Chronic lymphocytic leukemia modeled in mouse by
targeted miR-29 expression
Urmila Santanama, Nicola Zanesia, Alexey Efanova, Stefan Costineana, Alexey Palamarchuka, John P. Hagana,
Stefano Voliniaa, Hansjuerg Aldera, Laura Rassentib, Thomas Kippsb, Carlo M. Crocea,1, and Yuri Pekarskya,1
aHuman Cancer Genetics Program and Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University School of Medicine, Ohio
State University, Columbus, OH 43210; andbDepartment of Medicine, University of California at San Diego, La Jolla, CA 92093
Contributed by Carlo M. Croce, May 24, 2010 (sent for review May 10, 2010)
B-cell chronic lymphocytic leukemia (B-CLL), the most common
leukemia in the Western world, occurs in two forms, aggressive
(showing for the most part high ZAP-70 expression and unmutated
IgH VH) and indolent (showing low ZAP-70 expression and mutated
CLL as compared with aggressive B-CLL and normal CD19+B cells. To
study the role of miR-29 in B-CLL, we generated Eμ-miR-29 trans-
genic mice overexpressing miR-29 in mouse B cells. Flow cytometric
analysis revealed a markedly expanded CD5+population in the
spleen of these mice starting at 2 mo of age, with 85% (34/40) of
miR-29 transgenic mice exhibiting expanded CD5+B-cell popula-
tions, a characteristic of B-CLL. On average, 50% of B cells in these
transgenic mice were CD5 positive. At 2 y of age the mice showed
significantly enlarged spleens and an increase in the CD5+B-cell
population to ∼100%. Of 20 Eμ-miR-29 transgenic mice followed
to 24–26 mo of age, 4 (20%) developed frank leukemia and died
of the disease. These results suggest that dysregulation of miR-29
can contribute to the pathogenesis of indolent B-CLL.
∼10,000 new cases observed each year in the United States.
Characteristically, CLL is a disease of elderly people, with the in-
cidence increasing linearly with each decade above age 40 y (1, 2).
It is known that this disease is characterized by the clonal ex-
pansion of CD5+B cells (2).
which regulate gene expression at the transcriptional or trans-
lational level (3). Approximately half of human microRNAs are
located at fragile sites and genomic regions involved in alterations
in cancers (4), and alteration of microRNA expression profiles
occurs in most cancers, suggesting that individual microRNAs
could function as tumor suppressors or oncogenes (5).
The 13q14 deletion is the most common CLL aberration and is
detected by cytogenetic analysis in approximately half of the
cases (6). Analysis of a deletion at 13q14.3 led to the discovery of
two physically linked microRNAs, miR-15a and miR-16–1, as
targets of these deletions (7). Consequently, miR-15a and miR-
16–1 expression is reduced in the majority of CLL cases (7), and
further studies indicated that miR-15a/miR-16–1 negatively reg-
ulate Bcl2 expression (8). These findings indicated that micro-
RNAs play important roles in CLL and that down-regulation of
miR-15/16 and subsequent Bcl2 up-regulation contribute to CLL
pathogenesis (7). Because miR-15/16 was identified as a tumor
suppressor in indolent CLL, the microRNA expression profile in
CLL has been studied extensively, and a signature profile was
reported describing 13 microRNAs that differentiate aggressive
and indolent CLL (4).
We and others observed that miRNA-29 expression is down-
hronic lymphocytic leukemia (CLL) is the most common
human leukemia, accounting for ∼30% of all cases (1), with
and CDK6 (9, 11, 12). On the other hand, one report showed that
miR-29 expression is up-regulated in metastatic breast cancer, and
a very recent study reported that miR-29 overexpression can cause
acute myeloid leukemia(AML) inmice(13, 14).Toclarify therole
of miR-29 in B-cell leukemias, we generated transgenic mice over-
expressing miR-29 in B cells and now report the phenotype of this
MiR-29 Expression in CLL and Production of the Eμ-miR-29 Transgenic
Mouse Model. As noted above, we have reported previously that
indolent) CLL (9, 10), but data comparing miR-29 expression in
CLL and normal CD19+B cells was not available. To determine
expression levels of miR-29 in CLL and normal CD19+B cells, we
studied the expression of miR-29a and miR-29b in 29 aggressive
cell controls. Fig. 1 A and B shows real-time RT-PCR results in
these samples. MiR-29a expression was 4.5-fold higher in indolent
CLL than in normal CD19+B cells, whereas aggressive CLL
samples showed a 3.2-fold increase. Similarly, miR-29b expression
was increased 4-fold in indolent CLL and 3.5-fold in aggressive
CLL compared with normal CD19+B cells. Both miR-29a and
miR-29b were down-regulated in aggressive versus indolent CLL,
confirming previous observations (9), although in the case of miR-
29b this difference was not statistically significant (Fig. 1B). In-
terestingly, in all samples miR-29a expression level was more than
20-fold higher than that of miR-29b (Fig. 1 A and B).
