NSCLC癌干细胞TLR9的表达特点、信号级联与干性维持作用

The hypothesis of cancer stem cells has been proposed to explain the genesis and progression in a variety of cancers including non-small cell lung cancers（NSCLC）. Previously, we demonstrated that Toll-like receptor（TLR）-9 had a crucial role in growth, chemotherapy-resistance and metastatic dissemination of NSCLC cancer cells. Recently，we and other researchers showed that TLR9 also expressed in tissue stem cells and NSCLC cancer stem cells. Very interestingly，many downstream effectors of TLR9-mediated singnaling including NF-κB, PI3K, AKT and MAPK，posses regulating abilities on cancer stem-like cells (CSCs). So，we presume that TLR9 pathway has regulation potential on NSCLC CSCs. In this study， we will aim to identify an important mechanism for the maintenance of stemness in multiple distinct phenotypic subpopulations of NSCLC CSCs. Using CD166, CD133, CD44 and ALDH as surface markers, we will isolate CSCs from primary NSCLC tumors and cancer cell lines. Firstly, the expression of TLR9 and genes involved in stem cell pathways [i.e.,Sonic hedgehog (SHH,Gli-1),Notch(Notch-1, Notch-2, Hes-1), OCT4/3, and Nanog] will be invstigated by real-time PCR or FACS. Secondly, to investigate the impact of TLR9 knockin/knockdown and activation on self-renewing, tumorigenic potential, and chemoresistant properties of CSCs. Thirdly, to gain a deeper understanding of the molecular basis for tumorigenic capacity of TLR9, we will perform genome-wide trascriptome analysis on TLR9-activated CSCs, the highest-ranking genes will be validated by RT-PCR or western-blot. And then, choose the highest-ranking genes as therapeutic target，to evaluate the effect of TLR9 signaling inhibitors on the self-renewing, tumorigenic potential of CSCs. Our research will suggest that TLR9 pathway inhibition may be a new therapeutic strategy aiming at NSCLC CSCs.

近来癌干细胞(CSCs)对肿瘤进展的影响凸显。TLR9信号通路介导促进并维系NSCLC的恶性征，我们的后续工作发现TLR9不仅特异高表达于NSCLC-CSCs并维持CSCs的自我更新能力，且其下游分子多具干性调节潜能，故推测TLR9通路参与NSCLC-CSCs的干性维持。为此，课题组拟从不同病理类型的NSCLC临床标本/细胞系中分离CSCs，①获得CD166+/133+/44+或ALDH+等不同标识的干细胞亚群并行干性鉴定，②用实时定量 PCR或流式细胞仪检测TLR9和常见干性基因的表达及其分化前后的动态变化,③观察TLR9基因敲入/敲减及其信号激活对CSCs自我更新、化疗抵抗和成瘤性的影响,④芯片技术筛选TLR9信号级联,并用WB或RT-PCR等进行验证以遴选目标靶分子,⑤验证目标靶分子干预对CSCs干性的抑制作用。研究旨在完善TLR9的信号功能并探索CSCs的调控机制与治疗措施。

肿瘤干细胞具有自我更新、多向分化以及药物抵抗、高致瘤性和转移潜能，被认为是导致肿瘤复发与恶化的主要原因。TLR9以及下游信号分子NF-κB、PI3K、AKT、mTOR、CXCR4、MAPK均与癌干细胞的干性维持密切相关。本课题借助流式细胞技术，从不同病理类型的NSCLC患者手术标本或肿瘤细胞系中分离获得NSCLC-CSCs亚群，用Real-Time PCR、Western-Blot和流式细胞术检测比较分离的干细胞群、非干细胞群中TLR9的表达，在干细胞、非干细胞群中加入TLR9激活配体CpG DNA，观察加入TLR9激动剂后对干细胞自我更新、分化的影响，发现干细胞群中TLR9的表达明显高于非干细胞群，TLR9激动剂CpG ODN促进干细胞自我更新、抑制干细胞的分化。从NCI-H460、SPCA-1中分离的肺癌干细胞，用ShRNA干扰的方法，获得TLR9稳定低表达干细胞克隆，用悬浮球形成实验观察TLR9表达下调后干细胞的自我更新能力，Transwell方法观察细胞的侵袭性改变，流式细胞检测细胞周期的分布，TUNEL法检测细胞凋亡情况，MTS法检测TLR9下调后干细胞对化疗药物顺铂、阿霉素的敏感性改变情况，建立裸鼠移植瘤模型，观察TLR9下调后干细胞成瘤能力改变。结果发现TLR9基因下调后，形成的细胞悬浮球明显缩小、侵袭性减弱，S期细胞比例减少，凋亡细胞增加，对化疗药物顺铂、阿霉素敏感性增强，裸鼠体内成瘤能力减弱。ShRNA干扰方法下调TLR9的表达，观察TLR9下调后其他干细胞干性基因：Oct-4，Sox2，Gli1，Nanog，Notch1,β-catenin表达的改变，检测耐药基因ABCB1、ABCC1、ABCG2表达的改变，检测细胞周期相关基因Cyclin B1、CDK1、BCL2、TRET、Survivin表达的改变，发现TLR9下调后，干性基因Gli、Notch1、β-catenin明显下调，Cyclin B1、CDK1表达下降，耐药基因ABCB1、ABCC1、ABCG2明显下调。TLR9是肺癌干细胞干性维持的重要因子，有可能成为肺癌干细胞功能调控的关键靶位，进而控制肺癌的复发与转移。