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目的探讨盐酸吡柔比星增强宫颈癌He La细胞自噬水平的可能机制。方法采用CCK-8检测盐酸吡柔比星单独或联用自噬抑制剂氯喹对He La细胞增殖的影响;台盼蓝染色实验检测盐酸吡柔比星单独或联用自噬抑制剂氯喹对He La细胞死亡的影响;GFP-LC3质粒转染He La细胞后观察盐酸吡柔比星对自噬小体形成的影响;Western blot检测细胞中自噬标志分子Ⅱ型微管相关蛋白轻链3(microtuble-associated protein light chain 3-Ⅱ,LC3-Ⅱ)、自噬相关分子自噬相关蛋白4B(autophapy related 4B,ATG4B)的表达情况。CCK-8实验及台盼蓝染色实验观察在有或者无自噬抑制剂氯喹作用下以及shRNA下调He La细胞中的ATG4B后盐酸吡柔比星对He La细胞的杀伤效应的影响。结果盐酸吡柔比星能剂量依赖地抑制He La细胞的增殖并促进其死亡(P<0.05);盐酸吡柔比星处理能显著增加He La细胞中自噬小体的数量,并显著上调自噬标志分子LC3-II、自噬关键酶ATG4B的表达;自噬抑制剂氯喹可显著增强盐酸吡柔比星的杀细胞效应(P<0.05),shRNA干扰ATG4B可抑制盐酸吡柔比星对He La细胞中自噬的增强作用(P<0.05),并增强盐酸吡柔比星的杀细胞效应(P<0.05)。结论盐酸吡柔比星通过上调ATG4B而增强的自噬对He La细胞起保护作用。
Objective To investigate the possible mechanism of pirarubicin hydrochloride in enhancing the autophagy of cervical cancer He La cells. Methods CCK-8 was used to detect the effect of pirarubicin alone or in combination with chloroquine on the proliferation of HeLa cells. Trypan blue staining was used to detect the effect of pirarubicin alone or in combination with chloroquine La cells. The effect of pirarubicin hydrochloride on the formation of autophagic bodies was observed after GFP-LC3 plasmid was transfected into Hela cells. Western blot was used to detect the expression of autophagic marker molecule Ⅱ microtubule-associated protein light chain 3 ( microtuble-associated protein light chain 3-Ⅱ, LC3-Ⅱ) and the expression of autophagy-related protein 4B (ATG4B). CCK-8 and Trypan blue staining were used to observe the effect of pirarubicin hydrochloride on HeLa cells killing by HeLa cells with or without autophagy inhibitor chloroquine and shRNA down-regulation of ATG4B in HeLa cells. Results Pirarubicin could inhibit the proliferation and promote the death of HeLa cells in a dose-dependent manner (P <0.05). Pirarubicin hydrochloride significantly increased the number of autophagic bodies in HeLa cells and significantly up-regulated from LC3-II, the autophagy key enzyme ATG4B, and the autophagy inhibitor chloroquine significantly enhanced the cytocidal effect of pirarubicin hydrochloride (P <0.05). ShRNA interference ATG4B can inhibit the effect of pirarubicin hydrochloride on He (P <0.05), and enhance the cytotoxicity of pirarubicin hydrochloride (P <0.05). Conclusion Pirarubicin has a protective effect on He La cells by increasing autophagy induced by ATG4B up-regulation.