Because expression levels of miR-29a and miR-29b were sig-
hypothesized that miR-29 could contribute to the pathogenesis of
CLL. To investigate this possibility, we generated transgenic mice
by a VHpromoter-IgH-Eμ enhancer (15), along with humanized
renilla green fluorescent protein (hrGFP), and the simian virus 40
(SV40) poly(A) site. This promoter/enhancer combination drives
The miR-29a/b cluster sequence was inserted within the intron of
this construct (Fig. 1C). Two founders on FVB/N background,
designated “F1” and “F2,” were generated and bred to establish
the transgenic lines. Expression of miR-29a and miR-29b was ex-
amined by Northern blot analysis, using RNAs isolated from
of miR-29a and miR-29b in both transgenic lines (F1 and F2)
compared with nontransgenic (WT) siblings. To confirm that the
transgene is expressed in B cells, we performed flow cytometry
Author contributions: C.M.C. and Y.P. designed research; U.S., N.Z., A.E., S.C., A.P., J.P.H.,
and H.A. performed research; L.R. and T.K. contributed new reagents/analytic tools; U.S.,
N.Z., A.E., S.C., S.V., H.A., C.M.C., and Y.P. analyzed data; and Y.P. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or pekarsky.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 6, 2010
| vol. 107
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using CD19 as a B-cell marker. Fig. 1F shows that all CD19+cells
in both transgenic lines also express GFP (F1 and F2), whereas no
GFP expression was detected in WT littermates.
Eμ-miR-29 Transgenic Mice Show CLL Phenotype. We used flow
cytometry to determine the immunophenotypic profile of spleen
lymphocytes from miR-29 transgenic mice. At the age of 12–24
B-cell population (a characteristic of CLL) in the spleen of 34
of 40 (85%) miR-29 transgenic mice; ∼50% of B cells in these
transgenic mice were CD5+. Fig. 2A (Left) shows a representative
example. Although almost all spleen B cells from this animal were
CD5+CD19+IgM+, these cells represented only 25–30% of all
spleen lymphocytes. A more advanced CLL case is shown in Fig.
2A (Center). Almost all normal lymphocytes in the spleen of this
animalwere replaced by malignant CD5+CD19+IgM+B cells.As
expected, almost no CD5+CD19+IgM+B cells were detected in
spleens of WT littermates (Fig. 2A, Right). The expanded pop-
ulation of CD5+CD19+B cells also was detected in peripheral
blood and bone marrow from miR-29 transgenic mice, but not
from WT littermates (Fig. 2, B and C). Fig. 2 D–F shows the
number of animals with increased CD5+CD19+IgM+pop-
ulations in spleen. Although only 7 of40 (17%)miR-29 transgenic
more CD5+CD19+IgM+cells. In addition, miR-29 transgenic
mice showed significant increases in the percentage of CD5+
splenic B cells with age (Fig. 2F). In animals younger than 15 mo,
CD5+Bcellsrepresentedonly∼20%oftotalB cells;by15–20 mo
of age, that percentage increased to ∼40% (Fig. 2F). At the age of
20–26 mo, on average, >65% of all B cells were CD5+(Fig. 2F).
These data suggest gradual progression of indolent CLL in miR-
29 transgenic mice. We followed 20 Eμ-miR-29 mice to the age of
24–26 mo. Almost all these mice showed significantly enlarged
spleens, and 4 of 20 (20%) developed frank leukemia and died of
disease. Fig. 2G shows a representative case of frank leukemia
presenting with an enlarged spleen and liver and advanced
Clonal IgH gene rearrangements are typical in human CLL
cases (1). These rearrangements also were observed in the Tcl1-
driven mouse model of CLL (15). To determine if CD5+B cells
from Eμ-miR-29 transgenic mice show clonality, we carried out
Southern blot hybridization using spleen lymphocyte DNA iso-
lated from cases showing at least 50% CD5+CD19+IgM+B cells.
