MiRNA-125a-5p inhibits glioblastoma cell proliferation and promotes cell differentiation by targeting TAZ

doi:10.1016/j.bbrc.2014.12.078

Biochemical and Biophysical Research Communications

Volume 457, Issue 2, 6 February 2015, Pages 171-176

https://doi.org/10.1016/j.bbrc.2014.12.078 Get rights and content

Highlights

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Expression of miR-125a-5p is inversely correlated with that of TAZ in glioma cells.
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MiR-125a-5p represses TAZ expression in glioma cells.
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MiR-125a-5p directly targets the 3′ UTR of TAZ mRNA and promotes its degradation.
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MiR-125a-5p represses CTGF and survivin via TAZ, and inhibits glioma cell growth.
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MiR-125a-5p inhibits the stem cell features of HFU-251 MG cells.

Abstract

Glioblastoma (GBM) is the most lethal brain tumor due to the resistance to conventional therapies, such as radiotherapy and chemotherapy. TAZ, an important mediator of the Hippo pathway, was found to be up-regulated in diverse cancers, including in GBM, and plays important roles in tumor initiation and progression. However, little is known about the regulation of TAZ expression in tumors. In this study, we found that miR-125a-5p is an important regulator of TAZ in glioma cells by directly targeting the TAZ 3′ UTR. MiR-125a-5p levels are inversely correlated with that of TAZ in normal astrocytes and a panel of glioma cell lines. MiR-125a-5p represses the expression of TAZ target genes, including CTGF and survivin, and inhibits cell proliferation and induces the differentiation of GBM cells; whereas over-expression of TAZ rescues the effects of miR-125a-5p. This study revealed a mechanism for TAZ deregulation in glioma cells, and also demonstrated a tumor suppressor role of miR-125a-5p in glioblastoma cells.

Introduction

Glioblastoma (GBM) is the most lethal brain tumor with median survival of about 15 months using current standard of care for patients [1]. A subgroup of stem cell-like cells in GBMs (known as glioma stem cells) was identified and proved to be responsible for the initiation of glioma and the resistance of GBMs to conventional therapies [2], [3]. Therefore, it is important to reveal the mechanisms regulating glioma stem cell, and the initiation and progression of GBMs.

TAZ is an important transducer of the Hippo tumor-suppressor pathway. Upon activation of the Hippo pathway, TAZ was phosphorylated by LATS1/2, which promotes its cytoplasmic sequestration and then functional inhibition [4]. Moreover, phosphorylation by the Hippo pathway also promotes TAZ degradation [5]. Deregulation of TAZ has been reported at a high frequency in many kinds of cancers and often correlates with poor prognosis [6], [7], [8], [9], [10]. In addition, TAZ was required to sustain the self-renewal of cancer stem cells (CSCs) and induce the tumorigenic potential of CSCs [11], [12]. In glioma, TAZ expression was lower in low grade gliomas and proneural GBMs compared with GBMs that had a mesenchymal phenotype [13]. Knockdown of TAZ in glioma stem cell decreases the expression of mesenchymal markers, as well as invasion, self-renewal and tumor formation [13]. However, the regulation of TAZ expression in high grade tumors is largely unknown.

MicroRNAs (miRNAs) are a large family of endogenous small RNAs (19–24 nt) and act as important post-transcriptional regulators of gene expression [14]. It has been firmly established that miRNAs were involved in the regulation of diverse physiological and pathological processes [15], especially in tumorigenesis [16], [17]. MiR-125a-5p is derived from the 5′ end of pre-miR-125a. It was reported that miR-125-5p was deregulated in some kinds of cancers, and acts as a tumor suppressor by targeting genes associated with cell proliferation and tumor progression [18], [19]. In glioblastoma, miR-125a-3p, another mature miRNA derived from pre-miR-125a, was proved to be down-regulated and inhibited the invasion and proliferation of GBM cells [20]. Moreover, miR-125a-3p was down-regulated in CD133⁺ GBM cells compared with the CD133⁻ GBM cells, suggesting its involvement in the regulation of glioma stem cells [20]. However, little is known about the expression and roles of miR-125a-5p in glioblastoma.

In this study, we revealed the regulation of TAZ by miR-125a-5p in glioma cells. We found that expression of TAZ and miR-125a-5p are inversely correlated in normal astrocytes and different glioma cell lines. Mechanically, miR-125a-5p directly targets the 3′ UTR of TAZ mRNA and represses TAZ expression. MiR-125a-5p inhibits GBM cell proliferation and promotes cell differentiation by repressing the TAZ target genes including CTGF and survivin. Our study revealed a mechanism for TAZ deregulation in glioma cells, and also demonstrated a tumor suppressor role of miR-125a-5p in glioblastoma cells.

Section snippets

Cell culture and transfection

Normal Human Astrocytes (NHA), glioma cell lines (HS683, SW1783, LN229, U87MG, T98G, HFU-251MG) and HEK293 cells were all cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin in a 5% CO₂ humidified incubator at 37 °C. Transient cell transfections of plasmids or small RNAs were performed using Lipofectamine 2000 (Life Technologies). siRNA and miRNA mimics were transfected at a final concentration of 50

TAZ expression is repressed by miR-125a-5p in human glioma cells

To reveal the functional relationship between miR-125a-5p and TAZ, we detected the levels of miR-125a-5p and TAZ protein in different glioma cell lines. We used seven cell lines including one cell line derived from normal human astrocytes (NHA), two cell lines from low-grade glioma (HS683 and SW1783), and four from high-grade glioma (LN229, U87 MG, T98G and HFU-251 MG). MiR-125a-5p was highest in NHA cells, relative lower in HS683, SW1783 and LN229 cells (Fig. 1A). However, miR-125a-5p had the

Discussion

TAZ is an important transducer of the Hippo pathway and play important roles in tumor initiation and progression. However, little is known about the regulation of TAZ expression in tumors. In this study, we revealed that miR-125a-5p represses TAZ expression by directly targeting the 3′ UTR of TAZ mRNA. MiR-125a-5p also represses the expression of CTGF and survivin, two important target genes of TAZ, and inhibits glioma cell proliferation and promotes GBM cell differentiation (Fig. 4D). This

Conflict of interest

There are no conflict of interest for all authors.

