Zhang J, Bai H J
Department of Gynecological Oncology, Zhengzhou University Affiliated Cancer Hospital, Henan Cancer Hospital, Zhengzhou 450008, China.
Zhonghua Zhong Liu Za Zhi. 2021 Oct 23;43(10):1062-1068. doi: 10.3760/cma.j.cn112152-20200427-00384.
To explore the effect of circBIRC6 on cisplatin resistance of ovarian cancer cells and the molecular mechanism. The ovarian cancer cell line SKOV3 and ovarian cancer cisplatin-resistant cell line SKOV3 / DDP were cultured in vitro, and treated with different concentrations of cisplatin. SKOV3 and SKOV3/DDP cells were transfected with si-NC, si-circBIRC6, si-circBIRC6+ anti-miR-NC, si-circBIRC6+ anti-miR-367-3p by liposome-mediated method, which were denoted as DDP+ si-NC group, DDP+ si-circBIRC6 group, DDP+ si-circBIRC6+ anti-miR-NC group and DDP+ si-circBIRC6+ anti-miR-367-3p group, respectively, and then were treated with 2 μg/ml cisplatin for 24 hours. The cell proliferation inhibition rate was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide method, and the half inhibitory concentration (IC(50)) value of cisplatin was calculated. Real-time fluorescent quantitative polymerase chain reaction was used to detect the transcriptional levels of circBIRC6 and miR-367-3p. Flow cytometry was used to detect the apoptotic rate. Dual luciferase report experiment verified the targeting relationship of circBIRC6 and miR-367-3p. Western blot was used to detect the expressions of Cyclin D1, Bcl-2, p21, Bax. The expression levels of circBIRC6 in SKOV3 cells were 1.00±0.05, significantly lower than 3.04±0.24 in SKOV3/DDP cells (<0.001). The expression levels of miR-367-3p in SKOV3 cells were 1.00±0.08, significantly higher than 0.54±0.05 in SKOV3/DDP cells (<0.001). The cell proliferation inhibition rates of SKOV3 cells and SKOV3/DDP cells were (22.47±2.04)% and (8.84±0.71)%, the IC(50) values of SKOV3 cells and SKOV3/DDP cells were 6.65±0.94 and 28.18±4.91, respectively, with significant difference (<0.05). The proliferation inhibition rate and apoptosis rate of SKOV3 cells in DDP+ si-NC group[(22.19±2.19)% and (10.98±1.12)%] were lower than those in DDP+ si-circBIRC6 group [(74.18±5.36)% and (32.91±3.19)%, all <0.05]. The proliferation inhibition rate and apoptosis rate of SKOV3/DDP cells[(8.71±0.87)% and (7.39±0.63)%] were lower than those of DDP+ si-circBIRC6 group [(40.85±4.07)% and (25.31±2.53)%, all <0.05]. The protein expression levels of Cyclin D1 and Bcl-2 in SKOV3 and SKOV3/DDP cells in DDP+ si-circBIRC6 group were lower than those in DDP+ si-NC group, and the protein expression levels of p21 and Bax were higher than those in DDP+ si-NC group (all <0.05). The dual luciferase report experiment confirmed that circBIRC6 targeted miR-367-3p. Inhibition of miR-367-3p expression reduced the effect of circBIRC6 deletion on ovarian cancer cell proliferation, apoptosis and cisplatin resistance. Knockdown of circBIRC6 may inhibit the proliferation of ovarian cancer cisplatin-resistant cells and induce apoptosis by up-regulating the expression of miR-367-3p, therefore impair the cisplatin resistance of these cells.
探讨环状BIRC6对卵巢癌细胞顺铂耐药性的影响及其分子机制。体外培养卵巢癌细胞系SKOV3和卵巢癌顺铂耐药细胞系SKOV3/DDP,并用不同浓度的顺铂处理。采用脂质体介导法将si-NC、si-circBIRC6、si-circBIRC6 + 抗miR-NC、si-circBIRC6 + 抗miR-367-3p转染至SKOV3和SKOV3/DDP细胞,分别记为DDP + si-NC组DDP + si-circBIRC6组、DDP + si-circBIRC6 + 抗miR-NC组和DDP + si-circBIRC6 + 抗miR-367-3p组,然后用2 μg/ml顺铂处理24小时。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H溴化四氮唑法检测细胞增殖抑制率,并计算顺铂的半数抑制浓度(IC(50))值。采用实时荧光定量聚合酶链反应检测环状BIRC6和miR-367-3p的转录水平。采用流式细胞术检测凋亡率。双荧光素酶报告实验验证环状BIRC6与miR-367-3p的靶向关系。采用蛋白质免疫印迹法检测细胞周期蛋白D1、Bcl-2、p21、Bax的表达。SKOV3细胞中环状BIRC6的表达水平为1.00±0.05,显著低于SKOV3/DDP细胞中的3.04±0.24(<0.001)。SKOV3细胞中miR-367-3p的表达水平为1.00±0.08,显著高于SKOV3/DDP细胞中的0.54±0.05(<0.001)。SKOV3细胞和SKOV3/DDP细胞的增殖抑制率分别为(22.47±2.04)%和(8.84±0.71)%,SKOV3细胞和SKOV3/DDP细胞的IC(50)值分别为6.65±0.94和28.18±4.91,差异有统计学意义(<0.05)DDP + si-NC组SKOV3细胞的增殖抑制率[(22.19±2.19)%]和凋亡率[(10.98±1.12)%]低于DDP + si-circBIRC6组[(74.18±5.36)%和(32.91±3.19)%,均<0.05]。SKOV3/DDP细胞的增殖抑制率[(8.71±0.87)%]和凋亡率[(7.39±0.63)%]低于DDP + si-circBIRC6组[(40.85±4.07)%和(25.31±2.53)%,均<0.05]。DDP + si-circBIRC6组SKOV3和SKOV3/DDP细胞中细胞周期蛋白D1和Bcl-2的蛋白表达水平低于DDP + si-NC组,p21和Bax的蛋白表达水平高于DDP + si-NC组(均<0.05)。双荧光素酶报告实验证实环状BIRC6靶向miR-367-3p。抑制miR-367-3p表达可降低环状BIRC6缺失对卵巢癌细胞增殖、凋亡及顺铂耐药性的影响。敲低环状BIRC6可能通过上调miR-367-3p的表达抑制卵巢癌顺铂耐药细胞的增殖并诱导其凋亡,从而削弱这些细胞的顺铂耐药性。