氯喹通过上调miR129增强顺铂抗鼻咽癌作用

Chloroquine enhances cisplatin-induced apoptosis of nasopharyngeal carcinoma cells by inhibiting autophagy via upregulating miR129

目的探究miR129在氯喹增强顺铂促鼻咽癌细胞HNE1凋亡的作用及机制。方法实验分为空白对照组、氯喹组、顺铂组、氯喹+顺铂组。MTT法观察HNE1细胞存活率;结晶紫染色法观察HNE1细胞集落克隆形成;采用HNE1细胞接种BALB/C裸鼠建立移植瘤模型,随机分为4组,6只/组,3 d给药1次,连续用药4周,观察各组肿瘤生长情况;q-PCR检测HNE1细胞中miR129表达;质粒转染下调HNE1细胞miR129的表达;Western blot法检测自噬相关蛋白p62、LC3B、Beclin1和耐药蛋白P-gp的表达;Annexin V/PI双染法,检测细胞凋亡率。结果体内外实验结果均显示氯喹+顺铂较顺铂单用能显著抑制肿瘤的生长(P<0.01)。体外实验表明,下调抑癌基因miR129能显著促进HNE1细胞自噬、上调P-gp的表达(P<0.01);氯喹显著抑制顺铂诱导的HNE1细胞自噬,上调HNE1细胞中miR129的表达(P<0.01);转染抑制miR129后,氯喹抑制顺铂诱导HNE1细胞自噬的作用被取消、氯喹和顺铂联合作用后HNE1细胞存活率显著增加(P<0.05),细胞凋亡率显著降低(P<0.01)。结论氯喹通过上调miR129抑制HNE1细胞自噬和耐药,增强顺铂的促凋亡作用。

Objective To investigate the role of miR129 in mediating the effect of chloroquine to enhance cisplatin-induced apoptosis in nasopharyngeal carcinoma cells(HNE1). Methods 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, LC3 B, 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. Results Chloroquine combined with cisplatin significantly inhibited HNE1 cell proliferation in vitro and the growth of HNE1 cell-derived tumor in nude mice as compared with cisplatin alone(P<0.01). In cultured HNE1 cells, inhibition of the expression of miR129 significantly promoted autophagy and up-regulated P-gp expression(P<0.01);Chloroquine obviously inhibited cisplatin-induced autophagy and up-regulated the expression of miR129 in HNE1 cells(P<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(P<0.05) and lower the cell apoptotic rate(P<0.01) after combined treatment with chloroquine and cisplatin. Conclusion Chloroquine enhances the pro-apoptotic effect of cisplatin by up-regulating miR129 to inhibit autophagy and drug resistance in HNE1 cells.