Fig. 2H shows clonal rearrangements of the IgH gene in three of
five cases analyzed. These results further indicate that the expan-
sion of CD5+B cells in Eμ-miR-29 mice resembles human CLL.
To confirm further that Eμ-miR-29 mice develop CLL-like
disease, we carried out histological and immunohistological
analysis. Fig. 3 A–C shows representative smears from blood of
Eμ-miR-29 transgenic mice and a WT control. The smear from
a WT mouse showed rare lympho-monocytes with a normal ap-
pearance (Fig. 3A). In contrast, the smear from a Eμ-miR-29
mouse with low-grade CLL exhibited an increased number of
from a miR-29 transgenic mouse with advanced CLL presented
numerous malignant lymphoid cells (Fig. 3C), including smudge
cells, typical of CLL (Fig. 3C, Inset; smudge cells are indicated by
arrowheads). Fig. 3 D–L shows representative histological images
of Eμ-miR-29 transgenic mice and a WT control. The spleen of
the WT mouse shows preserved architecture and several normal-
looking lymphoid follicles (Fig. 3D, green arrow). In contrast, the
spleen of a diseased miR-29 transgenic mouse with CLL exhibits
with advanced CLL shows total obliteration of the normal ar-
chitecture by malignant lymphoid proliferation (Fig. 3F). B220
staining of the same sections shows a lymphoid follicle of a WT
mouse presenting a normal B-cell disposition (Fig. 3G). In con-
trast, transgenic spleens show lymphoid follicles in disarray be-
or CLL with diffuse distribution of a B-cell malignant population
(Fig. 3I). Fig. 3 J–L shows low expression of cyclin D1 in a WT
spleen (Fig. 3J) and moderate to high cyclin D1 expression in low-
grade CLL (Fig. 3K) and advanced CLL (Fig. 3L). In summary,
histological and immunohistological examination confirmed that
Eμ-miR-29 mice develop CLL-like disease.
As noted above, only 20% of Eμ-miR-29 transgenic mice de-
veloped advanced leukemia and died from the disease. Fig. S1
shows a representative advanced case of CLL that invaded liver
and kidney. Histological examination showed total obliteration
of the normal spleen architecture with high expression of B220,
cyclin D1, and Ki67 (Fig. S1 A–D). These B220+malignant B
cells invaded liver (Fig. S1 E–H) and kidney (Fig. S1 I–L).
Recent investigations showed that accumulation of CLL lym-
phocytes can result not only from prolonged survival but also from
proliferating CD5+B220+cells originating in the bone marrow,
lymph nodes, or spleen (16–18). To determine whether CLL cells
from Eμ-miR-29 mice proliferate, we used cell cycle analyses based
on BrdU incorporation. We assessed the proliferative capacity of
B220+CD5+, as well as B220+CD5−transgenic splenic lympho-
shows that B220+CD5+B cells from Eμ-miR-29 mice proliferate,
(2.7%and5.6% cells inS-phase fortransgenic B cells versus0.3%
and 0.5% for WT B cells (Fig. 4 I and J versus C and D). Interest-
proliferation compared with B220+WT B cells, with 1.0% and
founder mice. (A) MiR-29a and (B) miR-29b expression in aggressive and
indolent CLL. (C) Eμ-miR-29 construct. (D and E) Expression of (D) miR-29a
and (E) miR-29b in splenic lymphocytes of Eμ-miR-29 founders. (F) Expression
of GFP in splenic lymphocytes of Eμ-miR-29 founders.
MiR-29 expression in CLL and production of Eμ-miR-29 transgenic
Santanam et al.PNAS
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| vol. 107
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G and H versus C and D. These data suggest that miR-29 over-
expression promotes B-cell proliferation, even in CD5−cells.
Human CLL is characterized by immune incompetence and
progressive severe hypogammaglobulinemia that eventually de-
velops in almost all patients (19). To determine if Eμ-miR-29 mice
develop hypogammaglobulinemia, we compared levels of serum Ig
in transgenic mice and in WT littermates at age ∼18 mo. Fig. 4K
shows that the levels of IgG1, IgG2a, and IgG2b were decreased
2- to 4-fold in Eμ-miR-29 transgenic mice as compared with WT
controls. To determine if Eμ-miR-29 mice show impaired im-
mune response, we compared levels of anti-sheep RBC (SRBC)
antibodies after injection of SRBC in miR-29 transgenic mice
and WT siblings. Fig. 4L shows that serum levels of anti-SRBC
antibodies were decreased ∼4-fold in serum of miR-29 trans-
genic mice compared with age-matched WT mice. These data
clearly indicate that, as in human CLL, the CLL-like disease in
Eμ-miR-29 mice is characterized by hypogammaglobulinemia
and immune incompetence.
