miR-29a调控PER1-LAMR1相互作用影响放射后肺腺癌细胞侵袭迁移的研究

The circadian gene Per1 plays an important role on physiological function by protein-protein interaction. LAMR1 is one of the proteins interacted with PER1. Their combining site is coincide with the interacted site between LAMR1 and laminin which could regulate cell adhesion and invision. It is suggested that PER1 might interacted with LAMR1 competing with laminin, and the interaction intensity might influence the regulating ability of Per1 on tumor cell invasion and migration. Moreover, Per1 expression could be down-regulated by miR-29a which could affect cell proliferation and invasion of lung cancer. Per1 also could affect radiation killing of many kinds of tumor by influencing cell radiosensitivity. The previous study found that the migration of the residual lung adenocarcinoma cell A549 was changed after radiation follow the time. Therefore, the circadian gene Per1 is related with tumor invasion and metastasis, radiation, miR-29a and LAMR1; the invasion and metastasis of residual lung adenocarcinoma cell could changed after radiation. The study will focused on the change of cell invasion and migration of lung adenocarcinoma after radiation regulated by miR-29a through PER1-LAMR1 interaction mediated by Per1. The study will be help to illustrate the related mechanism of biological behavior change of lung adenocarcinoma cell after radiation.

节律基因Per1通过蛋白间相互作用发挥生理功能，LAMR1是其作用蛋白之一。其与PER1结合的位点恰与调控细胞粘附、侵袭的层粘连蛋白作用位点重合，提示PER1与层粘连蛋白竞争结合LAMR1，PER1-LAMR1相互作用强弱可能影响Per1对肿瘤细胞侵袭迁移等调控的能力。而Per1又可受到miR-29a调控表达下调，miR-29a本身也会影响肺癌细胞增殖侵袭。Per1还能影响多种肿瘤细胞对射线的敏感性，调控肿瘤放射杀灭。项目组前期发现，放射后残留肺腺癌A549细胞的迁移能力随时间延续发生不同改变。因此，结合节律基因Per1与肿瘤侵袭转移、放射、miR-29a及LAMR1的关系，以及放射后肺腺癌细胞侵袭转移的改变。本项目拟采用体内外实验研究miR-29a通过调控Per1表达介导PER1-LAMR1相互作用影响放射后肺腺癌细胞侵袭迁移能力的变化，以期揭示放射后肺腺癌细胞生物学行为变化的相关机制。

放射治疗是肺癌的主要治疗方法之一，但放疗后的复发转移却难以避免。节律基因Per1与肿瘤细胞增殖、侵袭、转移有关，并与可影响放射生物效应。本项目结合通过TPA刺激检测体外培养A549细胞后明确了细胞中Per1、miR-29a和LAMR1表达节律，并发现干扰Per1后A549细胞放射后侵袭、转移能力增强，MMP-2表达升高，而Per1过表达细胞放射后侵袭能力减弱。而转染 miR-29a mimics入A549细胞放射后侵袭、迁移能力减弱，Per1和MMP-2表达也降低；转染miR-29a inhibitor后结果相反。进一步通过回复实验发现miR-29a可抑制Per1表达，而二者均可抑制放射后肺腺癌细胞侵袭迁移。为探究其调控机制，将不同荧光蛋白标记的PER1和LAMR1过表达质粒共转染入A549细胞，再转染miR-29a mimics， X线照射。荧光共振能量转移检测发现PER1和LAMR1能够发生相互作用，转入miR-29a mimics后PER1-LAMR1作用减弱；PER1、LAMR1共转染细胞放射后侵袭能力减弱，而miR-29a、PER1、LAMR1三转染细胞的侵袭能力最弱；miR-29a可以影响Per1和LAMR1表达，转染了miR-29a、Per1、LAMR1的细胞MMP2、LN表达降低。构建肺腺癌裸鼠原位移植瘤模型，发现移植瘤组织中Per1、miR-29a表达呈现24h近日节律变化。分别在Per1表达峰值及谷值时间行8Gy X线胸部照射，活体荧光成像结果显示肿瘤生长延迟，Per峰值组差异较谷值组明显，Per1的表达变化也不同。研究提示miR-29a可能通过影响PER1、LAMR1的表达来调控PER1-LAMR1相互作用影响了放射后A549细胞的侵袭迁移能力。有助于探究肺腺癌放射生物学特性和行为，探索肺腺癌复发转移的防治策略，为肺癌的综合治疗疗效的提高提供理论指导。