MIC1 Inhibits Gastric Cancer Cell Migration, Proliferation, and Promotes Macrophage Polarization

MIC1 Inhibits Gastric Cancer Cell Migration, Proliferation, and Promotes Macrophage Polarization

This study investigated the impact of MIC1 on the behavior of MFC gastric cancer cells. Through a series of experiments, we explored the role of MIC1 in cell migration, proliferation, and its influence on macrophage polarization.

3.1 Vector Construction and MIC1 Protein Expression

The vector construction process for expressing MIC1 is outlined in Figure 1. PCR amplification confirmed the successful amplification of the MIC1 target fragment (Fig. 2B), which was subsequently ligated with the pMD18-T vector, resulting in visible positive colonies after transformation (Fig. 2C). The positive recombinant plasmid, named pMD18-T-MIC1, was further digested with QuickCut HindIII and QuickCut EcoRI, confirming the correct plasmid connection (Fig. 2D). The pEGFP-N1-MIC1 vector was also digested, resulting in bands consistent with the expected target fragment and vector fragments (Fig. 2E). Notably, MIC1 protein expression was observed in the pEGFP-N1-MIC1 group, with a molecular weight of approximately 34 kD, confirming successful expression in MFC cells (Fig. 2F).

3.2 Cell Cycle Analysis

Flow cytometry analysis revealed that MFC cells transfected with the pEGFP-N1-MIC1 plasmid exhibited a significant increase in the proportion of cells arrested in the G0/G1 phase, accompanied by a reduction in cells entering the S phase (Fig. 3 **P<0.01). This suggests that MIC1 overexpression may induce cell cycle arrest, potentially inhibiting cell proliferation.

3.3 Cell Migration Assay

Cell scratch assays were performed to assess the impact of MIC1 on the migration capability of MFC cells. Results demonstrated that overexpression of the MIC1 gene significantly impaired the migration ability of MFC cells (Fig. 4 **P<0.01). Quantitative analysis using TScratch software confirmed that MFC cells with MIC1 gene overexpression exhibited weaker scratch healing ability compared to the control group. This implies that MIC1 negatively regulates the migration of MFC cells, potentially hindering their invasion and metastasis.

3.4 Colony Formation Assay

Colony formation assays showed that the pEGFP-N1-MIC1 transfected group displayed a reduced number of colony-forming cells compared to the control group (Fig. 5 *P<0.05). These findings suggest that MIC1 expression is linked to the inhibition of colony formation by MFC cells, indicating a potential role in suppressing tumor growth.

3.5 MIC-1 Promotes M2 to M1 Polarization of Mouse Macrophages

Treatment of mouse M2 macrophages with varying concentrations of MIC1 resulted in a dose-dependent increase in the proportion of M1 macrophages. This indicates that MIC1 promotes the polarization of M2 macrophages towards an M1 phenotype, suggesting a potential role in promoting anti-tumor immunity.

Overall, these findings demonstrate that MIC1 exhibits anti-tumor effects in MFC gastric cancer cells by inhibiting cell migration and proliferation, and by promoting macrophage polarization towards an M1 phenotype. These results highlight the potential therapeutic implications of MIC1 in the treatment of gastric cancer.