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MicroRNA-21 Is an Antiapoptotic Factor in Human Glioblastoma Cells

Departments of ¹ Pathology and ² Neurology, Brigham and Women's Hospital and ³ Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts; and ⁴ Neuroscience Research Institute, University of California Santa Barbara, Santa Barbara, California

Requests for reprints: Anna M. Krichevsky, Neurology, Brigham and Women's Hospital, Harvard Medical School, 4 Blackfan Circle HIM 760, Boston, MA 02115. Phone: 617-525-5195; Fax: 617-525-5305; E-mail: krichevsky{at}cnd.bwh.harvard.edu.

	Abstract

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MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate protein expression by targeting the mRNA of protein-coding genes for either cleavage or repression of translation. The roles of miRNAs in lineage determination and proliferation as well as the location of several miRNA genes at sites of translocation breakpoints or deletions has led to the speculation that miRNAs could be important factors in the development or maintenance of the neoplastic state. Here we show that the highly malignant human brain tumor, glioblastoma, strongly overexpresses a specific miRNA, miR-21. Our studies show markedly elevated miR-21 levels in human glioblastoma tumor tissues, early-passage glioblastoma cultures, and in six established glioblastoma cell lines (A172, U87, U373, LN229, LN428, and LN308) compared with nonneoplastic fetal and adult brain tissues and compared with cultured nonneoplastic glial cells. Knockdown of miR-21 in cultured glioblastoma cells triggers activation of caspases and leads to increased apoptotic cell death. Our data suggest that aberrantly expressed miR-21 may contribute to the malignant phenotype by blocking expression of critical apoptosis-related genes.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The discovery of microRNAs (miRNAs) has broadened our understanding of the mechanisms that regulate gene expression with the addition of an entirely novel level of regulatory control. These small noncoding transcripts of 18 to 25 nucleotides modulate protein expression by binding to complementary or partially complementary target mRNAs and thereby targeting the mRNA for degradation or translational inhibition (1). Although knowledge of the specific mRNA targets for the 250 to 300 known mammalian miRNAs is limited, one emerging function of this extensive regulatory network is its control over processes that underlie cell proliferation and differentiation in diverse organisms during normal development (2). In Caenorhabditis elegans, miRNAs regulate temporal transitions between developmental stages (3, 4). In Drosophila, the miRNA bantam both prevents apoptosis and stimulates cell proliferation by suppressing the proapoptotic gene hid (5). In mammals, functions for specific miRNAs have been described in the processes of hematopoietic (6) and adipocyte differentiation (7) and insulin secretion (8). miRNAs are precisely regulated and characteristic patterns of miRNA expression appear during brain development and neuronal differentiation in vitro (9–11). A potential role for miRNAs in malignancy has been suggested by the location of the genes for several miRNAs at sites of translocation breakpoints or deletions linked to human leukemias (12). Indeed, altered miRNA expression has now been reported in leukemia, lung cancer, and colon cancer (13–15). The functional significance of these changes, however, has yet to be addressed. Here we examined the expression of miR-21 in glioblastoma multiforme. We show that high expression of miR-21 is a common feature of glioblastoma multiformes and that miR-21 can function as an antiapoptotic factor in cultured glioblastoma multiforme cells.

	Materials and Methods

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Human tissue samples. Fresh frozen human nonneoplastic brain tissue and human tumor samples were obtained from the Department of Pathology at Brigham and Women's Hospital. All human materials were used in accordance with the policies of institutional review board at Brigham and Women's Hospital.

Cell lines and culturing conditions. Early-passage (passage 3) cultures from four independent human high-grade gliomas were a generous gift from Dr. David Louis (Massachusetts General Hospital). From each of the four high-grade gliomas, three cultures were established to give a total of 12 early-passage cultures (passage 3) used in our studies. Human glioblastoma cell lines A172, U87, LN229, U373, LN428, and LN308 were kindly donated by Drs. Azad Bonni and Rosalind Segal (Harvard University). All glioblastoma cell lines were maintained in DMEM supplemented with 10% fetal bovine serum (FBS). Undifferentiated P19 mouse embryonal carcinoma cells were cultured in MEM supplemented with 10% FBS and were routinely passaged at 2- to 3-day intervals. Primary rat cortical astrocyte cultures were prepared from the cerebral cortices of newborn pups according to established protocols (16).

