Objective To explore the effect of down-regulated methionine synthase reductase (MTRR) gene on the apoptosis and autophagy pathway, and offer a possible approach for the MTRR to reverse the multi-resistant ovarian cancer. Methods (1) The experiment was divided into 3 groups, SKOV3/DDP-MTRRi (down-regulated MTRR group), SKOV3/DDP-NC (negative control group), and SKOV3/DDP (blank control group). Different concentration of cisplatin (0, 1, 2, and 4 μg/ml) treated on 3 groups cells. The apoptosis rate was measured by flow cytometry (FCM). Autophagy was detected by immunofluorescence. Autophagy microtubule associated protein light chain 3β(LC3B) and p62 were detected by western blot. The formation of autophagosome of cells was observed by transmission electron microscope. (2) Detection of autophagy and apoptosis of SKOV3/DDP-MTRRi induced by rapamycin. The experiment was divided into 4 groups included rapamycin group (5 nmol/L rapamycin), rapamycin+cisplatin group (5 nmol/L rapamycin+4μg/ml cisplatin), cisplatin group (4μg/ml cisplatin) and blank control group. LC3B and p62 protein were detected by western blot. The survival rate cells were detected by methyl thiazolyl tetrazolium (MTT) method. The apoptosis rate was measured by FCM. (3) The 3 groups cells (SKOV3/DDP, SKOV3/DDP-NC and SKOV3/DDP-MTRRi) induced by a certain concentration of cisplatin (4 μg/ml) after 48 hours, then detecting the protein expression of caspase, Bcl-2 family in apoptosis pathway and the key proteins in phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) autophagy pathways by western blot, getting the time when the proteins′expression changed. Results (1) The 3 groups cells (SKOV3/DDP, SKOV3/DDP-NC, and SKOV3/DDP-MTRRi) induced by a certain concentration of cisplatin (4 μg/ml) after 48 hours, apoptosis and autophagy of 3 groups of cells were gradually increased with the increased concentration of cisplatin. The apoptosis rate of SKOV3/DDP-MTRRi cells [(26.2 ± 1.4)%] were significantly increased compared with the SKOV3/DDP-NC cells or SKOV3/DDP cells [(14.8 ± 2.4)%, (14.2 ± 2.4)%;all P<0.05] at 2μg/ml cisplatin. Immunofluorescence tests revealed that the aggregates of LC3B in SKOV3/DDP-MTRRi cells were more than that of SKOV3/DDP-NC cells and SKOV3/DDP cells. The expression of LC3B of SKOV3/DDP-MTRRi cells was lower than those of SKOV3/DDP-NC cells and SKOV3/DDP cells (P<0.05). The expression of p62 of SKOV3/DDP-MTRRi cells was higher than those of SKOV3/DDP-NC cells and SKOV3/DDP cells (P<0.05). The structure of chloroplast was integrity and autophagosome was dispersing in plastids of SKOV3/DDP-NC cells and SKOV3/DDP cells. Organelles disappear and vacuoles increased obviously in SKOV3/DDP-MTRRi cells, no autophagosome was observed. (2) The expression of LC3B of rapamycin+cisplatin group was higher than those of other 3 group cells (1.72±0.08,1.43±0.04, 1.37±0.11, and 1.11 ± 0.09;P<0.05). The expression of p62 of rapamycin + cisplatin group was significant decreased (0.58 ± 0.10,0.94 ± 0.12, 1.21 ± 0.11, and 1.57 ± 0.10; P<0.05). The survival rate of rapamycin + cisplatin group was higher than that of cisplatin group [(0.78±0.03)%vs (0.62±0.03)%;P=0.018], the apoptosis rate was significant decreased in rapamycin+cisplatin group [(59.0 ± 3.9)% vs (40.4 ± 3.0)%, P=0.019]. (3) The 