM stage,VM presence and worse prognosis.HIF-1? expression was positively correlated with LOXL2 in HCC samples.2.In HCC cells,HIF-1? can affect LOXL2 expression.HIF-1? can promoted HCC cells migration,invasion and VM formation by promoting LOXL2 expression.3.Expression profile analysis showed that some of the biological functions associated with the tumor were activated.Among the 12 genes,the expression of HLTF,CENPF,ASPM,NIPBL,SLK,PIK3C2 A,SMC2,TAF9 B and ATAD2 were consistent with the results of microarray analysis and the expression of AGL,LRPPRC and ATP5 E were inconsistent with the results of microarray analysis.