Constructionand Preliminary Functional Identification Of Plasmid PcDNA6-GATA4 P.S335X

Aim:GATA4 is an important transcription factor which is involved in the heart development. We previously found a special type of GATA4 mutation (p.S335X) in the myocardium in patients with ventricular septal defect (VSD), that results in premature termination codons and thus lead to C terminal deletion of GATA4. In the present study, the vectors of GATA4 wildtype (pcDNA6-GATA4-WT, WT) and mutation (pcDNA6-GATA4-MU, MU) were constructed, while the transfection efficiency was evaluated in mouse embryonic carcinoma cell line P19 cells, and the effects on apoptosis with the possible molecular mechanisms were preliminarily studied.Method:Polymerase chain reaction (PCR) amplification of human GATA4 cDNA was performed with 5’-end incorporation of restriction enzyme cutting site, and relevant sequences encoding full-length or mutant GATA4 fragments was obtained by using PCR and overlap-PCR.Fragments were retrieved by using agarose electrophoresis, enzyme cleaved, and ligated to the vector pcDNA6 (EV), and thus GATA4 WT and MU vectors were constructed. The vectors were validated by sequencing results and transfected to mouse embryonic carcinoma cell line P19 cells. The RNA and total protein were then collected from transfected cells, followed by examination of GATA4 mRNA (WT or MU) and protein by using qPCR and Western blot, respectively. The successful construction of WT and MU vectors was demonstrated. The effects of MU on apoptosis was assessed by using TUNEL, while the involvement of apoptosis molecular pathway was preliminarily studied by using Western blot.Results:1. The successful construction of WT and MU vectors was identified by sequencing and further demonstrated by using PCR and Western blot.2. TUNEL functional assay showed that, compared to that of WT, significant apoptosis was observed in the P19 cells transfected with MU.3. Western blot demonstrated that MU led to up-regulation of pro-apoptotic signal bax, down-regulation of anti-apoptotic signal Bcl-2, as well as inhibition of PI3K proliferative signal pathway.Conclusion:1. GATA4 WT and MU vectors were successfully constructed in the present study.2. Compared to that of GATA4 WT, MU (p.S335X) enhanced apoptosis in mouse embryonic carcinoma cell line P19 cells.3. The mutation (p.S335X) involved in apoptosis pathway, could affect the expression of Caspase-3 and Caspase-9, which are the key proteins in classic apoptosis pathway; and made the apoptosis related proteins expression of Bcl-2, Bax, Bim and FoxO3a change, may involve mitochondrial apoptotic pathways.