沉默PPP3CA对后肾间充质细胞MET转化、凋亡、增殖及迁移的影响

PPP3CA silence regulates MET process, cell apoptosis, proliferation and migration in metanephric mesenchyme cells

摘要:

肾脏是人体重要器官，肾脏发育对肾脏的形成和功能至关重要，其中后肾间充质细胞 (Metanephric mesenchyme，MM) 间质-上皮转化 (Mesenchymal-epithelial transition，MET) 是肾单位形成的关键环节。qRT-PCR和Western blotting实验检测蛋白质磷酸酶3催化亚基α (Protein phosphatase 3 catalytic subunit alpha，PPP3CA) 在不同状态MM细胞株mK3、mK4中的表达谱及对MET标志蛋白调控作用；采用慢病毒包装方式构建稳定敲低PPP3CA的mK4细胞株；采用CCK-8、EdU实验、细胞划痕实验、流式细胞技术分别检测PPP3CA对上皮样后肾间充质细胞株mK4细胞生长、迁移、凋亡的调控作用。PPP3CA在mK4细胞中表达量较间质样后肾间充质细胞mK3更高，敲低PPP3CA后，检测MET标志物及细胞生物学行为，结果显示敲低PPP3CA显著上调上皮细胞标志物E-cadherin表达，促进MET过程，且促进细胞凋亡，抑制细胞增殖和迁移。此外，敲低PPP3CA促进ERK1/2磷酸化，提示PPP3CA生物学功能的调控机制可能与其去磷酸化ERK1/2蛋白相关。以上结果提示PPP3CA在MM细胞MET转化和生物学行为调节中发挥重要功能，为发现和解析肾发育过程中潜在的关键调节因子提供了新的理论基础。

Abstract:

Kidney is one of the most important organs of the body and the mammalian kidney development is essential for kidney unit formation. The key process of kidney development is metanephric development, where mesenchymal-epithelial transition (MET) plays a crucial role. Here we investigated the biological function of PPP3CA in metanephric mesenchyme (MM) cells. qRT-PCR and Western blotting were used to detect PPP3CA and MET makers expression in mK3, mK4 cells respectively at mRNA and protein level. Subsequently, PPP3CA was stably knocked down via lentivirus infection in mK4 cells. Flow cytometry, EdU/CCK-8 assay, wound healing assay were conducted to clarify the regulation of PPP3CA on cell apoptosis, proliferation and migration respectively. PPP3CA was expressed higher in epithelial-like mK4 cells than mesenchyme-like mK3 cells. Thus, PPP3CA was silenced in mK4 cells and PPP3CA deficiency promoted E-cadherin expression, cell apoptosis. Moreover, PPP3CA knock down attenuated cell proliferation and cell migration in mK4 cell. The underlying mechanism was associated with the dephosphorylation of PPP3CA on ERK1/2. Taken together, our results indicated that PPP3CA mediated MET process and cell behaviors of MM cells, providing new foundation for analyzing potential regulator in kidney development process.