| Objective: The aim was to investigate the expression levels of nerve growth factor (NGF), vascular endothelial growth factor (VEGF), E-cadherin (E-CD) in different tissues of the liver and the expression levels of NGF in different metastatic potential cells. Through analysis the expression levels among NGF, VEGF, E-CD and clinicopathological features of human hepatocellular carcinoma(HCC) to further explore a new possible target on molecular level to early diagnose and treat the invasion/metastasis of HCC.Methods: Immunohistochemistry was used to respectively detect the expression of NGF, VEGF and E-CD in 45 specimens of HCC, 45 specimens of adjacent non-cancerous cirrhosis hepatic tissue and 15 specimens of normal liver tissue in liver angioma (as controls). NGF expression was also studied in two different metastasis potential cell lines, SMMC-7721, HepG2, using flow cytometry (FCM). We followed up the overall surial rate of postoperative HCC patients. We analyzed the correlation among NGF, VEGF, E-CD and the association of their expression with clinicopathological features (sex, age, the size of tumor, AFP, HbsAg, HbeAb, clinical stage, the pathological classification and different metastatic potential groups) and the overall surial rate of postoperative HCC patients.Results:1 Compared the expression levels of NGF, VEGF and E-CD in HCC tissues with adjacent non-cancerous cirrhosis hepatic tissues and normal liver tissues in liver angioma:Immunohistochemistry examination showed that NGF, VEGF and E-CD were positively expressed in HCC tissues, adjacent non-cancerous cirrhosis hepatic tissues and normal liver tissues in liver angioma, which were mainly restricted to cytoplasm and cellular membrane. The positive rates of NGF expression in HCC tissues was 68.9% (31/45), which was significantly higher than that in adjacent non-cancerous hepatic tissues 33.3% (15/45)(Z=-4.249, P=0.000<0.05)and in normal liver tissues in liver angioma 13.3% (2/15) (Z=-3.889, P=0.000<0.05), and no statistical difference existed between adjacent non-cancerous hepatic tissues and normal liver tissues in liver angioma (Z=-1.586, P=0.113>0.05). The positive rates of VEGF expression in HCC tissues was 77.8% (35/45), which was significantly higher than that in adjacent non-cancerous hepatic tissues 37.8% (17/45) (Z=-5.297, P=0.000<0.05) and in normal liver tissues in liver angioma 60.0% (9/15) (Z=-3.281, P=0.000<0.05), and no statistical difference existed between adjacent non-cancerous hepatic tissues and normal liver tissues in liver angioma (Z=-1.297, P=0.194>0.05). The positive rates of E-CD expression in HCC tissues was 40.0% (18/45), which was significantly lower than that in adjacent non-cancerous hepatic tissues 88.9% (40/45) (Z=-5.504, P=0.000<0.05) and in normal liver tissues in liver angioma 93.3% (14/15) ( Z=-4.753, P=0.000<0.05), and no statistical difference existed between adjacent non-cancerous hepatic tissues and normal liver tissues in liver angioma(Z=-1.305, P=0.192>0.05).2 The correlation among the expression levels of NGF, VEGF, E-CD in HCC tissues: Statistical analysis showed that the expression level of NGF in HCC tissues was positively correlated with the expression level of VEGF (rs =0.482, P=0.001<0.05), and negatively correlated with the expression level of E-CD (rs=-0.467, P=0.001<0.05). The expression level of VEGF was negatively correlated with the expression level of E-CD (rs=-0.509, P=0.000<0.05).3 The correlation between the expression levels of NGF, VEGF, E-CD in HCC tissues and clinicopathological features of postoperative HCC patients: Statistical analysis showed that the expression of NGF in postoperative HCC patients, which was later clinical stage, poorly differentiated and high-invasive group, was significantly stronger than that in earlier clinical stage (χ~2=11.463, P=0.022<0.05), well differentiated (χ~2=12.730, P=0.002<0.05) and low-invasive group (Z=-2.267, P=0.023<0.05), and was positively correlated with the clinical stage (rs=0.444, P=0.002<0.05), pathological classification ( rs =0.529, P=0.000<0.05), low-invasive and high-invasive groups ( rs=0.342, P=0.022<0.05) of postoperative HCC patients. The expression of VEGF in postoperative HCC patients, which was later clinical stage, poorly differentiated and high-invasive group, was significantly stronger than that in earlier clinical stage (χ~2=14.858, P=0.005<0.05), well differentiated (χ~2=6.579, P=0.037<0.05) and low-invasive group (Z=-1.987, P=0.047 <0.05), and was positively correlated with the clinical stage (rs =0.570, P=0.000<0.05), pathological classification (rs=0.366, P=0.011<0.05), low-invasive