Construction of IL-24 mutant expression vector and research its therapic effect on HepG2 cells

Objective To construct an expression vector of IL-24 mutant RGD-IL-24, which a Glycine residue was inserted into the mda-7/IL-24 to form a Arg-Gly-Asp (RGD)motif and research its therapic effect on HepG2 cells.Methods Constructed RGD-IL-24 by means of overlapping PCR, which resulted in the extra codon GTT encoding the Glycine between Arg164 and Asp165 to form a RGD motif.The RGD-IL-24 was inserted inframe into pCDNA3.1 (+) expression vector to construct the pCDNA3.1/RGD-IL-24.HepG2 cells were transfected with pCDNA3.1/RGD-IL-24, as compare with pCDNA3.1/IL-24,which were collected at the expected time.IL-24 mRNA expression and protein expressions were observed by RT-PCR analysis and Western blot respectively.The proliferation and apoptosis were detected by MTT assays and FITC-Annexin V assays respectively.The pro-apoptotic protein bax and anti-apoptotic protein bcl-2 were detected by Western blot.Results The fragement encoded RGD-IL-24 was confirmed by nucleotide sequencing.IL-24 mRNA was detected by RT-PCR analysis 24 hours after transfected.IL-24 protein was detected by western blot analysis 48 hours after transfected.Transfected with pCDNA3.1/IL-24 and pCDNA3.1/RGD-IL-24 48 hours later, FITC-annexin V staining assays showed high levels of FITC-Annexin V positive cells in pCDNA3.1/RGD-IL-24 group.The significantly up-regulated pro-apoptotic protein bax and down-regulated anti-apoptotic protein bcl-2 were detected in pCDNA3.1/RGD-IL-24 treatment group than pCDNA3.1/IL-24 treatment group.Conclusion RGD-IL-24 significantly enhanced IL-24 therapeutic efficacy in HepG2 cells in vitro. Key words: IL-24;  Mutant;  RGD peptide;  Apoptosis;  Hepatoma therapy