Zhang Haoxuan, Yu Yun, Cai Weiwei, Lu Huaqiu, He Rui, Zhang Renhao, Pei Feilong, Wang Xiaodie, Fang Yini, Wei Fang
Department of Basic Medical Sciences, Bengbu Medical College, Bengbu 233030, China.
School of Pharmacy, Bengbu Medical College, Bengbu 233030, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Mar 30;40(3):361-369. doi: 10.12122/j.issn.1673-4254.2020.03.13.
To investigate the role of miR129 in mediating the effect of chloroquine to enhance cisplatin- induced apoptosis in nasopharyngeal carcinoma cells (HNE1).
MTT assay was used to detect the viability of HNE1 cells treated with different concentrations of cisplatin. Colony formation of HNE1 cells treated with cisplatin and chloroquine, alone or in combination, was observed using crystal violet staining. BALB/C unde mice were inoculated with HNE1 cells and randomly divided into 4 groups with 6 mice in each group. The mice received intraperitoneal injections of cisplatin and chloroquine, alone or in combination once every 3 days for 4 consecutive weeks, and the tumor growth was observed in each group. The expression of miR129 in HNE1 cells treated with chloroquine, cisplatin, or both was detected with qPCR. The effects of miR129 suppression with a miR129 inhibitor on the expressions of autophagy related proteins p62, LC3B, Beclin1 and the drug-resistant related protein P-glycoprotein (P-gp) were examined using Western blotting in HNE1 cells treated with chloroquine, cisplatin, or both; the changes in cell apoptosis were detected Annexin V/PI double staining.
Chloroquine combined with cisplatin significantly inhibited HNE1 cell proliferation and the growth of HNE1 cell-derived tumor in nude mice as compared with cisplatin alone ( < 0.01). In cultured HNE1 cells, inhibition of the expression of miR129 significantly promoted autophagy and up-regulated P-gp expression ( < 0.01); Chloroquine obviously inhibited cisplatin-induced autophagy and up-regulated the expression of miR129 in HNE1 cells ( < 0.01). Transfection of the cells with the miR129 inhibitor abolished the inhibitory effect of chloroquine on cisplatin-induced autophagy, and significantly increased the cell survival rate ( < 0.05) and lower the cell apoptotic rate ( < 0.01) after combined treatment with chloroquine and cisplatin.
Chloroquine enhances the pro-apoptotic effect of cisplatin by up-regulating miR129 to inhibit autophagy and drug resistance in HNE1 cells.
探讨miR129在介导氯喹增强顺铂诱导的鼻咽癌细胞（HNE1）凋亡中的作用。
采用MTT法检测不同浓度顺铂处理的HNE1细胞活力。用结晶紫染色观察顺铂和氯喹单独或联合处理的HNE1细胞的集落形成。将BALB/C裸鼠接种HNE1细胞，随机分为4组，每组6只。小鼠每3天腹腔注射顺铂和氯喹，单独或联合给药，连续4周，观察每组肿瘤生长情况。用qPCR检测氯喹、顺铂或两者处理的HNE1细胞中miR129的表达。在氯喹、顺铂或两者处理的HNE1细胞中，用Western印迹法检测用miR129抑制剂抑制miR129对自噬相关蛋白p62、LC3B、Beclin1和耐药相关蛋白P-糖蛋白（P-gp）表达的影响；用Annexin V/PI双染法检测细胞凋亡的变化。
与单独使用顺铂相比，氯喹联合顺铂显著抑制HNE1细胞增殖和裸鼠体内HNE1细胞源性肿瘤的生长（P<0.01）。在培养的HNE1细胞中，抑制miR129的表达显著促进自噬并上调P-gp表达（P<0.01）；氯喹明显抑制顺铂诱导的自噬并上调HNE1细胞中miR129的表达（P<0.01）。用miR129抑制剂转染细胞消除了氯喹对顺铂诱导的自噬的抑制作用，并显著提高了氯喹和顺铂联合处理后的细胞存活率（P<0.05），降低了细胞凋亡率（P<0.01）。
氯喹通过上调miR129抑制HNE1细胞的自噬和耐药性，增强顺铂的促凋亡作用。
