4.1B Regulate Proliferation, Migration And Epithelial-Mesench Ymal Transition In Mouse Melanoma Cells

ObjectiveThe incidence of malignant melanoma is one of the fastest growing cancer worldwide. The traditional treatment methods can not achieve the desired effects due to metastasis, therefore, the effective approaches of inhibiting tumor metastatsis may improve the effect for treatment of melanoma. EMT plays a critical role in different aspects of cancer progression, such as metastasis, stem cell traits, and chemoresistance. The exploration and discovery of negative regulators of EMT is critical for the further understanding of tumor metastasis, the improvement of the early prevention and prognosis work. Protein 4.1B, a cytoskeletal protein, have been confirmed to be lost or down-regulated in a variety of malignant and benign tumors and it is a potential tumor suppressor. In the preliminary study, 4.1B expression was lost in B16 and B16-F10 cells. In order to explore the role of the 4.1B protein in melanoma, the expression plasmid vector pEGFP-N1-EPB41L3 was constructed which carrying the EPB41L3 gene sequence and transiently transfected into mouse melanoma cells. The effects of 4.1B on the proliferation and migration of mouse melanoma were assessed in vitro in our work. The effect of 4.1B on B16 melanoma model and B16-F10 pulmonary metastasis model was detected. Also, we initial explored the molecular mechanism of 4.1B in regulating melanoma cell EMT. Methods1.PCR and western blot were used to detecte to the expression of protein 4.1 family members on mRNA and protein levels. EPB41L3 gene expression was further detected by real time PCR.2. The expression plasmid vector pEGFP-N1-EPB41L3 was constructed carrying the EPB41L3 gene sequence and transiently transfected into mouse melanoma cells. Then we observed 4.1B intracellular localization by fluorescence microscopy. The expression of 4.1B was detected by real time PCR and western blot.3.MTT assay was used for detection of cell proliferation and migration in vitro.4.Real time PCR and western blot were used to detecte the E-cadherin, vimentin, MMP-2, MMP-9. The TGF-β, Snail, Slug, Twist 2 mRNA were detected by real time PCR.5.Real time PCR was used for detection the gene expression of integrin β1. Total β1 integrin and active β1 integrin were detected with flow cytometry.6.The effect of 4.1B on B16 melanoma model and B16-F10 pulmonary metastasis model was detected by mouse model. Results1.Protein 4.1 family members 4.1R/G/N were detectable in two kinds of melanoma cells, but 4.1B expression was lost.2. Eukaryotic expression vector pEGFP-N1-EPB41L3 was successfully constructed, green fluorescen were observed by fluorescence microscopy after transfection. 4.1B was detectable in two kinds of melanoma cells.3.Protein 4.1B can inhibit the proliferation and migration of B16 and B16-F10.4.4.1B inhibited EMT by down regulating the expression of Snail, Slug, Twist 2 and effecting E-cadherin, MMP-9, vimentin.5.The protein 4.1B had no effect on expression and activation of integrin β1 on cell surface.6.4.1B had a certain inhibitory effect on B16 melanoma model and B16-F10 pulmonary metastasis model. ConclusionProtein 4.1B can suppress B16 and B16-F10 cell proliferation and migration, and inhibit EMT by down regulating the expression of Snail, Slug, Twist 2 and effecting E-cadherin, MMP-9, vimentin. 4.1B can inhibit EMT and tumor cell metastasis in melanoma cells. 4.1B has partly inhibitory effect on B16 melanoma model and B16-F10 pulmonary metastasis model.