Downregulation ofPdcd4 by mir-21 facilitates
glioblastoma proliferation in vivo
Arti B. Gaur, Susan L. Holbeck, Nancy H. Colburn, and Mark A. Israel
Departments of Pediatrics (A.B.G., M.A.I.) Genetics (M.A.I.), Norris Cotton Cancer Center Dartmouth Medical
School, Lebanon, New Hampshire; Developmental Therapeutics Program (S.L.H.) and Laboratory of Cancer
Prevention (N.H.C.), National Cancer Institute, Bethesda, Maryland
MicroRNAs (miRNAs) are small, noncoding RNAs that
play a critical role in developmental and physiological
processes and are implicated in the pathogenesis of
several human diseases, including cancer. They function
by regulating target gene expression post-transcription-
ally. In this study, we examined the role of oncogenic
mir-21 in the pathogenesis of glioblastoma, the most
aggressive form of primary brain tumor. We have
higher levels in primary glioblastoma-tissue and glio-
blastoma-derived cell lines than in normal brain tissue.
We demonstrate that downregulation of mir-21 in glio-
expression of its target, programmed cell death 4
(Pdcd4), a known tumor-suppressor gene. In addition,
our data indicate that either downregulation of mir-21
or overexpression of its target, Pdcd4, in glioblastoma-
derived cell lines leads to decreased proliferation,
increased apoptosis, and decreased colony formation in
soft agar. Using a glioblastoma xenograft model in
immune-deficient nude mice, we observe that glioblasto-
maderived cell lines in which mir-21 levels are downre-
gulated or Pdcd4 is over-expressed exhibit decreased
tumor formation and growth. Significantly, tumors
grow when the glioblastoma-derived cell lines are trans-
fected with anti-mir-21 and siRNA to Pdcd4, confirming
that the tumor growth is specifically regulated by Pdcd4.
These critical in vivo findings demonstrate an important
functional linkage between mir-21 and Pdcd4 and
further elucidate the molecular mechanisms by which
the known high level of mir-21 expression in glioblas-
toma can attribute to tumorigenesis—namely, inhibition
of Pdcd4 and its tumor-suppressive functions.
Keywords: glioblastoma, mir-21, Pdcd4, tumor
mRNAs, causing mRNA deadenylation and degradation
or translational repression.1–3Alterations in the pattern
of gene expression resulting from aberrant expression of
miRNAs are associated with numerous diseases, includ-
ing cancer.4–7We previously assessed the expression
patterns of 241 mature human miRNAs in 59 of the
human tumor-derived cell lines that comprise the
NCI60 panel and in a set of corresponding normal
tissues.8These tumor-derived cell lines had been estab-
lished from melanoma, various leukemias, and from
cancers of the gastrointestinal tract, kidney, ovary,
breast, prostate, lung, and central nervous system
(CNS).8We found that miRNA expression patterns
may mark specific biological characteristics of tumors
and/or mediate biological activities important for their
pathobiology. We also identified miRNAs for which
the expression level in specific tumor cell lines was
either significantly increased or decreased compared
with levels observed in corresponding normal tissue,
suggesting that their function was either enhanced or
diminished in association with tumorigenesis.
Evaluating cell lines derived from glial tumors of the
CNS, we found that 5 miRNAs were expressed at higher
levels and 59 miRNAs were expressed at lower levels
than those detected in normal brain tissue.8We desig-
nated these miRNAs “candidate oncogenes” or “tumor
suppressors,” respectively. CNS tumors of glial origin
constitute the majority of primary brain tumors in
adults and are called gliomas, the most malignant of
which are known as glioblastoma. In this study, we eval-
uated the role of one candidate oncogenic miRNA,
miR-21, in mediatingthe pathologyassociated with glio-
blastoma. MiR-21 has been identified as one of the most
commonly overexpressed miRNAs in solid tumors.9,10
In glioblastoma, mir-21 is overexpressed, and its
iRNAs regulate gene expression after recog-
nition of sequence-specific binding sites, typi-
cally in the 3′-untranslated region of target
Corresponding Authors: Arti B. Gaur or Mark A. Israel, Norris Cotton
Cancer Center, Dartmouth Medical School, Lebanon, NH 03755 (arti.
firstname.lastname@example.org or email@example.com).
