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

Abstract

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

CLC Number:

R737.31

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.

Figures/Tables 9

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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

Inhibitory effects of centromere protein U knockdown on self-renewal, cisplatin resistance and Wnt/β-catenin signaling activity in cisplatin resistant ovarian cancer cells

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