摘要
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(q RT-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(30μg·kg^(-1),ip给药)+TAX(8 mg·kg^(-1))组、inhibitor-NC(5 nmol,瘤内注射)组、TPL(30μg·kg^(-1))+TAX(8 mg·kg^(-1))+miR-212-3p inhibitor(5 nmol,瘤内注射)组,干预2周。q RT-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 m RNA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,inhibitor-NC(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,inhibitor-NC(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(30μg·kg^(−1),ip)+TAX(8 mg·kg^(−1))group,inhibitor-NC(5 nmol,intratumoral injection)group,and TPL(30μg·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.
作者
薛光辉
丁肖华
周传亚
沈晶
XUE Guanghui;DING Xiaohua;ZHOU Chuanya;SHEN Jing(Puyang Medical College,Puyang 457005,China;Sanquan College of Xinxiang Medical University,Xinxiang 453000,China;Department of Gynecology,Puyang Maternity and Child Care Centers,Puyang 457005,China)
出处
《药物评价研究》
北大核心
2025年第8期2145-2153,共9页
Drug Evaluation Research
基金
河南省医学科技攻关计划联合共建项目(LHGJ20210940)。
