| Objective Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors worldwide, and its therapy still remains dismal. Carcinogenesis of hepatocytes is a multi-factor, multi-step, and complex process, which includes multiple genetic alterations such as activation of oncogenes, inactivation of tumor suppressor genes, and so on. Human insuline-like growth factor-Ⅱ(IGF-Ⅱ) is a kind of fetal growth factor, and overexpressed during the occurrence and development of HCC. It is related to continuous proliferation, abnormal growth, differentiation, and carcinogenesis of hepatocytes. However, how to regulate IGF-Ⅱgene expression for inhibiting hepatoma cell proliferation, and whether IGF-Ⅱgene is a potential molecular-target for HCC theray is not clear. The aim of this study was to investigate the levels of IGF-Ⅱexpression in cancerous, paracancerous, noncancerous tissues, and circulating blood in HCC patients, and the clinicopathological features of IGF-Ⅱabnormality, and analyze the effect of specific siRNA-mediated inhibition of IGF-Ⅱexpression on apoptosis of human hepatoma cell lines (HepG2 cells).Methods Cancerous, paracancerous (3 cm to cancerous margin), and noncancerous ( over 5 cm to cancerous margin) tissues were collected from 35 patients who underwent operations for liver cancer. The expression and distribution of IGF-Ⅱin different liver tissues was analyzed by an immunohistochemical teachology. The levels of IGF-Ⅱexpression in peripheral blood of 146 patients with HCC, 25 with non-liver cancers, 30 with liver cirrhosis, 30 with chronic hepatitis, 17 with acute hepatitis, and 30 healthy individuals were detected by an enzyme-linked immunosorbent assay (ELISA). The relationships between IGF-Ⅱexpression and clinical pathological characteristics were also investigated. IGF-ⅡsiRNA was used to down-regulate IGF-Ⅱexpression in human HepG2 cells; reverse-transcription polymerase chain reaction (RT-PCR) and ELISA were used to examine IGF-Ⅱexpression; Annexin V-FLUOS/PI was used to test cell apoptosis.Results The positive staining of hepatic IGF-Ⅱappeared as brown particles mainly located in the cytosol, with a few in the nucleus and none in the cell membrane. The distribution of positive cells in cancerous tissues was clearly heterogeneous. The cytoplasmic IGF-Ⅱstaining was significantly higher in the HCC cells than in the paracancerous tissues, but non-positive expression was found in the distant cancerous tissues. The higher expression of hepatic IGF-Ⅱin HCC was associated with serosal invasion, tumor size, differentiation of tumor, and HBV infection (P < 0.05). However, no significant difference was found between IGF-Ⅱexpression and the number of tumors (P > 0.05). The expression of IGF-Ⅱin peripheral blood of HCC patients was significantly more than patients with non-liver tumors, acute or chronic hepatitis, liver cirrhosis, and normal subjects (P = 0.000). The overexpression of IGF-Ⅱin the cancerous group was associated with HBV infection. At 72 h after IGF-ⅡsiRNA transfection, IGF-Ⅱexpression in the 150 nM group in the HepG2 cells reduced 63% at mRNA and 44.5% at protein levels. The down-regulation of IGF-Ⅱexpression was depended on the dose of IGF-ⅡsiRNA and the action time after specific siRNA transfection. Interestingly, the apoptosis index of the HepG2 cells increased with IGF-Ⅱinhibition, and the down-regulation of IGF-Ⅱsensitized HepG2 cells to adriamycin.Conclusion The abnormal activation of hepatic IGF-Ⅱis closely associated with the occurrence and development of HCC, and IGF-Ⅱinhibition mediated by specific siRNA promotes HepG2 cells apoptosis. Therefore IGF-Ⅱis a potential target for HCC gene therapy. |
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