Effects On Matrix Metalloproteinases MMP2 And MMP9 In Melanoma Cells By The Transfection With T-cadherin

Objective:Malignant melanoma(MM)is a malignant tumor,which originates from melanocytes.The onset age of the patients were more than 30 years old.Its occurrence may be associated with race genetic,trauma stimulation,viral infection,sun exposure and immunization.The malignant degree of melanoma is very high,and the blood and lymph metastasis can occur at the early stage of the tumor.Once the metastasis occurs,the possibility of cure is greatly reduced.Bone and liver are the preferred organ for metastasis of metastatic melanoma,which has great influence on the quality of life,the success rate of treatment and the total survival rate.How to improve the cure rate and prolong the life of the patients is still the focus of the study of melanoma.T-cadherin also known as H-cadherin and Cadherin 13(CDH13),is different from other cadherin members in its molecular structure and biological function.It is now believed that the T-cadherin mainly affects the biological behavior of the cell through its signal characteristics.With the deep study of the relationship between T-cadherin and tumor,the relationship between T-cadherin and tumor is constantly being understood.In recent years,a large number of studies have shown that T-cadherin is closely related to the biological behavior such as proliferation,invasion and metastasis of malignant tumor.The expression of T-cadherin in malignant tumors is usually down regulated,such as lung cancer,ovarian cancer,bladder cancer,cervical cancer and prostate cancer,but it is upregulated in primary hepatocellular carcinoma and osteosarcoma.Matrix metalloproteinase family(MMPs)is a highly homologous protease system with sequence and structure.It plays an important role in the degradation process of basement membrane and extracellular matrix.MMPs,whose active center contains zinc ions,was mainly produced by vascular endothelial cells and tumor cells.MMPs was secreted into the extracellular matrix in the form of zymogen,and degraded by other MMPs or extracellular fibrinolytic systems in the matrix.The MMPs protein family plays an important role in regulating the formation of neovascularization and promoting the invasion and migration of tumor cells.Our previous studies have shown that the ability of invasion and immigration of melanoma cells B16F10 were obviously reduced after the T-cadherin gene transfected into melanoma cells B16F10.But the specific mechanism has not been studied.In this study,we took mouse melanoma cell B16F10 as the research object,and transfected the T-cadherin gene into B16F10 cells by liposome transfection,and detected the effect of T-cadherin on the expression of matrix metalloproteinase MMP2 and MMP9 in B16F10 cells.Methods:1.Cell source: the melanoma cell B16F10 used in this study is a malignant melanoma cell line derived from highly metastatic C57BL/6 mice.2.Experimental grouping and stable transfection: the experiments were divided into 3 groups,which were untransfected group,empty plasmid transfected group and T-cadherin transfected group.The B16F10 cells were seeded on 6 wells plate.When the cell fusion degree was about 90%,liposomes Lipotectamine2000,Opti-MEM and empty plasmid and T-cadherin vector plasmid were transfected.G418 was screened and stable transfected cell lines were obtained by limited dilution method.3.Real-time quantitative PCR(qRT-PCR)experiment was used to detected the mRNA level of T-cadherin,MMP2 and MMP9 of untransfected group,empty plasmid transfected group and T-cadherin transfected group.4.Protein immunoblotting Technology(Western blot)experiment was used to detected the level of transfection relative protein,MMP2 and MMP9 protein of untransfected group,empty plasmid transfected group and T-cadherin transfected group.5.Statistical software SPSS19.0 was used for data analysis,data was described by x ±s,and one-way ANOVA was used for statistical analysis.SNK-q test was used for pairwise comparisons between roups,and P < 0.05 was statistically significant.Results:1.The changes of mRNA and protein expression level of T-cadherin after T-cadherin gene was transfected into B16F10 cells.They were detected by qRT-PCR and Westeren-blot methods.1.1 qRT-PCR method was used to detect the mRNA expression of T-cadherin in untransfected group,empty plasmid transfected group and T-cadherin transfected group.The mRNA expression levels of T-cadherin were: 1.00±0.00,1.01±0.11,5.13±0.18.The expression levels of T-cadherin mRNA in the T-cadherin transfected group was significantly increased compared with the untransfected group and empty plasmid transfected group.There were significant differences among the three groups(P<0.001).1.2 Westeren-blot method was used to detect the protein expression of transfection relatived protein in untransfected group,empty plasmid transfected group and T-cadherin transfected.In the three group,the expression levels of T-cadherin protein were: 0.10±0.02,0.85±0.11,0.87±0.06.There were significant differences among the three groups(P<0.001).2.qRT-PCR method was used to detect the expression of mRNA of MMP2 and MMP9 in the untransfected group,empty plasmid transfected group and T-cadherin transfected group.2.1 qRT-PCR method was used to detect the mRNA expression levels of MMP2 in untransfected group,empty plasmitransfected d group and transfected T-cadherin group.The gene expression level of MMP2 were: 1.00±0.00,0.99±0.03 and 0.40±0.02.The expression level of mRNA in T-cadherin transfected group was significantly lower than that in the untransfected group and the empty plasmid transfected group,and the difference among the three groups was statistically significant(P<0.001).2.2 qRT-PCR method was used to detect the mRNA expression levels of MMP2 in untransfected group,empty plasmid transfected group and T-cadherin transfected group.The expression level of MMP9 gene in the groups were: 1.00±0.00,1.01±0.07,0.99±0.03.The difference among the three groups was not statistically significant(P>0.05).3.Westeren-blot method was used to detect the protein expression of MMP2 and MMP9 in untransfected group,empty plasmid transfected group and T-cadherin transfected group.3.1 Westeren-blot method was used to detect the protein expression of MMP2 in untransfected group,empty plasmid transfected group and T-cadherin transfected group.The protein expression level of MMP2 were 0.52±0.07,0.48±0.05 and 0.27±0.05.The expression level of MMP2 protein in T-cadherin transfected group was significantly lower than that the untransfected group and the transfected empty plasmid group,and the difference among the three groups was statistically significant(P<0.001).3.2 Westeren-blot method was used to detect the protein expression of MMP9 in untransfected group,empty plasmid transfected group and T-cadherin transfected group.The protein expression levels of MMP9 in the three groups were: 0.37±0.10,0.40±0.02,0.41±0.10.The difference among the three groups was not statistically significant(P>0.05).Conclusion:The expression of MMP2 was downregulated after transfection of T-cadherin gene to malignant melanoma cells.T-cadherin may have no effect on the expression of MMP9.