Melanoma MAGE-A1 Gene And EMT Relative MiRNA Function Research

Research backgroundMAGE-A1 gene (Melanoma Associated Antigen Family A1) belongs to the cancer-germline gene or cancer-testis antigen, which is locate in human X-chromosome q28 domain. In recent years, many researchs indicated that MAGE-A1 gene is high expression in various tumours include melanoma. Our revious research revealed that MAGE-A1 gene expression increased gradually, from the pigment nevus, and in-situ melanoma to metastatic melanoma.miRNA is endogenous small RNA in eukaryotic organisms, length of about 22bp, miRNA combines with target genes to form dimers, Cause the degradation of target genes or inhibit its translation, to achieve the purpose of regulating gene expression. In recent years, the study found that in the process of melanoma EMT, many important regulate genes could be regulated by multiple miRNAs. This study combining mRNA transcription with miRNA group data, analysis of miRNAs have an important regulation role to the melanoma EMT process.Objective1. We try to discover that in the process of melanoma deterioration and metastasis, the molecular control mechanism has change with MAGE-A1 gene alteration.2. We want to screen for miRNA has an important regulation role in melanoma EMT process, verify its function of cell biology and participate in the regulation of signal network.Methods1、MAGE-A1 gene over-expression and knockdown have effect on melanoma cell function and its mechanism analysis(1). qRT-PCR detect MAGE-A1 gene expression in cell lines and melanoma patient samples. (2). In A375 and A2058 cells construction MAGE-A1 gene over-expression and knockdown cell lines. (3). Application of clone formation, cell proliferation and cell cycle experiment observed MAGE-A1 gene over-expression and knocked have effect on melanoma cell proliferation ability. (4). Using Transwell Chambers experiment observed that over-expression and knockdown MAGE-Al gene have effect on invasion and migration ability in melanoma cell. (5). By RNA-sequence method study A375 cells MAGE-A1 gene over-expression and knockdown have impact on cell gene expression, and using the GO and IPA software to the analysis its impact of cell function and the corresponding control network.2、The miRNA regulation of melanoma molecular network and its regulation mechanism in melanoma EMT process.(1). Combining mRNA transcription and miRNAs group data, the application of bioinformatics methods search for miRNAs have a regulatory role of melanoma EMT process. (2). qRT-PCR method detect actual miRNAs expression in cell lines. (3). By transwell Chambers experimental, we observe if that over-expression of miRNAs affect melanoma migration ability in A375 cells. (4). Using the software of IPA analysis miRNA target genes involved in regulating cell function and network in the process of melanoma EMT.(5). qRT-PCR analysis over expression of miRNA has influence on EMT related key gene expression in cell lines.Results1、MAGE-Al gene over-expression and knockdown have effect on melanoma cell function and its mechanism analysis1. Compared pigmented nevus, with melanoma cell and tissue samples from patients, the MAGE-Al gene expression level is difference between the two groups (P<0.05).2. In melanoma A2058 and A375 cell lines, to over-express the MAGE-Al gene can obviously promote the melanoma clone formation and cell proliferation capacity, and knockdown MAGE-Al gene lead to decline, determination of the cell cycle in accordance with the above results (P<0.05).3. Over-expression of MAGE-Al gene in melanoma, the melanoma cell invasion and migration ability have enhance, and knockdown would lead to the lower (P>0.05).4. RNA-seq analysis found that the MAGE-A1 genes influence the EMT process of melanoma by regulation MAPK-ERK, PI3K-AKT, NF-κB, Hedgehog and Wnt signal pathway.2 、 The miRNA regulation of melanoma molecular network and its regulation mechanism in melanoma EMT process.(1). Mirbase website estimate the 11 miRNAs and 48 corresponding target genes. (2).In the three cell lines, tested qRT-PCR results declare that seven miRNAs have results are consistent with prediction. (3). Melanoma migration experiment found that except miR-195-5p, the other six miRNAs over-expression in melanoma has stimulative effect on the ability to melanoma migration. (4). Over-expression of miRNAs in 7 lines, verifies that mir-195-5p, mir-130a-3p, mir-509-5p and mir-509-3-5p have important effects on melanoma EMT process, by detection the mRNA level of EMT related important genes. (5). Through 11 miRNAs corresponding target gene construct molecular regulation pathways, found 11 miRNAs involved in several tumor EMT related the regulation pathwaysConclusion1. MAGE-A1 gene through MAPK-ERK, PI3K-AKT, NF-κB, Hedgehog and Wnt signaling pathways, involved in the EMT process of melanoma, and has important affect on clone formation, cell proliferation and migration ability of melanoma cells.2. Bioinformatics research found that 11 of miRNAs through promote or inhibit Wnt, VEGF, NF-κB, PTEN, PI3K-AKT pathway, and TP53, c-MYC and MMP9 genes, influence on the EMT process of melanoma. Through the transwell cell experiment in vitro, we have confirmed 6 of miRNAs have affect on melanoma cell migration ability and this change has statistically difference. qRT-PCR analysis get 4 miRNAs have important effect on melanoma gene expression in the process of EMT.