The Role Of Epithelial-mesenchymal Transition Induced By Twist Protein In Vasculogenesis In Hepatocelluar Carcinoma

Objective to study if there was vasculogenic mimicry(VM)in Hepatocelluar Carcinoma(HCC),and to investigate the role of Epithelial-mesenchymal transition (EMT)induced by twist protein in the formation of VM,to explain its potential mechanism in the process.Methods Immunohistochemical staining of CD31,Hepatocyte and PAS special histochemical staining were used to study if there were VM in 102 formalin-fixed and paraffin-embedded tissue samples of HCC.Immunohistochemistry method was used to detect the expression of Twist,E-cadherin,β-catenin,vimentin,MMP-9和MMP-2 in VM-positive group and VM-negative group.Staining of endothelial cells for CD31 was used to evaluate the microvessel density(MVD).Results1 There was vasculogenic mimicry in HCC1)There were some tubular structures in 19 of 102 samples which were outlined by tumor cells,not endothelial cells in HE slides.There present blood cells in this structures while there was no necrosis and inflammatory cells infiltrating near the structures.Hepatocelluar Carcinoma cells differentiate poorly which have the structures,indicating it correlates with biological behavior.2)CD31 did not stain the cells that outlined these tubular structures,which indicated these cells are not endothelial cells.Hepatocyte stained the cells which outlined the vasculogenic-like tubular wall,which indicated these cells are hepatocellular Carcinoma.cells.The PAS-positive material can be observed with PAS stain between the red blood cells and tumor cells.2 The expression of Twist and relative proteins1)Twist mainly was expressed in cytoplasm of the HCC cells,while twist were expressed in the nuclear of 10%samples.Furthermore we observed twist was distributed diffusely in the cancer tissues.The positive expression rate of twist in the VM-positive group was 78.95%(15/19),The positive expression rate of twist in the VM-negative group was 42.17%(35/83).There was significant difference between them(P＜0.05),indicating twist played an important role as a transcription fator in the sigal transduct in the formation of VM in HCC.2)E-cadherin mainly was expressed in the membranes of paracancerous hepatocytes.The expression of E-cadherin in HCC decreased or was even lost.The positive expression rate of E-cadherin in the VM-positive group was 15.79%(3/19), The positive expression rate of E-cadherin in the VM-negative group was 48.19% (40/83).There is significant difference between them(P＜0.05).β-catenin was expressed in the membranes,cytoplasm or nuclear.The expression ofβ-catenin in the membranes was positive,while the expression in the cytoplasm or nuclear was abnormal.We foundβ-catenin was expressed negatively in most of HCC samples.β-catenin positive expression could be detected in 16 of 102.The abnormal expression ofβ-catenin could be detected in 32 of 102,of which it was expressed in the nuclear in 8 samples.The positive expression rate ofβ-catenin in the VM-positive group was 5.26(1/19),The positive expression rate ofβ-catenin in the VM-negative group was 18.07%(15/83).There is no difference between them(P＞0.05).The abnormal expression rate ofβ-catenin in the VM-positive group was 52.63%(10/19).The abnormal expression rate ofβ-catenin in the VM-negative group was 26.51%(22/83).There was significant difference between them(P＜0.05),which indicated the abnormal expression ofβ-catenin had releation with the formation of VM in HCC.3)Vimentin was distributed in the mesenchymal tissue,mainly expressed in the endothelial cells.It was expressed negatively in the tumor cells.Vimentin was expessed in the mesenchymal cells normally,but sometimes it was expressed in the cancer cells,which had a relation with its malignant behavior.The results show that the malignant behavior of hepatocellular carcinoma couldn't relate with the expression of Vimentin.MMP-2 and MMP-9 were diffusely expressed in cytoplasm of the HCC cells, Also,MMP-2 and MMP-9 demonstrated a faint expression in the precancerous cirrhosis tissue and fibroblasts and leucocytes in the ECM.The positive expression rate of MMP-2 in the VM-positive group was 78.95%(15/19).The positive expression rate of MMP-2 in the VM-negative group was 69.88%(58/83).There is no difference between them(P＞0.05).However,compared with VM-negative group, the positive expression rate of MMP-9 in the VM-positive group was higher,which was 84.21%(16/19).There was significant difference between them(P＜0.05),which indicated MMP-9 could promote the fomation of VM by degradation ECM components.4)Correlative analysis indicated that there was negative correlation between the positive expression of Twist and E-cadherin(r=-0.227,P＜0.05),while positive correlation between the positive expression of Twist and MMP-9(r=0.347, P＜0.05).3 Correlation between Twist and relative proteins and MVDMicrovessel density(MVD)of patients with high Twist,was higher than that in the low Twist expression group,so was MMP-2 and MMP-9(p＜0.05).However,there was no difference between E-cadherin andβ-catenin high expression group and low expression group(P＞0.05).It indicated Twist,MMP-2 and MMP-9 play an important role in angiogenesis.4 Correlation between expression of Twist,E-cadherin,β-catenin,MMP-2 and MMP-9 and clinicopathological parameter in the Hepatocellular CarcinomaThere was no correlation between Twist,E-cadherin,β-catenin,MMP-2 and MMP-9 expression and patient's age,sex,classification,tumor size and Edmondson pathologic grade.The expression of Twist,E-cadherin,MMP-2 and MMP-9 was closely correlated to the stage and metastasis of HCC.The positive expression rate of Twist was 60.71%in the stageⅢⅣ,which was much higher than that in the stageⅠⅡ(34.78%)(p＜0.05).The positive expression rate of E-cadherin was 32.14%in the stageⅢⅣ,which was lower than that in the stageⅠⅡ(54.35%) (p＜0.05).The positive expression rate of Twist,MMP-2 and MMP-9 was 75%, 75%and 69.44%respectively in the metastasis group,which was higher than non-metastasis group(34.85%,42.12%and43.89%respectively)(P＜0.05).The positive expression rate of E-cadherin was lower in the metastasis group than that in non-metastasis group(P＜0.05). Conclusions 1 There was vasculogenic mimicry in hepatocelluar carcinoma,which make tumor cells acquire blood supply to sustain growth and metastasize by this special microcirculation pattern.The positive rate of vasculogenic mimicry in hepatocelluar carcinoma was 18.6%(19/102).Hepatocelluar Carcinoma with VM differentiate poorly,matastasis earlier and had a poor prognosis.2 The positive expression rate of Twist and MMP-9 in the VM-positive group was higher than that in the VM-negative group.However,The positive expression rate of E-cadherin in the VM-positive group was lower than that in the VM-negative group. There was negative correlation between the positive expression of twist and E-cadherin,while positive correlation between the positive expression of Twist and MMP-9.These indicate twist could suppress the expression of E-cadherin and up-regulate the expression of MMP-9,which contribute to the formation of VM.3 The MVD of HCC in the groups in which Twist,MMP-2 and MMP-9 was expressed positively was greater than that in negative group.Meanwhile,There was positive correlation between the positive expression of twist and MMP-9.There was no correlation between the positive expression of twist and MMP-2.Twist can up-regulate the expression of MMP-9,which contribute to tumor angiogenesis.4 Twist can play an important role in the formation of local microcirculation.The mechanism was that twist overexpression in HCC could induce EMT,in which the expression of E-cadherin was lower and the expression ofβ-catenin was abnormal.As a result,abundantβ-catenin enter the nuclear and promote the transcription of MMP-9.5 Twist promoted the formation of local microcirculation in HCC and accelerated the progression and metastasis.