Expression and purification of an FGF9 fusion protein in E. coli, and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration
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Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity. Both purified forms of rhFGF9, with and without fusion tags, significantly stimulated the proliferation of NIH3T3 cells. The FGF9 subfamily, including FGF9, FGF16, and FGF20, in addition to rhFGF16, rhFGF9, and rhFGF20, were shown to stimulate the proliferation and migration of HuH7 human hepatocellular carcinoma (HCC) cells. Mechanistic studies revealed that the stimulation of HuH7 cell proliferation and migration with rhFGF9 and rhFGF20 were associated with the activation of the extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) pathways and matrix metalloproteinase-26 (MMP26). Inhibition of the ERK and NF-κB pathways blocked cell migration, and NF-κB was demonstrated to be regulated by ERK. Therefore, the present study demonstrates a simple method for the preparation of biologically active rhFGF9 protein. Furthermore, the results indicate that exogenous rhFGF9- and rhFGF20-activated ERK/NF-κB signal transduction pathways play important roles in the regulation of HCC cell proliferation and migration, and this discovery helps to find the potential for new solutions of the treatment of liver cancer.
KeywordsRecombinant human FGF9 Fusion expression TEV protease cleavage Mitogen activity HuH7 cells Signal transduction mechanism
This work was supported by grants of National Natural Science Foundation of China (No. 81471075) and granted by the Opening Project of Zhejiang Provincial TOP Key Discipline of Pharmaceutical Sciences (No.201720).
Compliance with ethical standards
This article does not contain any studies with animals or human participants. All authors confirm that ethical principles have been followed in the experiments.
Conflicts of interest
The authors declare that they have no conflict of interest.
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