Construction of a Eukaryotic Expression Vector for Overexpression of MIC-1 and Its Effect on Macrophage Phenotype

Overexpression of MIC-1 in Macrophages using a Novel Eukaryotic Expression Vector

Abstract

This study focused on constructing a eukaryotic expression vector, pEGFP-N1-MIC1, designed for the overexpression of the mouse macrophage inhibitory cytokine-1 (MIC-1) gene. The constructed vector was then used to transfect the MFC cell line to achieve MIC-1 overexpression. The impact of increased MIC-1 expression on macrophage phenotype, specifically focusing on cell migration and colony formation, was then evaluated. This research provides valuable insights for future studies and development of MIC-1 targeted therapies in macrophages, particularly for diseases like gastric cancer.

Methods

The MIC-1 gene fragment was obtained by extracting RNA from mouse stomach tissue, followed by reverse transcription into cDNA and PCR amplification. The fragment was then cloned into the pMD18T vector using T-A cloning and confirmed through sequencing. The MIC-1 fragment was then excised using HindIII and EcoRI enzymes and subsequently ligated into the pEGFP-N1 plasmid vector. The resulting pEGFP-N1-MIC1 vector was validated via enzyme digestion and sequencing.

The constructed pEGFP-N1-MIC1 recombinant plasmid was introduced into mouse MFC cells using Lipofectamineﾮ 2000. Successful MIC-1 protein expression was confirmed through Western blot analysis. To assess the functionality of the vector and its impact on macrophage phenotype, transfected MFC cells were subjected to a series of assays, including cell proliferation, cell cycle analysis, wound healing, and colony formation assays.

Results

The successful construction of the pEGFP-N1-MIC1 vector was confirmed through both enzyme digestion and DNA sequencing analyses. Following transfection with the vector, MFC cells exhibited a significant increase in MIC-1 protein expression levels. Further analyses revealed that MFC cell migration and colony formation capabilities were significantly reduced after transfection with pEGFP-N1-MIC1.

Conclusion

This study successfully constructed the pEGFP-N1-MIC1 expression vector, enabling efficient overexpression of the MIC-1 gene in the MFC cell line. The results demonstrated that increased MIC-1 expression negatively impacted the migration and colony formation capabilities of MFC cells. These findings suggest that MIC-1 could be a potential therapeutic target for gastric cancer (GC), and this study paves the way for future research in this area.