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MicroRNA-26a inhibits the growth and invasiveness of malignant melanoma and directly targets on MITF gene.

ABSTRACT: Metastatic melanoma is the most aggressive form of skin cancer and is refractory to therapy. MicroRNAs have been recently discovered as novel molecules that provide therapeutic benefits against melanoma. This work aims to examine the effects of miR-26a and let-7a on the growth and invasiveness of malignant melanoma in vitro and in vivo. In addition, we elucidate the mechanism of action by identifying the target gene of miR-26a. Both miR-26a and let-7a inhibited proliferation and invasiveness and halted the cell cycle at the G₁/G₀ phase in SKMEL-28 and WM1552C malignant melanoma cell lines. Moreover, miR-26a potently induced apoptosis and downregulated the expressions of microphthalmia-associated transcription factor (MITF) and MAP4K3 in both cell lines. The luciferase reporter assay demonstrated that miR-26a suppresses MITF expression by binding the 3'-UTR, suggesting that MITF is a bona fide target of miR-26a. SiRNA knockdown of the MITF gene confirmed that miR-26a reduced cell viability and induced apoptosis by regulating MITF. Using a murine model, we also found miR-26a significantly retarded the growth of melanoma tumors in vivo. In conclusion, miR-26a and let-7a suppressed the growth and invasiveness of melanoma cells, suggesting that miR-26a and let-7a may represent novel therapies for malignant melanoma.

SUBMITTER: Qian H

PROVIDER: S-EPMC5502303 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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MicroRNA-26a inhibits the growth and invasiveness of malignant melanoma and directly targets on MITF gene.

Qian Hui H Yang Chen C Yang Yixin Y

Cell death discovery 20170710

Metastatic melanoma is the most aggressive form of skin cancer and is refractory to therapy. MicroRNAs have been recently discovered as novel molecules that provide therapeutic benefits against melanoma. This work aims to examine the effects of miR-26a and let-7a on the growth and invasiveness of malignant melanoma in vitro and in vivo. In addition, we elucidate the mechanism of action by identifying the target gene of miR-26a. Both miR-26a and let-7a inhibited proliferation and in ...[more]

PMID: 28698805

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Project description:Background & aimsMicroRNAs (miRNAs) have been implicated in the development and progression of human cancers. We investigated the roles and mechanisms of miR-26a in human cholangiocarcinoma.MethodsWe used in situ hybridization and quantitative reverse transcriptase polymerase chain reaction to measure expression of miR-26a in human cholangiocarcinoma tissues and cell lines (eg, CCLP1, SG231, HuCCT1, TFK1). Human cholangiocarcinoma cell lines were transduced with lentiviruses that expressed miR-26a1 or a scrambled sequence (control); proliferation and colony formation were analyzed. We analyzed growth of human cholangiocarcinoma cells that overexpress miR-26a or its inhibitor in severe combined immune-deficient mice. Immunoblot, immunoprecipitation, DNA pull-down, immunofluorescence, and luciferase reporter assays were used to measure expression and activity of glycogen synthase kinase (GSK)-3β, β-catenin, and related signaling molecules.ResultsHuman cholangiocarcinoma tissues and cell lines had increased levels of miR-26a compared with the noncancerous biliary epithelial cells. Overexpression of miR-26a increased proliferation of cholangiocarcinoma cells and colony formation in vitro, whereas miR-26 depletion reduced these parameters. In severe combined immune-deficient mice, overexpression of miR-26a by cholangiocarcinoma cells increased tumor growth and overexpression of the miR-26a inhibitor reduced it. GSK-3β messenger RNA was identified as a direct target of miR-26a by computational analysis and experimental assays. miR-26a-mediated reduction of GSK-3β resulted in activation of β-catenin and induction of several downstream genes including c-Myc, cyclinD1, and peroxisome proliferator-activated receptor δ. Depletion of β-catenin partially prevented miR-26a-induced tumor cell proliferation and colony formation.ConclusionsmiR-26a promotes cholangiocarcinoma growth by inhibition of GSK-3β and subsequent activation of β-catenin. These signaling molecules might be targets for prevention or treatment of cholangiocarcinoma.

Project description:MicroRNA (miR) are a class of small non-coding RNA that are able to inhibit gene expression by directly binding to the 3' untranslated region (UTR) of their target mRNA and thus promote translational repression or mRNA degradation. Recently, miR-9 was reported to have a suppressive role in malignant melanoma; however, the underlying mechanism remains largely unclear. In the present study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to examine the mRNA and protein expression levels in malignant melanoma tissues and cell lines. The MTT assay and wound healing assay were used to examine the cell viability, proliferation and migratory capacities. Bioinformatics prediction and luciferase reporter assay were performed to investigate the relationship between miR-9 and its potential target gene. The present data revealed that miR-9 expression was significantly downregulated in malignant melanoma tissues when compared with their matched adjacent non-tumor tissues. Furthermore, the expression levels of miR-9 were reduced in malignant melanoma cell lines when compared with human normal skin HACAT cells. Moreover, the ectopic expression of miR-9 significantly suppressed the proliferation and migration of malignant melanoma cells, accompanied with a remarkable decrease in the protein expression levels of sirtuin 1 (SIRT1), which were markedly upregulated in malignant melanoma tissues and cell lines. Additionally, restoration of SIRT1 reversed the suppressive effects of miR-9 on the proliferation and migration of malignant melanoma cells. Luciferase reporter assay data further identified SIRT1 as a direct target gene of miR-9. To conclude, the present findings indicate that miR-9 has a suppressive role in malignant melanoma cell viability and migration, at least in part, via directly inhibiting the protein expression of its target gene, SIRT1. Therefore, miR-9 may serve as a potential candidate for the treatment of malignant melanoma.

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MicroRNA-26a inhibits the growth and invasiveness of malignant melanoma and directly targets on MITF gene.

Publications

MicroRNA-26a inhibits the growth and invasiveness of malignant melanoma and directly targets on <i>MITF</i> gene.

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