NOS1在宫颈癌干细胞中的表达及对化疗敏感性的影响

Expression of nitric oxide synthase 1 in cervical cancer stem cells and its effect on chemotherapy sensitivity

目的探讨一氧化氮合酶1(NOS1)在宫颈癌干细胞(CCSCs)中的表达水平,并分析NOS1对CCSCs顺铂(Cisplatin,DDP)化疗敏感性的影响及作用机制。方法qRT-PCR检测NOS1在宫颈癌细胞系HeLa、Siha、Caski及人正常宫颈上皮细胞系Hucec中的表达水平。选择高表达NOS1的宫颈癌细胞培养CCSCs。Western blot检测肿瘤干细胞(CSCs)中Nanog、SOX2、OCT4干性标志蛋白及NOS1的表达。MTS检测宫颈癌细胞和CCSCs对DDP的IC50值。对照siRNA和NOS1 siRNA(si-NOS1)转染CCSCs分为NC组和si-NOS1组,MTS检测两组CCSCs对DDP的IC50值。DDP处理NC组和si-NOS1组CCSCs后流式细胞仪实验检测细胞凋亡,Western blot检测细胞中ABCG蛋白和凋亡相关蛋白的表达。结果qRT-PCR结果显示NOS1在宫颈癌细胞系中的表达高于在人正常宫颈上皮细胞系Hucec中的表达水平,在HeLa细胞中表达最高(P<0.05)。OCT4、Nanog、SOX2干细胞标志物NOS1在CSCs中表达上调(P<0.05)。MTS检测发现与HeLa细胞相比,CCSCs细胞对DDP的IC50值增加(P<0.05)。MTS检测发现与NC组相比,si-NOS1组CCSCs细胞对DDP的IC50值降低(P<0.05)。DDP处理CCSCs后,与NC组相比,si-NOS1组细胞凋亡率增加,细胞中ABCG2和Bcl-2表达降低,Bax蛋白表达增加(P<0.05)。结论NOS1在CCSCs中表达增加,NOS1可能通过上调ABCG2调控凋亡相关蛋白降低CCSCs对DDP的化疗敏感性。

Objective To study the expression level of nitric oxide synthase 1(NOS1)in cervical cancer stem cells(CCSCs)and to explore the effect of NOS1on CCSCs sensitivity to Cisplatin(DDP)chemotherapy.Methods qRT-PCR was used to detect the expression level of NOS1 in cervical cancer cell lines.Cervical cancer cells with high expression of NOS1 were selected to culture CCSCs.Western blot was used to detect the expression of Nanog,SOX2,OCT4 stem marker protein and NOS1 in CSCs.MTS detects the IC50 value of cervical cancer cells and CCSCs for DDP.Control siRNA and NOS1 siRNA(si-NOS1)transfected CCSCs were divided into NC group and si-NOS1 group.MTS detected the IC50 value of CCSCs in each group to DDP.Flow cytometry was used to detect apoptosis after treatment of CCSCs in NC group and si-NOS1 group by DDP,and the expression of ABCG protein and apoptosis-related protein in cells was detected by Western blot.Results qRT-PCR results showed that the expression of NOS1 in cervical cancer cell lines was higher than that in human normal cervical epithelial cell line Hucec,and the highest expression was in HeLa cells(P<0.05).OCT4,Nanog,and SOX2 stem cell markers were up-regulated in CSCs(P<0.05).NOS1 was up-regulated in CCSCs(P<0.05).MTS test showed that compared with HeLa cells,the IC50 value of CCSCs on DDP was increased.The MTS test found that compared with the NC group,the IC50 value of DDP of CCSCs cells in the si-NOS1 group was reduced.After CCSCs were treated with DDP,compared with the NC group,the apoptosis rate of si-NOS1 group was increased,ABCG2 and Bcl-2 expression were decreased,and Bax protein expression was increased(P<0.05).Conclusion NOS1 expression is increased in CCSCs.NOS1 may reduce the sensitivity of CCSCs to DDP by up-regulating ABCG2 to regulate apoptosis-related proteins.