The Expression Of EMT Regulators And Correlation With Recurrence And Metastasis And Prognosis In HCC

To invetigate the expression of EMT regulatory molecules and correlation with recurrence and metastasis in the hepatocelluar carcinoma(HCC), and to further explore EMT regulatory molecules interrelationship and the correlation between them and prognosis, and to preliminary study the mechanism of metastasis in HCC.We collected 97 HCC resection samples with complete clinical and prognostic data from Cancer Hospital of Tianjin Medical University. Immunohistochemical method was used to detect the expression of Twistl, Twist2, Snail, Slug, E-cadherin, MMP-2, MMP-9 in HCC.Scoring method reported by Mattern was applied to analyze the difference of them in the recurrence and metastasis(RM) group and the non-recurrence and non-metastasis(NRM) group, the interrelationship and the correlation between them and prognosis.In 97 hepatocellular carcinoma cases, RM group had 49 cases and NRM group had 48 cases. The mean survival time of patients in RM group was 26.423±3.279 months, and the median survival time of it was 21 months; The mean survival time of patients was 46.75±3.325 months and the median survival time was 56 months in NRM group.We can see that the survival time of the RM group was shorter than that of NRM group(P=0.000).Recurrence and metastasis was closely related to clinical stage.The survival time of patients in stage III and IVwas shorter than that in stageⅠandⅡ(χ2=41.399, P=0.000). The bigger the tumor, the higher the probability of recurrence and matastasis(χ2=7.506,P=0.006).If pathological grade was higher,the tumor would be prone to occur recurrenceandmetastasis(χ2=4.118, P=0.042). Recurrence and metastasis were unrelated with gender and age(χ2=0.639,P=0.424,x2=0.112,P=0.738).2. their expression of EMT regulatory molecules and invasion-related proteins in HCC and their correlation with recurrence and metastasis 2.1.1 Immunohistochemical expression of Twistl:Twistl was mainly expressed in cytoplasm of the HCC cell,extensively,showing yellow and brown.48.98%(24/49) of HCC specimens nuclear expressed Twistl in RM group,while the nuclear positive rate of Twistl was 18.75%(9/48) in NRM group.Twistl nuclear positive expressing rate in RM group was higher than that of NRM group,which had statistical significance(χ2=9.871,P=0.002). Using Scoring method reported by Mattern, we discovered that the difference of Twistl in the cytoplasm between the two groups had statistical significance.The expression of Twist1-cytoplasm in RM group was higher than that in NRM group(Z=-3.619,P=0.000).2.1.2 Twist2 was extensively expressed in the cytoplasm of HCC.57.14% (28/49)HCC specimens expressed Twist2 in the cytoplasm in RM group,while the positive rate of Twist2 in the cytoplasm was 50%(24/48)in NRM group, Mattern scoring method was selected to compare the difference of Twist2 between the two groups,and there was no statistical significance(Z=-0.239,P=0.811).2.1.3 As an important member of the family of zinc finger domains,Snail was extensively expressed in the nuclear of HCC. The positive rate of it in RM group was 69.39%,which was higher than that in NRM group,whose positive rate was 47.92%(χ2=4.613,P=0.032).2.1.4 Slug was the same household member as Snail,which was expressed at the membrane and partialy in cytoplasm of HCC,the positve rate of Slug in RM group was 67.35%(33/49), while it was 50%(24/48) in NRM group. Mattern scoring method was used to compare the difference of Slug expression in the cytoplasm between the two groups. the difference between them had no statistical significance(Z=-1.911, P =0.056). Slug was also expressed in vascular endothelial cell, so the function of Slug was to be further explored.E-cadherin was an important epidermal adhesion molecule, which was positive at the memberane of HCC.The expression of E-cadherin decreased in partial liver tumor tissue and even there was no expression in some liver tumor tissue. The positive rate of E-cadherin was 44.90%(22/49) in RM group, while the positive rate was 56.25% in NRM group. Mattern scoring method was applied to compare the difference of E-cadherin between the two groups.the difference between them had statistical significance (Z=-2.205,P=0.027).The expression in RM group was lower than that in NRM group.2.3.1 MMP-2 was an important member of MMP Household, which was closely related to invasion and metastasis, it was positive in the cytoplasm of HCC, the positive rate was 69.39%(34/49) in RM group, while it was 75% (36/48) in NRM group. Mattern scoing method was selected