Effects Of MiRNA-let-7a On The Biological Behaviors Of Melanoma Cell Line And Analysis Of Its Action Mechanism

Cutaneous malignant melanoma originates from melanocytes. It is one of the most deadly human cancers with no effective cure for metastatic disease. miRNAs is a new kind of small molecular RNA which play the role of oncogene or tumor suppressor gene. The study is to investigate the role of miRNA-let-7a in the pathogenesis of cutaneous melanoma and identify the new early diagnosis mark and therapy target of skin melanoma.Objective To investigate the expression of miRNA-let-7a in formalin-fixed paraffin-embedded melanoma tissue and melanoma cell line A375. To explore the effect and possible reason of mciroRNA-let-7a on proliferation and apoptosis in melanoma cell line A375. To detect the expression difference of BRAF protein in melanoma cell line A375 and primary melanin cells and study the effect of miRNA-let-7a on the expression of BRAF protein which is the key protein in melanoma processing.Methods13 FFPE malignant melanoma tissues and 10 FFPE benign nevus tissues were choosed. Melanoma cell line A375 and primary malanin cells were cultured with different culture medium. miRNAs were isolated from FFPE tissues by high pure miRNA isolation kit and total RNA were isolated from cells with RANiso Plus. miRNAs and total RNA were reverse transcripted to cDNA. Then real time quantitative PCR was performed with Taqman MGB probe and matched primer on ABI Prism 7500 fluorescence quantitative PCR instrument. The numerical values from the SDS software were calculated and analyzed by statistics.The expression of BRAF protein were detected in melanoma cell line A375 and primary melanin cells by western blotting.Cy3-labeled siRNA was transfected into A375 cell line, the transfection efficiency was examined under the fluorescense microscopy. Then, hsa-let-7a mimics and its negative control were transfected into A375 cell line respectively. Cell proliferation variances of the two groups were detected by CCK-8 assay and cell apoptotic percentage changes were examined by flow cytometry. The expression of Caspase-3 protein and BRAF protein in the two groups cells were analysed by western blot.Results miRNAs were successfully isolated from FFPE tissues and real time quantitative PCR results showed miRNA-let-7a is significantly reduced both in melanoma tissues and A375 cell line compared to nevus(P<0.05) and melanin cell respectively.The transfection efficiency of siRNA is about 80%-90% under fluorescense microscopy. Compared with negative control, the expression of miRNA-let-7a increased 2.067 folds after 50nM hsa-let-7a mimics transfected the A375 cell line. The cell proliferation was inhibited and the apoptosis of A375 cell is increased after hsa-let-7a mimics was transfected into A375 cell line comprared to negative control (P<0.05). The expression of BRAF protein is increased in melanoma cell line A375 compared with melanin cells. Compared with negative control, the protein expression of Caspase-3 and BRAF are both reduced after hsa-let-7a mimics transfected A375 cell line.Conclusions FFPE tissues are suitable to study the expression level of miRNA in melanoma. The expression of miRNA-let-7a is decreased in melanoma. Overexpression of miRNA-let-7a can inhibit proliferation of melanoma cell line A375 and promote its apoptosis. It also downregulate the expression of caspase-3. Its promoting apoptosis may be related with the downregulation of caspase-3. The expression level of BRAF protein which is a key factor in melanoma processing is obviously increased in melanoma cell line A375 to primary melanin cells. miRNA-let-7a can downregulate the expression of BRAF protein. In brief, miRNA-let-7a may play a role of cancer suppressor gene in melanoma developing and it may become a new early diagnosis mark and therapy target of melanoma.