摘要
目的探讨microRNA-154-5p(miR-154-5p)靶向枯灵素2(Cullin2,CUL2)对人乳头瘤病毒(HPV)16型阳性子宫颈癌细胞的作用。方法收集山西医科大学第二医院妇产科2018年1月至2020年12月单一HPV16阳性的正常子宫颈(对照组)、低级别鳞状上皮内病变(LSIL)、高级别鳞状上皮内病变(HSIL)和子宫颈鳞状细胞癌(CSCC)组织各20份。实时荧光定量PCR(RT-qPCR)和免疫组织化学(IHC)分别检测4组子宫颈组织中miR-154-5p和CUL2蛋白的表达情况。双荧光素酶报告分析明确miR-154-5p与CUL2的靶向调控关系。利用慢病毒载体介导的DNA重组技术构建稳定高、低表达miR-154-5p的SiHa细胞,分为miR-154-5p过表达组(Lv-miR-154-5p-OE组)和miR-154-5p沉默组(Lv-miR-154-5p-Sp组),对照组为转染空慢病毒载体的SiHa细胞(LvControl组)。RT-qPCR验证构建效果后,分别采用蛋白免疫印迹(Western blot)、细胞增殖-毒性检测试剂盒(CCK-8)、划痕和Transwell侵袭实验评估差异表达miR-154-5p对CUL2蛋白表达量及SiHa细胞增殖、迁移和侵袭的影响。结果RT-qPCR结果表明,LSIL、HSIL和CSCC 3组中miR-154-5p表达量均低于对照组(11.89±42.31、0.18±0.26、0.03±0.05 vs.288.97±486.64,P<0.05)。IHC结果显示,相较于对照组,LSIL、HSIL和CSCC 3组中CUL2蛋白的表达强度增强。双荧光素酶报告分析证实CUL2为miR-154-5p的下游调控基因。Lv-miR-154-5p-OE组SiHa细胞较Lv-Control组CUL2蛋白表达量少(0.87±0.01 vs.1.00±0.03,P<0.05)、增殖活力低(7.56±0.51 vs.24.80±1.98,P<0.05)、迁移指数小(0.41±0.07 vs.1.00±0.13,P<0.01)、侵袭能力弱(35.33±3.22 vs.66.00±10.15,P<0.01);而Lv-miR-154-5p-Sp组SiHa细胞较Lv-Control组CUL2蛋白表达量多(1.17±0.04 vs.1.00±0.03,P<0.05)、增殖活力高(31.16±0.70 vs.24.80±1.98,P<0.05)、迁移指数大(1.97±0.13 vs.1.00±0.13,P<0.001)、侵袭能力强(134.67±9.29 vs.66.00±10.15,P<0.01)。结论miR-154-5p通过靶向调控CUL2抑制HPV16阳性子宫颈癌细胞的生长,有望成为子宫颈癌治疗的潜在靶点。
Objective To probe the effect of microRNA-154-5p(miR-154-5p)on HPV16-positive cervical cancer cells by directly targeting Cullin2(CUL2).Methods Twenty normal cervical(control group)tissues,20 low-grade squamous intraepithelial lesions(LSIL)tissues,20 high-grade squamous intraepithelial lesions(HSIL)tissues and 20cervical squamous cell carcinoma(CSCC)tissues were collected from January 2018 to December 2020 in the Second Hospital of Shanxi Medical University.Quantitative realtime polymerase chain reaction(RT-qPCR)and immunohistochemistry were used to detect the expression level of miR-154-5p and CUL2 protein in four groups of cervical tissues,respectively.The targeted regulatory relationship of miR-154-5p with CUL2 was clarified via dual-luciferase reporter assay.SiHa cells with stable up-and down-expression levels of miR-154-5p were established and divided into Lv-miR-154-5p-OE group and Lv-miR-154-5p-Sp group by lentiviral vector-mediated DNA recombination technology,and the control group was SiHa cells transfected with empty lentiviral vecto(rLv-Control group).After validation by RT-qPCR,the effects of miR-154-5p on CUL2 protein expression and cell proliferation,migration and invasion were assessed by Western blot,Cell Counting Kit8(CCK-8),scratch and Transwell invasion assays.Results RT-qPCR showed that miR-154-5p expression was lower in LSIL,HSIL,and CSCC than in control group[(11.89±42.31)(,0.18±0.26)(,0.03±0.05)vs.(288.97±486.64),P<0.05].IHC indicated that CUL2 protein expression intensity was increased in LSIL,HSIL,and CSCC groups compared with control group.Dual-luciferase reporter assay confirmed CUL2 was a downstream regulatory gene of miR-154-5p.SiHa cells in Lv-miR-154-5p-OE group had lower CUL2 protein expression,lower proliferation vitality,smaller migration index and weaker invasion ability compared with Lv-Control group;Lv-miR-154-5p-OE group vs.LvControl group:[(0.87±0.01)vs.(1.00±0.03),P<0.05;(7.56±0.51)vs.(24.80±1.98),P<0.05;(0.41±0.07)vs.(1.00±0.13),P<0.01;(35.33±3.22)vs.(66.00±10.15),P<0.01].SiHa cells in Lv-miR-154-5p-Sp group had higher CUL2 protein expression,higher proliferation vitality,larger migration index and stronger invasion ability than LvControl group;Lv-miR-154-5p-Sp group vs.Lv-Control group:[(1.17±0.04)vs.(1.00±0.03),P<0.05;(31.16±0.70)vs.(24.80±1.98),P<0.05;(1.97±0.13)vs.(1.00±0.13),P<0.001;(134.67±9.29)vs.(66.00±10.15),P<0.01].Conclusion MiR-154-5p exerts a tumor-suppressing effect on the growth of HPV16-positive cervical cancer cells by targeting regulating CUL2,which is expected to be a potential target for the treatmen of cervical cancer.
作者
李亚芹
张宏蕾
卫怡妙
拜莹
李琦
王秀婷
王家璞
王淑玲
温松泉
李嘉荣
赵卫红
LI Ya-qin;ZHANG Hong-lei;WEI Yi-miao;BAI Ying;LI Qi;WANG Xiu-ting;WANG Jia-pu;WANG Shu-ling;WEN Song-quan;LI Jia-rong;ZHAO Wei-hong(不详;Department of Obstetrics and Gynecology,the Second Hospital of Shanxi Medical University,Taiyuan 030001,China)
出处
《中国实用妇科与产科杂志》
CAS
CSCD
北大核心
2022年第11期1120-1124,共5页
Chinese Journal of Practical Gynecology and Obstetrics
基金
国家自然科学基金(81702583)
山西省回国留学人员科研教研资助项目(2022-195)
山西省应用基础研究计划(201901D211506)
中国博士后科学基金(2019M651072)。