PTTG1通过TGFβ1/SMAD3通路调控放射后肺腺癌细胞侵袭和免疫改变的研究

The major causes of death in patients with non-small cell lung cancer after radiotherapy are cancer recurrence and metastasis. They are depended on the growth, invasion ability and immune function of residual tumor cells. One of the important causes is Epithelial-Mesenchymal Transition (EMT). Radiation could induce changes on TGFβ1/SMAD3 pathway and influence EMT process. TGFβ1/SMAD3 pathway was regulated by pituitary tumor-transforming gene 1(PTTG1). Meanwhile, TGF-β1 is an important immunologic suppressor released by tumor cells. TGF-β1 could inhibit immune system and enhanced tumor growth, invasion and metastasis. With radiation induced tumor death, changes on TGFβ1 activity would regulate tumor microenvironment, release death signal and influence immune response. It is related to radiation abscopal effect. Therefore, the study will investigate the effects of PTTG1 on changes of invasion and immune in residual lung adenocarcinoma cells after radiation in vivo and in vitro. And it will investigate the mechanism of changes on TGFβ1/SMAD3 pathway. The experimental techniques involved cell transfection, animal model construction, flow cytometry, western blot、Transwell will be used. The study will help to reveal the mechanisms of recurrence and metastasis of lung adenocarcinoma after radiotherapy. It will provide a theory for radiotherapy combined with immune therapy on cancer. And it is also in favour of prevention and treatment on multiple tumors in Yunnan province.

非小细胞肺癌放疗后主要死亡原因是复发与转移，取决于残余细胞的生长、侵袭转移能力与宿主对肿瘤细胞的免疫功能。重要原因之一是放射引起TGFβ1/SMAD3信号通路改变影响上皮-间质转化(EMT)过程，该通路受到垂体瘤转化基因1(PTTG1)调控。同时，TGFβ1又是重要的免疫抑制因子，肿瘤细胞会自身释放，抑制宿主免疫系统，促进肿瘤生长、侵袭和转移。通过放射肿瘤杀灭，TGFβ1活性改变，调节肿瘤微环境、释放死亡危险信号而影响机体免疫应答，与研究热点“放射远隔效应”有关。因此，项目拟通过细胞转染、动物模型构建、流式细胞术、western blot、Transwell等体内外实验，研究PTTG1对放射后肺腺癌残余细胞侵袭能力和免疫功能变化的影响，探讨TGFβ1/SMAD3通路变化的发生机制，以期揭示放疗与肺腺癌复发转移和放射免疫治疗肿瘤的相关机制，提供治疗指导，有利于促进云南地区多发性肿瘤的防治。

放射治疗是肺癌治疗的主要手段之一，但是放疗后的复发转移却难以避免，这可能与放射后残余细胞的生长、侵袭转移能力及机体抗肿瘤免疫相关。本项目通过体内外实验探究PTTG1对放射后肺腺癌细胞侵袭能力和免疫功能的影响及相关生物学机制。研究发现X线照射后，人肺腺癌A549和H1299细胞侵袭能力减弱，并与照射剂量相关；PTTG1基因沉默增强了射线对A549和H1299细胞侵袭能力的抑制作用；各组细胞中TGF-β1、SMAD3、MMP-2、MMP-9、E-cadherin、N-cadherin、Snail1表达也发生了不同的变化。A549/A549（PTTG1-）细胞与PBMNC共培养后照射，辐射降低了Tregs比例削弱免疫抑制，增加了MHC-I分子表达和CD8+T细胞比例，增强抗肿瘤免疫反应；PTTG1沉默可提高辐射诱导的免疫增强功能。进一步阻断TGF-β1/SMAD3通路，放射后细胞侵袭能力显著降低，Tregs比例降低，MHC-I分子和CD8+T细胞比例增加。构建PTTG1敲除的LLC-LUC小鼠肺癌原位模型，PTTG1组瘤体小于对照组，转移率也低于对照组。单次大剂量X线15Gy照射肿瘤后，对照组小鼠肿瘤组织、血液和外周淋巴器官中Tregs显著上调，MHC-I和CD8+T细胞比例上调；而PTTG1组中，X线照射后 Tregs无明显变化，各组织和免疫器官中MHC-I和/或CD8+T细胞比例进一步提高。PTTG1敲除组小鼠的肿瘤和肺组织中TGF-β1、SMAD3蛋白表达均在X线照射后降低。提示PTTG1可能通过TGF-β1/SMAD3通路影响EMT调控放射后肺腺癌细胞的侵袭能力，并影响Tregs、MHC-I分子调节放射后CD8+T细胞介导的抗肿瘤免疫反应，影响肿瘤转移。本研究有助于揭示放疗与免疫治疗协同作用和肺腺癌复发转移的机制，为肺癌的综合治疗策略制定提供参考，进而提高肺癌的治疗疗效。