目的 基于miR-212-3p/E盒結(jié)合鋅指蛋白2（ZEB2）通路探討雷公藤甲素（TPL）對卵巢癌細(xì)胞紫杉醇（TAX）耐藥的影響。方法 體外培養(yǎng)卵巢癌細(xì)胞SKOV3及其TAX耐藥細(xì)胞SKOV3/TAX，以實(shí)時(shí)熒光定量PCR（ qRT-PCR）檢測2者miR-212-3p、ZEB2表達(dá)。構(gòu)建SKOV3/TAX移植瘤裸鼠模型，以TPL干預(yù)SKOV3/TAX細(xì)胞及動(dòng)物后，CCK-8法檢測細(xì)胞存活率，測量腫瘤體積，篩選其最佳作用濃度/劑量。將SKOV3/TAX、SKOV3細(xì)胞隨機(jī)分為對照組、TPL（20 nmol·L^-1）組、inhibitor-NC（100 nmol·L^-1）組、TPL（20 nmol·L^-1）＋ miR-212-3p inhibitor（100 nmol·L^-1）組，同時(shí)以TAX（0、5、10、20、40、60 nmol·L^-1）處理細(xì)胞，檢測SKOV3/TAX細(xì)胞耐藥指數(shù)。將SKOV3/TAX細(xì)胞隨機(jī)分為對照組、TAX（30 nmol·L^-1）組、TPL（20 nmol·L^-1）＋TAX（30 nmol·L^-1）組、inhibitor-NC（100 nmol·L^-1）組、TPL（20 nmol·L^-1）＋TAX（30 nmol·L^-1）＋ miR-212-3p inhibitor（100 nmol·L^-1）組，干預(yù)24 h；將SKOV3/TAX移植瘤裸鼠隨機(jī)分為對照組、TAX（8 mg·kg^-1，ip給藥）組、TPL（30 μg·kg^-1，ip給藥） ＋TAX（8 mg·kg^-1）組、inhibitor-NC（5 nmol，瘤內(nèi)注射）組、TPL（30 μg·kg^-1） ＋TAX（8 mg·kg^-1） ＋miR-212-3p inhibitor（5 nmol，瘤內(nèi)注射）組，干預(yù)2周。qRT-PCR法檢測細(xì)胞和組織中miR-212-3p、ZEB2表達(dá)，以CCK-8、流式細(xì)胞實(shí)驗(yàn)分別檢測細(xì)胞增殖、凋亡；測量腫瘤體積； Western blotting檢測ZEB2、Bax、Bcl-2、多藥耐藥蛋白1（MDR1）蛋白表達(dá)。結(jié)果 與SKOV3細(xì)胞比較，SKOV3/TAX細(xì)胞miR-212-3p降低且ZEB2 mRNA表達(dá)升高（P＜0.05）。TPL可降低SKOV3/TAX細(xì)胞耐藥指數(shù)（P＜0.05） ，miR-212-3p inhibitor可逆轉(zhuǎn)TPL上述作用（P＜0.05）。與對照組相比，TAX組細(xì)胞存活率、腫瘤體積、Bcl-2蛋白表達(dá)降低（P＜0.05），凋亡率、Bax蛋白表達(dá)升高（P＜0.05）； TPL＋ TAX組細(xì)胞存活率、腫瘤體積、ZEB2 mRNA與蛋白表達(dá)、Bcl-2與MDR1蛋白表達(dá)降低（P＜0.05），凋亡率、miR-212-3p表達(dá)、Bax蛋白表達(dá)升高（P＜0.05）； inhibitor-NC組細(xì)胞各指標(biāo)無顯著變化。與TAX組相比，TPL＋TAX組細(xì)胞存活率、腫瘤體積、ZEB2 mRNA與蛋白表達(dá)、Bcl-2與MDR1蛋白表達(dá)降低（P＜0.05），凋亡率、miR-212-3p表達(dá)、Bax蛋白表達(dá)升高（P＜0.05）。與TPL＋TAX組相比，TPL＋TAX＋miR-212-3p inhibitor組細(xì)胞存活率、腫瘤體積、ZEB2 mRNA與蛋白表達(dá)、Bcl-2與MDR1蛋白表達(dá)升高（P＜0.05），凋亡率、miR-212-3p表達(dá)、Bax蛋白表達(dá)降低（P＜0.05）。結(jié)論 TPL可拮抗卵巢癌細(xì)胞的TAX耐藥性，機(jī)制可能與調(diào)控miR-212-3p/ZEB2信號(hào)通路相關(guān)。

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

河南省醫(yī)學(xué)科技攻關(guān)計(jì)劃聯(lián)合共建項(xiàng)目（LHGJ20210940）