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 Pdf file on Genetic variation of miRNA
sequence in pancreatic cancer
Zheng Zhu1†, Wentao
Gao2†, Zhuyin Qian2*, and Yi Miao2*
1Department of General Surgery, The 2Department of General Surgery, The First Affiliated
Hospital of
† These
authors contributed equally to this work.
*Correspondence address. Zhuyin
Qian: Tel: t86-25-83718836; Fax: t86-25-83724440; E-mail:
[email protected].
Yi Miao: Tel: t86-25-83718836; Fax: t86-25-83724440; E-mail: [email protected]
MicroRNAs (miRNAs)
are small non-coding RNAs of 20–22 nucleotides (nts)
and constitute a novel class of gene regulators that negatively regulate gene
expression at the post-transcriptional level. The expression of miRNA is deregulated in many types of cancers. Alterations
in miRNA expression may be an important contributor
to the development of pancreatic carcinoma. We hypothesized that genetic
variations in miRNA genes were associated with
pancreatic carcinoma and analyzed genomic sequences coding for the precursors
of eight miRNA genes in both pancreatic carcinoma
tissues and cancer cell lines. Four novel mutations in primary miRNA transcripts were identified. TaqMan
miRNA assays showed that miR-21 was significantly overexpressed in 20 pancreatic carcinomas and 6 cancer cell
lines compared with paired benign tissues and normal pancreas. Two mutations of
miR-21 did not notably alter the activity of the promoter of the miRNA gene. Although most of these mutations seem to have
no effect on miRNA processing, an A–G mutation at 29-nt downstream of pre-miR-21
led to a conformational change of the secondary structure close to the stem
reaching into the pre-miR-21 and a relative reduction of the mature miR-21
expression in vivo. These results suggested that miRNA might play an important role in pancreatic tumorigenesis, but the molecular mechanism underlying the
particular sequence variations in miRNA that can
cause aberrant expression remains to be determined.
Keywords microRNAs; pancreatic cancer; promoter; secondary structure
Received: November 23, 2008 Accepted: March 10, 2009
Introduction
Pancreatic cancer is a highly lethal disease,
characterized by late clinical presentation, early and aggressive local
invasion, metastatic potential, strong resistance to chemotherapy and radiation
therapy, and most importantly, a very poor overall prognosis [1,2]. Many
previous studies have attempted to elucidate the molecular mechanisms
underlying pancreatic tumorigenesis, but it is still
not fully understood. Therefore, a better understanding of the genes involved
in tumor growth and progression is necessary for the development of novel
diagnostic and therapeutic strategies that can improve the treatment of this
deadly disease.
A recently identified class of non-coding small RNAs, microRNAs (miRNAs), may provide new insights in cancer research. miRNAs are small non-coding RNAs
of 20–22 nucleotides and constitute an abundant class of gene
regulators that negatively regulate gene expression at the post-transcriptional
level. They bind with the 3′ untranslated regions of their mRNA targets
and repress target-gene expression by mRNA degradation or translational
repression. miRNAs are initially transcribed from
genomic DNA as large primary miRNA (pri-miRNA) transcripts that are then processed by the RNase III enzyme Drosha, in the
nucleus into hairpin-shaped pre-miRNAs. Pre-miRNAs are exported to the cytoplasm through Exportin-5,
and further processed by another RNase III enzyme
Dicer, into mature miRNAs [3–5]. A total of 678 human miRNAs
have been reported (April 2008 release of miRBase at
the Sanger Institute). Previous results indicated that miRNAs
could function as tumor suppressors and oncogenes,
and the miRNAs with a role in cancer were designated
as oncogenic miRNAs (oncomirs) [3]. At the present time, the main mechanism that
underlies changes in the function of miRNAs in cancer
cells seems to be aberrant gene expression, characterized by abnormal levels of
expression for mature and/or precursor miRNA
sequences compared with the corresponding normal tissues. The global miRNA expression patterns of many tumors have been
described [6–10] and
the miRNA expression signature that is associated
with pancreatic cancer has also been identified [11–13]. The precise mechanisms regulating miRNA expression and maturation are largely unknown, but
studies have suggested several mechanisms, including genetic and epigenetic
alterations [14,15]. A mutation or a single nucleotide polymorphism (SNP) at
the miRNA gene region might affect the transcription
of pri-miRNA transcripts, the processing of miRNA precursors to mature miRNAs,
or miRNA–target
interactions [16]. This study was designed to identify the genetic alterations
(mutations) of miRNAs in pancreatic cancer and to
investigate the function of these mutations.
