钙网织蛋白-短发夹RNA慢病毒载体构建及其对喉鳞状细胞癌细胞Hep-2中钙网织蛋白表达的影响  

Construction of the calreticulin-short hairpin RNA lentiviral vector and its effect on calreticulin expression in laryngeal squamous cell carcinoma Hep-2 cells

作　　者：高树峰[1] 杨明福[1] 张克辉[1] 游龙贵[1] 王富华[1] 王心涛[1] 刘遗斌[1] 罗冬[1] 蔡云香[1] GAO Shufeng;YANG Mingfu;ZHANG Kehui;YOU Longgui;WANG Fuhua;WANG Xintao;LIU Yibin;LUO Dong;CAI Yunxiang(Department of Otolaryngology,Head and Neck Surgery,Ganzhou People’s Hospital,Ganzhou 341000,Jiangxi,China)

机构地区：[1]赣州市人民医院耳鼻咽喉头颈外科,江西赣州3410000

出　　处：《癌症进展》2020年第24期2509-2512,共4页Oncology Progress

基　　金：江西省卫生计生委科技计划项目(20194058)。

摘　　要：目的构建钙网织蛋白(CRT)-短发夹RNA(shRNA)慢病毒载体,并检测其对喉鳞状细胞癌细胞Hep-2中CRT表达的影响。方法设计CRT特异性靶序列,合成含有靶序列的线性化载体pSIH1-H1-copGFP,通过转染喉鳞状细胞癌细胞Hep-2筛选基因表达效率最高的靶序列。包装生产CRT-shRNA慢病毒并测定滴度,转染喉鳞状细胞癌Hep-2细胞,通过实时荧光定量聚合酶链式反应(PCR)和蛋白质印迹法(Western blot)检测重组慢病毒载体对CRT基因在mRNA和蛋白水平的沉默效果。结果PCR阳性鉴定及测序分析结果显示,插入片段的序列及方向均与预期结果一致。含靶序列CRT siRNA1、CRT siRNA2、CRT siRNA3的载体转染喉鳞状细胞癌细胞Hep-2的表达量均低于空白对照组(P﹤0.05)。含靶序列CRT siRNA1、CRT siRNA2的载体转染喉鳞状细胞癌细胞Hep-2的基因表达效率均低于CRT siRNA3(P﹤0.05)。经测定,慢病毒载体的滴度为3.5×10^7 TU/ml,感染复数(MOI)=30时慢病毒转染至Hep-2细胞的转染率在90%以上。实时PCR结果显示,shRNA组中CRT基因的表达水平低于空白对照组和阴性对照组(P﹤0.05)。Western blot结果显示,与空白对照组和阴性对照组相比,shRNA组喉鳞状细胞癌细胞Hep-2中CRT蛋白的表达水平明显下降。结论成功构建针对CRT的重组慢病毒载体可有效抑制喉鳞状细胞癌细胞Hep-2中CRT的表达,为后续研究CRT基因的功能提供了实验工具。Objective To construct the calreticulin(CRT)-short hairpin RNA(shRNA)lentiviral vector and detect its effect on CRT expression in laryngeal squamous cell carcinoma Hep-2 cells.Method The specific target sequence of CRT was designed,and the linearized vector pSIH1-H1-copGFP containing the target sequence was synthesized.The target sequence with the highest efficiency of gene expression was screened by transfection into laryngeal squamous cell carcinoma Hep-2 cells.The titer of CRT-shRNA lentivirus was determined by packaging production and then transfected into laryngeal squamous cell carcinoma Hep-2 cells.The silencing effect of recombinant lentiviral vector on CRT gene at mRNA level and protein level was detected by real-time fluorescent quantitative polymerase chain reaction(PCR)and Western blot,respectively.Result The results of PCR positive and sequencing showed that the sequence and direction of the insert were consistent with the expectations.The expression levels of the vectors containing the target sequence CRT siRNA1,CRT siRNA2 and CRT siRNA3 transfected into laryngeal squamous cell carcinoma cells Hep-2 were all lower than the blank control group(P<0.05).The gene expression efficiency of vectors containing target sequence CRT siRNA1 and CRT siRNA2 transfected into laryngeal squamous cell carcinoma Hep-2 cells were all lower than CRT siRNA3(P<0.05).The titer of the lentiviral vector was 3.5×10^7 TU/ml after examination,and when the multiplicity of infection(MOI)=30,the infection rate of the lentivirus to Hep-2 cells was over 90%.Real-time PCR results showed that the CRT gene expression level in the shRNA group was lower than that of the blank control group and negative control group(P<0.05).Western blot results showed that compared with the blank control group and the negative control group,the expression level of CRT protein in laryngeal squamous cell carcinoma Hep-2 cells in the shRNA group was significantly decreased.Conclusion The recombinant lentiviral vector targeting CRT was successfully construc

关 键 词：钙网织蛋白 喉鳞状细胞癌 慢病毒载体 HEP-2 

分 类 号：R739.65[医药卫生—肿瘤]