Construction Of Human TWEAK Gene Eukaryotic Expression Vector And Preliminary Study Of Its Function In Vitro

Objective：To construct recombinant pcDNA3.1(+)-TWEAK vector andstudy the role of TWEAK in HepG2cells.Methods：The entire coding region of TWEAK gene was amplified frompORF5-hTWEAK plasmid by PCR,and then TWEAK gene fragments werepurified and cloned into prokaryotic expression vector pET32a(+). Therecombinant plasmids were transformed into E.coli JM109and identified withPCR, restriction enzyme digestion and sequencing. The recombinant plasmidswere named pET32a(+)-TWEAK.TWEAK gene from pET32a(+)-TWEAKby digesting with XholⅠand HindIII was subcloned into eukaryoticexpression vector pcDNA3.1(+) and identificated by double restrictionenzyme digestion. The recombinant eukaryotic expression plasmids werenamed pcDNA3.1(+)-TWEAK and transfected into HepG2cells usinglipofectamine2000transfection reagent. TWEAK expression inpcDNA3.1-TWEAK transfected cells was verified by RT-qPCR and Westernblot. MTT assay was carried out to evaluate the effect of TWEAK on HepG2cells viability in vitro. The mRNA of Fn14,caspase-8,bcl-2and bax weremeasured by RT-qPCR. The protein of TWEAK，NF-κB1and MAPK3weredetermined by Western blot.Results：The entire coding region of TWEAK gene was cloned intopET32a(+) plasmid and successfully subcloned into expression vectorpcDNA3.1(+). After the recombinant pcDNA3.1-TWEAK was transfectedinto HepG2, the mRNA of TWEAK,Fn14and caspase-8were expression higher than the negative control groups(P<0.05), and the protein of TWEAKand NF-κB1were expression higher than the negative control groups(P<0.05).Meanwhile,the proliferation of HepG2cells were inhibited.Conclusion： The recombinant eukaryotic expression vectorpcDNA3.1(+)-TWEAK was constructed successfully and transfected intoHepG2cells to overexpress TWEAK. Overexpression of TWEAK was able toactivate Fn14,caspase-8,bax and NF-κB1,and inhibit the proliferation ofHepG2. Collectively, TWEAK may play a key role in HepG2cells byaffecting cell proliferation/apoptosis. TWEAK Selective targeting of theTWEAK-dependent signaling pathway or augmenting TWEAK levels mayserve as novel therapeutic strategies for human hepatoma in future.