1、 王瑞官 等/核纤层蛋白 B1 通过影响端粒酶活性调控肝癌细胞生长 Chinese Journal of Biotechnology http:/ Apr.25,2023,39(4):1609-1620 DOI:10.13345/j.cjb.220625 2023 Chin J Biotech,All rights reserved 资助项目：解放军总医院第八医学中心课题(2021MS003)This work was supported by the Project of the Eighth Medical Center of Peoples Liberation Army General
2、 Hospital(2021MS003).*Corresponding authors.E-mail:LI Jiangbo,;QIN Lingmei, Received:2022-08-06;Accepted:2022-10-20;Published online:2022-10-24 1609 生物工程学报 核纤层蛋白 B1 通过影响端粒酶活性调控肝癌细胞生长 王瑞官1,2，陈思3，孙志佳4，王诗昆5，王杰6，秦玲玫7*，李江波7*1 中国人民解放军总医院 肝胆胰腺外科医学部，北京 100853 2 中国人民解放军总医院 第八医学中心 肝胆外科，北京 100091 3 湖南省职业病防治院 妇
3、产科，湖南 长沙 410021 4 中国人民解放军空军特色医学中心 放疗科，北京 100142 5 新疆军区信息通信旅，西藏 阿里 859499 6 石河子大学医学院，新疆 石河子 832000 7 军事科学院军事医学研究院 生物工程研究所，北京 100089 王瑞官,陈思,孙志佳,王诗昆,王杰,秦玲玫,李江波.核纤层蛋白 B1 通过影响端粒酶活性调控肝癌细胞生长J.生物工程学报,2023,39(4):1609-1620.WANG Ruiguan,CHEN Si,SUN Zhijia,WANG Shikun,WANG Jie,QIN Lingmei,LI Jiangbo.Lamin B1 re
4、gulates the growth of hepatocellular carcinoma cells by influencing telomerase activityJ.Chinese Journal of Biotechnology,2023,39(4):1609-1620.摘 要：核纤层蛋白 B1(nuclear lamina protein B1,LMNB1)高表达于肝癌组织中，通过敲低LMNB1 探讨其对肝癌细胞增殖的影响及其机制。利用 siRNA 在肝癌细胞中敲低 LMNB1，Western blotting 检测敲低效果，使用端粒重复序列扩增法(telomeric repe
5、at amplification protocol assay,TRAP)检测其端粒酶活性变化。利用实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,qPCR)检测其端粒长度变化。并通过 CCK-8、克隆形成、Transwell、划痕实验检测其生长，侵袭和迁移能力变化。利用慢病毒系统构建稳定敲低 LMNB1 的 HepG2 细胞，检测其端粒长度及端粒酶活性变化，采用 SA-gal 衰老染色检测细胞衰老情况，通过裸鼠皮下成瘤实验及对肿瘤后续的组化染色，SA-gal 衰老染色，端粒荧光原位杂交(fluorescence in s
6、itu hybridization,FISH)检测其对成瘤性的影响。最后利用生物信息分析的方法寻找 LMNB1 在临床肝癌组织中的表达情况，及其与临床分期、病人生存期的关系。HepG2 和 Hep3B 中敲低 LMNB1 后端粒酶活性显著降低，细胞增殖、迁移和侵袭能力显著降低，细胞和裸鼠成瘤实验证明稳定敲低 LMNB1 后端粒酶活性降低的同时端粒长度缩短，细胞发生衰老，此外细胞成瘤性降低，Ki-67 表达降低，生物信息分析结果显示，LMNB1 高表达于肝癌组织，且与肿瘤分期和患者生存相关。LMNB1 在肝癌细胞中过表达，其有望成为评估肝癌患者临床预后的指标和精准治疗的靶点。医药生物技术 ISS
7、N 1000-3061 CN 11-1998/Q 生物工程学报 Chin J Biotech http:/ 1610 关键词：核纤层蛋白 B1；肝癌；端粒酶；端粒；细胞衰老 Lamin B1 regulates the growth of hepatocellular carcinoma cells by influencing telomerase activity WANG Ruiguan1,2,CHEN Si3,SUN Zhijia4,WANG Shikun5,WANG Jie6,QIN Lingmei7*,LI Jiangbo7*1 Faculty of Hepato-Pancreat
8、o-Biliary Surgery,Chinese Peoples Liberation Army General Hospital,Beijing 100853,China 2 Department of Hepatobiliary Surgery,the Eighth Medical Center,Chinese Peoples Liberation Army General Hospital,Beijing 100091,China 3 Department of Obstetrics and Gynecology,Hunan Prevention and Treatment Insti
9、tute for Occupational Diseases,Changsha 410021,Hunan,China 4 Radiotherapy Department,Peoples Liberation Army Air Force Characteristic Medical Center,Beijing 100142,China 5 Physician of Information and Communication Brigade of Xinjiang Military Prefecture,Ngari Prefecture 859499,Tibet,China 6 School
10、of Medicine,Shihezi University,Shihezi 832000,Xinjiang,China 7 Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100089,China Abstract:Lamin B1(LMNB1)is highly expressed in liver cancer tissues,and its influence and mechanism on the proliferation of
11、 hepatocellular carcinoma cells were explored by knocking down the expression of the protein.In liver cancer cells,siRNAs were used to knock down LMNB1.Knockdown effects were detected by Western blotting.Changes in telomerase activity were detected by telomeric repeat amplification protocol assay(TR
12、AP)experiments.Telomere length changes were detected by quantitative real-time polymerase chain reaction(qPCR).CCK8,cloning formation,transwell and wound healing were performed to detect changes in its growth,invasion and migration capabilities.The lentiviral system was used to construct HepG2 cells
13、 that steadily knocked down LMNB1.Then the changes of telomere length and telomerase activity were detected,and the cell aging status was detected by SA-gal senescence staining.The effects of tumorigenesis were detected by nude mouse subcutaneous tumorigenesis experiments,subsequent histification st
14、aining of tumors,SA-gal senescence staining,fluorescence in situ hybridization(FISH)for telomere analysis and other experiments.Finally,the method of biogenesis analysis was used to find the expression of LMNB1 in clinical liver cancer tissues,and its relationship with clinical stages and patient su
15、rvival.Knockdown of LMNB1 in HepG2 and Hep3B cells significantly reduced telomerase activity,cell proliferation,migration and invasion abilities.Experiments in cells and tumor formation in nude mice had demonstrated that stable knockdown of LMNB1 reduced telomerase activity,shortened telomere length
16、,senesced cells,reduced cell tumorigenicity and KI-67 expression.Bioinformatics analysis showed that LMNB1 was highly expressed in liver cancer tissues and correlated with tumor stage and patient survival.In conclusion,LMNB1 is overexpressed in 王瑞官 等/核纤层蛋白 B1 通过影响端粒酶活性调控肝癌细胞生长 ：010-64807509： 1611 liver cancer cells,and it is expected to become an indicator for evaluating the clinical prognosis of liver cancer patients and a target for precise treatment.Keywords:nuclear lamina protein B1(LMNB1);h
