Background: Bone is a common site for metastasis in lung adenocarcinoma, but the mechanism behind lung adenocarcinoma bone metastasis is still unclear. And currently, there is a lack of easily traceable and stable lung adenocarcinoma bone metastasis cell models, which limits the research on the mechanism of lung adenocarcinoma bone metastasis. The establishment of human lung adenocarcinoma cell line that are highly metastatic to bone, labeled with green fluorescent proteins (GFP) and fireflies luciferase (LUC), along with transcriptomic characterization, would be beneficial for research on lung adenocarcinoma bone metastasis and provide new experimental methods.
Methods: The human lung adenocarcinoma cell line A549-GFP-LUC was injected into nude mice via the left ventricle to construct a bone metastasis model, and was domesticated in vivo for three consecutive times to obtain the human high bone metastasis lung adenocarcinoma cell line A549-GFP-LUC-BM3; cell counting kit-8 (CCK-8), colony formation assay, scratch wound assays, Transwell assay and Western blot were used to compare the proliferation and invasion abilities of A549-GFP-LUC-BM3 with the parental cells. A549-GFP-LUC-BM3 cells and parental cells were further analyzed by transcriptomic sequencing.
Results: Human high-bone metastatic lung adenocarcinoma cells A549-GFP-LUC-BM3 was successfully established. Compared to parental cells, this cells exhibited a significantly higher incidence of bone metastasis and enhanced in vitro proliferation, migration, and invasion abilities. Transcriptomic sequencing results revealed that the A549-GFP-LUC-BM3 cell line had 2954 differentially expressed genes compared to the parental cells, with 1021 genes up-regulated and 1933 genes down-regulated. Gene Ontology (GO) functional enrichment analysis indicated that the differentially expressed genes were primarily localized in cellular components such as the cell periphery. The molecular functions identified as significantly enriched included signaling receptor activity, calcium ion binding, and extracellular matrix structural constituent. Additionally, the biological processes found to be enriched were cell adhesion and biological adhesion. The enrichment analysis conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the differentially expressed genes were primarily involved in the metabolism of xenobiotics by cytochrome P450, retinol metabolism, drug metabolism-cytochrome P450, cell adhesion molecules, steroid hormone biosynthesis, and the nuclear factor kappa B (NF-κB) signaling pathway.
Conclusions: The highly bone-metastatic human lung adenocarcinoma cell line with GFP and luciferase double labeling was successfully established. The biological behavior and transcriptome sequencing of the cell line suggest that it has a high bone-metastatic potential.
【中文题目：双荧光标记的人高骨转移肺腺癌细胞株的 建立及其转录组学特征分析】 【中文摘要：背景与目的 骨是肺腺癌常见的转移部位，但肺腺癌骨转移的机制尚不明确。目前肺腺癌骨转移机制研究缺乏易于示踪且稳定高骨转移的肺腺癌细胞模型，因此，本研究旨在建立绿色荧光蛋白（green fluorescent protein, GFP）和萤火虫荧光素酶（firefly luciferase, LUC）双标记的人高骨转移肺腺癌细胞株，为肺腺癌骨转移的研究提供新的实验工具。方法 人肺腺癌细胞系A549-GFP-LUC经左心室注射至裸鼠体内构建骨转移模型，经连续3次体内驯化，获取人高骨转移肺腺癌细胞株A549-GFP-LUC-BM3；CCK-8（cell counting kit-8）、克隆形成实验比较A549-GFP-LUC-BM3细胞株和亲本细胞的体外增殖能力，划痕实验、Transwell实验以及Western blot比较迁移和侵袭能力；并进一步将A549-GFP-LUC-BM3细胞和亲本细胞行测序转录组学分析。结果 成功建立人高骨转移肺腺癌细胞A549-GFP-LUC-BM3，相较于亲本细胞，该细胞骨转移发生率显著提高，且体外增殖、迁移和侵袭能力显著增强。转录组学测序结果显示，相较于亲本细胞，A549-GFP-LUC-BM3细胞中共筛选到差异基因2954个，其中1021个基因上调，1933个基因下调；基因本体（Gene Ontology, GO）功能富集显示差异基因主要定位于细胞外周、质膜以及细胞外基质等细胞组分，分子功能主要富集在信号受体结合、钙离子结合和细胞外基质结构成分等，生物过程富集在细胞黏附和生物黏附等；京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析显示差异基因在细胞色素P450（cytochrome P450, CYP）对外源性物质的代谢、视黄醇代谢、细胞黏附分子、CYP对药物代谢、类固醇激素的生物合成以及核因子κB（nuclear factor kappa B, NF-κB）信号通路上显著富集。结论 成功建立GFP和LUC双标记的人高骨转移肺腺癌细胞株，该细胞株在生物学行为水平和转录组测序水平均提示具有高骨转移潜能。 】 【中文关键词：肺肿瘤；骨转移；动物模型；细胞株；转录组学分析】.
Keywords: Animal models; Bone metastasis; Cell line; Lung neoplasms; Transcriptomic analysis.