Received March 23, 2010; accepted March 9, 2011.
Neuro-Oncology 13(6):580–590, 2011.
Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved.
For permissions, please e-mail: firstname.lastname@example.org.
expression correlates with glioma grade.8,10–15Low
levels of mir-21 are expressed in grade II and grade III
Pdcd4, a known tumor suppressor gene, has been
Recently, mir-21 was shown to regulate Pdcd4 in glio-
blastoma.18However, the effect of mir-21 regulation of
Pdcd4 on specific biological activities of pathologic
potential (eg, apoptosis, proliferation, anchorage-inde-
pendent growth, or more significantly, in vivo growth
of glioblastoma xenografts) has, to our knowledge, not
pressive activities of mir-21 that are modulated by its
inhibition of Pdcd4.
levels areobserved in
Materials and Methods
Glioblastoma specimens and normal
samples were from the Neurosurgery Tissue Bank at
samples were obtained with informed consent after
approval of the human research committees at the
University of California and studied after approval of
the Committee for the Protection of Human Subjects
at Dartmouth-Hitchcock Medical Center.
Cell Lines and Culture Conditions
Glioblastoma-derived cell lines SNB19, U251, U87, and
SF767 were cultured in Dulbecco’s Modified Eagle’s
Medium/10% fetal bovine serum (FBS)/1% penicillin
cells were grown in a humidified incubator in 5% CO2
(10, 000 mg/mL).All
Derivation of Stable, Polyclonal Cultures and
Monoclonal Cell Lines Expressing Pdcd4
To derive stable Pdcd4-expressing polyclonal cultures,
U251 and U87 cell lines were transfected with
pcDNA-Pdcd4 (kindly provided by Dr Nancy Colburn,
National Cancer Institute, Bethesda, MD) and cells
were selected for 3 weeks with Geneticin (Invitrogen),
500 mg/mL. After this cultures were expanded and
maintained in Geneticin, 200 mg/mL. To derive stable
GFP- and Pdcd4-expressing polyclonal cultures, U87
and U251 cell lines were transfected first with pEGFP
(Clontech), and cells were selected for 3 weeks with
hygromycin, 100 mg/mL, after which cells were trans-
fected with pcDNA-Pdcd4 and selected as mentioned
above. Monoclonal U25Pdcd4or U87Pdcd4as well as
U251GFP+Pdcd4or U87GFP+Pdcd4cultures were derived
from single cells seeded in 96-well plates.
Transient Expression of Anti-mirs and siRNAs
Inhibitors-Negative/toxicity Control, and FAM dye-
labeled Anti-miR were
Biosystems/Ambion. Predesigned siRNA constructs
Ambion (Silencer Select, siRNA ID s26048). Transient
transfections were performed using siPORT NeoFX
Transfection Agent (Applied Biosystems/Ambion) in
During the transfection, cells were cultured in reduced
serum OptiMEM medium (Invitrogen).
miRNA Inhibitor,anti-miR miRNA
Real-time Quantification of microRNAs Using
Stem-loop Real-time Polymerase Chain Reaction
The expression profiles of 241 microRNAs were
measured as described previously.8This method uses
stem-loop primers for reverse transcription followed by
real-time PCR (TaqMan MicroRNA Assays; Applied
Biosystems). Expression of mature miRNAs was deter-
Biosystems). The Taqman primer-probe for quantifi-
cation of miR-21 (for the target sequence UAGCU
ous controls: 18S rRNA, b2M, glyceraldehyde-3-
phosphate dehydrogenase, and b-actin.
