Establishment Of Cell Line Stably Expressing ZAG And Its Effects On Relevant Factors Of Mitochondria Biogenesis

Objective: To construct pcDNA3.1(-)-mZAG recombinant mammalian expression vector , which was transfected into 3T3-L1 cell and observe its effects on relevant factors of mitochondria biogenesis after overexpression of ZAG.Methods: The total RNA was extracted from mouse liver cell, and the sequence of ZAG was amplified with RT-PCR.The PCR product was then cloned into the prokaryotic expression vector pcDNA3.1(-) after enzyme by XbaⅠand HindШ. After identification by double restriction enzyme digestion and DNA sequencing, the recombinant eukaryotic expression plasmid pcDNA3.1(-)-mZAG was transfected into 3T3-L1 cell by lipofectamine 2000. The stable transfected 3T3-L1 cell line was established after selection with G418. The expression of ZAG mRNA and PGC-1α,NRF-1/2,mtTFA mRNA were detected by RT-PCR. The expression of PGC-1α,NRF-1/2,mtTFA protein were detected by Western Blot.Results: Mouse ZAG coding sequence was amplified successfully and ligated with vector fragments by T4 ligase. The construction of pcDNA3.1(-)-mZAG plasmid containing mouse ZAG coding sequence of 5.4kb fragment and another 924bp fragment when digested by XbaⅠand HindШ. PcDNA3.1(-)-ZAG was transfected into the 3T3-L1 cell successfully, the expression of PGC-1α,NRF-1/2,mtTFA and ZAG mRNA were up-regulated in the transfected 3T3-L1 cell.Conclusion: The recombinant eukaryotic expression vector pcDNA3.1(-)-ZAG was constructed successfully. The stable 3T3-L1 cell line over-expression of ZAG was established. In the stable transfected 3T3-L1 cell, the expression of ZAG mRNA is correlated with mitochondria generation, which have promote effects on expression of relevant factors of mitochondria biogenesis. Objective: To explore whether PKA and p38MAPK could mediate the effects of ZAG on relevant factors of mitochondria biogenesis.Methods: 3T3-L1 cells were treated with ZAG,PKA inhibitor H89,H89+ZAG,p38MAPK inhibitor SB203580 and SB203580+ZAG. The expression of PGC-1α,NRF-1/2,mtTFA mRNA and protein were detected by the method of RT-PCR and Western Blot.Results: ZAG promoted the expression of relevant factors of mitochondria biogenesis, PKA inhibitor H89,p38MAPK inhibitor SB203580 inhibited the expression of relevant factors of mitochondria biogenesis, H89+ZAG,SB203580+ZAG decreased the promote effects of ZAG on mitochondria biogenesis.Conclusion: ZAG have a promote effect on relevant factors of mitochondria biogenesis, PKA and p38MAPK signal pathway can mediate this effect.