LINK-A RNAi慢病毒载体构建及其抑制U251胶质瘤细胞增殖的研究  

Construction of LINK-A RNAi lentivirus vector and its inhibition of proliferation of U251 glioma cells

作　　者：王军成[1] 兰彦平 赵岳阳 蒯涛[1] 马东明[1] 李敏 WANG Juncheng;LAN Yanping;ZHAO Yueyang;KUAI Tao;MA Dongming;LI Min(People’s Hospital of Ningxia Hui Autonomous Region,Yinchuan 750002,China)

机构地区：[1]宁夏回族自治区人民医院,宁夏银川750002

出　　处：《宁夏医学杂志》2023年第7期577-580,F0002,共5页Ningxia Medical Journal

基　　金：宁夏自然科学基金(2021AAC03313);宁夏医科大学校级科研项目(XM2020087);宁夏回族自治区人民医院国家自然科学基金预实验项目(2021GZRYSY017);2021年第六批宁夏青年科技人才托举工程项目(202130)。

摘　　要：目的构建lncRNA LINK-A的特异性RNAi慢病毒载体,并借此敲减人U251胶质瘤细胞株内的LINK-A,探究LINK-A对U251胶质瘤细胞增殖的影响。方法针对人LINK-A设计siRNA序列,构建RNAi慢病毒载体质粒并进行测序鉴定;利用pHelper 1.0、pHelper 2.0以及shRNAi 3种质粒分别转染293T细胞,筛选分离慢病毒并测定滴度;用PBS及构建好的慢病毒感染人U251胶质瘤细胞,实验分3组:空白组(PBS)、NC组(空载病毒)、LINK-A(-)组(LINK-A RNAi慢病毒),72 h后利用RT-PCR检测细胞中LINK-A的表达情况;取病毒感染成功后的U251胶质瘤细胞行平板克隆实验培养10 d,行CCK-8实验分别于24 h、48 h检测。结果RNAi慢病毒载体质粒测序结果与RNAi设计序列一致,提示LINK-A RNAi慢病毒载体构建成功,滴度为3×108 TU/mL;病毒感染U251胶质瘤细胞72h后,细胞生长状态良好,荧光持续稳定表达,LINK-A RNA相对表达量明显降低(P<0.05),表明病毒成功感染U251细胞;10 d后LINK-A(-)组细胞克隆形成数及形成率较空白组及NC组明显减少(P<0.05);与空白组及NC组相比,24 h后LINK-A(-)组OD值减少(P<0.05),72 h明显减少(P<0.05)。结论人LINK-A RNAi慢病毒构建成功;LINK-A表达降低可抑制U251胶质瘤细胞增殖。Objective To explore the effects of LINK-A on the proliferation of U251 glioma cells by constructing a specific RNAi lentivirus vector used to knock down the expression level of LINK-A in human U251 glioma cell line.Methods The siRNA sequence was designed for human LINK-A,and the RNAi lentivirus vector plasmid was constructed and sequenced;Three plasmids(pHelper 1.0,pHelper 2.0 and shRNAi)were used to transfect 293T cells,screen and isolate lentivirus and determine the titer.Human U251 glioma cells were infected with PBS and constructed lentivirus.The experiment was divided into blank group(PBS),NC group(empty virus)and LINK-A(-)group(LINK-A RNAi lentivirus).The expression of LINK-A in the cells was detected by RT-PCR after 72 hours;U251 glioma cells after successful infected were cultured in plate cloning experiment for 10 days,and detected in CCK-8 experiment for 24 hours and 48 hours,respectively.Results The sequencing results of RNAi lentivirus vector were consistent with the RNAi design sequence,suggesting that LINK-A RNAi lentivirus vector was successfully constructed with a titer of 3×108 TU/mL.The U251 cells grew well with the green fluorescence continued express stably after infected 72h,and the relative expression of LINK-A RNA decreased significantly(P<0.05),which indicated that the virus successfully infected U251 cells;The number and rate of cell clone formation in LINK-A(-)group were significantly lower than those in blank group and NC group(P<0.05)10 days later.Compared with the blank group and NC group,the OD value of lINK-A(-)group decreased after 24 hours(P<0.05)and significantly decreased after 72 hours(P<0.05).Conclusion Human LINK-A RNAi lentivirus is successfully constructed and the decreased expression of LINK-A can inhibits the proliferation of U251 glioma cells.

关 键 词：胶质瘤 增殖 LINK-A RNAI 

分 类 号：R749.94[医药卫生—神经病学与精神病学]