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[Effect of RKIP on proliferation and migration of malignant melanoma cells and potential mechanism]
1Department of Plastic and Burn Surgery, West China Hospital of Sichuan University, Chengdu 610041, China.
Abstract:
Objective: To explore the effect and mechanism of Rafkinase inhibitor protein (RKIP) on proliferation and migration of malignant melanoma cells in vitro. Methods: The RKIP overexpression and down-regulated stable transfected strains of mouse malignant melanoma cell line B16 were constructed by recombinant lentiviral transfection technique and established as RKIP overexpression group and RKIP down-regulation group, the mouse malignant melanoma B16 cells without any treatment were used as a blank control group, and the proliferation activity and migration ability of each group were detected by cell counting kit-8 (CCK-8) and cell scratch test. The relative expression levels of CyclinD1, Calcium-dependent cell adhesin, Ki-67, Matrix metalloproteinase (MMP)-9, MMP-13, MMP-2 and Phosphatidylethanolamine binding protein (PEBP-1) were detected by quantitative real-time polymerase chain reaction (qPCR). Western blot was used to detect the difference of RKIP expression and the protein expression level of nuclear factor-kappa B (NF-κB) signaling pathway in each group. Results: Comparison of RKIP overexpression group and blank control group shown cell proliferation and migration were significantly inhibited in RKIP overexpression group (0.794±0.038 vs 1.200±0.081) (P<0.001). However, there was no significant difference in cell proliferation between RKIP down-regulation group and blank control group (1.077±0.084 vs 1.200±0.081) (P>0.05), and the cell migration ability of RKIP down-regulation group was significantly higher than that of the blank control group (P<0.001). In addition, there was no significant difference between the RKIP down-regulation group and the blank control group in PEBP-1 expression (P>0.05), while the expression levels of the remaining genes in the RKIP overexpression group were significantly lower than those in the blank control group, and the expression levels in the RKIP down-regulation group were significantly higher than those in the blank control group (P<0.001). Furthermore, the protein level of phosphorylated P65 (p-P65) in RKIP overexpression group was significantly lower than that in blank control group (0.080±0.000 vs 0.236±0.000), and RKIP down-regulation group was significantly higher than that in blank control group (1.139±0.001 vs 0.236±0.000) (both P<0.001). Conclusion: RKIP overexpression can inhibit the proliferation and migration of malignant melanoma cells, which may be related to the regulation of NF-κB signaling pathway-related protein p-P65.
Insights
Rafkinase inhibitor protein (RKIP) overexpression significantly inhibits malignant melanoma cell proliferation and migration. This effect is linked to the regulation of the nuclear factor-kappa B (NF-κB) signaling pathway, specifically the p-P65 protein.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Malignant melanoma is an aggressive skin cancer with high mortality rates.
- Understanding the molecular mechanisms regulating melanoma cell proliferation and migration is crucial for developing effective therapies.
Purpose of the Study:
- To investigate the role of Rafkinase inhibitor protein (RKIP) in controlling the proliferation and migration of malignant melanoma cells in vitro.
- To elucidate the underlying molecular mechanisms, particularly the involvement of the NF-κB signaling pathway.
Main Methods:
- Stable RKIP-overexpressing and down-regulated B16 mouse melanoma cell lines were established using lentiviral transfection.
- Cell proliferation was assessed using the CCK-8 assay, and migration was evaluated by the cell scratch test.
- Gene expression levels (CyclinD1, E-cadherin, Ki-67, MMP-9, MMP-13, MMP-2, PEBP-1) were quantified by qPCR.
- Protein expression of RKIP and the NF-κB pathway (p-P65) was analyzed via Western blot.
Main Results:
- RKIP overexpression significantly inhibited melanoma cell proliferation and migration compared to control cells (P<0.001).
- Down-regulation of RKIP did not affect proliferation but significantly increased cell migration (P<0.001).
- RKIP overexpression decreased p-P65 levels, while RKIP down-regulation increased p-P65 levels, indicating NF-κB pathway modulation.
Conclusions:
- RKIP overexpression acts as an inhibitor of malignant melanoma cell proliferation and migration.
- The inhibitory effects of RKIP are potentially mediated through the modulation of the NF-κB signaling pathway, specifically targeting p-P65.
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