PPP1R14D靶向PP1/Mps1干预纺锤体组装调控宣威肺癌增殖的机制研究

The incidence of Xuanwei lung cancer is the highest, but so far its pathogenesis has not been elucidated. We obtained Xuanwei lung cancer-specific proliferative gene PPP1R14D (Result One ~ Two) using mRNA microarray, Real-time PCR and Cellomics high-throughput proliferation genes screening system; then detected it by immunohistochemistry, PPP1R14D is highly expressed in Xuanwei lung cancer, and significantly correlated with T stage (Result Three); in Xuanwei lung cancer cells line XLA-07, knockdown PPP1R14D, the proliferation ability of lung cancer was decreased, appeared G2/M phase arrest (Result Four). PPP1R14D may affect PP1/Mps1 signaling, then regulated spindle assembly and mitosis. Further experiments found that knockdown PPP1R14D, phosphorylation of PP1 was increased, Mps1 and pMps1 were decreased, immunoprecipitation confirmed PPP1R14D was associated with PP1 (Result Five), these results suggested that PP1/Mps1 is the downstream pathway of PPP1R14D. This project intends to clarify the mechanism on PPP1R14D affected PP1/MPS1 signaling, then intervented spindle assembly and regulated proliferation of lung cancer, which would provide a new target for the prevention and treatment of Xuanwei lung cancer.

云南宣威肺癌高发，然而其驱动基因和发病机制至今未见阐明。我们前期采用mRNA芯片、Real-time PCR和Cellomics高通量细胞增殖基因筛选系统，获得宣威肺癌特异促增殖基因PPP1R14D（结果一~二）；然后免疫组化检测，PPP1R14D在宣威肺癌高表达，并且与T分期显著相关（结果三）；在宣威肺癌XLA-07细胞，敲减PPP1R14D，肺癌增殖能力降低，出现G2/M期阻滞（结果四）。PPP1R14D 可能通过PP1/Mps1通路，调控纺锤体组装，干预有丝分裂。进一步实验发现，敲减PPP1R14D，磷酸化PP1 增多，Mps1和pMps1均减少，免疫共沉淀证实PPP1R14D与PP1存在关联（结果五），提示PP1/Mps1可能是PPP1R14D的下游通路。本项目拟阐明PPP1R14D介导PP1/MPS1，干预纺锤体组装，调控肺癌增殖的机制，为宣威肺癌的防治提供新的干预靶点。

云南宣威地区长期的二叠纪烟煤空气污染，使肺癌发病率持续上升，为全球肺癌高发研究现场，而其发病机制远未阐明。宣威肺癌具有特殊的内在驱动基因和发病机制。前期研究中，我们采用安捷伦mRNA芯片，对比研究宣威女性肺癌和非宣威女性肺癌的表达谱差异，筛选出 LYPD1、 SDC1、 PPP1R14D 等15个差异表达基因，Real-time PCR验证显示PPP1R14D在宣威肺癌中高表达；临床组织样本IHC结果显示，PPP1R14D在宣威肺癌高表达，与T分期显著相关；功能学实验结果显示，利用慢病毒敲减PPP1R14D，肺癌细胞增殖能力降低，集落形成能力下降，细胞周期出现G2/M 期阻滞；裸鼠成瘤实验结果显示，分别利用慢病毒敲减肺癌细胞PPP1R14D、PP1及MPS1，肿瘤生长明显减慢，肿瘤湿重明显低于对照组，以上结果提示PPP1R14D与肺癌发生密切相关。进一步机制研究表明，敲减PPP1R14D致磷酸化PP1增多、Mps1磷酸化减少，免疫共沉淀检测显示PPP1R14D与PP1存在直接结合关系。在稳定干扰PPP1R14D的肺癌细胞中，过表达PP1，其下游分子Mps1表达增加，细胞增殖得到恢复。在稳定干扰PPP1R14D的肺癌细胞中，过表达Mps1，pMps1表达增加，细胞增殖得到恢复，提示PPP1R14D通过级联磷酸化反应，作用于 PP1/MPS1通路，调控肺癌恶性增殖。.通过本课题研究，从细胞、动物、人体组织标本阐明PPP1R14D 通过级联磷酸化反应，作用于 PP1/MPS1 通路，干预纺锤体组装，调控肺癌增殖的作用机制，为肺癌的防治提供新的干预靶点和治疗思路。