敲低着丝粒蛋白U对顺铂耐药卵巢癌细胞自我更新、顺铂耐药和Wnt/β-catenin信号活性的抑制作用

doi:10.13481/j.1671-587X.20210309

摘要/Abstract

摘要： 目的

探讨下调着丝粒蛋白U（CENPU）对卵巢癌（OC）细胞顺铂耐药的影响，并分析其作用机制。

方法

采用Western blotting法检测OC细胞（OVCAR3和SKOV3细胞）和顺铂耐药OC细胞（OVCAR3/DDP和SKOV3/DDP细胞）中CENPU蛋白表达水平。将OVCAR3/DDP和SKOV3/DDP细胞分为shControl组（转染shControl质粒）和CENPU短发夹RNA质粒（shCENPU）组（转染shCENPU质粒）。CCK-8法检测0 ~ 100 μmol·L^－1顺铂刺激24 h后 2组细胞活性，流式细胞术检测20 μmol·L^－1顺铂刺激24 h后2组细胞凋亡率，肿瘤球形成实验检测2组细胞的细胞球形成率（SFE），实时荧光定量PCR（RT-qPCR）法检测2组细胞中自我更新相关基因性别决定区Y框蛋白2（Sox2）、Nanog和八聚体结合转录因子4（Oct4）mRNA表达水平，Western blotting法检测2组细胞中Wnt/β-catenin通路相关蛋白无翅型MMTV整合位点家族成员1（Wnt1）、细胞周期素D1（cyclinD1）、c-Myc和β-连环素（β-catenin）蛋白表达水平。

结果

与OVCAR3和SKOV3细胞比较，OVCAR3/DDP和SKOV3/DDP细胞中CENPU蛋白表达水平均明显升高（P<0.05）。顺铂作用后，与shControl组比较，shCENPU组OVCAR3/DDP和SKOV3/DDP细胞活性明显降低（P<0.05），细胞凋亡率明显升高（P<0.05）。与shControl组比较，shCENPU组OVCAR3/DDP和SKOV3/DDP细胞的SEF，细胞中Sox2、Nanog和Oct4 mRNA水平以及Wnt1、cyclin D1、c-Myc和β-catenin蛋白表达水平均明显降低（P<0.05）。

结论

敲低CENPU能抑制顺铂耐药OC细胞的自我更新、顺铂耐药和Wnt/β-catenin信号活性。

关键词: 着丝粒蛋白U, 卵巢肿瘤, 化疗耐药, 自我更新, Wnt/β-catenin信号通路, 顺铂

Abstract: Objective

To investigate the effect of centromere protein U （CENPU） on cisplatin resistance of ovarian cancer（OC） cells， and to analyze its mechanism.

Methods

The expression levels of CENPU protein in the OC cells （OVCAR3 and SKOV3 cells） and cisplatin resistant OC cells （OVCAR3/DDP and SKOV3/DDP cells） were detected by Western blotting method. The OVCAR3/DDP and SKOV3/DDP cells were divided into shControl group （transfected with shControl plasmid） and CENPU short hairpin RNA plasmid（shCENPU） group （transfected with shCENPU plasmid）.The cell viabilities in two groups after stimulated with 0－100 μmol·L^－1 cisplatin for 24 h were detected by CCK-8 assay； after stimulated with 20 μmol·L^－1 cisplatin for 24 h， the apoptotic rates of cells in two groups were detected by flow cytometry； the sphere formation efficiencies （SFE） of the cells in two groups were detected by tumor sphere formation assay； the expression levels of self-renewing related gene sex determining region Y （SRY）-related high-mobility-group （HMG）-box protein-2 （Sox2）， Nanog and octamer-binding transcription factor-4 （Oct4） mRNA in the cells in two groups were detected by Real-time fluorescence quantitative PCR（RT-qPCR） method； the expression levels of Wnt/β-catenin pathway related proteins wingless type MMTV integration site family member 1 （Wnt1）， cyclinD1， c-Myc， and β-catenin in the cells in two groups were detected by Western blotting method.

