Aim: To establish a murine secreted mature peptide IL-1β expression vector, transfect into Hepa1-6 hepatoma cells, and analyze the effect of recombinant IL-1β on proliferation, migration, and its specific expression in Hepa1-6 hepatoma cells.
Methods: The murine AFP signal peptide encoding sequence and mature IL-1β encoding fragment were linked together through overlapping PCR, and the chimeric DNA sequence was then inserted into a liver specific expression vector pLIVE(TM); to make a recombinant pLIVE-smIL-1β which expressed secreted murine IL-1β of classical pathway. pLIVE-smIL-1β, pLIVE(TM); and pLIVE-lacZ were transfected into Hepa1-6 by jetPEI respectively. Transfection of the vectors were detected by β-gal staining using pLIVE-lacZ transfectants. Cells treated with 5 μg/mL lipopolysaccharide were used as positive control and 3 μmol/L monesin was added into culture system to block classical pathway secretion, then sandwich ELISA was employed to detect the IL-1β levels both in supernatant and in cytoplasm of each group of transfected cells. The proliferation of Hepa1-6 hepatoma cells was determined by MTT assay and migration of Hepa1-6 cells was assessed by scratch test in vitro.
Results: pLIVE-smIL-1β vector successfully expressed murine IL-1β in Hepa1-6 cells. Expression of the recombinant vector peaked at day 3 as indicated in a β-gal staining method. After transfection, compared with Hepa1-6/mock cells, IL-1β expression levels both in supernatant and in cytoplasm of Hepa1-6/smIL-1β cell were significantly increased detected by ELISA. The proliferation of Hepa1-6/smIL-1β group was markedly promoted in vitro detected by MTT assay.
Conclusion: The recombinant expression vector can secret IL-1β through classical pathway which significantly promoted proliferation and migration of hepatoma cells in vitro.