目的 探究芬苯達(dá)唑?qū)Ψ蜗侔┘?xì)胞增殖、遷移、侵襲的影響以及自噬與遷移、侵襲的關(guān)系。方法 將人肺腺癌A549、H358細(xì)胞分為對(duì)照組及芬苯達(dá)唑（1、2.5、5、10、20 μmol/L）組。用CCK-8法及細(xì)胞計(jì)數(shù)法檢測(cè)細(xì)胞增殖水平，用劃痕實(shí)驗(yàn)及Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞遷移、侵襲能力，通過RT-qPCR及蛋白免疫印跡法檢測(cè)上皮-間充質(zhì)轉(zhuǎn)化（EMT）相關(guān)蛋白的mRNA及蛋白表達(dá)水平，通過轉(zhuǎn)錄組測(cè)序并分析自噬相關(guān)基因及其表達(dá)量。用自噬抑制劑氯喹（5、10 μmol/L）與芬苯達(dá)唑（5 μmol/L）處理細(xì)胞后再通過劃痕及Transwell實(shí)驗(yàn)檢測(cè)細(xì)胞遷移、侵襲能力。結(jié)果 與對(duì)照組相比，芬苯達(dá)唑各濃度處理組能顯著抑制A549和H358細(xì)胞活性及遷移、侵襲能力（P＜0.05、0.01、0.001）；隨著芬苯達(dá)唑藥物濃度增加，N-鈣黏蛋白（N-cadherin）、細(xì)胞角蛋白（Cytokeratin）、纖維連接蛋白（FN1）mRNA相對(duì)表達(dá)逐漸下降（P＜0.05、0.01、0.001）；緊密連接蛋白（Occludin）、波形蛋白（Vimentin）mRNA表達(dá)水平在低濃度時(shí)較高，隨著芬苯達(dá)唑藥物濃度增加Occludin、Vimentin mRNA表達(dá)水平降低（P＜0.05、0.01、0.001）。與對(duì)照組相比，隨著芬苯達(dá)唑藥物濃度增加，N-cadherin、Cytokeratin、FN1、Occludin蛋白相對(duì)表達(dá)量顯著降低，而Vimentin蛋白相對(duì)表達(dá)水平隨藥物濃度降低而增高（P＜0.05、0.01、0.001）。基因差異熱圖顯示，芬苯達(dá)唑處理后自噬相關(guān)基因（MAP1LC3B、ATG5、ULK3、Bax、ATG16L1）表達(dá)量增高，芬苯達(dá)唑的濃度增加使自噬相關(guān)蛋白重組人自噬效應(yīng)蛋白（Beclin-1）、微管相關(guān)蛋白輕鏈3（LC3）-II/LC3-I相對(duì)蛋白表達(dá)量升高，選擇性自噬接頭蛋白P62表達(dá)量明顯降低（P＜0.05、0.01、0.001）。在使用了氯喹阻斷自噬后，可以逆轉(zhuǎn)芬苯達(dá)唑引起的細(xì)胞增殖、遷移及侵襲（P＜0.05、0.01、0.001）抑制作用。結(jié)論 芬苯達(dá)唑可以通過促進(jìn)自噬從而抑制了肺腺癌細(xì)胞的增殖、遷移和侵襲能力。

Objective To explore the effects of fenbendazole on the proliferation, migration and invasion of lung adenocarcinoma cells and the relationship between autophagy and migration invasion. Methods A549 and H358 cells were divided into control group and fenbendazole group (1, 2.5, 5, 10, and 20 μmol/L). Cell proliferation level was detected by CCK-8 method and cell counting method, cell migration and invasion ability was detected by scratch assay and Transwell assay, mRNA and protein expression levels of epithelial-mesenchymal transformation (EMT) related proteins were detected by RT-qPCR and western blot. Autophagy related genes and their expression levels were analyzed by transcriptome sequencing. The cells were treated with autophagy inhibitors chloroquine (5, 10 μmol/L) and fenbendazole (5 μmol/L), and then the migration and invasion ability of the cells were detected by scratch and Transwell assay. Results Compared with control group, different concentration fenbendazole treatment groups significantly inhibited the activity, migration and invasion ability of A549 and H358 cells (P < 0.05, 0.01, 0.001). With the increase of fenbendazole concentration, the relative mRNA expressions of N-cadherin, Cytokeratin, and FN1 decreased gradually (P < 0.05, 0.01, 0.001). Occludin and Vimentin mRNA expression levels were higher at low concentration, and decreased with the increase of fenbendazole concentration (P < 0.05, 0.01, 0.001). Compared with control group, the relative expression of N-cadherin, Cytokeratin, FN1, and Occludin decreased significantly with the increase of fenbendazole concentration. The relative expression level of Vimentin protein increased with the decrease of drug concentration (P < 0.05, 0.01, 0.001). The thermal map of gene difference showed that the expression of autophagy related genes (MAP1LC3B, ATG5, ULK3, Bax, ATG16L1) increased after fenbendazole treatment, and the expression of autophagy related proteins Beclin-1 and LC3-II/LC3-I increased with the increase of fenbendazole concentration. The expression of P62 showed a decreased trend (P < 0.05, 0.01, 0.001). After chloroquine was used to block autophagy, the inhibitory effects of fenbendazole on cell proliferation, migration and invasion could be reversed (P < 0.05, 0.01, 0.001). Conclusion Fenbendazole can inhibit the proliferation, migration, and invasion abilities of lung adenocarcinoma cells by activating autophagy.

R966

國家自然科學(xué)基金資助項(xiàng)目（81972190）；陸軍軍醫(yī)大學(xué)第二附屬醫(yī)院青年博士人才孵化計(jì)劃（2022YQB012）