Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China.
Int J Mol Med. 2010 Sep;26(3):365-72.
The aim of this study was to investigate the effects of GATA-4 on the differentiation of P19 cells into cardiomyocytes and to examine the relationship between GATA-4 and cardiomyocytes. We constructed vectors to overexpress and silence GATA-4. These vectors, as well as empty ones were transfected into P19 cells. Subsequently, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were performed. The morphology of P19 cells during differentiation was observed using an inverted microscope. Total RNA was extracted from P19 cells. We used real-time PCR to evaluate the expression levels of 6 genes: GATA-4, GATA-6, transthyretin (TTR), alpha-fetoprotein (AFP), Nkx2.5, and alpha-myosin heavy chain (alpha-MHC). The gene expression pattern of these 6 genes is graphically shown for each group. The GATA-4 mRNA level in cells overexpressing GATA-4 was notably higher than that in the controls, whereas the levels in the controls were notably higher than those in the GATA-4-silenced P19 cells. The cell lines overexpressing GATA-4 expressed higher levels of Nkx2.5 and alpha-MHC than the controls. However, the controls expressed higher levels of AFP, GATA-6 and TTR than the cells overexpressing GATA-4. The RNAi group expressed lower levels of TTR, Nkx2.5, and alpha-MHC than the controls, but there were no differences in the RNAi group and the controls with regard to the expression levels of AFP and GATA-6. The gene expression pattern in the cells overexpressing GATA-4 was biased toward the Nkx2.5 and alpha-MHC. On the other hand, the gene expression pattern in GATA-4-silenced cells and the controls was biased toward the TTR and AFP. The overexpression of GATA-4 enhances the differentiation of P19 cells into cardiac myocytes, whereas its down-regulation suppresses this trend.
本研究旨在探讨 GATA-4 对 P19 细胞向心肌细胞分化的影响，并研究 GATA-4 与心肌细胞之间的关系。我们构建了过表达和沉默 GATA-4 的载体。将这些载体以及空载体转染到 P19 细胞中。随后，进行逆转录聚合酶链反应（RT-PCR）和 Western blot 分析。使用倒置显微镜观察 P19 细胞分化过程中的形态。从 P19 细胞中提取总 RNA。使用实时 PCR 评估 6 个基因的表达水平：GATA-4、GATA-6、转甲状腺素蛋白（TTR）、甲胎蛋白（AFP）、Nkx2.5 和α-肌球蛋白重链（α-MHC）。以图形方式显示每组这 6 个基因的表达模式。过表达 GATA-4 的细胞中 GATA-4 mRNA 水平明显高于对照组，而对照组中 GATA-4 沉默的 P19 细胞中 GATA-4 水平明显高于对照组。过表达 GATA-4 的细胞系表达的 Nkx2.5 和α-MHC 水平高于对照组。然而，对照组中 AFP、GATA-6 和 TTR 的表达水平高于过表达 GATA-4 的细胞。RNAi 组的 TTR、Nkx2.5 和α-MHC 表达水平低于对照组，但 RNAi 组与对照组在 AFP 和 GATA-6 的表达水平上无差异。过表达 GATA-4 的细胞基因表达模式偏向于 Nkx2.5 和α-MHC。另一方面，GATA-4 沉默细胞和对照组的基因表达模式偏向于 TTR 和 AFP。GATA-4 的过表达增强了 P19 细胞向心肌细胞的分化，而其下调则抑制了这种趋势。
