POLD1增强三阴性乳腺癌细胞MDA-MB-231对紫杉醇耐药性的作用研究

Effect of POLD1on the resistance of triple negative breast cancer cells MDA-MB-231to paclitaxel

目的探讨DNA聚合酶δ催化亚基基因(POLD1)在增强三阴性乳腺癌MDA-MB-231细胞耐受紫杉醇(PTX)抑制其活性及上皮间质转化的作用。方法细胞计数试剂盒8(CCK8)法检测PTX抑制MDA-MB-231细胞活性。使用重组慢病毒转染构建POLD1过表达的MDA-MB-231/POLD1-OE及空载对照的MDA-MB-231/POLD-NC细胞株。分设3个组,分别为无处理的MDA-MB-231/POLD-NC细胞(POLD1-NC组)、分别使用PTX刺激的MDA-MB-231/POLDNC(PTX+POLD1-NC组)及MDA-MB-231/POLD1-OE(PTX+POLD1-OE组)细胞。1μmol/L PTX刺激作用48h后,通过蛋白质印迹实验检测各组细胞P125(POLD1编码的蛋白)、增殖细胞核抗原(PCNA)、B淋巴细胞瘤-2(Bcl-2)、E-钙黏蛋白(E-cadherin)及波形蛋白(Vimentin)表达水平;CCK8、Transwell侵袭实验和划痕实验分别检测各组细胞的活性及侵袭迁移能力。结果CCK8结果显示,不同浓度(0.1、0.5、1、5、10、50μmol/L)的PTX刺激48h后,细胞的存活率分别为(87.71±2.13)%、(74.13±1.58)%、(56.94±1.21)%、(34.23±0.15)%、(10.55±0.02)%和(1.71±0.09)%,较对照组(0μmol/L)下降,各组数据间总的差异有统计学意义,F=2051.408,P<0.001。蛋白质印迹实验结果显示,1μmol/L PTX刺激48h后,与POLD1-NC组相比,PTX+POLD1-NC组细胞的P125、PCNA和Vimentin的表达降低(P值分别为<0.001、0.007和0.034),而E-cadherin的表达上升,P=0.026;与POLD1-NC组相比,PTX+POLD1-OE组细胞中PCNA、Bcl-2和Vimentin蛋白表达水平有所降低,但略高于PTX+POLD1-NC组,E-cadherin的表达则略高于POLD1-NC组细胞而略低于PTX+POLD1-NC组,但差异均无统计学意义。CCK8实验结果显示,以POLD1-NC组细胞存活率(100.00±8.83)%为对照,PTX+POLD1-NC组和PTX+POLD1-OE组存活率分别为(48.87±2.41)%和(80.78±5.47)%,总体差异有统计学意义,F=52.749,P<0.001。其中PTX+POLD1-NC组和PTX+POLD1-OE组存活率较POLD1-NC组降低(P值分别为<0.001和0.009),而PTX+POLD1-OE组细胞的存活率高于PTX+POLD1-NC组,P=0.001。Transwell侵袭实验结果显示,POLD1-NC组、PTX+POLD1-NC组及PTX+POLD1-OE组的穿膜细胞数分别为(238.33±12.01)、(122.33±9.07)和(192.67±3.21)个,总体差异有统计学意义,F=129.672,P<0.001。其中PTX+POLD1-NC组和PTX+POLD1-OE组穿膜细胞数少于POLD1-NC组(P值分别为<0.001和0.001),但PTX+POLD1-OE组的穿膜细胞数多于PTX+POLD1-NC组,P<0.001。划痕实验显示,POLD1-NC组、PTX+POLD1-NC组及PTX+POLD1-OE组细胞的24h愈合率分别为(30.93±3.05)%、(16.19±2.18)%和(23.47±2.27)%,总体差异有统计学意义,F=25.490,P=0.001。其中PTX+POLD1-NC组和PTX+POLD1-OE组愈合率低于POLD1-NC组(P值分别为<0.001和0.011),而PTX+POLD1-OE组的愈合率高于PTX+POLD1-NC组,P=0.012。结论PTX能够下调MDA-MB-231细胞中POLD1的表达,POLD1的高表达能够增强MDA-MB-231细胞对PTX的耐药性。