Western Blot Analysis to Detect Pdcd4 Protein
To obtain whole-cell lysates, cells were sonicated and
(50 mmol/L Tris-HCl, 150 mmol/L NaCl, 5 mmol/L
EDTA, 0.5% NP40, 1 mmol/L phenylmethylsulfonyl
fluoride, and complete protease inhibitor cocktail
mix [Roche]). Protein concentration was determined
with a bicinchoninic acid protein assay kit (Pierce).
For Western blot analysis, 40 mg of protein was separ-
ated on a 10% sodium dodecyl sulfate (SDS) poly-
acrylamide gel electrophoresis and transferred to a
nitrocellulose membrane. Pdcd4 was detected using a
(Rockland) at a 1:5000 dilution as the primary anti-
body, followed by a alkaline phosphatase-linked goat
1:50,000 dilution. To detect b-actin as a loading
control, a mouse monoclonal antibody to b-actin
(Abcam) was used at 1:5000 as the primary antibody,
followed by an alkaline phosphatase–linked rabbit
1:5000. After incubation of the membranes with the
specific antibodies, proteins were visualized by chemi-
luminescence (ECL; Amersham). To detect Pdcd4 in
tumors, tumor tissues were first homogenized using a
sonicator, and proteins were extracted and processed
as mentioned above.
30 minin lysis buffer
antibody used at
Gaur et al.: Downregulation of Pdcd4 by mir-21
†J U N E 2 0 1 1
22. Young MR, Yang HS, Colburn NH. Promising molecular targets for cancer
23. Allgayer H. Pdcd4, a colon cancer prognostic that is regulated by a
microRNA. Crit Rev Oncol Hematol. 2009;73:185–191.
24. Lankat-Buttgereit B, Go ¨ke R. The tumour suppressor Pdcd4: recent
advances in the elucidation of function and regulation. Biol Cell.
25. Go ¨ke R, Gregel C, Go ¨ke A, Arnold R, Schmidt H, Lankat-Buttgereit B.
Programmed cell death protein 4 (PDCD4) acts as a tumor suppressor
in neuroendocrine tumor cells. Ann N Y Acad Sci. 2004;1014:220–221.
26. Gao F, Wang X, Zhu F, et al. PDCD4 gene silencing in gliomas is associ-
ated with 5′CpG island methylation and unfavorable prognosis. J Cell
Mol Med. 2008;13:4257–4267.
27. Gao F, Zhang P, Zhou C, et al. Frequent loss of PDCD4 expression in
human glioma: possible role in the tumorigenesis of glioma. Oncol
28. Thaker NG, Pollack IF. Molecularly targeted therapies for malignant
glioma: rationale for combinatorial strategies. Expert Rev Neurother.
29. Li T, Li D, Sha J, Sun P, Huang Y. MicroRNA-21 directly targets
MARCKS and promotes apoptosis resistance and invasion in prostate
cancer cells. Biochem Biophys Res Commun. 2009;383:280–285.
30. Iorio MV, Visone R, Di Leva G, et al. MicroRNA signatures in human
ovarian cancer. Cancer Res. 2007;67:8699–8707.
31. Nam EJ, Yoon H, Kim SW, et al. MicroRNA expression profiles in serous
ovarian carcinoma. Clin Cancer Res. 2008;14:2690–2695.
32. Asangani IA, Rasheed SA, Nikolova DA, et al. MicroRNA-21 (miR-21)
post-transcriptionally downregulates tumor suppressor Pdcd4 and
stimulates invasion, intravasation and metastasis in colorectal cancer.
33. Dillhoff M, Liu J, Frankel W, Croce C, Bloomston M. MicroRNA-21 is
over expressed in pancreatic cancer and a potential predictor of survival.
J Gastrointest Surg. 2008;12:2171–2176.
34. Lui WO, Pourmand N, Patterson BK, FireA. Patterns of known and novel
smallRNAs in human cervical cancer. Cancer Res. 2007;67:6031–6043.