3 groups cells (SKOV3/DDP, SKOV3/DDP-NC, and SKOV3/DDP-MTRRi) induced by a certain concentration of cisplatin (4μg/ml) after 48 hours, the expression of Bax in 3 groups cell were not evidently changed (P=0.661). The expression of Bcl-2 was significantly decreased in SKOV3/DDP-MTRRi cells (P=0.030). The expression of caspase-3, caspase-7, caspase-9, and poly (ADP-ribose) polymerase (PARP) were not evidently changed (P>0.05), but cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, and cleaved PARP were significantly increased in SKOV3/DDP-MTRRi cells (P<0.05). For the autophagy pathway, the expression of phosphorylated Akt (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR) were significantly increased (P<0.05), but Akt and mTOR had no significant variation. The expression of phosphatase and tensin homologue deleted on chromosome ten (PTEN) was significantly decreased (P<0.05). Conclusions MTRR silencing significantly increase cisplatin-induced apoptosis and reduce the autophagy induced by cisplatin in SKOV3/DDP cells. Down-regulation of MTRR enhanced the chemosensitivity of cisplatin-resistant ovarian cancer cells may be by activating caspase and Bcl-2 apoptosis family and inhibiting the PI3K/Akt autophagy pathway.%目的探讨下调甲硫氨酸合成酶还原酶（MTRR）基因表达对顺铂耐药的卵巢上皮性癌（卵巢癌）细胞自噬和凋亡的影响及作用机制。方法（1）实验分为3组：下调MTRR基因表达的SKOV3/DDP细胞（SKOV3/DDP-MTRRi组）、转染阴性对照（NC）序列的SKOV3/DDP细胞（SKOV3/DDP-NC组，作为阴性对照）、未转染的SKOV3/DDP细胞（SKOV3/DDP组，作为空白对照）。不同浓度（0、1、2、4μg/ml）顺铂作用后，采用流式细胞仪检测3组细胞的凋亡率，免疫荧光法检测3组细胞的自噬现象，蛋白印迹（western blot）法检测3组细胞中自噬标志分子自噬微管相关蛋白轻链3β（LC3B）、p62蛋白的表达，透射电镜观察3组细胞中自噬体的形成情况。（2）雷帕霉素诱导后SKOV3/DDP-MTRRi细胞自噬及凋亡情况变化的检测，实验分为4组，雷帕霉素组（5 nmol/L雷帕霉素处理）、雷帕霉素+顺铂组（5 nmol/L雷帕霉素+4μg/ml顺铂处理）、顺铂组（4μg/ml顺铂处理）、空白对照组，采用western blot法检测4组细胞中自噬标志分子LC3B、p62蛋白的表达，四甲基偶氮唑蓝（MTT）比色法检测4组细胞的生存率，流式细胞仪检测4组细胞的凋亡率。（3）4μg/ml顺铂作用48 h后，western blot法检测3组（即SKOV3/DDP-MTRRi组、SKOV3/DDP-NC组、SKOV3/DDP组）细胞中凋亡相关因子半胱氨酸天冬氨酸蛋白酶（caspase）和Bcl-2家族以及磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）自噬通路关键蛋白的表达。结果（1）不同浓度（0、1、2、4μg/ml）顺铂作用48 h后，随着顺铂浓度的增加，3组细胞的凋亡逐渐增加，当顺铂浓度达到2μg/ml时，SKOV3/DDP-MTRRi组细胞的凋亡率[（26.2±1.4）%]明显高于SKOV3/DDP-NC组和SKOV3/DDP组[分别为（14.8±2.4）%、（14.2±2.4）%；P<0.05]；免疫荧光法检测显示，随着顺铂浓度增加，SKOV3/DDP-MTRRi组细胞中LC3B蛋白在细胞质内聚集成团，明显多于SKOV3/DDP组和SKOV3/DDP-NC组；且与SKOV3/DDP组和SKOV3/DDP-NC组比较， SKOV3/DDP-MTRRi组细胞中LC3B蛋白表达水平明显降低（P<0.05），p62蛋白表达水平明显升高（P<0.05）；当顺铂浓度达到4μg/ml时，SKOV3/DDP组和SKOV3/DDP-NC组细胞内的细胞器结构完整，有少量自噬线粒体出现；而SKOV3/DDP-MTRRi组细胞内的细胞器结构消失，空泡明显增多，且没有观察到自噬体的形成。（2）与雷帕霉素组、顺铂组及空白对照组相比，雷帕霉素+顺铂组SKOV3/DDP-MTRRi细胞中LC3B蛋白的表达水平（分别为1.43±0.04、1.37±0.11、1.11±0.09、1.72±0.08）明显升高（P<0.05），p62蛋白的表达水平（分别为0.94±0.12、1.21±0.11、1.57±0.10、0.58±0.10）明显降低（P<0.05）；与顺铂组比较，雷帕霉素+顺铂组SKOV3/DDP-MTRRi细胞的生存率[分别为（0.62±0.03）%、（0.78±0.03）%]明显升高（P=0.018），凋亡率[分别为（59.0±3.9）%、（40.4±3.0）%]明显降低（P=0.019）。（3）4μg/ml顺铂处理48 h后，与SKOV3/DDP-NC组、SKOV3/DDP组比较，SKOV3/DDP-MTRRi组细胞中Bcl-2家族成员Bax蛋白的表达水平无明显变化（P=0.661），而Bcl-2蛋白的表达水平明显降低（P=0.030）；caspase家族成员caspase-3、caspase-7、caspase-9、多聚二磷酸腺苷-核糖聚合酶（PARP）蛋白的表达水平无明显变化（P>0.05），而裂解的caspase-3、caspase-7、caspase-9、PARP蛋白的表达水平均明显升高（P<0.05）。SKOV3/DDP-MTRRi组细胞中PI3K/Akt自噬通路关键蛋白细胞外信号调节激酶1/2（ERK1/2）、磷酸化Akt（p-Akt）、磷酸化雷帕霉素靶蛋白（p-mTOR）蛋白的表达水平显著升高（P<0.05），而第10号染色体同源丢失性磷酸酶-张力蛋白（PTEN）蛋白的表达水平显著下降（P<0.05）。结论顺铂诱导可使MTRR基因下调的顺铂耐药卵巢癌细胞的凋亡增加、自噬减弱，其可能机制为通过调节caspase和Bcl-2凋亡家族蛋白以及PI3K/Akt自噬通路中关键蛋白的表达，从而增加细胞对顺铂的敏感性。
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