Previously, we reported development and characterization of
29 can target TCL1 expression (9). In this mouse model, the TCL1
ORF (lacking 3′ UTR) was under the control of a VHpromoter-
transgenic construct, miR-29could not inhibitTCL1expression in
these mice. Eμ-TCL1 transgenic mice develop aggressive CLL,
and all mice die of the disease at 12–15 mo of age (15). To de-
TCL1 transgenic mice, we crossed Eμ-miR-29 and Eμ-TCL1
transgenic mice. Eμ-miR-29/Eμ-TCL1 mice and their Eμ-TCL1
representative FACS analysis of spleen lymphocytes of these
genotypes. TCL1/miR-29 double transgenic mice showed signifi-
compared with Eμ-TCL1 mice (93.9% and 93.3% versus 48.3%
and 50%). On average, Eμ-miR-29/Eμ-TCL1 mice had 40% more
CD5+CD19+splenic B cells and 3-fold increases in spleen weight
blood, and (C) bone marrow. (D–F) Analysis of CD5+B-cell populations in miR-29 transgenic mice and WT controls. (G) Gross pathology of a representative Eμ-
miR-29 transgenic mouse showing advanced CLL and a WT control of the same age. (H) Analysis of IgH gene configuration by Southern blot: spleen lym-
phocyte DNA isolated from five representative cases showing at least 50% CD5+CD19+B cells. Clonal rearrangements are indicated by asterisks.
Eμ-miR-29 mice develop CLL. (A–C) Flow cytometric analysis of miR-29 transgenic (Tg) and control lymphocytes isolated from (A) spleen, (B) peripheral
by arrowheads. Atypical lymphoid cells are indicated by black arrowheads.
A normal lymphoid follicle is indicated by a green arrow.
Histopathological analysis of Eμ-miR-29 mice. Smudge cells indicated
| www.pnas.org/cgi/doi/10.1073/pnas.1007186107 Santanam et al.
that miR-29 can contribute to the pathogenesis of CLL in-
dependently of Tcl1.
Analysis of miR-29 Targets. To determine whether miR-29 over-
expression in mouse B cells affects expression of its targets, we
analyzed expression levels of several previously reported miR-29
targets, Cdk6 (11), Mcl1 (12), and DNMT3A (22), in sorted
B220+B cells from miR-29 transgenic mice and WT controls.
We found that two targets, Cdk6 and DNMT3A, are down-
regulated in miR-29 transgenic mice, whereas no differences in
Mcl1 and Pten were detected (Fig. 6A) [although Pten is not
a proven miR-29 target, it previously was predicted to be a po-
tential target (14)].
Because Cdk6 and DNMT3 are not known to be tumor sup-
potential miR-29 targets contributing to its oncogenic activity.
Using microarray analysis, we compared gene expression in
sorted B220+B cells from miR-29 transgenic mice and WT
controls. We then cross-referenced genes down-regulated in miR-
function with the list of potential miR-29 targets obtained from
Targetscan software (Whitehead Institute for Biomedical Re-
search at MIT). We identified three potential targets: peroxidasin
(PXDN), a p53-responsive gene down-regulated in AML (23, 24);
Bcl7A, a proapoptotic gene down-regulated in T-cell lymphomas
(25); and ITIH5, a member of the inter-α-trypsin inhibitor family
down-regulated in breast cancer (26). Fig. 6 B and C shows the
of miR-29a and corresponding 3′ UTRs. To determine if miR-29
indeed targets expression of PXDN, Bcl7A, and ITIH5, we inserted
3′ UTR fragments (including miR-29 homology regions) of these
cDNAs downstream of the luciferase ORF into pGL3 vector, as
previously described (8). HEK293 cells were cotransfected with
miR-29a, miR-29b, or scrambled negative control and a pGL3
construct containing fragments of PXDN, Bcl7A, and ITIH5
cDNAs, including a region homologous to miR-29, as indicated
decreased luciferase expression of the construct containing the
3′ UTR of PXDN, whereas no significant effect was observed for
Bcl7A and ITIH5 (Fig. 6D). Thus, we concluded that PXDN ex-
used full-length PXDN cDNA including 5′ and 3′ UTRs in a cyto-
megalovirus mammalian expression vector and investigated
whether miR-29 expression affects Pdxn protein expression levels.
