中性粒细胞（PMN）自发性凋亡调控机制仍不十分清楚。我们已经证实PMN自发性凋亡过程中，miR199a上调表达而PKCB和NF-κB下调表达。那么它们与PMN凋亡调控有何关系？表观遗传调控子Tet2具活化microRNA的功能，且与PMN凋亡有关。我们深入分析发现，miR199a启动子含CpG岛，具有被表观调控的可能；PKCB 3'-UTR区含miR199a靶向结合区。由此，我们提出PMN自发性凋亡调控新机制，即：PMN中存在Tet2-miR199a-PKCB-NF-κB轴，miR199a 被Tet2活化后抑制PKCB-NF-κB，进而介导PMN凋亡。本研究拟用RNAi、超表达、电转抗体和CHIP等方法，研究Tet2、miR199a、PKCB及NF-κB的相互关系，并找到几者互作调控PMN凋亡的直接证据,阐明Tet2-miR199a-PKCB-NF-κB轴调控PMN自发性凋亡的新机制。

The regulation mechanism of polymorphonuclear neutrophil (PMN) apoptosis is unclear. We have confirmed that during PMN spontaneous apoptosis, the transcription of miR199a is up-regulated and that the expression of PKCB and NF- κB is down-regulated. But the relationship of miR199a, PKCB, and NF-κB with spontaneous PMN apoptosis has not been studied. Epigenetic regulator Tet2 can activate microRNA by mediating DNA demethylation and is related to PMN apoptosis. Our further analysis revealed that miR199a promoter contained CpG Island and miR199a was likely to be epigenetically regulated. We also found that the PKCB 3'-UTR region contained a target binding region with miR199a. Thus, we proposed a new mechanism by which spontaneous PMN apoptosis was regulated. This mechanism involved a Tet2-miR199a-PKCB-NF-κB axis in PMN and a phenomenon wherein miR199a activated by Tet2 inhibited the PKCB-NF-κB pathway followed by PMN apoptosis mediation. The relationship among Tet2, miR199a, PKCB, and NF-κB was then studied, and direct evidence of interactions regulating PMN apoptosis were investigated using RNAi, overexpression, electroporation of antibody, and CHIP method. Our results elucidated the mechanism of the Tet2-miR199a-PKCB-NF-κB axis regulation of spontaneous PMN apoptosis.

中性粒细胞（PMN）自发性凋亡调控机制仍不十分清楚。前期我们已经证实PMN自发性凋亡过程中，miR199a上调表达而PKCB下调表达。那么它们与PMN凋亡调控有何关系？表观遗传调控子Tet2具活化microRNA的功能，且与PMN凋亡有关。我们深入分析发现，miR199a启动子含CpG岛，具有被表观调控的可能；PKCB 3'-UTR区含miR199a靶向结合区。由此，我们提出PMN自发性凋亡可能存在调控新机制，即：PMN中存在Tet2-miR199a-PKCB轴，miR199a 被Tet2活化后抑制PKCB，进而介导PMN凋亡。本研究采用脂质体转染和CHIP-sequence等方法，重点研究了miR199a对PKCB是否有抑制作用，Tet2是否结合并调控miR199a的基因序列，期望为进一步证实是否存在Tet2-miR199a-PKCB轴调控PMN自发性凋亡的新机制提供理论依据。但脂质体转染实验结果显示，miR199a与PKCB之间并无直接相互作用，脂质体转染实验结果为阴性；同时另一候选microRNA miR432与PKCB之间也无直接相互作用，上述结果提示对PKCB能够起到抑制作用的可能为其它microRNA。采用CHIP-sequence，分别比较和解析分离培养0h和12h时Tet2结合的基因序列，经过分析，Tet2并不结合miR199a的基因序列，但是Tet2结合多个其它基因序列，且部分基因序列提示转录后为microRNA。上述结果提示，Tet2可能通过结合调控其它microRNA的基因表达，进而对PCKB进行调控。下一步研究将继续分析Tet2究竟调控哪个（哪些）microRNA，而哪个（哪些）microRNA可抑制PKCB，进而调控PMN凋亡。