沉默诱骗受体3提高宫颈癌Caski细胞放疗敏感性的研究

Silencing DcR3 improves the radiosensitivity of cervical cancer Caski cells

目的探讨沉默诱骗受体3(DcR3)对宫颈癌Caski细胞放疗敏感性的影响。方法将宫颈癌Caski细胞分成Control组、sh-NC组(感染shRNA阴性对照)、sh-DcR3组(感染DcR3 shRNA)、RAD+sh-NC组(感染shRNA阴性对照,照射处理)、RAD+sh-DcR3组(感染DcR3 shRNA,照射处理);采用噻唑蓝(MTT)法检测细胞的增殖能力,碘化丙啶(PI)单染法测定细胞周期,膜联蛋白(Annexin)V-FITCV-FITC/PI双染法测定细胞的凋亡,Western blot测定周期蛋白p21、细胞周期蛋白D1(cyclin D1)和凋亡蛋白Bcl-2相关X蛋白(Bax)、剪切的含半胱氨酸的天冬氨酸蛋白水解酶3(C-caspase-3)的表达变化,克隆形成试验检测放射敏感性。结果与Control组、sh-NC组比较,sh-DcR3组、RAD+sh-NC组、RAD+sh-DcR3组细胞增殖能力下降(0.41±0.05、0.39±0.03 vs.0.26±0.03、0.24±0.01、0.16±0.02),G_(0)/G_(1)期比例升高[(52.32±2.15)%、(51.88±3.62)%vs.(60.21±4.27)%、(64.83±3.51)%、(72.15±4.39)%],细胞凋亡增加[(2.84±0.21)%、(2.95±0.38)%vs.(16.54±1.26)%、(20.51±1.37)%、(29.17±2.86)%],Bax、C-caspase-3、p21表达增多,cyclin D1表达减少(P<0.05)。与sh-DcR3组、RAD+sh-NC组比较,RAD+sh-DcR3组细胞增殖能力下降(0.26±0.03、0.24±0.01 vs.0.16±0.02),G_(0)/G_(1)期比例升高[(60.21±4.27)%、(64.83±3.51)%vs.(72.15±4.39)%],细胞凋亡增加[(16.54±1.26)%、(20.51±1.37)%vs.(29.17±2.86)%],Bax、C-caspase-3、p21表达增多,cyclin D1表达减少(P<0.05)。shRNA慢病毒感染后的宫颈癌细胞放射增敏比为1.496。结论沉默DcR3可通过阻滞细胞周期、诱导细胞凋亡提高宫颈癌Caski细胞的放疗敏感性。

Objective To investigate the effect of silencing decoy receptor 3(DcR3) on radiosensitivity of Caski cells in cervical cancer.Methods Cervical cancer Caski cells are divided into Control group,sh-NC group(infected with shRNA control),sh-DcR3 group(infected with DcR3 shRNA),RAD+sh-NC group(infected with shRNA control,irradiation treatment),RAD+sh-DcR3 group(infected with DcR3 shRNA,irradiation treatment).Methylthiazolyldiphenyl-tetrazolium bromide(MTT) was used to detect cell proliferation.Cell cycle was measured by propidium Iodide(PI) monochrome staining.Apoptosis was detected by Annexin V-FITC/PI double staining method.The expression of cyclin p21,cyclin D1 and apoptotic proteins Bcl-2 associated X protein(Bax)and Cleaved cysteinyl aspartate specific proteinase-3(C-caspase-3) were detected by Western blot.Radiosensitivity was measured by clonogenic assay.Results Compared with the Control and sh-NC groups,the cell proliferation ability of the sh-DcR3,RAD+sh-NC,and RAD+sh-DcR3 groups decreased(0.41±0.05,0.39±0.03 vs.0.26±0.03,0.24±0.01,0.16±0.02),the ratio of G_(0)/G_(1) phase increased [(52.32±2.15)%,(51.88±3.62)% vs.(60.21±4.27)%,(64.83±3.51)%,(72.15±4.39)%],cell apoptosis increased[(2.84±0.21)%,(2.95±0.38)% vs.(16.54±1.26)%,(20.51±1.37)%,(29.17±2.86)%],Bax,C-caspase-3,p21 protein expression increased,the expression of cyclin D1 decreased(P<0.05).Compared with sh-DcR3 and RAD+sh-NC groups,cell proliferation in RAD+sh-DcR3 group decreased(0.26±0.03,0.24±0.01 vs.0.16±0.02),and the ratio of G_(0)/G_(1) phase increased [(60.21±4.27) %,(64.83±3.51)% vs.(72.15±4.39)%],apoptosis increased [(16.54±1.26)%,(20.51±1.37)% vs.(29.17±2.86)%],the expression of Bax,C-caspase-3,p21 protein increased,and the expression of cyclin D1 decreased(P<0.05).The radiosensitization ratio of cervical cancer cells after shRNA lentiviral infection was 1.496.Conclusions Silencing DcR3 can improve the radiosensitivity of cervical cancer Caski cells by blocking the cell cycle and inducing cell apoptosis.