Lu Yue, Qiu Rong, Deng Yan, Liu Xingyu, Du Yuzhen
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
Department of Laboratory Medicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 201306, China.
Zhongguo Fei Ai Za Zhi. 2024 Apr 20;27(4):257-265. doi: 10.3779/j.issn.1009-3419.2024.101.09.
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.
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.
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.
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.
骨是肺腺癌转移的常见部位，但肺腺癌骨转移背后的机制仍不清楚。目前，缺乏易于追踪且稳定的肺腺癌骨转移细胞模型，这限制了对肺腺癌骨转移机制的研究。建立高骨转移潜能的人肺腺癌细胞系，并用绿色荧光蛋白（GFP）和萤火虫荧光素酶（LUC）标记，同时进行转录组学表征，将有助于肺腺癌骨转移的研究并提供新的实验方法。
将人肺腺癌细胞系A549-GFP-LUC经左心室注射到裸鼠体内构建骨转移模型，并在体内连续驯化3次，获得人高骨转移肺腺癌细胞系A549-GFP-LUC-BM3；采用细胞计数试剂盒-8（CCK-8）、集落形成实验、划痕实验、Transwell实验和蛋白质免疫印迹法比较A549-GFP-LUC-BM3与亲代细胞的增殖和侵袭能力。对A549-GFP-LUC-BM3细胞和亲代细胞进行转录组测序进一步分析。
成功建立了人高骨转移肺腺癌细胞系A549-GFP-LUC-BM3。与亲代细胞相比，该细胞骨转移发生率显著更高，体外增殖、迁移和侵袭能力增强。转录组测序结果显示，与亲代细胞相比，A549-GFP-LUC-BM3细胞系有2954个差异表达基因，其中1021个基因上调，1933个基因下调。基因本体（GO）功能富集分析表明，差异表达基因主要定位于细胞外周等细胞成分中。显著富集的分子功能包括信号受体活性、钙离子结合和细胞外基质结构成分。此外，富集的生物学过程为细胞黏附和生物黏附。使用京都基因与基因组百科全书（KEGG）进行的富集分析表明，差异表达基因主要参与细胞色素P450对外源生物的代谢、视黄醇代谢、药物代谢-细胞色素P450、细胞黏附分子、类固醇激素生物合成以及核因子κB（NF-κB）信号通路。
成功建立了具有GFP和荧光素酶双标记的高骨转移潜能人肺腺癌细胞系。该细胞系的生物学行为和转录组测序表明其具有较高的骨转移潜能。
