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MicroRNA-33b inhibits cell proliferation and glycolysis by targeting hypoxia-inducible factor-1α in malignant melanoma.

ABSTRACT: Malignant melanoma (MM) is the most aggressive type of skin cancer. MicroRNA (miR) has been implicated in the development and progression of MM; however, their underlying mechanism of action remains largely unknown. The present study aimed to investigate the role of miR-33b in MM. Reverse transcription-quantitative polymerase chain reaction data indicated that the expression of miR-33b was significantly reduced (P<0.01) in MM cell lines, including WM35, WM451 and SK-MEL-1, when compared with human melanocyte cells. Subsequently, WM451 and SK-MEL-1 cells were transfected with an miR-33b mimic or inhibitor. MTT assay data demonstrated that the viability of MM cells markedly decreased following miR-33b overexpression; however, viability was markedly upregulated following miR-33b knockdown. Additionally, the glycolysis level was examined. Results demonstrated that glucose consumption and lactic acid production were significantly downregulated (P<0.01) after miR-33b upregulation, whereas these levels significantly increased in MM cells transfected with miR-33b inhibitor (P<0.01), suggesting that miR-33b negatively mediates the glycolysis level in MM cells. Bioinformatics indicated that hypoxia-inducible factor (HIF)-1α was a putative target gene of miR-33b, and this was confirmed by a luciferase reporter assay, which demonstrated that miR-33b was able to directly bind to the 3' untranslated region of HIF-1α mRNA. Furthermore, the expression of HIF-1α was negatively regulated by miR-33b at the post-transcriptional level in MM cells. Overexpression of HIF-1α reversed the inhibitory effect of miR-33b on the proliferation and glycolysis in MM cells. Finally, the results of the present study demonstrated that hexokinase 2 and lactate dehydrogenase-A may be involved in miR-33b/HIF-1α mediated glycolysis in MM cells. In conclusion, these results suggest that miR-33b inhibits cell proliferation and glycolysis by targeting HIF-1α in MM.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC5526185 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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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-33b inhibits cell proliferation and glycolysis by targeting hypoxia-inducible factor-1α in malignant melanoma.

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