| Purpose Vasculogenic mimicry(VM) has been reported is formed by tumor cells and is a unique endothelial-independent tumor microcirculation patern. VMwas associated with invasion, matastasis, a high tumor grade and short survival. Amotl2, one of Amot family members, could positively regulate tumor migration and invasion in various types of cancers. We reported to proveid a new idea and target for the treatment of HCC through studying the role of Amotl2 on VMformation in hepatocellular carcinoma(HCC)Methods1?Clinical specimens of 89 patients identified as HCCwith complete follow-up data were obtained from Tianjin Cancer hospital. Immunohistochemical analysis was used to analyse the relationship of Amotl2 and VM, age, gender, tumor size,histological differentiation, stage, matastasis. Kaplan-Meier survival analysis was used to compare the prognosis of patients between Amotl2-positive group and Amotl2-negative group.2 ? Based on Amotl2 expression level, select the Hep G2 cells which Amotl2 underexpression and Bel7402 cells which Amotl2 overexpression. Hep G2 cells was transfected with Amotl2 upregualtion plasimid to acquire upregulation model;Bel7402 cells was transfected with Amotl2 downregulation plasimid to acquire downregulation model. Western blot and immunofluorescence was used to identifiy the Amotl2 transfection efficiency.3 ? In wound healing assays, cell motility was assessed by measuring the movement of cells into a scarped and the invasion assay was used to determine the role of invasive potential of Hep G2 cells after Amotl2 upregualtion; The two assays were also used to detect the migration and migration ability of Bel7402 cells after Amotl2 downregulation.4 ? Three-dimensional culture was performed to analyse the ability of the tube structure formaton in Hep G2 cells after Amotl2 upregulation and in Bel7402 cells after Amotl2 downregualtion.5 ? Western blot were used to detect the EMT- related protein(E-cadherin,Viementin) in Hep G2 cells after Amotl2 upregulation and in Bel7402 cells after Amotl 2 downrugulation.Result1 ? Immunohistochemical analysis showed that 49 samples presented positive Amotl2 expression and 40 samples presented negative expression. The clinicopathological data in patients with Amotl2 positive(n=49) were compared with those with Amotl2 negative(n=40) in HCC. The results showed that Amotl2 was positively associated with VM( c2=5.662, P=0.017); In aged over 45 years old samples, Amotl2 was found expressed higher than in samples which 45 years old or younger(c2=8.400,P=0.004);Among the four HCC histological differentiation and stage types, Amotl2 was found expressed more in ?- ? than ?- ?types( c2=5.475, P=0.019).However, the presence of Amotl2 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 Amotl2 expression were remarkably shorter compared with those of patients with negative Amotl2 expression(P=0.046)2 ? Statistical analysis showed that Amotl2 was related to VM and was closely related to VM(r=0.277,P=0.009). Also, Amotl2 was positively related to EMT-related characterizing protein VE-cadherin(r=0.631,P=0.000). Although MMP2 had no relationship with Amotl2 in all 89 samples, MMP2 was correlated with Amotl2 in?B types(r=0.418,P=0.024).3?Western blot showed that the expression of Amotl2 was significantly increased in Hep G2 cells after Amotl2 upregulation and the expression of Amotl2 was significantly decreased in Bel7402 cells after Amotl2 downregulation.4 ? Wound healing and invasion assay showed that the migration and invasion ability of Hep G2 cells was remarkly increased after Amotl2 upregulation; the two assays also showed that the migration and invasion ability of Bel7402 cells was remarkly decreased after Amotl2 downregulation.5 ? Three-dimensional culture showed that the ability of the tube structure formation in Hep G2 cells was significantly increased after Amotl2 upregulation andin Bel7402 cells was significantly decreased after Amotl2 downrugulation.6 ? Western blot showed that the expression level of the EMT-related protein E-cadherin was decreased, Viementin was increased and after Amotl2 upregulating in Hep G2 cells; the two assays also showed that the expression level of E-cadherin was increased, Vimentin was decreased after Amotl2 downregulationg in Bel7402 cells.Conclusions1?Amotl2 was positively associated with VM, age, histological differentiation and stage, but it did not show any association with other clnicopathological data. And the Amotl2-positive patients had shorter survival duration.2 ? Amotl2 could promote the migration, invasion and VM formation ability in HCC cells, it probably promote VM formation through inducing EMT markers expression. This research may provide a new target for anti-tumor angiogenesis therapy. |
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