MicroRNA array and Northern blot analysis. Total RNA from human tissue samples and cell lines was isolated with Trizol reagent (Invitrogen, Carlsbad, CA). Oligonucleotide arrays were printed with tri-mer oligonucleotide probes (antisense to miRNAs) specific for 180 human and mouse miRNAs on GeneScreen Plus (NEN) membranes, and miRNA expression profiling was done and analyzed as described previously (9). To ensure accuracy of the hybridizations, each labeled RNA sample was hybridized with three separate membranes. Northern blots were done with 15 µg of total RNA as previously described (9).

Oligonucleotide transfection. 2'-O-methyl (2'-OMe-) oligonucleotides and locked nucleic acid (LNA/DNA) oligonucleotides were chemically synthesized by Integrated DNA Technologies (Coralville, IA). 2'-O-Methyl oligos were composed entirely of 2'-O-methyl bases and had the following sequences: 2'OMe-EGFP antisense 5'-AAGGCAAGCUGACCCUGAAGU-3' and 2'OMe-miR-21 5'-GUCAACAUCAGUCUGAUAAGCUA-3'. LNA/DNA oligos contained locked nucleic acids at eight consecutive centrally located bases (indicated by underline) and had the following sequences: LNA-miR-21 5'-TCAACATCAGTCTGATAAGCTA-3', LNA-scrambled 5'-CATTAATGTCGGACAACTCAAT-3', LNA-miR124a 5'-GGCATTCACCGCGTGCCTTA-3', and LNA-miR125b 5'-TCACAAGTTAGGGTCTCAGGGA-3'.

Cells were transfected using LipofectAMINE 2000 reagent (Invitrogen) 24 hours after plating. Transfection complexes were prepared according to the manufacturer's instructions and added directly to the cells to a final oligonucleotide concentration of 10 nmol/L. Transfection medium was replaced 8 hours post-transfection. For studies of repeated transfection, surviving cells at 72 hours post-transfection were collected, replated, and transfected again 24 hours after replating (96 hours after initial transfection).

Cell number assay. A172, U87, LN229, and LN308 cells were plated at 3 x 10³ cells per well in 96-well plates with five replicate wells for each condition, transfected, and assayed 48 hours post-transfection. Metabolic activity of the cells was determined using a luminescent ATP-based assay (CellTiter GLO, Promega, Madison, WI) according to the manufacturer's instructions. Results were read with a fluorescent plate reader with a read time of 1 second per well.

Apoptosis assays. For detection of caspase-3 and caspase-7 activation, A172, U87, LN229, and LN308 cells were plated in replicates of five in 96-well plates, transfected as described above and analyzed using ApoONE Homogeneous Caspase 3/7 Assay (Promega) according to the manufacturer's instructions. Samples were read after 1 hour of incubation with the caspase substrate on a fluorescent plate reader using wavelengths of 480 and 535 for excitation and emission, respectively.

