Affection Of Leucine-rich ?2-glycoprotein-1 On Tumorigenesis And Angiogenesis In Head And Neck Squamous Cell Carcinoma And Its Mechanisms

Part I:Objective:To detect the expression of LRG1 in HNSCC and to explore its correlation with clinicopathological features of the HNSCC cases.Methods:The expression of LRG1 in cancer and normal tissues were assessed by immunohistochemical staining and western blotting.Tumor tissue samples were classified by clinicopathological characteristics such as age,gender,tumor loacation,histologic grade or clinical stage,to compare the difference of LRG1 expression.ELISA assay was applied in detection of serum LRG1 in HNSCC patients and normal controls.HNSCC patients were classified by tumor location or clinical stages to compare serum LRG1 concentrations.The ROC curve analysis was performed to determine the clinical usefulness of LRG1 for diagnosing HNSCC.Results:Compared to normal tissues,there was a significant increase in CES,assessing LRG1 staining,in the tumor tissues.The CES became higher with the enlargement of tumor size or the increase in malignancy degree.There was not significant difference between T3 and T4,or between Grade 2 and Grade 3,in LRG1 expression.There were no significant associations between LRG1 protein expression and clinicopathological parameters such as age,gender,tumor location,N stage or clinical stage.Serum LRG1 of HNSCC patients has an average level of 62.4 ?g/ml,0.5 times higher than that in normal controls.There were no significant associations between serum LRG1 level and clinicopathological parameters such as tumor location,occurance or clinical stage.When setting the serum CLRG1 of 60.66?g/ml as a threshold,specificity of diagnosis for HNSCC,LSCC,early or advanced HNSCC was 93.8%or higher,whereas sensitivity was from 46.2%to 71.4%.LRG1 was overexpressed in 75%(15/20)of the HNSCC patients.Conclusion:LRG1 was overexpressed in HNSCC,and the level increased with the enlargement of tumor size or the increase in malignancy degree,showing its involvment in regulation in HNSCC genesis and development.Meanwhile,there is a clinical value for the diagnosis of HNSCC to determine serum LRG1 level.Part ?:Objective:to explore the affection of LRG1 on HNSCC cell growth and its mechanisms.Methods:The expression of LRG1 in different cell lines were assessed by western blotting.LRG1 gene was silenced by RNA interference technology.Lentiviral vector plasmids were used to establish new HNSCC cell lines,which may overexpress LRG1.Effective siRNAs for silencing LRG1 were assessed by qRT-PCR and western blotting.CCK-8 and colony-forming assay were performed to assess cell proliferation activity when cells transfected by siLRG1 or infected by lentivirus containing LRG1 gene.Western blotting was performed to detect cyclins,pro-survival Bcl-2 family proteins,pro-apoptosis Bcl-2 family proteins,LC3A/B and Beclin 1.Results:In five HNSCC cell lines,LRG1 expression level was relatively higher in CAL-27,relatively lower in HEp-2,SCC-9 and FaDu.siLRG1-2 was selected effective for silencing LRG1.New HNSCC cell lines overexpressing LRG1 were established.Overexpression of LRG1 promoted the growth of HEp-2 cells and increased the expression level of Bcl-2.LRG1 silenceing inhibited the growth of CAL-27 cells,decreased the expression level of cyclin E2,led to the increasement in expression level of Bik,LC3A/B and Beclin 1.Conclusion:LRG1 positively regulates the growth of HNSCC cells,which is correlated with cyclin E2,LC3A/B,Beclin 1,pro-apoptosis-related protein Bik and pro-survival-related protein Bcl-2.Part ?:Objective:to explore the affection of LRG1 on HNSCC angiogenesis and its mechanisms.Methods:After treated with rLRGl,HUVEC and HNSCC cell migration was assessed by wound-healing assay,angiogenesis-related proteins were deteced by western blotting.Aortic ring sprouting assay was performed to assess effection of rLRG1 on endothelial cells.The affection of LRG1-overexpressed HNSCC cell lines on angiogenesis was tested by wound-healing assay,transwell method,endothelial cell tube formation assay and aortic ring sprouting assay.We analyzed VEGF-A?HIF-1??TGF-?1 and TAp73 gene expression in HNSCC by investigating data obtained from the GEO database.Then,angiogenesis-related proteins were deteced by western blotting.Results:Addition with rLRGl didn't show additional effect on HUVEC migration(P>0.05),whereas VEGFR2 level elevated with the increase in rLRGl concentration(P<0.05).rLRG1 promoted microvessel sprouting out and cell migration of HEp-2 and FaDu.The expression level of VEGF-A increased but HGF decreased after treatment with rLRGl on HEp-2 cells.Conditioned medium from LRG1-overexpressed HNSCC cell lines can promote not only cell migration and tube formation of HUVEC,but also microvessel sprouting of endothelial cells.Analysis of GSE58911 showed an increasement in gene level of VEGF-A,HIF-la and TGF-?1.The protein level of VEGF-A,HIF-1?,TGF-?1 signaling pathway related proteins increased and the mRNA level of TAp73 increased in LRG1-overexpressed HNSCC cell lines.Conclusion:LRG1 exterts pro-angiogenic effect on endothelial cells by activating VEGFR2 signaling,rather than via VEGF-A which is bound to VEGFR2.LRG1 promotes tumor angiogenesis,and this effect was produced by elevation of VEGF-A via TGF-?1,HIF-la and p73 signaling pathways.