Cancer Letters

Cancer Letters

Volume 272, Issue 2, 18 December 2008, Pages 197-205
Cancer Letters

MicroRNA-21 down-regulates the expression of tumor suppressor PDCD4 in human glioblastoma cell T98G

https://doi.org/10.1016/j.canlet.2008.06.034Get rights and content

Abstract

MicroRNAs have been linked to different cancer-related processes. The microRNA miR-21 appears to function as an anti-apoptosis factor in glioblastomas. However, the functional target genes of miR-21 are largely unknown in glioblastomas. In this study, bioinformatics analysis was used to identify miR-21 target sites in various genes. Luciferase activity assay showed that a number of genes involved in apoptosis, PDCD4, MTAP, and SOX5, carry putative miR-21 binding sites. Expression of PDCD4 protein correlates inversely with expression of miR-21 in a number of human glioblastoma cell lines such as T98G, A172, U87, and U251. Inhibition of miR-21 increases endogenous levels of PDCD4 in cell line T98G and over-expression miR-21 inhibits PDCD4-dependent apoptosis. Together, these results indicate that miR-21 expression plays a key role in regulating cellular processes in glioblastomas and may serve as a target for effective therapies.

Introduction

Glioblastomas are the most common type of malignant primary brain tumors. Despite the advances in surgery, radiation therapy, and chemotherapy, the prognosis of patients with glioblastomas has not improved significantly over the past 20 years [1]. Understanding how the signaling pathways involved in migration, invasion, and apoptosis of glioblastomas are regulated is important for the development of more effective tumor therapies.

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that regulate the stability or translational efficiency of target messenger RNAs[2]. A number of miRNAs have been identified from different human tumors and appear to play crucial roles in proliferation, differentiation, and apoptosis. One of these microRNAs, miR-21, is a key player in human cancers such as breast cancer, liver cancer, pancreatic cancer, colorectal cancer, and glioblastomas [3]. Chan et al. found that miR-21 is an anti-apoptosis factor in human glioblastoma cells. Knock-down of miR-21 in cultured glioblastoma cells triggers activation of caspases and leads to increased apoptotic cell death [4]. miR-21 knock-down disrupts glioma growth and displays synergistic cytotoxicity with S-TRAIL with in vivo xenografts of U87 cells [5]. Aberrantly expressed miR-21 down-regulates tumor suppressor PTEN and modulates gemcltabbine-induced apoptosis by PTEN-dependent activation of phosphoinositide 3-kinase (PI3K) signaling in cholangiocarcinoma and hepatocellular cancer [6], [7]. Post-transcriptional down-regulation of tumor suppressor PDCD4 by miR-21 stimulates invasion, intravasation, and metastasis in colorectal cancer [8]. Expression of tumor suppressor Tropomyosin 1 (TPM1) and PDCD4 in breast cancer cell MCF-7 are down-regulated by expression of miR-21 [9], [10] and knock-down of miR-21 expression suppresses both MCF-7 cells growth in vitro and tumor growth in the xenograft mouse model [11].

In this study, bioinformatics analysis was used to screen and identify various genes with miR-21 target sites. Luciferase activity assays indicated that PDCD4, MTAP, and SOX5 carry putative miR-21 binding sites. Furthermore, PDCD4 protein levels correlate inversely with miR-21 levels in human glioblastoma cell lines A172, T98G, U87, and U251. Reducing miR-21 increases PDCD4 in T98G cell line and over-expression of miR-21 inhibits PDCD4-dependent apoptosis.

Section snippets

Cell lines

Human glioblastoma established cell lines A172, T98G, and U87 were purchased from American Type Culture Collection (Manassas, VA) and U251 was purchased from Cell center of Peking Union Medical College. Cell line 293ET was obtained from Dr. Chengyu Jiang (Peking Union Medical College). The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. A172, T98G, U87, and U251 cells were maintained in Dulbecco’s modified Eagle’s Medium, and 293ET in Iscove’s modified Dulbecco’s

Identification of miR-21 possible target genes

It is established that miR-21 is aberrantly expressed in various types of cancers [3], [15]. Inhibition of miR-21 leads to increased apoptosis in glioblastomas [4] and attenuated intravasation and metastatic capacity in colon cancer [8]. To examine the potential involvement of miR-21 and its targets in glioblastomas, MiRanda, TargetScan, and Pictar were used to analyze the possible target genes of miR-21 (Fig. 1A). Our analysis showed that there were 119 candidates in the intersection of at

Discussion

A better understanding of the molecular mechanisms occurring in glioblastomas has the potential of helping the development of new and various targeted molecular therapies. In this study, we identified functional targets of an anti-apoptosis factor miR-21. Although more than several hundred candidate target genes which might carry miR-21 binding sites based on the combined analysis of miRanda, PicTar, and TargetScan programs, not every putative site is a functional miRNA target. Our results

Conflict of interest

None declared.

Acknowledgements

This work was supported by funds from National Natural Sciences Foundation of China (Nos. 30608022, 90612019, 3043000, 30721063), “863” project (Nos. 2006AA0Z137, 006AA02A304), “973” project (Nos. 2004CB518604, 2005CB2507, 2006CB504100, 2007CB946902, 2007CB946900), Program for New Century Excellent Talents in University (Nos. NCET-07-0505). We thank Prof. Samuel Schacher, Department of Neuroscience, Columbia University for proofreading the manuscript.

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