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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

26卷

期数:

2020年06期

页码:

505-509

栏目:

基础医学

出版日期:

2020-11-20

文章信息/Info

Title:

Construction of lncRNA(AC073934.6) lentiviral plasmid and its effects on SND1 expression in HEY and HeLa cell lines

作者:

扈利红; 邱宝晨; 辛灵彪; 张纬

天津医科大学基础医学院生物化学与分子生物学系，天津300070

Author(s):

HU Li-hong;  QIU Bao-chen;  XIN Ling-biao;  ZHANG Wei

Department of Medical Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China

关键词:

lncRNA; 质粒构建; 同源重组; SND1

Keywords:

lncRNA;  plasmid construction;  homologous recombination;  SND1

分类号:

Q784

DOI:

-

文献标志码:

A

摘要:

目的：构建长链非编码RNA AC073934.6（lncRNA-AC）的慢病毒质粒，在HEY和HeLa细胞中稳定表达，并观察其对邻近基因SND1表达的影响。方法：提取基因组DNA，扩增出lncRNA-AC外显子1（F1R1）和外显子2（F2R2），化学合成外显子3（F3R3）。利用重叠延伸PCR获得上述3个DNA片段的连接产物（F1R3）；通过同源重组的方法将F1R3和慢病毒载体pLVX-IRES-Puro连接起来，构建pLVX-lncRNA-AC重组质粒。用慢病毒包装的方法获得过表达lncRNA-AC的慢病毒，随后感染HEY和HeLa细胞系，PCR检测lncRNA-AC的转录，通过实时荧光定量PCR和Western印迹检测SND1表达。结果：通过菌液PCR和测序验证了重组质粒pLVX-lncRNA-AC构建成功；利用慢病毒感染细胞后，通过PCR检测过表达lncRNA-AC的稳定株构建成功；在稳定株中通过实时荧光定量PCR检测SND1的mRNA水平，在HEY细胞系中，WT和过表达lncRNA-AC后SND1的mRNA相对表达量分别为1.00±0.14和1.24±0.19，两者比较，差异无统计学意义（t=1.761，P=0.07）；在HeLa细胞系中，两者的表达量分别为1.02±0.09和0.83±0.13，差异无统计学意义（t=2.081，P=0.14），同时Western 印迹检测SND1的蛋白水平无明显差异。结论：在HEY和HeLa细胞系中过表达lncRNA-AC不影响SND1的表达。

Abstract:

Objective: To construct a lentiviral plasmid of long non-coding RNA AC073934.6（lncRNA-AC） stably expressed in HEY and HeLa cell lines, and observe its effects on the expression of SND1. Methods: LncRNA-AC exon 1（F1R1） and exon 2（F2R2） were amplified by using the extracted genomic DNA as template, and exon 3（F3R3） was chemically synthesized. The target product（F1R3） of lncRNA-AC was obtained by overlap extension PCR that ligated the above three DNA fragments. F1R3 and the lentiviral vector pLVX-IRES-Puro were ligated by homologous recombination to construct recombinant pLVX-lncRNA-AC plasmid. Virions, over-expressing lncRNA-AC，was obtained by lentivirus packaging, and subsequently infected with HEY and HeLa cell lines. PCR was used to test the transcription of lncRNA-AC. Real-time fluorescent quantitative PCR and Western blotting were used to detect the expression of SND1. Results: The recombinant plasmid pLVX-lncRNA-AC was successfully constructed by bacterial PCR and sequencing. Lentivirus was used to infect cells to construct a stable overexpressing lncRNA-AC strain which was tested by PCR. The mRNA level of SND1 was detected by real-time fluorescent quantitative PCR in stable strains. In the HEY cell line, the relative expression of SND1 mRNA in WT and overexpression of lncRNA-AC were 1.00±0.14 and 1.24±0.19, there was no statistical difference（t=1.761，P=0.07）. In the HeLa cell line, the expression levels were 1.02±0.09 and 0.83±0.13, the difference was not statistically significant（t=2.081，P=0.14），and there was no significant difference in the protein level of SND1 detected by Western blotting. Conclusion: In HEY and HeLa cell lines, overexpression of lncRNA-AC can not affect the expression of SND1.

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备注/Memo

备注/Memo:

文章编号 1006-8147（2020）06-0505-05
基金项目 国家自然科学基金青年项目（81802588）；天津市自然科学基金青年项目（17JCQNJC12600）；天津市高等 教育委员会科技发展基金一般项目（2016YD18）
作者简介 扈利红（1996-），女，硕士在读，研究方向：SND1在海马体中的作用；通信作者：张纬，E- mail:zhangjingyi98@163.com。

常用功能

更新日期/Last Update: 2020-11-20