| PurposeApart from the vessel tunnels which are lined with endothelial cells,a new microcirculation mechanism: channels are externally lined with tumor cells,with no endothelial cells,this phenomenon was describe as vasculogenic mimicry(VM).Runx2 is a transcription factor that is a member of the Runx2 family.Recent studies have found that Runx2 is overexpressed in cancer cells,enhancing their migration and invasion.We hope to provide a new method for clinical treatment of hepatocellular carcinoma by studying the effect of Runx2 on VM formation.Methods1? HCC specimens were obtained from 89 patients who received surgical excision in Tianjin General Hospital and Tianjin Medical University Cancer Hospital from 2005 to 2015.All specimens came from patients who received no radiotherapy or chemotherapy.Immunohistochemical staining was performed to inspect the relationship between Runx2 and VM,E-cadherin,Vimentin,VE-cadherin,Galectin-3,gender,age,tumor size,histological differentiation,metastasis,stage and survival duration.We performed Kaplan-Meier survival analysis to inspect Runx2-positive and Runx2-negative patients' prognosis.2? Western Blot assays were performed to screen cell lines based on the Runx2 expression level in Bel7402,SMMC7721,HepG2,97 L,97H cells lines.HepG2 cells and SMMC7721 cells were choosed for HepG2 cell had lowest Runx2 expression level and SMMC7721 cell had highest Runx2 expression level.We transfected HepG2 cells with Runx2 overexpression plasmid to raise Runx2 expression level,and transfected SMMC7721 cells with Runx2 knockdown plasmids to down-regulate the Runx2 expression level.After Runx2 overexpression or knockdown plasmid transfection,HepG2 cells were transfected with Galectin-3 gene LGALS3 knockdown plasmids,and SMMC7721 cells were transfected with LGALS3 overexpression plasmids.We used western blot and RT-PCR assays to check the transfection efficiency.3? We used western blot and RT-PCR assays to investigate Galectin-3,EMT-related markers: Vimentin,E-cadherin;VM-related marker: VE-cadherin,expression level in HepG2-Runx2,SMMC7721-shRunx2,and HepG2-Runx2-shLGALS3,SMMC7721-shRunx2-LGALS3.4? Migration assay and invasion assay were performed to investigate the migration ability and invasion ability of HepG2-Runx2,SMMC7721-shRunx2,and HepG2-Runx2-shLGALS3,SMMC7721-shRunx2-LGALS3 cells.5? Three-dimensional assay was performed to investigate the ability of forming VM-like structures of HepG2-Runx2,SMMC7721-shRunx2 and HepG2-Runx2-shLGALS3,SMMC7721-shRunx2-LGALS3 cells.6? VE-cadherin immunofluorescence and confocal microscopy was performed to investigate whether those tube-like VM-like structres express VE-cadherin to support the real formation of VM.Results1? Immunohistochemical analyses results showed that 55 Runx2-positive samples and 34 Runx2-negative samples.And Runx2 was positively related with VM(?2=10.337,p=0.002)E-cadherin(?2= 5.848,p=0.027),Vimentin(?2=10.837,p=0.002),VE-cadherin(?2=14.008,p=0.001)and Galectin-3(?2=5.92,p=0.018).VM was found in 53 of 89 HCC samples(59.6%).In the 89 HCC samples,VM was detected in 40 out of 55(72.7%)samples in the Runx2 positive group and 13 out of 34(38.2%)samples in the Runx2 negative group.The positive rate of Vimentin,VE-cadherin and Galectin-3 expression in the presence of Runx2 was higher than in cancer cells that did not express Runx2,while the positive rate of E-cadherin in Runx2-positive cells was lower than in the Runx2 negative group.Clinicopathological data analysis showed Runx2 was associated with Histological differentiation(?2=6.994,p=0.008),metastasis(?2= 8.106,p=0.004).Runx2 expression was higher in ?/? grade than?