miR-34a对NanogP8调控肺癌启动细胞功能的抑制作用

Lung cancer is among the most prevalent and lethal malignancies throughout the world and in China. Although major efforts are now directed towards early detection and prevention, therapeutic efficacy in diagnosed lung cancer patients remains dismal mainly due to our poor understanding of the molecular underpinnings of the disease. Newly accumulated experimental and clinical evidence strongly suggests that a population of stem cell-like cells called cancer stem cells (CSCs) play critical roles in driving tumor development, fueling tumor maintenance, and mediating therapy resistance. Little is known about how lung CSCs are regulated at the molecular level. CSCs have been shown to share a gene expression profile with embryonic stem (ES) cells. Nanog1 (generally called Nanog), located on chromosome 12, is one of the most important core transcription factors for the pluripotency and self-renewal of ES cells. Of interest, CSCs in colon, prostate, breast, and other tumors as well as in glioblastoma multiforme express a variant Nanog, i.e., the retrotransposed gene (or retrogene) called NanogP8 (i.e., NP8), which is located on chromosome 15. NP8 is very similar but not identical to Nanog1 with the two sharing ~98% identity at the amino acid level. NP8 expression is cancer cell/CSC specific and NP8 may possess unique functions in CSCs. Lung cancer also contains CSCs and lung CSCs have also been reported to express Nanog. However, the key question remains whether lung CSCs express Nanog1 or NP8. Furthermore, whether Nanog-expressing lung cancer cells truly have CSC properties has not been fully addressed. Most importantly, how Nanog is regulated in lung CSCs and in the context of lung cancer development remains unclear. In this project, we address all these unanswered questions by testing the hypotheses that: 1) various lung CSC populations preferentially express NP8; 2) NP8 plays an essential role in conferring lung CSC properties by regulating self-renewal and asymmetric cell division; and 3) the microRNA 34a, i.e., miR-34a, negatively restricts NP8 expression in lung CSCs thus inhibit lung cancer development. We will employ a spectrum of state-of-the-art cell biological and molecular approaches to test the above hypotheses. Accomplishment of the goals proposed herein will significantly advance our knowledge on how lung CSCs are regulated at the molecular level by NP8 and how NP8 itself is regulated by a potent tumor-suppressive microRNA. The current project will establish a new paradigm connecting NP8 and miR-34a as a positive and negative, respectively, regulator of lung CSCs and lung cancer development and should lay a solid ground work for developing novel therapeutics targeting drug-resistant and metastasis-prone lung CSCs.

肺癌的发生和难治与癌启动细胞/癌干细胞（CSC）有关。转录因子Nanog（Nanog1）还是NanogP8（NP8）调控CSC功能特性和介导肺癌的机制不明确。我们前期实验提示①肺癌细胞表达NP8，而良性病变则为Nanog1；②miR-34a负调控CD44hi肺CSC和实验性抑制肺癌，并可负调控NP8+CSC中NP8表达。本项目在前期基础上用NP8功能获得与缺失、NP8-GFP 细胞分选、miR-34a示踪 /转染及系列先进的细胞与分子技术，在体内外研究①不同的肺CSC功能群NP8的表达；②NP8调控肺CSC自我更新功能和促进肺癌的分子机制；③ miR-34a抑制NP8操控的肺CSC功能和抑制肺癌作用。通过NP8和miR-34a分别正负调控肺CSC的研究，将阐释NP8启动肺癌和miR-34a/NP8信号通路抑制肺癌的分子机制，对探讨肺癌的新疗法新靶点将具有重要的科学意义和转化应用价值。

肺癌的发生和难治与癌启动细胞/癌干细胞（CSCs）有关。本研究中，我们研究了人肺癌及肺CSC的NanogP8（以下简称NP8）表达、NP8调控肺CSC自我更新功能和miR-34a抑制NP8操控肺CSCs功能的作用靶点。①先检测了人肺癌和肺癌细胞系NP8表达。免疫组化结果显示，肺腺癌和鳞癌中Nanog1/NP8在低分化癌组显著高于高-中分化癌组，提示Nanog1/NP8的表达与肺癌分化程度密切相关；从A549、H460和H1299细胞中流式分选ALDH+/ ALDH-、CD90+/ CD90-、CD44hi和CD44low、SP/非SP亚群，RT-PCR和Western blot检测显示NP8 mRNA及蛋白表达水平在CSCs中显著高于非CSCs；②用可诱导性（doxycycline, dox）NP8慢病毒质粒，并设对照质粒Plvx-Teton-only、Nanog shRNA和RLL3.7，转染A549、H446、H460和H1299肺癌细胞。结果显示，正负双向调控NP8表达分别对肺癌细胞增殖、克隆形成和侵袭能力具有明显促进和抑制作用，提示在gain-of-function水平NP8具有调控肺癌启动细胞自我更新潜能；③用NP8启动子驱动的表达报告质粒NP8-GFP转染H1299细胞。NOD/SCID小鼠体内移植成瘤结果显示，NP8+组成瘤显著高于NP8-组（P<0.0001），而H460和A549细胞均显示NP8有促成瘤趋势。体外实验显示，NP8+组的细胞增殖、克隆扩增能力与侵袭能力显著增强；培养中活细胞实时间断显微摄影显示NP8+的H1299细胞自我更新时呈非对称分裂模式。流式分析显示，NP8+的H1299细胞G2/M期细胞明显增多；④转染过表达mir34a可抑制肺癌细胞NP8、SOX2和OCT4的表达。双荧光素酶报告基因分析显示NP8是mir34a靶分子。总之，NP8持有促进CSC自我更新和肿瘤启动功能，是miR-34a的靶点。后续增补的CRISPR/CAS9技术对NP8敲除与敲入的Mir-34a体内外实验和生物信息分析将进一步深化和扩大研究成果。通过NP8和miR-34a分别正负调控肺CSCs的研究，将对阐释肺癌CSCs自我更新的信号机制和寻找肺癌治疗新靶点与靶向CSCs新疗法有重要的科学意义和转化应用价值。