[RBM5载体构建、A549细胞稳定转染株的建立及RBM5基因功能的初步研究]

[Construction of RBM5 vector, establishment of stably transfected A549 cell line and preliminary research on the function of RBM5 gene].

作者信息

Xiao Jian, Li Nian, Xing Xiaowei, He Bixiu

机构信息

Department of Geriatrics, Xiangya Hospital, Central South University, Changsha 410008, China.

出版信息

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2014 Oct;39(10):994-1000. doi: 10.11817/j.issn.1672-7347.2014.10.002.

DOI:10.11817/j.issn.1672-7347.2014.10.002

PMID:25355266

Abstract

OBJECTIVE

To establish a stable A549 cell line transfected by RNA binding motif 5 (RBM5) expression vector, and to investigate the effect of RBM5 gene on proliferation of A549 cell line and the expression of DEAH box polypeptide 15 (DHX15).

METHODS

The eukaryotic expression vector pcDNA3.1 (+)/RBM5 was constructed by a twostep PCR technique. Then, the recombinant plasmid pcDNA3.1 (+)/RBM5 was verified by DNA sequencing and transfected into the lung adenocarcinoma cell A549. The positive cells with overexpression of RBM5 gene were identified by Western blotting. Flow cytometry was used to analyze the cell cycles of the positive A549 cells [pcDNA3.1 (+)/RBM5-A549] and the negative controls [pcDNA3 .1 (+)- A549]. Finally, RT-PCR was used to detect the expression of DHX15, a splicing-related factor, in the positively transfected A549 cells and the negative controls.

RESULTS

A pcDNA3.1 (+)/RBM5 eukaryotic expression vector has been constructed successfully, and the A549 cell line that stably transfected with RBM5 gene has been established. Compared with negative control cells, the percentage of G1 phase cells in the positive cells was increased, while the percentage of S phase was decreased (both P<0.01), and the expression of DHX15 is upregulated (P<0.01).

CONCLUSION

RBM5 gene can inhibit the cell cycle and upregulate the expression of DHX15 in A549 cells.

摘要

目的

构建稳定转染RNA结合基序5（RBM5）表达载体的A549细胞系，探讨RBM5基因对A549细胞系增殖及DEAH盒多肽15（DHX15）表达的影响。

方法

采用两步PCR技术构建真核表达载体pcDNA3.1(+)/RBM5。然后，通过DNA测序验证重组质粒pcDNA3.1(+)/RBM5，并将其转染至肺腺癌细胞A549。通过蛋白质免疫印迹法鉴定RBM5基因过表达的阳性细胞。采用流式细胞术分析阳性A549细胞[pcDNA3.1(+)/RBM5-A549]和阴性对照[pcDNA3.1(+)-A549]的细胞周期。最后，采用逆转录-聚合酶链反应（RT-PCR）检测阳性转染A549细胞和阴性对照中剪接相关因子DHX15的表达。