Construction and Functional Characterization of pEGFP-N1-MIC-1 Expression Vector for Overexpression of Mouse Macrophage Inhibitory Cytokine-1 in MCF Cells

Construction and Functional Characterization of pEGFP-N1-MIC-1 Expression Vector for Overexpression of Mouse Macrophage Inhibitory Cytokine-1 in MCF Cells

Abstract

This study aimed to construct a eukaryotic expression vector, termed pEGFP-N1-MIC-1, for the overexpression of the mouse macrophage inhibitory cytokine-1 (MIC-1) gene. The study also aimed to perform transfection of the MCF cell line to observe the upregulation of MIC-1 gene expression and evaluate its impact on macrophage phenotype conversion. The results of this study may contribute to future research and the development of therapeutic interventions targeting MIC-1 in macrophages.

Methods

We extracted RNA from mouse stomach tissue, conducted reverse transcription to obtain cDNA, and performed PCR amplification of the MIC1 gene fragment. The fragment was then ligated into the pMD18T vector through T-A cloning and confirmed by sequencing. Following this, we excised the fragment with HindIII and EcoRI enzymes, ligated it into the pEGFP-N1 plasmid vector, and analyzed the product through enzyme digestion and sequencing. We transfected the resulting pEGFP-N1-MIC1 recombinant plasmid into mouse MCF cells using Lipofectamineﾮ 2000 and verified MIC1 protein expression through Western blot analysis. In order to validate the functionality of the carrier, MCF cells were transfected with the vector and subjected to various assays including cell proliferation, cell cycle analysis, wound healing, and colony formation assay.

Results

Enzyme digestion and DNA sequencing verified the successful construction of the pEGFP-N1-MIC1 vector. After transfection, the MCF cells demonstrated a significant increase in MIC1 protein expression levels.

Conclusion

Our study successfully constructed the pEGFP-N1-MIC1 expression vector for the MIC-1 gene, which efficiently upregulated MIC-1 expression in the MCF cell line. The migration and colony formation capabilities of MFC cells were reduced after transfection with pEGFP-N1-MIC1. These findings may contribute to the development of therapeutic treatments targeting MIC-1 in gastric cancer in future research.