Study On The Expression Profiles,function And Molecular Mechanism Of MAT2B In Human Malignant Melanoma

Object: Melanoma is a kind of malignancy originated from the tissues that can produce pigment.This disease can occur in various of tissues,such as skin,mucous membrane surface,conjunctiva,uveal and other structures.Malignant melanoma,especially for advanced melanoma,is a kind of highly aggressive,highly lethal malignancy.With the improvement understanding of melanoma,molecular targeted therapy has become one of the effective ways of treating malignant melanoma.Although it has a good effect,while the emergence of drug-resistance calls for new,more reliable tumor-associated genes and therapeutic targets.With the development of molecular biology,molecular genetics,gene function,proteomics and second-generation sequencing technology,identification of new tumor-associated virulence genes has become more feasible and efficient.In this study,lentivirus-mediated RNA interference and microarray technology lead us to explore the expression profile,function and molecular mechanism of MAT2 B in human malignant melanoma.Methods: Download and Analyze data from Gene Expression Omnibus,a public functional genomics databases,and screened for differences in gene expression contained in melanoma cell lines or melanoma tissue and normal skin tissue.After a review of literature focus on gene function,MAT2 B had been determined as the candidate gene.We check the expression profile of MAT2 B in 5 melanoma cell lines,primary or metastatic malignant melanoma tissue and control tissue nevis.Based on the MAT2 B gene sequences,sh RNA was designed and transport into melanoma cell line A375 and Mel-RM via lentivirus vector.Constructed melanoma cell lines which have stable low expression of MAT2 B.Then we detected the functional changers such as cell proliferation,colony formation,cell cycle and apoptosis in vitro and tumor growth in nude mice.For further study about the underlying mechanisms,we took a gene expression microarray detection.After bioinformatics analysis,some molecules related to cell death in tumer cell lines have been checked in the level of m RNA and protein.Results: The MAT2 B gene has expression in human melanoma cell lines Mel-CV,Mel-RM,A375,C918 and OCM-1A.It is appropriate for knock-down through RNA interfering.After the analysis of human melanoma tissue samples,the MAT2 B gene has a higher expression profiles in 49 cases of primary malignant melanoma and 38 cases of metastatic malignant melanoma contrast with the contrl nevis group.Stratified analysis showed that the expression of MAT2 B was associatied with the histological grade.However,there is no expression difference between factors,such as gender,age,primary site and metastasis.We designed and synthesized MAT2 B sh RNA interference fragment.Then constructed lentivirus and infected human melanoma cell line A375 and Mel-RM.The expression of MAT2 B in A375 and Mel-RM was significantly down-regulated in both m RNA and protein levels detected by Real-time PCR and Western Blot.Celigo and MTT assay confirmed that the proliferation of cells was inhibited after infected by the lentivirus containning MAT2 B sh RNA interference fragment.The flow cytometry analysis showed a significant increase in apoptosis tatio and a redistribution in cell cycles.In vivo experiment,when the expression of MAT2 B gene was knocked down,Xenograft model showed a significantly augmentation in tumor growth time and has an effect of tumor inhibition.The gene expression microarray detection confirmed that there are 235 gene upregulated and 132 gene downregulated in human melanoma cell line A375 after MAT2 B knocked down.According to the results from the Ingenuity Pathway Analysis,several genes were detected in the m RNA and protein levels by Real-time PCR and Western Blot.XAF1 and IRF1 were upregulated in both m RNA and protein levels,BCL2 were downregulated in both m RNA and protein levels.However EGR1 was upregulated in m RNA levels but not changed in protein levels.Conclusion: Compared with nevus,MAT2 B gene has an augmented expression in primary and metastatic melanoma tissues.After down-regulated by lentivirus-mediated RNA interference in the human melanoma cell line A375 and Mel-RM,the cell proliferation was inhibited,cell cycle was redistributed,apoptosis was increased in vitro and Xenograft was inhibited in vitro.These evidences indicatied that the possible mechanisms are related to the up-regulation of anti-tumor genes XAF1,IRF1 and down-regulation of anti-apoptosic gene BCL2.These results indicated that MAT2 B is critical for melanoma cell proliferation and tumorigenicity.It may become a probable target of anti-melanoma therapy.