/? grade,patients with metastasis had higher Runx2 expression than patients without metastasis.On the other hand the expression of Runx2 did not related with age,gender,Hepatitis B,cirrhosis and tumor size.A Kaplan-Meier survival analysis revealed that patient with Runx2 expression had a shorter survival period than those without expression.2? HepG2 cells were transfected with the Runx2 overexpression plasmid.Western blot and RT-PCR revealed an increase in the Runx2 protein and mRNA levels in the HepG2-Runx2 transfectants compared with the control.In SMMC7721 cells,knockdown by shRNA decreased the Runx2 expression detected with RT-PCR and western blot.The results revealed a high gene knockdown efficiency.3? Western blot and RT-PCR assays results showed E-cadherin expression was repressed,however Vimentin expression was raised in HepG2-Runx2 cells compared with control.E-cadherin expressions in SMMC7721-shRunx2 were elevated,and the expression of Vimentin was suppressed.We also found the up-regulation of Galectin-3 and VE-cadherin expression in HepG2-Runx2 cells compared to the control,and its down-regulation in SMMC7721-shRunx2 cells compared to the control.4? Quantitative analysis following a wound healing assay suggested a significant difference in the speed of wound healing between the transfected groups and the control group.Following a Matrigel invasion assay,an increase in cell invasion was observed in the Runx2-transfected HepG2 cell line compared with the control,and a decrease in cell invasion in the Runx2-shRNA-transfected SMMC7721 cells compared with the control(P<0.05).5? The Matrigel 3D culture results demonstrated that when up-or downregulated by Runx2,HepG2-Runx2 and SMMC7721 cells formed a typical pipe-like structure on the surface of the Matrigel medium,while HepG2 cells failed to form pipe-like structure and the number of those structres formed by SMMC7721-shRunx decreased significantly.6? RT-PCR and western blot showed that the Galectin-3 expression level in HepG2-Runx2 cells decreased following LGALS3 knockdown compared with the control,and increased as a result of the LGALS3 transfection of SMMC7721-shRunx2 cells compared with control.However,no significant change in Runx2 expression was observed after LGALS3 transfection or knockdown compared with control.7? E-cadherin expression was increased while Vimentin expression was down-regulated in HepG2-Runx2-shLGALS3 cells compared with control,and Ecadherin expression in SMMC7721-shRunx2-LGALS3 cells decreased,while theexpression of Vimentin increased.8? Wound healing assays showed that LGALS3 knockdown slowed the wound healing rapidity in HepG2-Runx2 cells compared with the control,and the introduction of LGALS3 increased wound healing rapidity in SMMC77210-shRunx2 cells.Matrigel invasion assay,we observed a decrease in LGALS3-transfected HepG2-Runx2 cells,and an increase in SMMC7721-shRunx2 cells following LGALS3 up-regulation compared with the control.9? Western blot assay result showed VE-cadherin expression was down-regulated after LGALS3 knockdown in HepG2-Runx2 cells,and up-regulated following LGALS3 up-regulation in SMMC7721-shRunx2 cells.Three-dimensional culture experiments results demonstrated that HepG2-Runx2-shLGALS3 cells lost the ability to form tube-like structures compared with control,while the number of such tube-like structures was raised in SMMC7721-shRunx2 cells compared with the control.10? VE-cadherin immunofluorescence and confocal microscopy results showed those tube-like structures overexpressed the endothelial-specific marker VE-cadherin,especially on the wall of these tubes in vitro.Conclusions1? Runx2 expression was positively related with Histologic differentiation,metastasis and grouping.And patient with Runx2 expression had a shorter survival period than those without Runx2 expression.Also,Runx2 expression with E-cadherin,Vimentin,VE-cadherin,Galectin-3 and VM.2? Runx2 might promote the ability of migration,invasion and VM-forming of HCC cells.Runx2 could promote VM formation through inducing EMT and Galectin-3 might function intermittently. |
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