Identiifcation Analysis of Eukaryotic Expression Plasmid Rap2a and Its Effect on the Migration of Lung Cancer Cells
10.3779/j.issn.1009-3419.2014.09.01
- VernacularTitle:Rap2a真核表达质粒的鉴定及其对肺癌细胞迁移能力的影响
- Author:
WU JINXIA
1
,
2
;
SANG MIAOMIAO
;
CAO WENJIA
;
ZHENG JUNNIAN
;
PEI DONGSHENG
Author Information
1. 221002 徐州，徐州医学院生理学教研室
2. 221002徐州，徐州医学院肿瘤生物治疗实验室
- Keywords:
Rap2a;
Gene cloning;
Migration;
A549;
H1299
- From:
Chinese Journal of Lung Cancer
2014;(9):643-648
- CountryChina
- Language:Chinese
-
Abstract:
Background and objective Rap2a, a member of the small GTPase superfamily, plays a critical role in regulating the function of integrin and cell adhesion, thereby controlling cell motility and cell/matrix interactions. However, the function of Rap2a in carcinogenesis is still poorly understood. To clone Rap2a cDNA, which belongs to human Ras-related small G protein superfamily, we constructed its eukaryotic expression vector and determined its expression in lung cancer cells. hTe aim of this study is to explore the role of Rap2a in carcinogenesis. Methods hTe levels of endogenous Rap2a protein in lung cancer cells were measured by Western blot. Total RNA of human osteosarcoma cells U2OS was extracted and reverse-transcribed into cDNA by RT-PCR. hTen, Rap2a gene was ampliifed by PCR and inserted into pcDNA3.1(+). hTe re-constructed plasmid was identiifed by restricted enzyme digestion and sequencing. pcDNA3.1(+)-Rap2a was transfected into H1299 and A549 cells, the expression of Rap2a was detected by Western blot. In addition, the migratory abilities of lung cancer cells were evaluated by Transwell assay. Matrix metalloproteinase (MMP)2 enzyme activity was evaluated by gelatin zymogra-phy. Results Rap2a is signiifcantly upregulated in lung cancer cells. hTe results of enzyme digestion and sequencing showed that the coding sequence of pcDNA3.1(+)-Rap2a was right and was inserted into the vector correctly. hTe results of Western blot showed that H1299 and A549 cells were transfected successfully. Transwell assay indicated that the ectopic expression of Rap2a promotes lung cancer cells migration. Correspondly, enzyme activity of MMP2 also increased. Conclusion Eukaryotic expression plasmid pcDNA3.1(+)-Rap2a was constructed successfully. Rap2a could be expressed in lung cancer cells effciently and promotes lung cancer cell migration.
