目的 基于miR-212-3p/E盒结合锌指蛋白2（ZEB2）通路探讨雷公藤甲素（TPL）对卵巢癌细胞紫杉醇（TAX）耐药的影响。方法 体外培养卵巢癌细胞SKOV3及其TAX耐药细胞SKOV3/TAX，以实时荧光定量PCR（ qRT-PCR）检测2者miR-212-3p、ZEB2表达。构建SKOV3/TAX移植瘤裸鼠模型，以TPL干预SKOV3/TAX细胞及动物后，CCK-8法检测细胞存活率，测量肿瘤体积，筛选其最佳作用浓度/剂量。将SKOV3/TAX、SKOV3细胞随机分为对照组、TPL（20 nmol·L^-1）组、inhibitor-NC（100 nmol·L^-1）组、TPL（20 nmol·L^-1）＋ miR-212-3p inhibitor（100 nmol·L^-1）组，同时以TAX（0、5、10、20、40、60 nmol·L^-1）处理细胞，检测SKOV3/TAX细胞耐药指数。将SKOV3/TAX细胞随机分为对照组、TAX（30 nmol·L^-1）组、TPL（20 nmol·L^-1）＋TAX（30 nmol·L^-1）组、inhibitor-NC（100 nmol·L^-1）组、TPL（20 nmol·L^-1）＋TAX（30 nmol·L^-1）＋ miR-212-3p inhibitor（100 nmol·L^-1）组，干预24 h；将SKOV3/TAX移植瘤裸鼠随机分为对照组、TAX（8 mg·kg^-1，ip给药）组、TPL（30 μg·kg^-1，ip给药） ＋TAX（8 mg·kg^-1）组、inhibitor-NC（5 nmol，瘤内注射）组、TPL（30 μg·kg^-1） ＋TAX（8 mg·kg^-1） ＋miR-212-3p inhibitor（5 nmol，瘤内注射）组，干预2周。qRT-PCR法检测细胞和组织中miR-212-3p、ZEB2表达，以CCK-8、流式细胞实验分别检测细胞增殖、凋亡；测量肿瘤体积； Western blotting检测ZEB2、Bax、Bcl-2、多药耐药蛋白1（MDR1）蛋白表达。结果 与SKOV3细胞比较，SKOV3/TAX细胞miR-212-3p降低且ZEB2 mRNA表达升高（P＜0.05）。TPL可降低SKOV3/TAX细胞耐药指数（P＜0.05） ，miR-212-3p inhibitor可逆转TPL上述作用（P＜0.05）。与对照组相比，TAX组细胞存活率、肿瘤体积、Bcl-2蛋白表达降低（P＜0.05），凋亡率、Bax蛋白表达升高（P＜0.05）； TPL＋ TAX组细胞存活率、肿瘤体积、ZEB2 mRNA与蛋白表达、Bcl-2与MDR1蛋白表达降低（P＜0.05），凋亡率、miR-212-3p表达、Bax蛋白表达升高（P＜0.05）； inhibitor-NC组细胞各指标无显著变化。与TAX组相比，TPL＋TAX组细胞存活率、肿瘤体积、ZEB2 mRNA与蛋白表达、Bcl-2与MDR1蛋白表达降低（P＜0.05），凋亡率、miR-212-3p表达、Bax蛋白表达升高（P＜0.05）。与TPL＋TAX组相比，TPL＋TAX＋miR-212-3p inhibitor组细胞存活率、肿瘤体积、ZEB2 mRNA与蛋白表达、Bcl-2与MDR1蛋白表达升高（P＜0.05），凋亡率、miR-212-3p表达、Bax蛋白表达降低（P＜0.05）。结论 TPL可拮抗卵巢癌细胞的TAX耐药性，机制可能与调控miR-212-3p/ZEB2信号通路相关。

