Development of IGF signaling antibody arrays for the identification of hepatocellular carcinoma biomarkers.

PURPOSE: Our objective was to develop a system to simultaneously and quantitatively measure the expression levels of the insulin-like growth factor (IGF) family proteins in numerous samples and to apply this approach to profile the IGF family proteins levels in cancer and adjacent tissues from patients with hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Antibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) were immobilized on the surface of a glass slide in an array format to create an IGF signaling antibody array. Tissue lysates prepared from patient's liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized with a fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. The expression levels of the ten IGF family proteins in 25 pairs of HCC and adjacent tissues were quantitatively measured using this novel antibody array technology. The differential expression levels between cancer tissues and adjacent tissues were statistically analyzed. RESULTS: A novel IGF signaling antibody array was developed which allows the researcher to simultaneously detect ten proteins involved in IGF signal pathway with high sensitivity and specificity. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues. CONCLUSION: Our IGF signaling antibody array which can detect the expression of ten IGF family members with high sensitivity and specificity will undoubtedly prove a powerful tool for drug and biomarker discovery.

[Effect of siRNA silenced human tissue factor gene on hepatocellular carcinoma cell invasion and metastasis].

OBJECTIVE: To study the effect of tissue factor (TF) on human hepatocellular carcinoma cells when their inner TF gene was knocked down by RNA interference. METHODS: The eukaryotic expression vector bearing siRNA sequence that targeted at TF gene was transfected into human hepatocellular carcinoma cell line HepG2. RT-PCR and Western blot were used to detect the changes of TF gene expression. Transwell invasion assay invitro were used to show the invasive ability of HepG2 cells with transfected pGPU6/GFP/Neo-TFsiRNA. RESULTS: RT-PCR and Western blot analysis showed that HepG2 cells with pGPU6/GFP/Neo-TFsiRNA transfected decreased the endogenous TF gene expression. Correspondingly the mean invading cells was 14 ± 10 for pGPU6/GFP/Neo-TFsiRNA transfection and 128 ± 18 for the NCsiRNA transfection, respectively by the Transwell invasion test in vitro (P < 0.05). The results indicated that the invasive ability of the HepG2 cells with pGPU6/GFP/Neo-TFsiRNA transfected decreased obviously. CONCLUSIONS: TF is involved in the progression of hepatocellular carcinoma and inhibiting the expression of TF can decrease the invasion and metastasis ability of tumor cell in vitro.

Role of tissue factor in hepatocellular carcinoma genesis, invasion and metastasis.

BACKGROUND: Numerous studies indicate that tissue factor (TF), namely tissue thromboplastin, has a close relationship with malignant tumor genesis and progress. It contributes to blood coagulation as well as the regulation of cellular differentiation, the formation of blood vessels, and also tumor recurrence and metastasis. The present study aimed to detect TF expression in hepatocellular carcinoma (HCC) patients and to elucidate its association with prognosis and clinical features of the disease. METHODS: The plasma TF levels of 50 HCC patients and 30 controls were assayed by ELISA. The expressions of TF mRNA and protein in HCC tissues, adjacent tissues and normal tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The acquired data were analyzed with related clinic-pathological documents. The patients were followed up for five years, and the relationship between TF and prognosis was analyzed. RESULTS: The plasma TF levels were significantly increased in HCC compared to the controls (P < 0.05), presenting a close relationship with differentiation level, tumor size and hepatocirrhosis occurrence (P < 0.05). There were remarkably higher values in cases of lymphatic metastasis, extrahepatic metastasis and portal tumor thrombus (PTT) (P < 0.05) compared to non-metastasis or non-tumor thrombus, but no significant difference with different focus number or envelope (P > 0.05). The positive rates and the relative expression of TF mRNA in HCC tissue were 63.0% (17/27) and 0.567 ± 0.268, respectively, significantly higher than that in adjacent tissues or normal tissues (P < 0.05). In the patients with positive results, the relative expression intensity varied significantly with different tumor size and index of local invasion and metastasis (P < 0.05). The positive rates and the relative expression intensities of TF protein in HCC tissue were 74.1% (20/27) and 4.093 ± 1.256, respectively, significantly higher than those in adjacent tissue or normal tissue (P < 0.05). In the patients with positive results, the relative expression intensity showed significant difference in different tumor size, differentiation level, and index of local invasion and metastasis (P < 0.05). CONCLUSIONS: The TF levels were significantly higher in plasma and tissues of HCC patients, presenting a close relationship with the index of invasion and metastasis. It indicated that TF might be related to differentiation and metastasis of HCC.