Objective To investigate the effect of DNA polymerase delta catalytic subunit gene 1(POLD1)on enhancing the resistance to the viability and epithelial-mesenchymal transition inhibition of paclitaxel(PTX)in triple negative breast cancer cells MDA-MB-231.Methods Cell counting kit 8(CCK8)test was used to detect the viability inhibition of MDA-MB-231cells induced by PTX.Recombinant lentivirus transfection was used to construct the MDA-MB-231/POLD1-OE and MDA-MB-231/POLDNC cell lines.A total of three groups were established,including MDA-MB-231/POLD-NC without treatment(POLD-NC group),MDA-MB-231/POLD-NC cells treated with PTX(PTX+POLD1-NC group),and MDA-MB-231/POLD1-OE cells treated with PTX(PTX+POLD1-OE group).After 48hof 1μmol/LPTX stimulation,western blots were performed to detect the expression levels of P125(the protein encoded by POLD1),PCNA,Bcl-2,E-cadherin,and Vimentin.CCK8test was used to measure the viability inhibited rate of cells stimulated with PTX.To determine the invasion and migration abilities of each group of cells,the transwell invasion assay and scratch assay were used.Results The CCK8test indicated that the cell viability in each group after 48hof PTX stimulation(0.1,0.5,1,5,10,50μmol/L)were(87.71±2.13)%,(74.13±1.58)%,(56.94±1.21)%,(34.23±0.15)%,(10.55±0.02)%,(1.71±0.09)%,respectively,lower than the control group(0μmol/L),and the differences were statistically significant(F=2051.408,P<0.001).Western blots analysis showed that,after 48hof 1μmol/L PTX stimulation,the expression of P125,PCNA,and Vimentin in the PTX+POLD1-NC group was significantly reduced as compared to the POLD1-NC group(P<0.001,P=0.007,P=0.034),while the expression of E-cadherin increased(P=0.026).The expression levels of PCNA,Bcl-2,and Vimentin in the PTX+POLD1-OE group were lower than those in the POLD1-NC group,but were higher than those in the PTX+POLD1-NC group,without statistical difference.The expression of E-cadherin in the PTX+POLD1-OE group was higher than that in the POLD1-NC group but lower than that in the PTX+POLD1-NC group,without statistical difference.The results of CCK8demonstrated that the cell viability of the POLD1-NC group was(100.00±8.83)%as control,and that of the PTX+POLD1-NC group and PTX+POLD1-OE group were(48.87±2.41)%and(80.79±5.47)%,respectively,and the differences were statistically significant(F=52.749,P<0.001).Among them,the survival rate of the PTX+POLD1-NC group and PTX+POLD1-OE group were significantly lower than that of the POLD1-NC group(P<0.001,P=0.009),and the cell viability of the PTX+POLD1-OE group was significantly higher than that of the PTX+POLD1-NC group(P=0.001).Transwell invasion results showed that the number of transmembrane cells in the POLD1-NC group,PTX+POLD1-NC group,and PTX+POLD1-OE group were 238.33±12.01,122.33±9.07and 192.67±3.21,respectively,the differences were statistically significant(F=129.672,P<0.001).Among them,the number of transmembrane cells in the PTX+POLD1-NC group and PTX+POLD1-OE group was significantly less than that in the POLD1-NC group(P<0.001,P=0.001),while the number of transmembrane cells in PTX+POLD1-OE group was significantly higher than that in PTX+POLD1-NC group(P<0.001).The results of the scratch assay demonstrated that the 24h healing rates of the POLD1-NC group,PTX+POLD1-NC group,and PTX+POLD1-OE group were(30.93±3.05)%,(16.19±2.18)%and(23.47±2.27)%,respectively,the differences were statistically significant(F=25.490,P=0.001).Among them,the healing rate of the PTX+POLD1-NC group and PTX+POLD1-OE group was significantly lower than that of the POLD1-NC group(P<0.001,P=0.011),however,the healing rate of the PTX+POLD1-OE group was significantly higher than that of the PTX+POLD1-NC group(P=0.012).Conclusion Paclitaxel can suppress the expression of POLD1in MDAMB-231cells,and the high expression of POLD1can enhance their tolerance to paclitaxel.