and high-invasive groups (rs=0.300, P=0.046<0.05) of postoperative HCC patients. The expression of E-CD in postoperative HCC patients, which was earlier clinical stage, well differentiated and low-invasive group, was significantly stronger than that in later clinical stage (χ~2=9.577, P=0.048<0.05), poorly differentiated (χ~2=23.139, P=0.000<0.05) and high-invasive group (Z=-2.134, P=0.033<0.05), and was negatively correlated with the clinical stage ( rs=-0.382, P=0.010<0.05), pathological classification (rs=-0.656, P=0.000<0.05), low-invasive and high-invasive groups ( rs=-0.322, P=0.031<0.05) of postoperative HCC patients. The expressions of NGF, VEGF and E-CD had no significant differences refered to sex, age, tumor size, AFP, HBeAb and HBsAg of postoperative HCC patients based on statistical analysis(P=>0.05).4 The expressions of NGF in different metastatic potential cells: FCM showed that the FI value of NGF in high-invisive human hepatocellular carcinoma cell SMMC-7721 was 0.609±0.071, which was higher than in low-invisive human hepatocellular carcinoma cell HepG2(0.537±0.092). There was statistically significant difference between the two compared (P=0.030<0.05).5 Survival analysis: All 45 specimens were followed up for 1.5-31.0 months, the median survival was 22.1±1.6 months. 1-year survival rate was 71.1%(32/45). The overall survival of NGF(-) group and NGF(+~+++)group were 78.6% (11/14) and 45.2%(14/31), respectively, and mean survival time were 28.1±1.0 months and 19.1±2.0 months, respectively(P<0.05). The overall survival of VEGF(-) group and VEGF(+~+++)group were 90.0%(9/10) and 45.7%(16/35), respectively, and mean survival time were 30.3±0.7 months and 19.8±1.9 months, respectively(P<0. 05). The overall survival of E-CD(-) group and E-CD (+~+++)group were 37.0%(10/27) and 83.3%(15/18), respectively, and mean survival time were 17.7±2.1 months and 28.8±1.2 months (P<0.05), respectively. Univariate surial analysis revealed that the prognosis of postoperative HCC patients had no significant differences refered to sex, age, tumor size, HBeAb and HBsAg based on statistical analysis(P>0.05), while, the survival rates of postoperative HCC patients, which were earlier clinical stage, well differentiated and low-invasive group, were significantly higher than the components (By single variable Log rank test, P<0.05). The survival rates of postoperative HCC patients, which were NGF(-), VEGF(-) and E-CD(+), were significantly higher than the components (By single variable Log rank test, P<0.05). Multivariate analysis revealed that only the expression of NGF and VEGF in HCC tissues, aggressiveness of the cancer were independent prognostic factors of HCC patients.Conclusions:1 The positive rates of NGF and VEGF expression in HCC tissues were significantly higher than that in adjacent non-cancerous hepatic tissues and in normal liver tissues in liver angioma. The expression of NGF and VEGF were stronger in the late clinical stage, poorly differentiated and high-invasive group, while the survival rate was shorter, and the prognosis was poorer.2 The positive rate of E-CD expression in HCC tissues was significantly lower than that in adjacent non-cancerous hepatic tissues and in normal liver tissues in liver angioma. The expression of E-CD was lower in the late clinical stage, poorly differentiated and high-invasive group, while the survival rate was shorter, and the prognosis was poorer.3 The clinical stage, the pathological classification, metastatic potential and the portal vein tumor thrombosis were the significant prognostic factors in postoperative HCC patients. The survival rates of postoperative HCC patients, which were earlier clinical stage, well differentiated, low-invasive group and lower portal vein tumor thrombosis, were significantly higher than the components, the prognosis was better.4 There were positively correlations between the expression of NGF and VEGF, while nagertively correlations between the expression of NGF and E-CD. It suggested that NGF play an important role in liver cancer occurrence and development, especially liver cancer recurrence and metastasis. The expression of NGF in HCC tissues and clinicopathological features could be regarded as valuable indicators for prediction of recurrence (or metastasis) risk in HCC patients. For high recurrence(or metastasis) risk HCC patients, some specific interventions could be used to reduce or prevent recurrence (or metastasis). It is expected to become the treatment and assessment of prognosis of liver cancer targets.
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