Objective To investigate the effect of triptolide (TPL) on paclitaxel (TAX) resistance in ovarian cancer cells based on miR- 212-3p/zinc finger E-box binding homeobox 2 (ZEB2) pathway. Methods SKOV3 ovarian cancer cells and their TAX-resistant cells SKOV3/TAX were cultured in vitro. The expression of miR-212-3p and ZEB2 in both cell lines was detected by real-time fluorescence quantitative PCR (qRT-PCR). A SKOV3/TAX xenograft nude mouse model was established. After TPL intervention in SKOV3/TAX cells and animals, the cell survival rate was detected by CCK-8 assay, and the tumor volume was measured to screen the optimal concentration/dose. SKOV3/TAX and SKOV3 cells were randomly divided into control group, TPL (20 nmol·L^-1) group, inhibitorNC (100 nmol·L^-1) group, and TPL (20 nmol·L^-1) + miR-212-3p inhibitor (100 nmol·L^-1) group. Meanwhile, the cells were treated with TAX (0, 5, 10, 20, 40, 60 nmol·L^-1), and the drug resistance index of SKOV3/TAX cells was detected. SKOV3/TAX cells were randomly divided into control group, TAX (30 nmol·L^-1) group, TPL (20 nmol·L^-1) + TAX (30 nmol·L^-1) group, inhibitorNC (100 nmol·L^-1) group, and TPL (20 nmol·L^-1) + TAX (30 nmol·L^-1) + miR-212-3p inhibitor (100 nmol·L^-1) group, and intervened for 24 h. SKOV3/TAX xenograft nude mice were randomly divided into control group, TAX (8 mg·kg -1, ip) group, TPL (30 μg·kg^-1, ip) + TAX (8 mg·kg^-1) group, inhibitor-NC (5 nmol, intratumoral injection) group, and TPL (30 μg·kg^-1) + TAX (8 mg·kg^-1) + miR-212-3p inhibitor (5 nmol, intratumoral injection) group, and intervened for two weeks. The expression of miR-212-3p and ZEB2 in cells and tissues was detected by qRT-PCR. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. The tumor volume was measured. The expression of ZEB2, Bax, Bcl-2, and multidrug resistance protein 1 (MDR1) was detected by Western blotting. Results Compared with SKOV3 cells, the expression of miR-212-3p in SKOV3/TAX cells was decreased, and the expression of ZEB2 mRNA was increased (P < 0.05). TPL could reduce the drug resistance index of SKOV3/TAX cells (P < 0.05), and miR-212-3p inhibitor could reverse the above effect of TPL (P < 0.05). Compared with the control group, the cell survival rate, tumor volume, and Bcl-2 protein expression in the TAX group were decreased (P < 0.05), and the apoptosis rate and Bax protein expression were increased (P < 0.05); in the TPL + TAX group, the cell survival rate, tumor volume, ZEB2 mRNA and protein expression, Bcl-2 and MDR1 protein expression were decreased (P < 0.05), and the apoptosis rate, miR-212-3p expression, and Bax protein expression were increased (P < 0.05); there were no significant changes in the inhibitor-NC group. Compared with the TAX group, the cell survival rate, tumor volume, ZEB2 mRNA and protein expression, Bcl-2 and MDR1 protein expression in the TPL + TAX group were decreased (P < 0.05), while the apoptosis rate, miR-212-3p expression and Bax protein expression were increased (P < 0.05). Compared with the TPL + TAX group, the cell survival rate, tumor volume, ZEB2 mRNA and protein expression, Bcl-2 and MDR1 protein expression in the TPL + TAX + miR-212-3p inhibitor group were increased (P < 0.05), while the apoptosis rate, miR-212-3p expression and Bax protein expression were decreased (P < 0.05). Conclusion TPL can antagonize TAX resistance in ovarian cancer cells, and regulating the miR-212-3p/ZEB2 signaling pathway may be its pharmacological mechanism.

R285.5

河南省医学科技攻关计划联合共建项目（LHGJ20210940）