TXNDC5-Prx2途径对前列腺癌细胞耐药性的调控

doi:10.3760/cma.j.cn371439-20210324-00090

摘要/Abstract

摘要：

目的研究硫氧还原蛋白5(TXNDC5)-过氧化物还原酶2(Prx2)对前列腺癌细胞耐药性的影响。方法体外培养前列腺癌PC3细胞,采用化疗药物环磷酰胺(5、10、15 μmol/L)干预24 h,另设空白对照组,采用实时荧光定量PCR(RT-qPCR)和蛋白质印迹法检测PC3细胞中TXNDC5的表达水平。在PC3细胞中给予不同浓度环磷酰胺处理的同时沉默TXNDC5,CCK-8法检测siTXNDC5组和siNC组细胞增殖活力,活性氧检测试剂盒测定活性氧自由基含量。在PC3细胞株及其环磷酰胺耐药细胞株中给予10 μmol/L环磷酰胺处理并同时沉默TXNDC5表达,检测细胞增殖活力。蛋白质印迹法检测在PC3细胞中沉默TXNDC5对Prx2蛋白表达的影响。在过表达TXNDC5的PC3细胞中沉默Prx2表达,检测Vec-Ctrl组、pcTXNDC5组、siNC组、siPrx2组、pcTXNDC5+siPrx2组的细胞增殖活力和活性氧自由基含量。结果与空白对照组相比,环磷酰胺处理可显著提高前列腺癌PC3细胞中TXNDC5 mRNA和蛋白的表达量。10、15 μmol/L环磷酰胺处理PC3细胞12 h后,与siNC组相比,siTXNDC5组细胞增殖活力明显受抑(0.44±0.08 vs. 0.74±0.10,t=3.647,P=0.031;0.30±0.04 vs. 0.53±0.06,t=6.115,P=0.006)。10 μmol/L环磷酰胺处理PC3细胞6、12 h,与siNC组相比,siTXNDC5组细胞内活性氧自由基的生成量显著增加(2.68±0.19 vs. 1.58±0.26,t=-6.027,P=0.005;4.56±0.37 vs. 2.73±0.26,t=-6.995,P=0.003)。采用10 μmol/L环磷酰胺处理PC3及其耐药细胞株12 h,与siNC组相比,siTXNDC5组细胞增殖活力均明显受抑。蛋白质印迹法结果表明沉默TXNDC5可显著抑制Prx2的表达。沉默Prx2表达可显著抑制因TXNDC5过表达导致的细胞增殖活力升高和活性氧自由基含量降低的趋势。10 μmol/L环磷酰胺处理PC3细胞12 h,Vec-Ctrl组、pcTXNDC5组、siNC组、siPrx2组和pcTXNDC5+siPrx2组细胞增殖活力分别为0.52±0.07、0.69±0.03、0.56±0.05、0.43±0.05、0.58±0.07,差异有统计学意义(F=8.868,P=0.003),pcTXNDC5+siPrx2组细胞增殖活性低于pcTXNDC5组(P=0.045);上述5组细胞活性氧自由基含量分别为3.26±0.46、2.09±0.49、3.16±0.38、4.62±0.26、2.87±0.36,差异有统计学意义(F=16.037,P<0.001),pcTXNDC5+siPrx2组活性氧自由基含量高于pcTXNDC5组(P=0.036)。结论 TXNDC5可通过调控Prx2的表达,降低前列腺癌细胞中活性氧自由基的水平,从而增强前列腺癌细胞对化疗药物的耐药性。

关键词: 前列腺肿瘤, 硫氧还原蛋白5, 过氧化物还原酶2, 细胞增殖, 活性氧

Abstract:

Objective To study the effect of thioredoxin domain containing protein 5 (TXNDC5)-peroxiredoxin 2 (Prx2) on the drug resistance of prostate cancer cells.Methods Prostate cancer PC3 cells were cultured in vitro, treated with the chemotherapy drug cyclophosphamide (5, 10, 15 μmol/L) for 24 hours, and PC3 cells without any treatment was served as the control group. The expression levels of TXNDC5 in PC3 cells were detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blotting. PC3 cells with TXNDC5 knocking down were exposed by cyclophosphamide and CCK-8 was used to detect the cell viability of siTXNDC5 group and siNC group. The content of reactive oxygen free radicals was determined by reactive oxygen detection kit. PC3 cells and its parental cyclophosphamide-resistant ones with TXNDC5 knocking down were treated by 10 μmol/L cyclophosphamide and subjected for CCK8 assay. The expression of Prx2 in PC3 cells was detected by Western blotting after TXNDC5 was silenced. Prx2 expression was silenced in PC3 cells overexpressing TXNDC5, and cell viability and reactive oxygen free radical content were detected in Vec-Ctrl group, pcTXNDC5 group, siNC group, siPrx2 group and pcTXNDC5+siPrx2 group.Results Compared with the control group, cyclophosphamide treatment significantly increased the expression of TXNDC5 at mRNA and protein levels in PC3 cells. After PC3 cells were treated with cyclophosphamide (10, 15 μmol/L) for 12 h, compared with the siNC group, the cell viability in the siTXNDC5 group was significantly suppressed (0.44±0.08 vs. 0.74±0.10, t=3.647, P=0.031; 0.30±0.04 vs. 0.53±0.06, t=6.115, P=0.006). When PC3 cells were treated with 10 μmol/L cyclophosphamide for 6 and 12 h, compared with the siNC group, the production of reactive oxygen free radicals in the siTXNDC5 group was significantly increased (2.68±0.19 vs. 1.58±0.26, t=-6.027, P=0.005; 4.56±0.37 vs. 2.73±0.26, t=-6.995, P=0.003). When PC3 cells and its cyclophosphamide-resistant ones were treated with 10 μmol/L cyclophosphamide for 12 h, compared with the siNC group, the cell viability was significantly inhibited in the siTXNDC5 group. Western blotting analysis showed that the expression of Prx2 was significantly reduced when TXNDC5 was silenced. Silencing Prx2 could significantly attenuate the increase of cell viability and the decrease of reactive oxygen content resulting from TXNDC5 overexpression. PC3 cells were treated with 10 μmol/L cyclophosphamide for 12 h, and the cell viabilities of the Vec-Ctrl group, pcTXNDC5 group, siNC group, siPrx2 group and pcTXNDC5+siPrx2 group were 0.52±0.07, 0.69±0.03, 0.56±0.05, 0.43±0.05, 0.58±0.07, respectively, and there was a statistically significant difference (F=8.868, P=0.003). Furthermore, the cell viability in the pcTXNDC5+siPrx2 group decreased significantly when compared to that of the pcTXNDC5 group (P=0.045). The contents of reactive oxygen free radicals in the above 5 groups were 3.26±0.46, 2.09±0.49, 3.16±0.38, 4.62±0.26, 2.87±0.36, respectively, and there was a statistically significant difference (F=16.037, P<0.001). The content of reactive oxygen radicals in the pcTXNDC5+siPrx2 group was higher than that of the pcTXNDC5 group (P=0.036).Conclusion TXNDC5 can reduce the level of reactive oxygen free radicals in prostate cancer cells by regulating the expression of Prx2, so as to promote the drug resistance of prostate cancer cells.

Key words: Prostate neoplasms, Thioredoxin domain containing protein 5, Peroxiredoxin 2, Cell proliferation, Reactive oxygen species

张永丽, 张若佳, 范焕彩, 葛鲁娜, 王林. TXNDC5-Prx2途径对前列腺癌细胞耐药性的调控[J]. 国际肿瘤学杂志, 2021, 48(8): 473-478.

Zhang Yongli, Zhang Ruojia, Fan Huancai, Ge Luna, Wang Lin. TXNDC5-Prx2 axis regulates drug resistance of prostate cancer cells[J]. Journal of International Oncology, 2021, 48(8): 473-478.

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组别	环磷酰胺浓度(μmol/L)
	正常细胞株				耐药细胞株
	0	10			0		10
siNC组	1.00±0.12		0.75±0.15	1.03±0.11		0.90±0.09
siTXNDC5组	0.97±0.11		0.42±0.10	0.97±0.07		0.41±0.06
t值	-0.350		3.608	0.890		7.991
P值	0.744		0.049	0.433		0.002

组别	0 h	6 h	12 h	24 h
siNC组	1.02±0.12	1.58±0.26	2.73±0.26	4.06±0.62
siTXNDC5组	0.99±0.09	2.68±0.19	4.56±0.37	4.99±0.49
t值	0.973	-6.027	-6.995	-2.021
P值	0.417	0.005	0.003	0.117

组别	0 h	6 h	12 h	24 h
Vec-Ctrl组	1.00±0.12	0.76±0.10	0.52±0.07	0.42±0.05
pcTXNDC5组	0.98±0.11	0.89±0.10	0.69±0.03^a	0.58±0.03^a
siNC组	0.99±0.12	0.79±0.07	0.56±0.05	0.46±0.03
siPrx2组	0.96±0.10	0.69±0.04	0.43±0.05	0.36±0.06
pcTXNDC5+ siPrx2组	1.02±0.09	0.79±0.05	0.58±0.07^b	0.49±0.06^b
F值	0.436	2.946	8.868	9.485
P值	0.780	0.076	0.003	0.002

组别	0 h	6 h	12 h	24 h
Vec-Ctrl组	1.00±0.09	2.36±0.38	3.26±0.46	4.53±0.39
pcTXNDC5组	1.00±0.16	1.62±0.39^a	2.09±0.49^a	2.96±0.25^a
siCtrl组	1.09±0.07	2.22±0.59	3.16±0.38	4.31±0.29
siPrx2组	1.16±0.06	3.26±0.08	4.62±0.26	6.15±0.38
pcTXNDC5+ siPrx2组	1.15±0.15	1.76±0.12	2.87±0.36^b	3.86±0.31^b
F值	1.190	9.549	16.037	38.362
P值	0.373	0.002	<0.001	<0.001