Novel Long non-coding RNA MAGEA6-DT1 Promotes Expression of the Melanoma Antigen Family 6 by Demethylating its Enhancer

Abstract

Abstract Background The occurrence and progression of various solid tumors are associated with the melanoma-associated antigen A (MAGE-A) family. Although it was demonstrated that demethylation at the promoter region usually causes the over-expression of the MAGE-A family, there has been very few research about the detailed mechanisms of how the genetic modification of promoter region promotes MAGE-A expression.Methods A new non-coding RNA (ncRNA) with the ability of binding with melanoma-associated antigen-A6 (MAGE-A6) promoter region was discovered. The expression consistency between MAGE-A6 and this novel ncRNA in different MAGE-A6 highly expressed malignant cell lines was analyzed by RT-qPCR. The full length of this ncRNA was acquired through RACE and were subsequently named as MAGEA6-DT1. Then up- and down-regulation of MAGEA6-DT1 in human malignant melanoma cells were achieved by lentivirus transduction and siRNA transfection respectively and the transcription and expression of MAGE-A6 was detected by RT-qPCR and Western Blot for verifying MAGE-A6 expression regulating function of MAGEA6-DT1. The exact binding site of MAGEA6-DT1 in MAGE-A6 promoter region was analyzed by dual-luciferase reporter system assay after MAGEA6-DT1 transfection in 293T cells. Moreover, by DNA methylation analysis, we tested whether MAGEA6-DT1 has the ability of MAGE-A6 expression regulation by manipulating its promoter region’s methylation. Finally, RNA pull-down assay was performed to identify the functional binding partner of MAGEA6-DT1.Results MAGEA6-DT1 was identified as a long non-coding RNA (lncRNA) with the length of 771 nucleotides and was abnormally expressed in consistency with MAGE-A6 among various cancer cell lines. Manipulation of MAGEA6-DT1 expression level would positively regulates MAGE-A6 expression. Specific binding site of MAGEA6-DT1 located near the enhancer of MAGE-A6, and its function was revealed to demethylate DNA near its binding site, probably with the assistance of relevant binding partners.Conclusion MAGEA6-DT1, as a lncRNA abnormally expressed in different malignant cell lines, could positively regulate MAGE-A6 expression via specifically combining with and subsequently demethylating MAGE-A6 enhancer. This function may be assisted by some of its binding protein such as DNA (cytosine-5)-methyltransferase 1 (DNMT1).