目的：探讨高迁移率族蛋白1（high-mobility group box 1， HMGB1 ）基因沉默对化疗药物多柔比星（doxorubicin, DOX）诱导BGC-823胃癌细胞发生自噬和凋亡的影响。方法：MTT、Western blotting和激光共聚焦显微镜分别检测DOX对BGC-823细胞增殖及微管相关蛋白轻链3-Ⅰ （microtubule light chain-3Ⅰ，LC3Ⅰ）、LC3Ⅱ表达和自噬小体形成的影响。构建靶向 HMGB1 的shRNA载体pSuper-shHMGBl及其对照载体pSuper-shNC并转染BGC-823胃癌细胞，建立 HMGB1 稳定沉默的细胞系。DOX处理不同质粒转染组的BGC-823胃癌细胞，激光共聚焦显微镜观察 HMGB1 沉默对DOX诱导的自噬小体形成的影响，MTT法、流式细胞术分别检测对细胞增殖和凋亡的影响，Western blotting检测对自噬及凋亡相关蛋白（HMGB1、LC3、Bcl-2、Bcl-XL、Mcl-1和激活型caspase-3）表达的影响。结果：DOX可上调BGC-823细胞中LC3-Ⅱ蛋白的水平和促进细胞自噬体的形成（ P <0.01 ）。与对照组相比， HMGB1 基因沉默可减少DOX诱导的胃癌细胞自噬\[(19.33±2.96)% v (71.67±3.38)%， P <0. 01 \]，促进胃癌细胞发生凋亡\[（46.12±3.15）% vs （12.37±2.84）%， P <0.05\]，上调激活型caspase-3的表达水平。进一步分析发现，DOX诱导胃癌细胞发生自噬时，Mcl-1的降解增加，而Bcl-2和Bcl-XL表达无明显变化； HMGB1 基因沉默可抑制DOX诱导的Mcl-1降解。结论：DOX可诱导BGC-823胃癌细胞发生自噬， HMGB1 基因沉默有助于逆转胃癌细胞对DOX的耐药性，促进胃癌细胞发生凋亡。

Objective: To investigate the effect of high-mobility group box 1 ( HMGB1 ) gene silencing on chemotherapeutic agent Doxorubicin (DOX)-induced autophagy and apoptosis in human gastric cancer BGC-823 cells. Methods: Cell viability, microtubule light chain 3 (LC3-Ⅰ, LC3-Ⅱ) protein content and autophagic vesicle formation in DOX-induced BGC-823 cells were assessed by MTT assay, Western blotting and confocal microscopy, respectively. BGC-823 cells were transfected with pSuper-shHMGBl (a shRNA targeting the HMGB1 gene) and pSuper-shNC (a control vector) respectively and subjected to G418 selection. BGC-823 cells stably expressing pSuper-shHMGBl or pSuper-shNC were treated with DOX. After treatment, autophagic vesicle formation was assessed by confocal microscopy. Cell viability and cell apoptosis were determined by the MTT assay and flow cytometry respectively. HMGB1, LC3, Bcl-2, Bcl-XL, Mcl-1 and cleaved caspase-3 proteins were assessed by Western blotting. Results: Treatment with DOX increased the protein content of LC3-Ⅱ and the formation of autophagic vesicles in BGC-823 cells ( P <0.01). As compared to the control, HMGB1 silencing inhibited DOX-induced autophagy ( P <0.01), promoted apoptosis (\[46.12±3.15\]% vs \[12.37±2.84\]%, P <0.05) and up-regulated the expression of cleaved caspase-3 in BGC-823 cells. Furthermore, antiapoptotic Mcl-1 protein degradation was increased in BGC-823 cells after induction of autophagy, however, Bcl-2 and Bcl-XL protein levels did not change significantly. HMGB1 gene silencing significantly attenuated the degradation of Mcl-1 protein. Conclusion: Autophagy can be induced by Doxorubicin in BGC-823 cells, which may contribute to DOX resistance. Knockdown of HMGB1 attenuates the DOX-induced autophagy and promotes apoptosis in gastric cancer cells.

国家自然科学基金资助项目（No.81101558）；福建省自然科学基金资助项目（No.2012J01364）；教育部博士点基金资助项目（No. 20113518120002）