35. Kutay H, Bai S, Datta J, et al. Downregulation of miR-122 in rodent and
human hepatocellular carcinomas. J Cell Biochem. 2006;99:671–678.
36. Fulci V, Chiaretti S, Goldoni M, et al. Quantitative technologies establish
a novel microRNA profile of chronic lymphocytic leukemia. Blood.
37. Jongen-Lavrencic M, Sun SM, Dijkstra MK, Valk PJ, Lowenberg B.
MicroRNA expression profiling in relation to the genetic heterogeneity
of acute myeloid leukemia. Blood. 2008;111:5078–5085.
38. Yang HS, Jansen AP, Komar AA, et al. The transformation suppressor
Pdcd4 is a novel eukaryotic translation initiation factor 4A binding
protein that inhibits translation. Mol Cell Biol. 2003;23:26–37.
39. LaRonde-LeBlanc N, Santhanam AN, Baker AR, Wlodawer A, Colburn
NH. Structural basis for inhibition of translation by the tumor suppressor
Pdcd4. Mol Cell Biol. 2007;27:147–156.
40. Chang JH, Cho YH, Sohn SY, et al. Crystal structure of the
41. Yao Q, Xu H, Zhang QQ, Zhou H, Qu LH. MicroRNA-21 promotes cell
proliferation and down-regulates the expression of programmed cell
death 4 (PDCD4) in HeLa cervical carcinoma cells. Biochem Biophys
Res Commun. 2009;388:539–542.
42. BourguignonLY, SpevakCC, WongG,Xia W,Gilad E.
Hyaluronan-CD44 interaction with protein kinase C (epsilon) promotes
oncogenic signaling by the stem cell marker Nanog and the Production
of microRNA-21, leading to down-regulation of the tumor suppressor
protein PDCD4, anti-apoptosis, and chemotherapy resistance in breast
tumor cells. J Biol Chem. 2009;284:26533–26546.
43. Selaru FM, Olaru AV, Kan T, et al. MicroRNA-21 is overexpressed in
human cholangiocarcinoma and regulates programmed cell death 4
andtissue inhibitorof metalloproteinase3.
44. Frankel LB, Christoffersen NR, Jacobsen A, Lindow M, Krogh A, Lund
AH. Programmed cell death 4 (PDCD4) is an important functional
target of the microRNA miR-21 in breast cancer cells. J Biol Chem.
45. Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T.
MicroRNA-21 regulates expression of the PTEN tumor suppressor
gene in human hepatocellularcancer.
46. Schmid T, Jansen AP, Baker AR, Hegamyer G, Hagan JP, Colburn NH.
Translation inhibitor Pdcd4 is targeted for degradation during tumor
promotion. Cancer Res. 2008;68:1254–1260.
47. Zhu S, Si ML, Wu H, Mo YY. MicroRNA-21 targets the tumor
suppressor genetropomyosin 1 (TPM1). J Biol Chem. 2007;282:
48. Zhu S, Wu H, Wu F, Nie D, Sheng S, Mo YY. MicroRNA-21 targets
tumor suppressor genes in invasion and metastasis. Cell Res.
49. Wickramasinghe NS, Manavalan TT, Dougherty SM, Riggs KA, Li Y,
Klinge CM. Estradiol downregulates miR-21 expression and increases
miR-21 target gene expression in MCF-7 breast cancer cells. Nucleic
Acids Res. 2009;37:2584–2595.
50. Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY. miR-21-mediated tumor
growth. Oncogene. 2007;26:2799–2803.
51. Zhou X, Ren Y, Moore L, et al. Downregulation of miR-21 inhibits EGFR
pathway and suppresses the growth of human glioblastoma cells inde-
pendent of PTEN status. Lab Invest. 2010;90:144–155.
Gaur et al.: Downregulation of Pdcd4 by mir-21
†J U N E 2 0 1 1