The Study Of BMP4 In Vasculogenic Mimicry Formation Of Hepatocellular Carcinoma

PurposeVasculogenic mimicry(VM) has been reported is formed by tumor cells and is a unique endothelial-independent tumor microcirculation pattern. VM was associated with invasion, metastasis, a high tumor grade and short survival. BMP4, one of TGF-β family menmebers, could positively regulate tumor migration and invasion in various types of cancers. We expect to provide a new idea and target for the treatment of HCC through studying the role of BMP4 on VM formation in hepatocellular carcinoma(HCC). Methods1、Clinical specimens of 92 patients identified as HCC with complete follow-up data were obtained from Tianjin Medical University General Hospital and Cancer Institue. Immunohistochemical analysis was used to analyse the relationship of BMP4 and VM, age, gender, tumor size, histological differentiation, stage, metastasis. Kaplan-Meier survival analysis was used to compare the prognosis of patients between BMP4-positive group and BMP4-negative group.2、Based on BMP4 expression level, select the HepG2 cells which BMP4 underexpression and the SMMC7721 cells which BMP4 overexpression. HepG2 cells was transfected with BMP4 upregulation plasmid to acquire upregulation model; SMMC7721 cells was transfected with BMP4 downregulation plasmid to acquire downregulation model. Western blot and immunofluorescence was used to identify the BMP4 transfection efficiency.3、Wound healing assay and invasion assay were used to detect the migration and invasion ability of HepG2 cells after BMP4 upregulation; the two assays were also used to detect the migration and migration ability of SMMC7721 cells after BMP4 downregulation.4、Three-dimensional culture was performed to analyse the ability of the tube structure formation in HepG2 cells after BMP4 upregulation and in SMMC7721 cells after BMP4 downregulation.5、Western blot and immunofluorescence were used to detect the EMT-related protein(E-cadherin, Vimentin), the stem cells-associated markers(OCT4, SOX2) in HepG2 cells after BMP4 upregulation and in SMMC7721 cells after BMP4 downregulation.6、Western blot and immunofluorescence were used to detect the VM-related protein EphA2, VE-cadherin, MMP2 in HepG2 cells after BMP4 upregulation and in SMMC7721 cells after BMP4 downregulation.7、Gelatin zymography assay was used for detecting the activity of MMP2 in HepG2 cells after BMP4 upregulation and in SMMC7721 cells after BMP4 downregulation. Results1、Immunohistochemical analysis showed that 52 samples presented positive BMP4 expression and 40 samples presented negative BMP4 expression. The clinicopathological data in patients with BMP4 positive(n=52) were compared with those with BMP4 negative(n=40) in HCC. The results showed that BMP4 was positively associated with VM(χ2=5.662, P=0.017); In aged over 45 years old samples, BMP4 was found expressed higher than in samples which 45 years old or younger(χ2=8.400,P=0.004); Among the four HCC histological differentiation and stage types, BMP4 was found expressed more in Ⅲ/ tⅣ hanⅠ/ typesⅡ(χ2=4.048,P=0.044; χ2=5.475,P=0.019). However, the presence of BMP4 did not show any association with other clinicopathological characteristics, such as gender, tumor size, and metastasis(P > 0.05). Kaplan-Meier analysis showed that the overall survival time of patients with positive BMP4 expression were remarkably shorter compared with those of patients with negative BMP4 expression(P= 0.046).2、Western blot and immunofluorescence showed that the expression of BMP4 was significantly increased in HepG2 cells after BMP4 upregulation and the expression of BMP4 was significantly decreased in SMMC7721 cells after BMP4 downregulation.3、Wound healing and invasion assay showed that the migration and invasion ability of HepG2 cells was remarkly increased after BMP4 upregulation; the two assays also showed that the migration and invasion ability of SMMC7721 cells was remarkly decreased after BMP4 downregulation.4、Three-dimensional culture showed that the ability of the tube structure formation in HepG2 cells was significantly increased after BMP4 upregulation and in SMMC7721 cells was significantly decreased after BMP4 downregulation.5、Western blot and immunofluorescence showed that the expression level of the EMT-related protein E-cadherin was decreased, Vimentin was increased and the stem cells-associated markers(OCT4, SOX2) were increased after BMP4 upregulating in HepG2 cells; the two assays also showed that the expression level of E-cadherin was increased, Vimentin was decreased and the stem cells-associated markers(OCT4, SOX2) were decreased after BMP4 downregulating in SMMC7721 cells.6 、 Western blot and immunofluorescence showed that the expression of VM-related protein EphA2, VE-cadherin and MMP2 were increased in HepG2 cells after BMP4 upregulation; the two assays also showed that the expression of EphA2, VE-cadherin and MMP2 were decreased in SMMC7721 cells after BMP4 downregulation.7、The gelatin zymography assay showed that the activity of MMP2 in HepG2 cells was increased after BMP4 upregulation and in SMMC7721 cells was decreased after BMP4 downregulation. Conclusions1、BMP4 was positively associated with VM, age, histological differentiation and stage, but it did not show any association with other clinicopathological data. And the BMP4-positive patients had shorter survival duration.2、BMP4 could promote the migration, invasion and VM formation ability in HCC cells, it probably promote VM formation through inducing EMT and stem cells-associated markers expression and by the way of EphA2/VE-cadherin/MMP2. This research may provide a new target for anti-tumor angiogenesis therapy.