Effects Of Exogenous NF-IL6 On Reversing Malignant Phenotype Of Human BEL-7404 Hepatoma Cells

The nuclear factor for interleukin-6 (NF-IL6 or C/EBP) is an important immunological regulation factor. It is a member of the C/EBP protein family. Its binding sequence exists in genes encoding acute-phase proteins, genes responsible for differentiation, genes of cytokines, and even viral genes; therefore, it plays important roles in regulation of immunity, cellular differentiation, inflammation and tumor formation.At the beginning of 90's of 20th century, domestic studies found the tumor suppressor function of the 3' untranslated region of NF-IL6 to malignant cells, a property similar to that of tumor suppressor genes. Studies on the effects of the coding region in NF-IL6 on the cellular phenotype changes revealed that exogenous expression of NF-IL6 could induce apoptosis of murine myeloma cells, and transfection of NF-IL6 coding region to murine abdominal resident macrophages could significantly enhance their cytotoxicity to tumor cells. On the other hand, studies abroad showed that NF-IL6 was essential for the antitumor cytotoxicity of murine macrophages. These results indicate that artificially enhanced expression of NF-IL6 is feasible to inhibit tumoral activity and to maintain the normal cellular phenotype.This study was designed to explore the effects of NF-IL6 gene on the malignant phenotype of BEL-7404 cell line. The eukaryotic expression vector pCN and pCN/vacant were constructed and transfected into BEL-7404 by calcium phosphate-mediated transfection. Stable G418-resistant clones were obtained, and the integration and the expression of NF-IL6 gene were detected by Southern and Northern blotting. Finally the cytobiological characterization of positive clone was analyzed by cell proliferation assay, soft agar assay and nude mice tumorigenesis assay. The cell cycle and apoptotic rate of BEL-7404 transfectants was measured with flow cytometry.The Southern and Northern blotting experiments showed that BEL-7404 transfectants stably expressing full-length coding region of NF-IL6 were established by calcium phosphate -mediated transfection. Cell proliferation and soft agar assays demonstrated that the NF-IL6 transfectants grew obviously more slowly than control cells, and the NF-IL6 transfectants exhibited markedly lower colony formation rate than control cells. Tumorigenesis assay revealed that the tumorvolumes from NF-IL6 transfectant clones in nude mice were significantly smaller than that from control cell lines. Flow cytometry assay showed that the exogenous NF-IL6 could induce apoptosis of BEL-7404 cells.Above results indicate that expression of the exogenous NF-IL6 could favor the malignant phenotype reversal of BEL-7404 cell line and inhibit the proliferation and tumorgenesis of BEL-7404. The results also suggest that NF-IL6 gene may be a tumor suppressor gene in human hepatoma cells. The present investigation can provide new insights into the development of gene therapy of hepatoma cellular carcinoma.