A Preliminary Study On The Expression Of Homo Sapiens MicroRNAs In Human Cutaneous Malignant Melanoma And On Action Mechanisms Of Homo Sapiens MicroRNA-21 In Human Malignant Melanoma Cell Lines

ObjectiveTo select homo sapiens microRNAs associated with human cutaneous malignant melanoma(MM) and to investigate their action mechanism,so as to provide theoretical and experimental evidence for the elucidation of pathogenesis of MM as well as development of therapeutic strategies for MM.MethodsTotal RNA was extracted from 6 tissue samples of MM and 9 control samples of pigmented nevi,and small RNA(～200bp) was isolated.Then we detected hsa-microRNAs profile in these samples by microRNA microarray,which contained 468 known homo sapiens microRNA(http://microrna.sanger.ac.uk SangermiRNA. Feb,2007.).Meanwhile,we examined the expression of homo sapiens microRNAs candidates in these samples by fluorescence based real-time quantitative PCR(qPCR). Those hsa-miRNAs that were identified as differentially expressed by both microRNA microarray and qPCR were considered as significant hsa-miRNAs.The expression of hsa-miRNA,which was most differentially expressed,was detected in cell lines A375 and M14.A375 and M14 cells were transfected with various concentrations(90,180, 270 nmol/L) of MicroRNA-21 inhibitor by Lipofectamine 2000.Following the transfection,the changes of cell activity was examined by CCK-8 kits.The cells experiencing the most changes of activity were subjected to the analysis of cell cycle and apoptosis via flow cytometry as well as to the detection of mRNA expression of BRAF and p27. Results1.The expression of miRNA-21 was obviously up-regulated,while that of miRNA-320 and miRNA-494 was down-regulated in the MM samples.2.MiRNA-21 was expressed at various degree in A375 and M14 cells.3.The cellular activity of A375 and M14 cells was apparently suppressed by the transfection with miRNA-21 inhibitor of 90nmol/L.And with the increase in the concentration of miRNA-21 inhibitor,the suppressing effect was enhanced.4.Among the transfected A375 cells experiencing the most changes in activity,the apoptosis rate and percentage of cells in G1 phase increased,while the percentage in S phase decreased.No significant change was noted in the mRNA expression of BRAF or p27 mRNA in these cells.ConclusionsOur results suggest that the expression of homo sapiens microRNA-21 is up-regulated,while that of homo sapiens microRNA-320 and -494 are down-regulated in MM.Hsa-miRNA-21 may function like an oncogene by inhibiting cell apoptosis and promoting cell proliferation in MM.However,the mRNA expression of BRAF and p27 seems not to be affected by hsa-miRNA-21.