Study On The Correlation Of ATF3 And Its Target Genes Expression To Invasion And Metastasis Of Hepatocellular Carcinoma

Objective: To detect the expressions of ATF3,MMP-2 and maspin in hepatocellular carcinoma (HCC) and study the correlation of ATF3, MMP-2 and maspin expression to invasion and metastasis of HCC by tissue chips; and to detect the DNA binding activity of ATF3 in HCC and study the DNA binding activity of ATF3 to invasion and metastasis of HCC by electrophoretic mobility shift assay(EMSA).Methods: The processing of tissue chip would be modified. Tissue chips were constructed, which contained HCC, non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues, normal liver tissues and intrahepatic metastasis lesions. The expressions of ATF3,MMP-2 and maspin were detected by tissue chips, immunohistochemistry(IHC) and semi-quantitative image analysis in 126 HCC samples, 126 non-neoplastic adjacent liver tissue samples, 81 non-neoplastic distant liver tissue samples, 20 normal liver tissue samples and 33 intrahepatic metastasis lesion samples. The correlations of ATF3, MMP-2 and maspin to clinicopathologic parameters of HCC were also analyzed. The DNA binding activity of ATF3 in 26 cases of HCC samples and 10 non-neoplastic adjacent liver tissue samples were detected by EMSA, and the correlations of ATF3 to clinicopathologic parameters of HCC were analyzed.Results:1,The processing of tissue chips was manually modified to construct successfully 7 paraffin blocks of tissue chips. Every paraffin blocks contained 185 small tissues including HCC, non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues, normal liver tissues and intrahepatic metastasis lesions. The five points represent positive control points and marker points in the middle of tissue chips, All point lattices in paraffin blocks of tissue chips are neatly arranged, no cracks and can be completely cut. After the tissue chips were stained by H-E and IHC, the effective rate was over 95% respectively. Tissue chips were suitable for the histological observation by microscopy and further study.2,The results of IHC showed that the expression of ATF3 and MMP-2 in HCC was significantly higher than that in non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues and normal liver tissues(P<0.01), but the expression of maspin in HCC was significantly lower than that in non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues and normal liver tissues(P<0.01).3,The expression of ATF3 in HCC with intrahepatic metastasis was significantly higher than that in those without intrahepatic metastasis(P<0.01), The expression of ATF3 in HCC in female was significantly higher than that in male(P<0.01), but it was not associated with tumor size, capsule invasion, histological type and histological grade(P>0.05). The expression of MMP-2 in HCC with capsule invasion was higher than that in those without capsule invasion (P<0.05), and the expression of MMP-2 in HCC primary lesions was lower than that in the corresponding metastasis lesions, the expression of MMP-2 in HCC greater than 3cm in Diameter was higher than that in those less than 3cm in Diameter (P<0.05), the expression of MMP-2 decreased following histological grade increasing(P<0.05), but it was not associated with sex and histological type(P>0.05). With the progression of HCC, the expression of ATF3 and MMP-2 increased, but the expression of maspin decreased following histological grade increasing(P<0.05), the expression of maspin was not associated with sex, tumor size, capsule invasion, histological type and tumor metastasis (P>0.05).4,ATF3 expression was positively correlated with MMP-2 expression (P<0.05), and negatively correlated with maspin expression (P<0.01); MMP-2 expression was negatively correlated with maspin expression (P<0.01).5,The results of EMSA showed that compared with the non-neoplastic adjacent liver tissues, the DNA binding activity of ATF3 in HCC was significantly highe(rP<0.01), the DNA binding activity of ATF3 in HCC with metastasis was higher than that in those without metastasis(P<0.05). but it was not associated with sex, age, histological type, tumor size, capsule invasion and histological grade(P>0.05).Conclusion:1,The expression of ATF3 in HCC with intrahepatic metastasis was significantly higher than that in those without intrahepatic metastasis, the expression of ATF3 in HCC in female was significantly higher than that in male, but it was not associated with tumor size, histological types, capsule invasion and histological grade.2,The expression of MMP-2 in HCC with capsule invasion was higher than that in those without capsule invasion (P<0.05), and the expression of MMP-2 in HCC primary lesions was lower than that in the corresponding metastasis lesions, the expression of MMP-2 in HCC greater than 3cm in Diameter was higher than that in those less than 3cm in Diameter (P<0.05), the expression of MMP-2 decreased following histological grade increasing(P<0.05), but it was not associated with sex and histological type(P>0.05). With the progression of HCC, the expression of ATF3 and MMP-2 increased, but the expression of maspin decreased following histological grade increasing(P<0.05), the expression of maspin was not associated with sex, tumor size, capsule invasion, histological type and tumor metastasis (P>0.05).3,overexpression of ATF3, MMP-2 and low expression of maspin were detected in HCC, ATF3 expression was positively correlated with MMP-2 expression, and negatively correlated with maspin expression; MMP-2 expression was negatively correlated with maspin expression. so ATF3, MMP-2 and maspin expression might interactively co-regulate HCC invasion and metastasis.4,The results of EMSA showed that compared with the non-neoplastic adjacent liver tissues, the DNA binding activity of ATF3 in HCC was significantly higher, the DNA binding activity of ATF3 in HCC with metastasis was higher than that in those without metastasis. the DNA binding activity of ATF3 was closely associated with HCC invasion and metastasis.5,The low expression of maspin and overexpression of ATF3 and MMP-2 played important roles in the proceeding of HCC invasion and metastasis, and might predict an aggressive clinical behavior and associate with metastatic potential in HCC.