CASC19调控miR-449b-5p表达对子宫颈癌细胞增殖、凋亡和辐射敏感性的影响  

Effect of CASC19 on proliferation,apoptosis and radiation sensitivity of cervical cancer cells by regulating miR-449b-5p expression

作　　者：刘艳杰[1] 郭瑞霞[1] 韩丽萍[1] 顾浩[2] 刘明珠[3] Liu Yanjie;Guo Ruixia;Han Liping;Gu Hao;Liu Mingzhu(Department of Gynecology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Department of Radiology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Department of Traditional Chinese Medicine and Gynecology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院妇科,450052 [2]郑州大学第一附属医院放射科,450052 [3]郑州大学第一附属医院中医妇科,450052

出　　处：《中华妇产科杂志》2020年第1期36-44,共9页Chinese Journal of Obstetrics and Gynecology

摘　　要：目的探讨癌易感性候选基因19(CASC19)调控微小RNA-449b-5p(miR-449b-5p)的表达后对子宫颈癌细胞增殖、凋亡和辐射敏感性的影响。方法(1)培养子宫颈癌细胞系HeLa细胞,予不同剂量(分别为0、2、4、6、8 Gy)的X线照射,实时荧光定量PCR技术检测HeLa细胞中CASC19 mRNA和miR-449b-5p的表达水平。(2)不同条件处理后HeLa细胞增殖、凋亡、辐射敏感性及其相关蛋白表达的检测,其中,不同处理条件包括沉默CASC19表达、过度表达miR-449b-5p、下调miR-449b-5p并沉默CASC19表达,相关蛋白包括细胞周期蛋白D1(cyclin D1)、活化的半胱氨酸天冬氨酸蛋白酶3(cleaved-caspase-3)和组蛋白变异体H2AX(γ-H2AX);采用四甲基偶氮唑蓝(MTT)比色法检测细胞增殖情况,流式细胞仪检测细胞凋亡情况,细胞克隆形成实验检测细胞的辐射敏感性(以存活分数表示)。(3)采用双荧光素酶报告基因实验(结果以荧光素酶活性表示)和实时荧光定量PCR技术验证CASC19和miR-449b-5p之间的靶向调控关系。结果(1)随着X线照射剂量(0、2、4、6、8 Gy)的增加,HeLa细胞中CASC19 mRNA的表达水平逐渐升高(F=502.681,P=0.000),miR-449b-5p的表达水平逐渐降低(F=202.936,P=0.000)。(2)沉默CASC19表达、过度表达miR-449b-5p后,HeLa细胞的存活率显著降低(P<0.05),凋亡率显著升高(P<0.05),存活分数显著降低(P<0.05),cyclin D1蛋白的表达水平显著降低(P<0.05),cleaved-caspase-3和γ-H2AX蛋白的表达水平显著升高(P<0.05);下调miR-449b-5p并沉默CASC19表达后,HeLa细胞的存活率显著升高(P<0.05),凋亡率显著降低(P<0.05),存活分数显著升高(P<0.05),cyclin D1、γ-H2AX蛋白的表达水平显著升高(P<0.05),cleaved-caspase-3的表达水平显著降低(P<0.05)。(3)与对照比较,过度表达miR-449b-5p可显著降低CASC19基因野生型HeLa细胞的荧光素酶活性(分别为1.00±0.09、0.37±0.05,P<0.01),而对CASC19基因突变型HeLa细胞的荧光素酶活性无显著影响(分别为0.92±0Objective To investigate the effects of cancer susceptibility candidate gene 19(CASC19)regulating the expression of microRNA-449b-5p(miR-449b-5p)on the proliferation,apoptosis and radiation sensitivity of cervical cancer cells.Methods(1)HeLa cells of cervical cancer cell line were cultured.HeLa cells were irradiated with X-ray at different doses(0,2,4,6,8 Gy,respectively),then the expression level of CASC19 mRNA and miR-449b-5p were detected by real-time quantitative PCR.(2)HeLa cell proliferation,apoptosis,radiation sensitivity(expressed as a survival fraction)and its related protein expression included cyclin D1,cleaved-caspase-3,and histone variant H2AX(γ-H2AX)were examined after different treatment including silencing CASC19 expression,over-expressing miR-449b-5p,down-regulating miR-449b-5p and silencing CASC19 expression.(3)The dual luciferase reporter gene experiment and real-time quantitative PCR technology were used to verify the targeting relationship between CASC19 and miR-449b-5p.Results(1)With the increase of X-ray irradiation different dose(0,2,4,6,8 Gy),the expression level of CASC19 mRNA in HeLa cells gradually increased(F=502.681,P=0.000),and the expression level of miR-449b-5p gradually decreased(F=202.936,P=0.000).(2)After silencing CASC19 expression or over-expressing miR-449b-5p,the survival rate of HeLa cells was significantly reduced(P<0.05),the apoptosis rate was significantly increased(P<0.05),the survival fraction was significantly reduced(P<0.05),the expression level of cyclin D1 protein was significantly reduced(P<0.05),and the expression levels of cleaved-caspase-3 andγ-H2AX protein were significantly increased(P<0.05).After down-regulating miR-449b-5p and silencing CASC19 expression,the survival rate of HeLa cells was significantly reduced(P<0.05),the apoptosis rate was significantly increased(P<0.05),the survival fraction was significantly reduced(P<0.05),the expression levels of cyclin D1 andγ-H2AX protein were significantly increased(P<0.05),and the expression level of cleaved-c

关 键 词：宫颈肿瘤 RNA 长链非编码 微RNAS 细胞增殖 细胞凋亡 

分 类 号：R737[医药卫生—肿瘤]