Identification Of MicroRNAs Differentially Expressed In Malignant Melanoma B16F0 And B16F10 Cells By MicroRNAs Microarray And The Effect Of MiR-763 On The Proliferation Of B16F10 Cells

BackgroundMalignant melanoma(malignant melanoma,MM)is caused by abnornal proliferation of melanocyte derived from skin neural crest.Malignant melanoma is the most deadly skin cancer due to its highly metastatic potential and accounts for 80% of mortality of skin cancer.Once it progresses into metastatic stage,there is currently no effective treatment.MicroRNA(miRNA)is a class of endogenous 21-25 nt,short non-coding,single-stranded RNAs which play essential regulatory roles by targeting mRNA for cleavage or translational repression.This study was to investigate the differential expression of miRNAs in malignant melanoma B16F0 and B16F10 cells,and to investigate the effect of miR-763 on B16F10 cells.Objectives1.To investigate the differential expression of miRNAs in malignant melanoma B16F0 and B16F10 cells.2.To determine the effect and the potential mechanism of miR-763 with different variability on malignant melanoma B16F10 cells.MethodsThe malignant melanoma B16F0 and B16F10 cells is used as the research object to construct the animal model.1.To observe the differences in migration,invasion and proliferation of malignantmelanoma B16F0 and B16F10 cells in vivo and in vitro;2.The expression of miRNA in malignant melanoma B16F0 and B16F10 cells is detected and the differences are analyzed by bioinformatics;3.To verify the accuracy of the microarray results of miRNA,we detected expression level of miR-763,miR-431-5p,miR-205-5p,let-7C-2-3p,miR-26a-1-3p,miR-28 b,miR-29b-3p and miR-24-2-5p on mouse melanoma cells by real-time PCR;4.miR-763 is overexpressed in malignant melanoma B16F10 cells and its effect of the proliferation on B16F10 cells is observed;5.The target gene predicted by bioinformatics of mi R-763 is detected and the phosphorylation levels of key kinases in each signaling pathway is detected by kinase phosphorylation specific antibody and analyzed to determine which signal pathways is affected by the target gene of miR-763 and then affects B16F10 cells.Results:1.migration,invasion and proliferation ability of malignant melanoma B16F10 cells in vitro and in vivo was significantly stronger than B16F0 cells.2.the miRNA expression levels of malignant melanoma B16F0 and B16F10 cells are significantly different through bioinformatics analysis.3.Real-time PCR detection found that the expressions levels of miR-763,miR-431-5p,miR-205-5p and let-7C-2-3p were down-regulated,and the expressions levels of miR-26a-1-3p,miR-28 b,miR-29b-3p and miR-24-2-5p were up-regulated.The results consistent with the chip results,miRNA chip results accurate and credible.4.The overexpression of mi R-763 significantly inhibits the proliferation of malignant melanoma B16F10 cells.5.miR-763 down-regulates the expression of latent target gene Grb2,which affects the downstream molecular Cyclin D1.Conclusion:1.Bioinformatics analysis showed that there were significant differences in miRNA expression between B16F10 and B16F0 cells,which may be an important reason for the difference in migration,invasion and proliferation.2.miR-763 may inhibit the proliferation of malignant melanoma B16F10 cells by targeting the expression of Grb2.