The Molecular Mechanism Of VD₃/VDR Regulating Nsun2 Expression And Affecting Melanoma Cells Proliferation And Migration

Melanoma is a malignant tumor with high metastasis and aggressiveness.The main feature is rapid progression and invasion to other tissues and organs.Once diagnosed,it is mostly at a late stage.After the skin is irradiated with ultraviolet rays,7-dehydrocholesterol in epithelial cells is converted into cholesterol,which is then transported to the liver through the vitamin D binding protein in the serum,and it could be hydroxylated to form 25（OH）D₃,then combined withαreceptors and transported to the kidneys.It is catalyzed by 1,αhydroxylase to form the biologically active vitamin D₃（VD₃）--1,25（OH）₂D₃.Active VD₃ is a fat-soluble steroid hormone that specifically binds to the vitamin D Receptor（VDR）to form a VD₃/VDR complex that mediates target gene transcription,regulating cell proliferation,differentiation,and apoptosis and other life activities.However,the potential molecular pathway of VD₃ on melanoma occurrence and development has not yet be revealed.In recent years,a large number of preclinical studies and epidemiological studies have shown that serum vitamin D levels in patients with melanoma are negatively correlated with tumor malignancy and prognostic survival,and VDR gene polymorphisms are also correlated with the incidence of melanoma.Sun domain family protease 2（NOP2/Sun RNA methyltransferase 2,NSUN2）is a typical RNA methyltransferase,which not only participates in cell proliferation,cell migration and other life processes,but also high-level expression of Nsun2 is associated with a variety of tumor malignancies.Nsun2 is positively correlated with cancer cell migration,and negatively correlated with patient prognosis.Our previous study found that active VD₃ significantly inhibited the proliferation of melanoma cells and reduced the expression level of Nsun2 gene.Based on this,we speculate that active VD₃ and VDR form a transcription complex to regulate the expression of Nsun2 gene,thereby inhibiting the proliferation of melanoma cells.In order to verify the above hypothesis,this topic has carried out research on the mechanism of active VD₃ inhibiting the proliferation and migration of melanoma cells.First,we compare the effect of active VD₃at different concentrations and duration of action on the proliferation efficiency of melanoma cells;Analyze the Nusn2 gene expression level after active VD₃treatment of melanoma cells;Use the CRISPR nucleic acid system to target the Vdr gene of melanoma cells to detect Vdr Knock out the cell line proliferation efficiency;Then,we constructed a melanoma cell line that interfered with the Nsun2 gene expression stably and efficiently,and tested its proliferation and migration ability;Using RNA-seq technology to detect the genomic expression level of the interfering Nsun2cell,searching f differentially expressed genes,and analyze the GO and KEGG of differential genes;finally,we use online software to predict the VDRE of the promoter region of the Nsun2 gene,construct a dual luciferase reporter vector with different promoter segments,and analyze the potential binding sites of active VD₃/VDR regulated target genes point.The main experimental results obtained in this study are following:1.After treating melanoma cells with different concentrations of active VD₃for different times,it was found that 100 n M active VD3 treated melanoma cells at 72 h to exert the best inhibitory cell proliferation and migration efficiency;then it was found that after active VD₃treated melanoma cells,NSUN2 protein expression level decrease;The proliferation efficiency of B16 cells is significantly improved after Vdr gene knockout.2.The efficiency of B16 cell proliferation and migration was significantly inhibited after stable interference with Nsun2;RNA-seq results showed that differentially expressed genes were mainly enriched in related pathways such as cell proliferation,signal transduction,and public opinion regulation.It was further found that B16 cells were inhibited after stable interference with Nsun2.The expression of cell proliferation gene cyclin inhibitor p21 is up-regulated,and the expression of cell proliferation gene extracellular regμLated protein kinase ERK（Extracellular regulated protein kinases,ERK）is down-regulated.3.Using the online website to predict the presence of two VDREs in the promoter region of the Nsun2 gene,and then construct a dual luciferase reporter system to further verify that the VD₃/VDR complex binds to VDRE to inhibit Nsun2 transcription.