[Identification analysis of eukaryotic expression plasmid Rap2a and its effect on the migration of lung cancer cells]
Jinxia Wu1, Miaomiao Sang2, Wenjia Cao2
1Department of Physiology, Xuzhou Medical College, Xuzhou 221002, China;Research Institute of Cancer Prevention and Therapy, Xuzhou Medical College, Xuzhou 221002, China.
Background:
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. The aim of this study is to explore the role of Rap2a in carcinogenesis.
Methods:
The 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. Then, Rap2a gene was amplified by PCR and inserted into pcDNA3.1(+). The reconstructed plasmid was identified 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 zymography.
Results:
Rap2a is significantly upregulated in lung cancer cells. The results of enzyme digestion and sequencing showed that the coding sequence of pcDNA3.1(+)-Rap2a was right and was inserted into the vector correctly. The 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.
Conclusions:
Eukaryotic expression plasmid pcDNA3.1(+)-Rap2a was constructed successfully. Rap2a could be expressed in lung cancer cells efficiently and promotes lung cancer cell migration.
Insights
Rap2a, a small GTPase, is upregulated in lung cancer and promotes cell migration and MMP2 activity. This study constructed an expression vector to investigate Rap2a
Area of Science:
- Molecular Biology
- Oncology
- Cell Biology
Background:
- Rap2a, a member of the small GTPase superfamily, regulates integrin function, cell adhesion, motility, and cell/matrix interactions.
- The role of Rap2a in carcinogenesis remains poorly understood, necessitating further investigation into its function in cancer development.
Purpose of the Study:
- To clone Rap2a cDNA and construct a eukaryotic expression vector for Rap2a.
- To investigate the expression of Rap2a in lung cancer cells.
- To explore the role of Rap2a in lung carcinogenesis and its impact on cell migration.
Main Methods:
- Western blot was used to measure endogenous Rap2a protein levels in lung cancer cells.
- Reverse transcription-polymerase chain reaction (RT-PCR) and PCR were employed to amplify Rap2a cDNA, followed by insertion into the pcDNA3.1(+) vector.
- Transwell assays and gelatin zymography were utilized to assess lung cancer cell migration and matrix metalloproteinase (MMP)2 activity, respectively.
Main Results:
- Rap2a was found to be significantly upregulated in lung cancer cells.
- Successful construction and transfection of the pcDNA3.1(+)-Rap2a plasmid into H1299 and A549 lung cancer cells were confirmed.
- Ectopic expression of Rap2a promoted lung cancer cell migration and increased MMP2 enzyme activity.
Conclusions:
- The eukaryotic expression plasmid pcDNA3.1(+)-Rap2a was successfully constructed and validated.
- Rap2a can be efficiently expressed in lung cancer cells.
- Rap2a expression promotes lung cancer cell migration, suggesting its potential role in cancer progression.