Acknowledgments

The study was supported by grants from the Planned Science and Technology Project of Hunan Province (No. 2012SK2020) and the Postgraduate Degree Thesis Innovation Projects of Central South University (No. 2011ssxt207).

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Citation Excerpt :
The miRNAs play a dual role during tumor development through modulating YAP/TAZ. Such as, miR-125a-5p inhibits cell proliferation and promotes cell differentiation in glioblastoma and retinoblastoma by directly targeting TAZ [53,54]; conversely, in breast cancer cells, miR-125a-5p indirectly upregulates TAZ expression by restraining leukemia inhibitory factor receptor (LIFR), a YAP/TAZ activity inhibitor, which increases the proportion of cancer stem cells [55]. Moreover, miR-9 promotes breast cancer cell migration and metastasis by directly targeting LIFR, which inhibits YAP1 activity [56], while miR-9-3p acts as a tumor suppressor to promote cell proliferation in hepatocellular carcinoma by directly targeting TAZ [57].
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Our report suggests potential clinical implications as radiotherapy is the backbone of treatment for patients with head and neck cancer, commonly used either in the definitive or adjuvant setting. The tumor suppressive function of miR-125a-5p in HNSCC, as demonstrated by our results, is concordant with previous studies in other malignancies, such as lung cancer [23], breast cancer [24], multiple myeloma [25], gastric cancer [26], hepatocellular carcinoma [27], chronic lymphocytic leukemia [28], nasopharyngeal carcinoma [29], glioblastoma [30], colon cancer [31], prostate cancer [32], cervical carcinoma [33], and thyroid cancer [34], implicating miR-125-5p as a general tumor suppressor miRNA, across a broad spectrum of malignancies. This report has also identified three mRNA targets of miR-125a-5p in head and neck squamous cell carcinoma, specifically, ERBB2, which encodes for the erbB-2, otherwise known as HER2/neu, receptor tyrosine kinase, TXNRD1, which encodes for thioredoxin reductase 1, and EIF4EBP1, which encodes for the 4E-BP1 translation initiation factor.
MicroRNAs (miRNAs) are short single-stranded RNAs, measuring 21 to 23 nucleotides in length and regulate gene expression at the post-transcriptional level through mRNA destabilization or repressing protein synthesis. Dysregulation of miRNAs can lead to tumorigenesis through changes in regulation of key cellular processes such as cell proliferation, cell survival, and apoptosis. miR-125a-5p has been implicated as a tumor suppressor miRNA in malignancies such as non-small cell lung cancer and colon cancer. However, the role of miR-125a-5p has not been fully investigated in head and neck squamous cell carcinoma (HNSCC). We performed microRNA microarray profiling of HNSCC tumor samples obtained from a prospective clinical trial evaluating the role of postoperative radiotherapy in head and neck cancer. We also mined through The Cancer Genome Atlas to evaluate expression and survival data. Biological experiments, including cell proliferation, flow cytometry, cell migration and invasion, clonogenic survival, and fluorescent microscopy, were conducted using HN5 and UM-SCC-22B cell lines. miR-125a-5p downregulation was associated with recurrent disease in a panel of high-risk HNSCC and then confirmed poor survival associated with low expression in HNSCC via the Cancer Genome Atlas, suggesting that miR-125a-5p acts as a tumor suppressor miRNA. We then demonstrated that miR-125a-5p regulates cell proliferation through cell cycle regulation at the G₁/S transition. We also show that miR-125a-5p can alter cell migration and modulate sensitivity to ionizing radiation. Finally, we identified putative mRNA targets of miR-125a-5p, including ERBB2, EIF4EBP1, and TXNRD1, which support the tumor suppressive mechanism of miR-125a-5p. Functional validation of ERBB2 suggests that miR-125a-5p affects cell proliferation and sensitivity to ionizing radiation, in part, through ERBB2. Our data suggests that miR-125a-5p acts as a tumor suppressor miRNA, has potential as a diagnostic tool and may be a potential therapeutic target for the management and treatment of squamous cell carcinoma of the head and neck.

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MiRNA-125a-5p inhibits glioblastoma cell proliferation and promotes cell differentiation by targeting TAZ

Highlights

Abstract

Introduction

Section snippets

Cell culture and transfection

TAZ expression is repressed by miR-125a-5p in human glioma cells

Discussion

Conflict of interest

Acknowledgments

J. Biol. Chem.

J. Thorac. Oncol.

Cell

Curr. Opin. Cell Biol.

Dev. Biol.

Cell

Class III beta-tubulin and gamma-tubulin are co-expressed and form complexes in human glioblastoma cells

Neurochem. Res.

Cancerous stem cells can arise from pediatric brain tumors

Proc. Natl. Acad. Sci. U.S.A.

Tumour-initiating cells: challenges and opportunities for anticancer drug discovery

Nat. Rev. Drug Discov.

TAZ promotes cell proliferation and epithelial–mesenchymal transition and is inhibited by the hippo pathway