Results

Compared with the OVCAR3 and SKOV3 cells， the expression levels of CENPU protein in the OVCAR3/DDP and SKOV3/DDP cells were significantly increased （P<0.05）. After treated with cisplatin， compared with shControl group， the viabilities of OVCAR3/DDP and SKOV3/DDP cells in shCENPU group were significantly decreased（P<0.05）， and the apoptotic rates were significantly increased （P<0.05）. Compared with shControl group， the SFE and the expression levels of Sox2， Nanog， and Oct4 mRNA， and the expression levels of Wnt1， cyclinD1， c-Myc， and β-catenin proteins in the OVCAR3/DDP and SKOV3/DDP cells in shCENPU group were significantly decreased（P<0.05）.

Conclusion

CENPU knockdown can inhibit the self-renewal，cisplatin resistance， and Wnt/β-catenin signaling activity in cisplatin resistant OC cells.

Key words: centromere protein U, ovarian tumor, chemotherapy resistance, self-renewal, Wnt/β-catenin signaling pathway, cisplatin

中图分类号:

R737.31

任英俊, 张慧, 周颖. 敲低着丝粒蛋白U对顺铂耐药卵巢癌细胞自我更新、顺铂耐药和Wnt/β-catenin信号活性的抑制作用[J]. 吉林大学学报(医学版), 2021, 47(3): 608-614.

Yingjun REN, Hui ZHANG, Ying ZHOU. Inhibitory effects of centromere protein U knockdown on self-renewal, cisplatin resistance and Wnt/β-catenin signaling activity in cisplatin resistant ovarian cancer cells[J]. Journal of Jilin University(Medicine Edition), 2021, 47(3): 608-614.

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Group	Cell viability after treated with DDP
	[c_B/(μmol·L^－1)] 0	4	8	16	32	64	100
shControl	100.00±5.67	96.04±12.38	94.15±13.42	91.23±11.25	85.54±9.86	68.26±5.45	45.37±6.53
shCENPU	95.02±9.64	90.58±9.89	65.07±8.64^*	52.48 ±7.65^*	41.65±5.38^*	25.24±3.25^*	15.13±3.59^*

Group	Cell viability after treated with DDP
	[c_B/(μmol·L^－1)] 0	4	8	16	32	64	100
shControl	100.00±7.89	99.13±11.04	95.26±9.83	92.67±10.12	88.54±11.04	62.15±8.32	41.21±7.02
shCENPU	93.38±14.56	93.13±8.02	73.26±7.02^*	51.35 ±11.26^*	32.73±4.54^*	18.76±3.84^*	6.89±1.23^*

Group	Apoptotic rate
	OVCAR3/DDP cells	SKOV3/DDP cells
shControl	11.25±2.38	8.26±2.13
shCENPU	48.56±5.31	53.27±6.31
t	12.454	16.783
P	<0.05	<0.05

Group	OVCAR3/DDP cells				SKOV3/DDP cells
	SFE (η/%)	Sox2 mRNA	Nanog mRNA	Oct4 mRNA	SFE (η/%)	Sox2 mRNA	Nanog mRNA	Oct4 mRNA
shControl	12.21±2.12	1.00±0.00	1.00±0.00	1.00±0.00	10.39±1.68	1.00±0.00	1.00±0.00	1.00±0.00
shCENPU	4.25±0.94	0.15±0.06	0.45±0.08	0.53±0.05	2.96±0.73	0.31±0.03	0.21±0.05	0.36±0.04
t	8.726	19.804	7.435	6.986	10.315	9.824	13.265	9.063
P	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05

Group	OVCAR3/DDP cells				SKOV3/DDP cells
	CyclinD1	c-Myc	Wnt1	β-catenin	CyclinD1	c-Myc	Wnt1	β-catenin
shControl	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
shCENPU	0.56±0.06	0.78±0.05	0.39±0.07	0.36±0.04	0.45±0.02	0.32±0.03	0.58±0.03	0.24±0.07
t	8.024	5.876	11.206	13.425	10.115	15.316	9.403	18.432
P	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05	<0.05