For labeling nuclei of apoptotic cells, A172 cells were plated on glass coverslips in 24-well plates, transfected, fixed in 4% paraformaldehyde 48 hours post-transfection, and terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) staining was done using the DeadEnd fluorometric TUNEL system (Promega) according to the manufacturer's protocol. The number of TUNEL-positive cells was divided by the number of 4',6-diamidino-2-phenylindole–stained cells to yield the percent apoptotic nuclei. Two 40x objective fields containing 300 cells each were counted per coverslip, with three coverslips analyzed per condition.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
We first identified miR-21 as a candidate miRNA dysregulated in gliomas during a screen for miRNA expression using miRNA arrays. We used an oligonucleotide array prepared in our laboratory (12) capable of determining expression patterns of 180 mammalian miRNAs (Fig. 1A; Supplementary Data 1). In our analyses of RNA extracted from three primary high-grade gliomas and eight nonneoplastic fetal and adult brain tissues, one consistent feature of the miRNA expression pattern was the striking up-regulation of miR-21 in all three tumors compared with its expression in nonneoplastic controls (P < 0.05). Several additional miRNA candidates differentially expressed by the arrays (Supplemental Data 2) have not been validated as significantly dysregulated in the Northern blot analysis of multiple glioblastoma multiforme samples and/or derived cell lines.

View larger version (40K): [in this window] [in a new window]   Figure 1. miR-21 is overexpressed in glioblastoma tissues and cells. A, representative view of glioblastoma multiforme (GBM) RNA hybridized to an oligonucleotide array containing multiple control probes and probes specific for 180 miRNAs. Position of the miR-21 probe (arrow). B, Northern blot showing expression of miR-21 in nonneoplastic human control tissues (fetal brain, adult cortex, and adult white matter) and tissue from five human glioblastoma samples (GBM tissues 1-5). C, Northern blot showing expression of miR-21 in control tissues (fetal brain, adult cortex, adult white matter), control cell cultures (P19 cell line and primary astrocytes), early-passage cultures from four high-grade gliomas (GBM cultures 1-4), and six established glioblastoma model cell lines (U373, A172, LN229, U87, LN428, LN308). Blots were reprobed for U6 snRNA as a loading control. D, Northern blot showing miR-21 expression in control tissues, glioblastoma multiformes, one anaplastic astrocytoma, one oligoastrocytoma, four oligodendrogliomas, and four medulloblastomas.

To study the biological significance of miR-21-elevated expression, we used a loss-of-function approach in glioma cell lines. We employed the U87, A172, LN229, and LN308 human glioblastoma cell lines as these representative lines have genetic abnormalities common in glioblastoma (17), show rapid growth in culture, and have strongly elevated expression of miR-21 (Fig. 1C). In addition, these cell lines are readily transfectable with small synthetic oligonucleotides in small lipid micelles, with 90% to 100% of cells showing uptake of a fluorescent-tagged marker oligonucleotide at 8 hours post-transfection (data not shown). For miR-21 suppression, we employed two similar strategies, both of which use chemically synthesized modified oligonucleotides. Recently, a strategy was reported that uses 2'-O-methyl-oligoribonucleotides as sequence-specific inhibitors of miRNA function and miRNA-directed RISC activity (18, 19). These molecules stoichiometrically bind and irreversibly inactivate miRNAs, providing a valuable tool to disrupt the function of a single miRNA in vitro and in vivo. We transfected different concentrations of the 2'-O-methyl-oligonucleotide complementary to miR-21 into the cell lines and the cells were analyzed by Northern blots 2 days post-transfection. These analyses confirmed that the target miRNA became undetectable after introduction of the 2'-O-methyl-oligonucleotide in the low-nanomolar range (Fig. 2), likely due to the formation of highly stable complexes with the blocking oligonucleotide that prevents miRNA detection even under the strong denaturing conditions used in the Northern blot. The effect was sequence-specific; miR-21 was blocked by the corresponding antisense 2'-O-methyl-oligonucleotide (2'OMe-miR-21) but not by a scrambled or unrelated 2'-O-methyl-oligonucleotide (2'OMe-EGFP). Other miRNAs tested were not affected by 2'-O'-methyl-oligonucleotide complementary to the miR-21.

View larger version (53K): [in this window] [in a new window]   Figure 2. Sequence-specific knockdown of miR-21 by 2'-O-methyl- and DNA/LNA-oligonucleotides. Northern blot showing miR-21 expression after transfection with vehicle control (Mock), an irrelevant 2'-O-methyl-oligonucleotide (2'OMe-EGFP), a 2'-O-methyl-oligonucleotide complementary to miR-21 (2'OMe-miR21), an irrelevant DNA/LNA-oligonucleotide (LNA-scrambled), and a DNA/LNA-oligonucleotide complementary to miR-21 (LNA-miR-21). The same blot was reprobed for miR-103 and U6 snRNA as specificity and loading controls.

