长链非编码RNA LOC146880在三手烟暴露导致女性非吸烟肺癌中的作用研究

LOC146880 has been proved to be a cancer-promoting long non-coding RNA through both epidemiology study and cell functional experiments for the first time, which promote lung cancer invasion and metastasis through its target gene KPNA2 involved in DNA damage response pathway. Moreover, we found that DNA damage could upregulate the expression of LOC146880 induced by CSE. With the change of the smoking habit, the major risk factor for non-smoking lung cancer is third-hand smoke with low level long time exposure instead of the second hand smoke. Now, THS Exposure Risk gradually become an important public health problem. Based on above results, we therefore hypothesized that LOC146880 might promote the prognosis of lung cancer induced by Third-hand smoke among female non-smoking people through upregulate the expression of KPNA2 and subsequently recruiting the key DNA damage response genes. In this hypothesis, firstly, we design to confirmed plasma LOC146880 expressed in a high level in female non-smoking lung cancer compared with control by qRT-PCR, evaluate the indoor Third-hand smoke exposure by GC-IT-MS/MS and detect DNA damage level in vivo by γ-H2AX using flow cytometry. Furthermore, through establishing the cell DNA damage model treated by THS, knocking down the expression of LOC146880 in lung Epithelial cell lines and lung cancer cell lines, detecting γ-H2AX, evaluating expression of LOC146880/KPNA2 and DNA damage response genes by qRT-PCR or Western Blot, and assessing the capacity for proliferation/migration/invasion, we design to explore the effect of LOC146880 in lung cancer induced by Third-hand smoke. This study is the original source of innovation and may provide new potential biomarkers for the prevention and early diagnosis of non-smoking lung cancer and have great significance in public health.

课题组前期采用芯片及生物信息学技术新发现长链非编码RNA LOC146880，其表达在336例非小细胞肺癌患者癌组织中显著高于癌旁；细胞学实验发现烟草提取物处理细胞会导致DNA损伤，LOC146880表达显著升高；敲低LOC146880表达后会抑制细胞增殖、迁移及侵袭。现阶段三手烟暴露风险逐渐成为主要公共卫生问题，因此，本项目拟通过人群病例对照研究，重点调查研究对象的三手烟暴露情况，分析三手烟的关键成分，检测DNA损伤指标 γ-H2AX及LOC146880表达，探索三手烟暴露与非吸烟肺癌的关联；同时采用细胞学实验研究三手烟暴露导致DNA损伤诱导LOC146880表达，进而调控靶基因KPNA2，影响DNA损伤应答基因，最终影响细胞表型的可能机制。本项目为三手烟暴露与非吸烟肺癌发病风险的关联提供前期依据，探索LOC146880作为非吸烟肺癌早期诊断标志物的应用价值，具有重要公共卫生学意义。

三手烟作为持久性的低浓度污染物对非吸咽人群的健康影响已受到社会关注，三手烟暴露风险逐渐成为主要的公共卫生问题，体外实验已发现其可能具有潜在致癌作用，但缺乏深入的机制研究，本项目对三手烟与非吸咽肺癌之间的相关性进行了深入研究，并发现了可能的致癌机制。首先从细胞实验层面，选用肺正常上皮细胞Beas-2B细胞建立了三手烟暴露致DNA损伤的细胞模型，结果发现Beas-2B细胞暴露于三手烟溶液后，r-H2AX表达增强，lncRNA-LOC146880表达升高，表明三手烟暴露可导致lncRNA-LOC146880表达改变。前期研究发现lncRNA-LOC146880在肺癌组织中高表达，下调LOC146880表达影响细胞周期、抑制细胞迁移和侵袭，因此我们深入研究lncRNA-LOC146880在三手烟影响非吸咽肺癌的发生发展的可能作用机制。我们分别从细胞实验和动物实验两个层面，研究发现lncRNA-LOC146880可通过外显子区域的基因多态性位点rs140618127影响其与miR-539-5p结合，进而影响ENO1的磷酸化水平，通过下游PI3K/Akt信号通路，最终影响肺癌的发生发展。同时，我们在人群研究层面发现 lncRNA-LOC146880外显区域的基因多态位点rs140618127 （G>A），相比GG野生型，GA杂合型具有保护性作用。特别是在女性和非吸烟人群中，GA杂合型及AA突变纯和型具有保护性作用。不过，在进行lncRNA-LOC146880外显子区域的rs140618127位点不同基因型的生存分析时，未发现有显著差异。综上所述，本项目的研究结果揭示了lncRNA-LOC146880影响非吸咽肺癌发生发展的可能机制，提示lncRNA-LOC146880可做为三手烟暴露导致非吸烟肺癌预防及早期诊断的分子生物标志物，为进一步研究非吸烟肺癌的预防策略及早期诊断方法提供前期基础。