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Microphthalmia-associated transcription factor/T-box factor-2 axis acts through Cyclin D1 to regulate melanocyte proliferation.

ABSTRACT: OBJECTIVES:Control of cell proliferation is critical for accurate cell differentiation and tissue formation, during development and regeneration. Here, we have analysed the role of microphthalmia-associated transcription factor MITF and its direct target, T-box factor TBX2, in regulating proliferation of mammalian neural crest-derived melanocytes. MATERIALS AND METHODS:Immunohistochemistry was used to examine spatial and temporal expression of TBX2 in melanocytes in vivo. RNAi and cell proliferation analysis were used to investigate functional roles of TBX2. Furthermore, quantitative RT-PCR, western blot analysis and flow cytometry were used to further scrutinize molecular mechanisms underlying TBX2-dependent cell proliferation. RESULTS:TBX2 was found to be co-expressed with MITF in melanocytes of mouse hair follicles. Specific Tbx2 knockdown in primary neural crest cells led to inhibition MITF-positive melanoblast proliferation. Tbx2 knockdown in melan-a cells led to reduction in Cyclin D1 expression and G1-phase cell cycle arrest. TBX2 directly activated Ccnd1 transcription by binding to a specific sequence in the Ccnd1 promoter, and the defect in cell proliferation could be rescued partially by overexpression of Cyclin D1 in Tbx2 knockdown melanocytes. CONCLUSIONS:Results suggest that the Mitf-Tbx2-Cyclin D1 pathway played an important role in regulation of melanocyte proliferation, and provided novel insights into the complex physiology of melanocytes.

SUBMITTER: Pan L

PROVIDER: S-EPMC6496872 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Microphthalmia-associated transcription factor/T-box factor-2 axis acts through Cyclin D1 to regulate melanocyte proliferation.

Pan L L Ma X X Wen B B Su Z Z Zheng X X Liu Y Y Li H H Chen Y Y Wang J J Lu F F Qu J J Hou L L

Cell proliferation 20151021 6

<h4>Objectives</h4>Control of cell proliferation is critical for accurate cell differentiation and tissue formation, during development and regeneration. Here, we have analysed the role of microphthalmia-associated transcription factor MITF and its direct target, T-box factor TBX2, in regulating proliferation of mammalian neural crest-derived melanocytes.<h4>Materials and methods</h4>Immunohistochemistry was used to examine spatial and temporal expression of TBX2 in melanocytes in vivo. RNAi and ...[more]

PMID: 26486273

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Project description:Background Based on existing evidence, microRNAs (miRs) are gene regulators that undertake key functions in the oncogenesis and tumor progression of every single human malignant disease, such as thyroid carcinoma (TC). Previous clinical findings showed that expression of miR-195 is down-regulated in TC, which implies that miR-195 may be practically involved in TC pathogenesis. Nevertheless, the function of hsa-miR-195-5p in TC is still largely unclear. Herein, we detected the conceivable involvement of hsa-miR-195-5p in TC cell proliferation. Methods Real time PCR examination was performed to assess the expression level of hsa-miR-195-5p in TC cell lines TPC-1 and B-CPAP. TPC-1 cells were transfected with either hsa-miR-195-5p mimics or hsa-miR-195-5p inhibitor. After confirmation of transfection efficiency, the effect of hsa-miR-195-5p on proliferation and cell cycle of TPC-1 cells was assessed. The expression of cyclin D1 and p21 was simultaneously detected by western blotting. Moreover, targetScan 6.2 was used to predict hsa-miR-195-5p target genes. Subsequently, luciferase reporter was performed to examine whether there is a possible binding of hsa-miR-195-5p to 3’-UTR of cyclin D1 mRNA. Furthermore, cyclin D1 mRNA and protein levels were measured to check whether hsa-miR-195-5p exerts its function at the post-transcriptional level. In addition, to explore the function of cyclin D1 in TPC-1 cells overexpressing hsa-miR-195-5p, cyclin D1 siRNA was used to silence the expression of cyclin D1 in TPC-1 cells overexpressing hsa-miR-195-5p. Results We quantified the expression of hsa-miR-195-5p in TC cells and normal thyroid cells and found a remarkable decrease in hsa-miR-195-5p expression in TC cells. Over-expression of hsa-miR-195-5p obviously resulted in downgraded proliferation of TC cells. Moreover, hsa-miR-195-5p caused cell arrest at the GO/G1 phase. Further in silico analyses and the dual-luciferase reporter assay confirmed that 3’-UTR of cyclin D1 is a direct target of hsa-miR-195-5p. Western blot analysis uncovered that hsa-miR-195-5p over-expression led to decreased levels of cyclin D1 and p21. In mechanistic analyses, we found that silencing of cyclin D1 reversed the inhibitory effect of hsa-miR-195-5p on the proliferation of TC cells, which indicates that hsa-miR-195-5p suppresses TC cell proliferation by adversely regulating cyclin D1. Conclusions We concluded that hsa-miR-195-5p is a candidate tumor-suppressor miRNA in TC and that the hsa-miR-195-5p/p21/cyclin D1 pathway could be a potential therapeutic target for TC.

Dataset Information

Microphthalmia-associated transcription factor/T-box factor-2 axis acts through Cyclin D1 to regulate melanocyte proliferation.

Publications

Microphthalmia-associated transcription factor/T-box factor-2 axis acts through Cyclin D1 to regulate melanocyte proliferation.

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