In determining the biological effects of miR-21 inhibition, we focused on two fundamental characteristics of neoplastic cells: the propensity of tumor cells to proliferate and their resistance to apoptosis. We detected a significant drop in cell number in cultures transfected with either 2'-O-methyl-miR-21 or LNA/DNA-miR-21, but not with unrelated 2'-O-methyl- or LNA/DNA-oligonucleotides (Fig. 3A). The metabolic activity of these cultures, reflecting the number of viable cells, was severely reduced with suppression of miR-21 but not with the control oligonucleotides or with suppression of two other miRNAs (miR-124a and miR-125b) that are abundant in normal and tumor brain, and this effect was seen across multiple cell lines (Fig. 3B). Repeated targeting of miR-21 in cell culture by transfections of LNA/DNA oligonucleotides further reduced the cell number. However, the cell number reduction was more modest in cells previously treated with LNA-miR21 compared with the effects on naive glioblastoma cells (Fig. 3C). Presumably, the subsequent diminished effect is due to increasing selection of resistant cells. The effect of the second transfection on remaining cells could indicate that either some cells were not efficiently transfected in the previous treatment or that some surviving cells could reexpress miR-21 and be vulnerable to later targeting.

View larger version (24K): [in this window] [in a new window]   Figure 3. Inhibition of miR-21 function leads to decreased cell number. A, cell number assay done on A172 cells after transfection with the indicated oligonucleotides. B, effect of miR-21 knockdown on cell number in several glioblastoma cell lines. C, effect of miR-21 knockdown by LNA-miR21 on naive A172 cells versus A172 cells that survived previous treatment with LNA-miR21. A, columns, mean (calculated from three separate experiments of five replicates each); bars, SE. B and C, columns, mean of four replicates in one experiment; bars, SD. P < 0.01 (z test) for all comparisons of LNA-miR-21 and 2'OMe-miR-21 with their respective controls in (A) and (B).

View larger version (27K): [in this window] [in a new window]   Figure 4. Suppression of miR-21 results in caspase activation and increased apoptosis. A, caspase-3 and caspase-7 activation after transfection with the indicated oligonucleotides. B, quantification of % apoptotic nuclei visualized by TUNEL staining after transfection with the indicated oligonucleotides. C, representative images of apoptotic nuclear TUNEL staining visualized with Cy3 (red) at 20x magnification. 4',6-Diamidino-2-phenylindole counterstain (blue) highlights all nuclei. Transfection with 2'OMe-miR21 and LNA-miR21 increased apoptosis compared with the control oligonucleotides, 2'OMe-EGFP and LNA-scrambled. LNA-miR-21 also caused focal lifting of cells (two representative panels shown). A, columns, mean (calculated from three separate experiments of five replicates each); bars, SE. B, bars, SD. P < 0.01 (z test) for all comparisons of LNA-miR-21 and 2'OMe-miR-21 with their respective controls in (A) and (B).

