MiR-148b Suppresses Malignant Biological Behavior Of Melanoma By Targeting Sirtuin7

Objective: To explore the effects of miR-148 b on the malignant biological behavior of melanoma and to investigate the potential mechanisms it participates.Provide basic theory for miR-148 b as a diagnostic/prognostic biologic target for melanoma.Methods:1.Human tissues and cells.Tissue specimens from 30 melanoma patients and 8 benign disease patients.3 melanoma cells were used and a primary melanocyte cell line was established.2.Bioinformatics analysis.To investigate the predicted target genes,we used the Target Scan program.SIRT7 is a direct target of miR-148 b.3.The expression of miR-148 b in melanoma cells and melanocyte was determined by Real time RT-PCR.4.The expression of miR-148 b in melanoma tissues and benign disease tissues were determined by fluorescence in situ hybridization.5.The expression and location of SIRT7 in melanoma tissues were observed by immunofluorescence staining.6.SIRT7 wild-type and mutant-type plasmid construction,Lentivirus-miR-148 b production and transduction.Inhibition or overexpression of mi R-148 b by using mimics or inhibitors.the control cells were infected with empty vector constructs.7.Luciferase reporter assay,Western blot and Real time RT-PCR assay.8.Cell growth,wound-healing,colony formation and invasion assays.A cell proliferation assay was performed with the Cell Counting Kit-8.Cell migration activity was evaluated by wound-healing assay.A cell invasion assay was carried out using Chambers.colony formation ability was carried out using colony formation assay.9.Statistical analysis.Results:1.Real time RT-PCR was performed to detect the expression of miR-148 b in melanoma cells and its levels were significantly lower than normal melanocyte.2.FISH was performed to examine the expression of miR-148 b in melanoma tissues and benign tissues,the expression of miR-148 b is frequently decreased in human melanoma tissues.3.SIRT7 expression was increased in melanoma tissues compared with benign disease tissues by IF staining.SIRT7 is mostly located in the nucleus and partly in cytoplasm.4.Luciferase assay revealed that miR-148 b directly bound to SIRT7 3'-UTR,and by which it remarkably influenced luciferase activities.These findings confirmed the predicted binding site of miR-148 b to the 3'UTR of SIRT7.5.By real time RT-PCR and western blot assay,we found that SIRT7 mRNA and protein levels were down-regulated in A375 cells.Overexpression of mi R-148 b in A375 cells resulted in substantial decreases in SIRT7 m RNA and protein expression.6.As expected,overexpression of miR-148 b significantly suppressed cell migration and invasion abilities.Moreover,ectopic miR-148 b expression inhibited colony formation ability and proliferation ability.Conclusions:1.Real time RT-PCR and FISH confirmed that miR-148 b is down-regulated in melanoma and IF confirmed that SIRT7 is up-regulated in melanoma.These results suggest that miR-148 b may function as a tumor suppressor in melanoma,mi R-148 b negatively regulate the expression of SIRT7 in melanoma.2.We identified SIRT7 as miR-148 b putative target gene.It is one of the focuses in the research of molecular biology mechanism.Up-regulation of miR-148 b significantly reduced SIRT7 mRNA and protein levels.MiR-148 b inhibits malignant biological behavior of melanoma by suppressing Sirtuin7.3.Up-regulation of miR-148 b could correspondingly affected melanoma malignant phenotype,including suppressing cell proliferation,reducing cell migration and invasion ability,reducing the colony formation capacity in vitro.Taken together,SIRT7 is a direct target of miR-148 b.The data is adequate to confirm that miR-148 b inhibits malignant biological behavior of melanoma by suppressing the expression of Sirtuin7.SIRT7 and mi R-148 b may provide a new target for early diagnosis,prognosis and molecular therapy of melanoma.