Wecotransfectedthisconstruct with miR-29a,miR-29b,orPremiR
6E. These experiments revealed that coexpression of PXDN with
miR-29a or miR-29b almost completely inhibited Pxdn expression
could play a role in the pathogenesis of human CLL, we studied
B-cell controls. Fig. 6F shows real-time RT-PCR results in these
samples.PXDNexpression was drastically down-regulated (50-fold
or more) in CLL samples compared with normal CD19+B cells.
These results suggest that the oncogenic role of miR-29 in B cells
might be, at least in part, dependent on targeting peroxidasin.
Although many microRNAs are up- or down-regulated in a num-
ber of solid tumors and hematological malignancies, and several
are postulated to function as tumor suppressors or oncogenes (5),
only three reports have used mouse models to demonstrate that
dysregulation of microRNAs can cause cancer. The first report
showed that overexpression of miR-155 in B cells results in pre–B-
cell leukemia in mice (27). The knockout of miR-15/16 led to
development of CLL with low penetrance (28, 29). Finally, a very
recent report showed that overexpression of miR-29 can cause
AML in mice (14). Although nearly all published reports conclude
that miR-155 functions as an oncogene and that miR-15/16 is
a tumor suppressor, the function of miR-29 in this respect has not
been clearly defined. Previous reports showed that miR-29 in-
hibited tumorigenicity in lung cancer (22), its expression was
down-regulated and correlated with poor prognosis in mantle cell
lymphoma (11), and its expression caused apoptosis in AML (30).
On the other hand, miR-29 expression was up-regulated in meta-
static breast cancer (13), its overexpression in mouse myeloid cells
caused AML (14), and in this report we show that miR-29 over-
expression in B cells results in CLL. For AML, several reports
defined miR-29 as a tumor suppressor that functions by targeting
oncogenes such as MCL1 and CDK6 and promoting apoptosis in
AML cells (30, 31). A very recent study reported directly opposite
results, showing that miR-29 is overexpressed in some AML
into transgenic B220+CD5+and B220+CD5−B-cell DNA. (K) Ig levels in serum of WT and transgenic animals. (L) Levels of anti-SRBC–specific antibodies in serum
of WT and transgenic animals 7 d after SRBC injection.
Cell-cycle analysis of leukemic cells from Eμ-miR-29 transgenic mice. (A–D) BrdU incorporation into DNA of WT B220++B cells. (E–J) BrdU incorporation
Santanam et al.PNAS
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| vol. 107
| no. 27
and that its up-regulation causes AML in a mouse model (14).
Although the role of miR-29 in AML needs to be sorted out, it is
likely that miR-29 can function as a tumor suppressor or oncogene
depending on the cellular context.
We and others previously reported that miR-29 is down-regulated
in aggressive CLL as compared with indolent CLL; we also
demonstrated that miR-29 is one of the microRNAs targeting
TCL1, a critical oncogene in the pathogenesis of aggressive CLL
(9, 10). Here we report that miR-29 is overexpressed in indolent
CLL compared with normal B cells. Because only 20% of Eμ-
miR-29 transgenic mice died of leukemia in old age, but almost
all mice showed expanded CD5+CD19+B-cell populations, the
phenotype of Eμ-miR-29 is similar to that of indolent CLL.
Therefore up-regulation of miR-29 initiates or at least signifi-
cantly contributes to the pathogenesis of indolent CLL. On the
other hand, TCL1 is mostly not expressed in indolent CLL (9)
and probably does not play an important role in indolent CLL.