Expression of miR-21 is not unique to glioblastoma. Indeed, our own data show that a low level of expression is present in normal white matter in the mature brain and in a variety of other primary brain tumors. Other adult human tissues likely express miR-21 to some extent as well, as is the case in a variety of murine tissues. In the mouse, miR-21 can be detected in liver, heart, ovary, kidney, and lung at levels ranging from 1.5- to 2-fold higher than that found in the brain (22). In comparison, however, the 5- to 100-fold change in expression we have found in glioblastoma samples versus nonneoplastic brain is striking. Interestingly, miR-21 and its precursor also have been reported to be strongly expressed in cell lines established from colorectal carcinoma (HCT-116) and cervical adenocarcinoma (HeLa) but are relatively weakly expressed in cell lines from promyelocytic leukemia (HL-60), chronic myelogenous leukemia (K562), and prostatic adenocarcinoma (LNCAP; ref. 23). A recent unpublished technical note from Ambion (November 2004) has suggested that miR-21 is overexpressed in some other tumor types as well. Our analysis of colon, kidney, and breast tumor RNA, although limited for very few samples, suggests a more modest up-regulation of miR-21 expression in these tumors compared with glioblastoma multiformes (Fig. 1B; Supplementary Data 3). Interestingly, a recently published article by Cheng et al. reported that several miRNAs, including miR-21, can regulate cell growth and apoptosis in HeLa cells (24). However, in contrast to the effect in glioblastoma cells that we report here, miR-21 suppression increased growth of HeLa cells without affecting their apoptosis. In other cells lines tested, however, such a growth effect was not detected. The different biological effects of any particular miRNA, including miR-21, in different cells are likely to be highly dependent on the cell-specific repertoire of target genes. Thus, a high level of miR-21 expression and perhaps its antiapoptotic activity may seem common for some but not all types of tumors.

In summary, we have found that miR-21 is commonly and markedly up-regulated in human glioblastoma and that inhibiting miR-21 expression leads to caspase activation and associated apoptotic cell death in multiple glioblastoma cell lines. These findings suggest that overexpressed miR-21 may function as a micro-oncogene in glioblastomas by blocking expression of key apoptosis-enabling genes. To our knowledge, the presented work is the first to ascribe a function to an aberrantly expressed miRNA in human neoplasia. Moreover, it is now clear that miRNAs cannot be overlooked as an entire class of molecules critical to the biology and possibly to the treatment of human malignancies.

	Acknowledgments

 
Grant support: Stanley Robbins Memorial Research Fund from the Department of Pathology at Brigham and Women's Hospital (J.A. Chan), NIH grant NS46569 (K.S. Kosik), Childhood Brain Tumor Foundation (A.M. Krichevsky), and Mahoney Center for Neuro-Oncology Fellowship at the Dana-Farber Cancer Institute (J.A. Chan).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. D. Louis for providing glioblastoma multiforme cells, K. Sonntag and O. Isacson (Neuroregeneration Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA) for mouse ES-derived neural precursors and differentiated neural cells, F. Gage for rat astrocytic and oligodendrocytic cells differentiated from adult hippocampal progenitor cells, and M. Hemming for technical assistance.

	Footnotes

 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

J.A. Chan and A.M. Krichevsky contributed equally to this work.

The authors have no conflicting interests to report.

⁴ I. Naguibneva et al., "MicroRNAs in terminal muscle differentiation," abstract in "siRNAs and miRNAs" 2004 Keystone Symposium and article submitted for publication.

Received 1/15/05. Revised 3/31/05. Accepted 4/20/05.

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				J. Jiang, Y. Gusev, I. Aderca, T. A. Mettler, D. M. Nagorney, D. J. Brackett, L. R. Roberts, and T. D. Schmittgen Association of MicroRNA Expression in Hepatocellular Carcinomas with Hepatitis Infection, Cirrhosis, and Patient Survival Clin. Cancer Res., January 15, 2008; 14(2): 419 - 427. [Abstract] [Full Text] [PDF]

				L. B. Frankel, N. R. Christoffersen, A. Jacobsen, M. Lindow, A. Krogh, and A. H. Lund Programmed Cell Death 4 (PDCD4) Is an Important Functional Target of the MicroRNA miR-21 in Breast Cancer Cells J. Biol. Chem., January 11, 2008; 283(2): 1026 - 1033. [Abstract] [Full Text] [PDF]

				P. E. Blower, J.-H. Chung, J. S. Verducci, S. Lin, J.-K. Park, Z. Dai, C.-G. Liu, T. D. Schmittgen, W. C. Reinhold, C. M. Croce, et al. MicroRNAs modulate the chemosensitivity of tumor cells Mol. Cancer Ther., January 1, 2008; 7(1): 1 - 9. [Abstract] [Full Text] [PDF]

