MiR-26a And Let-7a Inhibit Proliferation And Invasiveness Of Human Malignant Melanoma

Background:Metastatic melanoma,a malignant tumor originated from pigment-producing melanocytes,is the most aggressive type of skin cancer and is refractory for its therapy.microRNAs(miRNAs),a class of small non-coding RNAs,are recently discovered as novel molecules that provide potential diagnostic tools and therapeutic targets in melanoma.microRNAs are widely engaged in post-transcriptional regulation of gene expression and mediate the multiple biological processes,including the differentiation,apoptosis and proliferation.This work aimed to investigate the potentials of miR-26a and let-7a as novel therapeutics against human malignant melanoma.It examined the effect of miR-26a and let-7a on proliferation and invasiveness of malignant melanoma cells and examined the effect of miR-26a and let-7a on the cell cycle progression of human malignant melanoma cells.It also identified and validated the potential targets of miR-26a and let-7a in melanoma cells.Methods:The expression levels of miR-26a and let-7a in two melanoma cell lines,SKMEL-28 and WM1552C,were determined by qRT-PCR.The effect of miR-26a and let-7a on cell proliferation,migration and invasion has been investigated by using an MTT assay,wound healing assay and Transwell assay.The effect of miR-26a and let-7a on the cell cycle progression of human malignant melanoma cells was determined by flow cytometry.Apoptosis induced by miR-26a was also detected by flow cytometry.Three algorithms(TargetScan,miRDB,and microma.org)were used to predict potential gene targets for miR-26a and let-7a in human malignant melanoma.Clustal Omega was employed to generate alignments between miRNA and potential targets.Western blot has been applied to examine the expression of gene targets for miR-26a and let-7a at protein level.Dual luminescence assay was applied to investigate whether miR-26a and let-7a directly regulate the expression of their targets in melanoma.Results:Cell viability assay indicated that miR-26a and let-7a(50 nM and 100 nM) suppress cell proliferation in both malignant melanoma cell lines.Wound healing and Transwell invasion assay illustrated that miR-26a significantly inhibited the ability of migration and invasion.In addition,miR-26a induced cell cycle arrest at G1/G0 phase as shown by flow cytometry.The high concentration of miR-26a(100 nM)significantly induced apoptosis in SKMEL-28,Three microRNA target gene prediction algorithms identified MAP4K3 and MITF as potential targets for miR-26a and let-7a.Clustal Omega was employed to generate alignments,finding that miR-26 and let-7a target with 3’ UTR of MITF or 3’ UTR of MAP4K3,respectively.However,Western blot results showed that the expression of only MITF was down regulated by miR-26a.The luciferase reporter assays validate that MITF is a bona-fide gene target of miR-26a.In vivo experiment showed that miR-26a significantly retarded the growth of melanoma tumors in the mouse model.Our findings indicate that let-7a and miR-26a significantly inhibited the growth and invasiveness of melanoma cells and could function as novel therapeutic small molecules against human malignant melanoma.In addition,this study identifies and validates that MITF is a target gene of miR-26a and therefore a possible suitable therapeutic target.