lncRNA-AK303027协同Smad4促使PPP2R5B转录进而抑制AKT作为新信号通路在胰腺癌发生中的作用机制研究

TGF-β-Smad4 was considered as one of the most important pathways which inhibited pancreatic carcinogenesis. However, the antitumor effects were suppressed under specific condition, and the mechanism remained unknown,which suggested Smad4 may be regulated by other unknown genes. As a massive degree of cellular functionality was programmed in the noncoding genome, we discovered that lncRNA-AK303027 may bind to Smad4, and may have “crosstalk” with PPP2R5B and AKT through microarray analysis and subsequent verification in our preliminary work. Therefore, in the study, RNA immunoprecipitation , chromatin immunoprecipitation and in vitro dephosphorylation assay will be performed to explore whether lncRNA-AK303027 binds to Smad4 and Smad4 binds to PPP2R5B promoter and stimulates PPP2R5B expression, which may activate dephosphorylation of AKT and inhibit malignant transformation in pancreatic carcinogenesis. The study will implicate that lncRNA-AK303027/Smad4-PPP2R5B-AKT axis, a new signaling pathway, may play an important tumor suppressive role in pancreatic carcinogenesis, and may serve as a potential treatment target for patients with PDAC.

TGF-β-Smad4是抑制胰腺癌发生发展重要通路之一。然近有学者提出，在特定状态下，Smad4抗癌作用可能被抑制，其具体机制不明，提示Smad4可能被其它未知因子调控，或与其它通路存在“交叉对话”。细胞多种功能受长链非编码RNA调控，在前期工作中我们通过基因芯片分析及验证，发现lncRNA-AK303027 可能与Smad4结合，并协同Smad4促进PPP2R5B表达，且PPP2R5B与AKT结合。基于此，本项目拟进一步采用RNA结合蛋白免疫沉淀、染色质免疫沉淀、蛋白磷酸化等技术证实lncRNA-AK303027结合Smad4，协同Smad4促使PPP2R5B转录，介导AKT去磷酸化，阻断AKT通路，抑制胰腺肿瘤发生发展。本研究阐明lncRNA-AK303027/Smad4-PPP2R5B-AKT作为一新信号通路，为胰腺癌靶向治疗提供新理论依。

近来有研究显示，Smad4可能被其它未知因子调控，与其它通路存在“交叉对话”。在本课题中，我们通过高通量测序，发现lncRNA-AK303027；通过real-time PCR验证以及Pearson’s相关分析检验，发现lncRNA-AK303027、Smad4与PPP2R5B在胰腺癌组织中的表达显著低于癌旁组织，且无论在胰腺癌组织中或是癌旁组织中，lncRNA-AK303027与Smad4，以及lncRNA-AK303027与PPP2R5B均呈显著正相关。通过生物学功能实验发现，lncRNA-AK303027抑制胰腺癌细胞增殖、克隆形成、侵袭能力、成瘤能力以及尾静脉肺转移能力，提示lncRNA-AK303027起到抑癌基因作用。通过RIP实验显示：人正常胰腺导管上皮细胞HPDE6-C7细胞中，lncRNAAK303027可与Smad4蛋白直接结合，却不与PPP2R5B蛋白直接结合，提示lncRNA-AK303027可能直接调控Smad4。通过ChIP实验显示，在AK303027高表达的条件下，Smad4蛋白与PPP2R5B直接结合，提示PPP2R5B可能是Smad4的下游靶点，且Smad4可能依赖AK303027作用于PPP2R5B；通过蛋白免疫沉淀实验（IP）发现，PPP2R5B可与AKT直接结合。进一步，我们通过体外磷酸化实验以及免疫荧光实验检测发现，lncRNA-AK303027-Smad4通过PPP2R5B抑制AKT磷酸化；结合尾静脉肺转移实验，我们证实lncRNA-AK303027结合Smad4，协同Smad4促使PPP2R5B转录，介导AKT去磷酸化，阻断AKT通路，抑制胰腺肿瘤发生发展。本研究阐明lncRNA-AK303027/Smad4-PPP2R5B-AKT作为一新信号通路，为胰腺癌诊断及靶向治疗提供新理论依。