				D. Y. Lee, Z. Deng, C.-H. Wang, and B. B. Yang MicroRNA-378 promotes cell survival, tumor growth, and angiogenesis by targeting SuFu and Fus-1 expression PNAS, December 18, 2007; 104(51): 20350 - 20355. [Abstract] [Full Text] [PDF]

				Q. Pan, X. Luo, T. Toloubeydokhti, and N. Chegini The expression profile of micro-RNA in endometrium and endometriosis and the influence of ovarian steroids on their expression Mol. Hum. Reprod., November 1, 2007; 13(11): 797 - 806. [Abstract] [Full Text] [PDF]

				J. S. Satterlee, S. Barbee, P. Jin, A. Krichevsky, S. Salama, G. Schratt, and D.-Y. Wu Noncoding RNAs in the Brain J. Neurosci., October 31, 2007; 27(44): 11856 - 11859. [Abstract] [Full Text] [PDF]

				M. F. Corsten, R. Miranda, R. Kasmieh, A. M. Krichevsky, R. Weissleder, and K. Shah MicroRNA-21 Knockdown Disrupts Glioma Growth In vivo and Displays Synergistic Cytotoxicity with Neural Precursor Cell Delivered S-TRAIL in Human Gliomas Cancer Res., October 1, 2007; 67(19): 8994 - 9000. [Abstract] [Full Text] [PDF]

				Y. Xi, G. Nakajima, E. Gavin, C. G. Morris, K. Kudo, K. Hayashi, and J. Ju Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples RNA, October 1, 2007; 13(10): 1668 - 1674. [Abstract] [Full Text] [PDF]

				M. V. Iorio, R. Visone, G. Di Leva, V. Donati, F. Petrocca, P. Casalini, C. Taccioli, S. Volinia, C.-G. Liu, H. Alder, et al. MicroRNA Signatures in Human Ovarian Cancer Cancer Res., September 15, 2007; 67(18): 8699 - 8707. [Abstract] [Full Text] [PDF]

				W. Zhang, J. E. Dahlberg, and W. Tam MicroRNAs in Tumorigenesis: A Primer Am. J. Pathol., September 1, 2007; 171(3): 728 - 738. [Abstract] [Full Text] [PDF]

				D. Loffler, K. Brocke-Heidrich, G. Pfeifer, C. Stocsits, J. Hackermuller, A. K. Kretzschmar, R. Burger, M. Gramatzki, C. Blumert, K. Bauer, et al. Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer Blood, August 15, 2007; 110(4): 1330 - 1333. [Abstract] [Full Text] [PDF]

				S. Galardi, N. Mercatelli, E. Giorda, S. Massalini, G. V. Frajese, S. A. Ciafre, and M. G. Farace miR-221 and miR-222 Expression Affects the Proliferation Potential of Human Prostate Carcinoma Cell Lines by Targeting p27Kip1 J. Biol. Chem., August 10, 2007; 282(32): 23716 - 23724. [Abstract] [Full Text] [PDF]

				P. Sathyan, H. B. Golden, and R. C. Miranda Competing Interactions between Micro-RNAs Determine Neural Progenitor Survival and Proliferation after Ethanol Exposure: Evidence from an Ex Vivo Model of the Fetal Cerebral Cortical Neuroepithelium J. Neurosci., August 8, 2007; 27(32): 8546 - 8557. [Abstract] [Full Text] [PDF]

T. Thum, P. Galuppo, C. Wolf, J. Fiedler, S. Kneitz, L. W. van Laake, P. A. Doevendans, C. L. Mummery, J. Borlak, A. Haverich, et al. MicroRNAs in the Human Heart: A Clue to Fetal Gene Reprogramming in Heart Failure Circulation, July 17, 2007; 116(3): 258 - 267. [Abstract] [Full Text]