Our current hypothesis is that miR-29 overexpression is not
sufficient to initiate aggressive CLL. In contrast, up-regulation of
Tcl1 is a critical event in the pathogenesis of the aggressive form
of CLL. Because miR-29 targets TCL1, its down-regulation in
aggressive CLL (compared with the indolent form) contributes
to up-regulation of Tcl1 and the development of an aggressive
Materials and Methods
Eμ-miR-29 Transgenic Mice and Human CLL Samples. A 1.0-kb fragment con-
taining mouse miR-29ab clusterwascloned into theBamHIand SalIsites ofthe
plasmid containing a mouse VHpromoter (V186.2) and the IgH-Eμ enhancer
(15) along with the hrGFP and the SV40 poly(A) site. The miR-29a/b cluster
sequence was inserted within the intron of this construct. Transgenic mice
were produced in Ohio State University transgenic mouse facility. Genotyping
was performed on tail DNAs by PCR using theprimers: miR29d: gctgac gtt gga
gcc aca ggt aag; miR29r: aca aat tcc aaa aat gac ttc cag. Human CLL samples
were obtained from the Chronic Lymphocytic Leukemia Research Consortium
after informed consent was obtained from patients diagnosed with CLL. Re-
search was performed with the approval of the Institutional Review Board of
The Ohio State University. RNA extraction was carried out as previously de-
scribed (32). Real-time PCR experiments were carried out using miR-29a, miR-
29b, and PXDN assays for real-time PCR (Applied Biosystems) according to the
manufacturer’s protocol. Control human cord blood CD19+B cells were pur-
chased from Allcells and Lonza.
Characterization of miR-29 Transgenic Lymphocytes. Lymphocytes from
spleens and bone marrow were isolated as previously described (27). Flow
cytometry measurements of SRBC immune response, Ig levels, and pro-
liferation of B-cell populations were carried out as previously described (21).
To analyze IgH gene rearrangements, Southern blot analysis of spleen
lymphocyte DNA was carried out using EcoRI (Roche) digestions and mouse
JH4 probe as previously described (15). For histology and immunohisto-
chemistry, mice were necropsied, and spleens, livers, and kidneys were fixed
in 10% buffered formalin, included in paraffin, and then cut in 4-μm sections
blot analysis of Cdk6, DNMT3A, PTEN, and Mcl1 expression in CD19+B cells
of miR-29 transgenic and WT mice. (B) Microarray expression data for PXDN,
BCL7A, and ITIH5 in CD19+B cells of miR-29 transgenic and WT mice. (C)
Sequence alignments of miR-29a and 3′ UTRs of PXDN, BCL7A, and ITIH5. (D)
miR-29 targets PXDN but not BCL7A and ITIH5 expression in luciferase assays.
(E) Effect of miR-29 on Pxdn protein expression. (F) PDXN expression in CLL.
Analysis of miR-29 targets in Eμ-miR-29 transgenic mice. (A) Western
Flow cytometric analysis of Eμ-TCL1/Eμ-miR-29 and Eμ-TCL1 transgenic lym-
phocytes from spleen. (B) Percentage of CD5+B cells in Eμ-TCL1/Eμ-miR-29
and Eμ-TCL1 transgenic spleen lymphocytes. (C) Spleen weight from Eμ-TCL1/
Eμ-miR-29 and Eμ-TCL1 transgenic mice.
Mir-29 transgene expression accelerates CLL in Eμ-TCL1 mice. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1007186107 Santanam et al.
as previously described (27). Sections were stained with H&E according to Download full-text
standard protocols (27).
Analysis of miR-29 Targets. B cells were isolated using a B-cell isolation kit
(Miltenyi Biotec) according to the manufacturer’s instructions. Proteins from
spleens were extracted as previously described (33). Western blot analysis
was carried out using Cdk6 (H-96; Santa Cruz Biotechnology), DNMT3A
(2160; Cell Signaling Technology), Pten (mmac1; Lab Vision), Mcl1 (S-19;
Santa Cruz Biotechnology), Pdxn (Novus), and GAPDH (2118; Cell Signaling
Technology) antibodies. For luciferase assays, fragments of PXDN, BCL7A,
and ITIH5 cDNA, including regions complimentary to miR-29, were inserted
into a pGL3 vector using the XbaI site immediately downstream from the
stop codon of luciferase, as previously described (9). MiR-29a, miR-29b, and
scrambled control RNA duplexes were purchased from Ambion. The ex-
pression construct containing full-length human PXDN was purchased from
OriGene. Transfections were carried out as previously described (34).
ACKNOWLEDGMENTS. We thank Dr. Kay Huebner for critically reviewing
the manuscript and Vadim Maximov for technical assistance. The research
was supported by an American Cancer Society Research Scholar grant to Y.P.
and by National Institutes of Health Grant PO1-CA81534 of the Chronic
Lymphocytic Leukemia Research Consortium to L.R., T.K., and C.M.C.
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| vol. 107
| no. 27