to compare the difference of MMP-2 between the two groups, there was no stastical significance(Z=-2.623,P=0.009). The expression of MMP-2 in RM group was higher than that in NRM group.2.3.2 MMP-9 was mainly positive in the cytoplasm of HCC, And the minority was positive in the nuclear, the positive rate being 67.35%(33/49) in RM group, while it was58.33%(28/48) in NRM group.Mattern scoing method was selected to compare the difference of MMP-9 between the two groups,and there was no stastical significance(Z=-2.028,P=0.043).MMP-9 expression in RM group was higher than that in NRM group.From the positive rate of these factors in the recurrence and matastasis group,we can learn that:the expression of Twist1 in the nuclear was negatively correlated with E-cadherin(P=0.043) and positively correlated with MMP-9(P=0.047).The higher the nuclear positive rate of Twist1, the higher the positive rate of MMP-9 and the lower the positive rate of E-cadherin.The nuclear expression of Snail was negatively correlated with E-cadherin(P=0.048),the higher the nuclear positive rate of Snail, the lower the positive rate of E-cadherin. However, Twist2 and Slug were not related to E-cadherin, MMP-2 and MMP-9.4.1 single factor survival analysis:It was found that the difference of the survival time between the positive group and the negtive group about Twist-nuclear,Twist-cytoplasm,Snail,E-cadherin and MMP-9 had statistical significance,The survival time of the positive groups about Twist1,Snail and MMP-9 was shorter than that of the negtive group(P=0.000,P=0.002,P=0.028,P=0.039), and the survival time of negtive group about E-cadherin was longer than that of the positive group(P =0.002).There was no statisticly significant difference of survival time between the two groups about Twist2,Slug and MMP-2 (P=0.336,P=0.293,P=0.805)4.2 combined survival analysis of EMT regulators:Twist1,Twist2,Snail and Slug were all regulators and the upper stream factors in EMT, As for the coexpression of Twist 1 or Snail with other factors,one group of patiens were both positive,while the other group were both negative.we found that the survival time of the former group was shorter than the latter one. the coexpression of Comparing with the group whose Twist1-nuclear and Snail-nuclear were both positive, the survival time of the patients whose Twist1-nuclear and Snail-nuclear were both positive was shorter than the both negative group (P=0.000),so was the group of Twist1-cytoplasm and Snail-nuclear(P=0.001),the survival time of the group whose Twistl and Snail were both positive was shorter than that any other two factor who were both positive4.3 COX regression analysis of EMT regulators:the risk degree of Twistl-nuclear and Twistl-cytoplasm were 2.110 and 1.952 respectively,and Which had statistical significance,(P=0.015,P=0.029).The risk degree of Snail was 1.743,but the difference had not statistical significance (P=0.063).1. Recurrence and metastasis were closely related to clinical stage, pathology grade and tumor size, Recurrence and metastasis were not related to gender and age. The bigger the size of the tumor, the higher the rate of recurrence and metastasis, the later the clinical stage and the higher the pathological grade, the more possible recurrence and metastasis.2. In HCC,bHLH family transcriptional factor twistl was able to down-regulate E-cadherin expression through binding to E-boxes located in the proximal promoter of E-cadherin,decreasing or missing expression of E-cadherin may induce the missing of HCC cell polarity, intercellular adhesion descend, meanwhile it can promote matrixmetalloproteinase (MMPs)activation,decline matrix outside of cell,and promote recurrence and metastasis of HCC,leading to poor clinical prognoses.Zinc-finger protein Snail was able to down-regulate E-cadherin expression through binding to E-boxes located in the proximal promoter of E-cadherin,Missing expression of E-cadherin may induce missing of HCC cell polarity, intercellular adhesion descent, and promote recurrence and metastasis of HCC, leading to poor clinical prognoses. However, Twist2 and Slug were not related to recurrence and metastasis of HCC and adverse prognosis.3.Of EMT regulators, expression of Twistl and Snail was closed related with prognosis,while expression of Twist2 and Slug was unrelated with prognosis.What's more coexpression of Twistl and Snail led to worse prognosis.We could consider the coexpression of regulators the two regulators as a sign of poor prognosis. Twistl played a greater role.in HCC recurrence and metastasis and poor prognosis of patients.