Materials and Methods
Materials
Twenty pancreatic carcinoma tissues were collected from
patients who had undergone resection for ductal adenocarcinoma of the pancreas between 2006 and 2008 from
the First Affiliated
Identification of miRNA
mutations
First we performed bioinformatics analyses of the
available cDNA-microarray data [11–13], array-based comparative genomic hybridization (aCGH) data [17–21], miRBase miRNA target-gene data
(http://microrna. sanger.ac.uk/targets/v5/), and relative articles in other
tumors [6–10]. Then we chose the following eight miRNAs that might be relevant to pancreatic tumorigenesis for sequence analysis: miRAll specimens were sent to Invitrogen
Corporation in
TaqMan quantitative RT-PCR
Quantitative RT-PCR was used to determine the expression
of miRNA hsa-miR-21 (homo-sapiens miR-21). cDNA was synthesized from 10 ng
of total RNA and real-time PCR was performed with TaqMan
2´ Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems,
Promoter assay
The sequencing region of the interested miRNA gene was amplified by PCR and inserted into
pGL3-Basic luciferase reporter vector (Promega,
Prediction of secondary structure of miRNA precursor
The RNAfold web server
(http://rna.tbi.univie.ac.at/ cgi-bin/RNAfold.cgi)
was used to predict the secondary structure of pre-miRNA
[23]. Default parameters were employed. We used this program to predict the
most stable secondary structure of both the wild-type (WT) and variant
sequences. The sequence applied for prediction analysis included pre-miRNA and about 200 bp upstream
and about 100 bp downstream flanking sequences at each
end of the precursor. In all cases, folding structures with minimal free energy
were depicted.
Results
MiRNA mutations identified
from cancer cell lines and human tumor tissues
To investigate whether mutations in miRNA
genes play a role in the development of pancreatic cancer, we screened
approximately 20 human pancreatic cancer tissues and 6 human cancer cell lines
for analysis of 8 human miRNAs in the present study.
We amplified the genomic DNA containing the miRNA
precursor for each miRNA gene, and its flanking
sequences at both ends, and used the amplified PCR products for sequence
analysis. When a mutation was identified in a cancer cell line, the
human tumor tissues were also further screened. A total of four novel mutations
in two miRNAs, including miR-21 and miR-155, from two
pancreatic cancer cell lines (BXPC-3, SW1990) and one human tumor specimen
(Case 233) were identified in the screening (Table 2). These
mutations were confirmed by repeated PCR amplification and sequencing of the
re-amplified products. We further analyzed genomic DNA from five normal
individuals to determine whether the identified mutations are associated with
human cancer. We found no mutations in the normal subjects, suggesting that the
mutations may be potentially associated with human cancer. Further studies of
the adjacent unaffected tissues from the same patients (Case 233) suggested
that the mutation was germline mutation. Among these
four mutations, all of them were in the pri-miRNA
regions (Fig. 1).
Expression profile of miRTotal RNAs were extracted from
the 6 cancer cells lines, 19 pancreatic cancer tissues, 12 benign adjacent
pancreatic tissues and 4 normal pancreatic tissues, and the expression level of
mature miR-21 was evaluated by TaqMan miRNA assays. The results revealed that miR-21 was overexpressed in both cancer cell lines and pancreatic
cancer tissues relative to benign pancreatic tissue with P, 0.05 [Fig. 2(A)]. These
data correspond to the cDNA-microarray data [11–13], indicating that miR-21 may function as a
proto-oncogene Effect of mutations on miRNA
transcription Functional analysis was performed to determine whether
the identified mutations of miRNA genes could affect
the expression (transcription) of the mutated miRNA in
vivo. We
cloned both WT and mutated alleles of the miR-21 gene into a mammalian
expression vector. BXPC-3 cells were transfected with
the constructed expression vectors. Total luciferase
reporter enzymes were extracted from the transfected
cells and the luminescent signal from each sample was quantified by Dual-Luciferasew Reporter Assay (Promega). The
average intensities of the luminescent signal of the three specimens (relative
to pRL-SV40 control vector) were, respectively, 0.8818 (A t
Effects of sequence variations on the
secondary structures of miRNA precursors
The RNase III-mediated
processing and maturation of a miRNA precursor in
vivo lead to
the final product, a functional mature miRNA. Thus,
it is expected that the processing and maturation of a miRNA
precursor require appropriate secondary structures and specific sequence
elements within the miRNA precursor or pri-miRNA [15,24–26]. Interestingly,
while most of the mutations identified in this study did not cause major
conformational changes (data not shown), the A t
Discussion
A strong link between mutations, aberrant expression, or
altered mature miRNA processing and cancer
susceptibility and progression has been demonstrated over the past few years. A
germline mutation in the primary precursor of
miR-16-1–miRmiR-21 and miR-155, were identified.
It has also been shown that miRNA
expression patterns are relevant to the biologic and clinical behavior of human
solid tumors. miR-21 was highly expressed in many kinds of human tumors, and
the elevated expression might directly decrease the phosphatase
and tensin homolog deleted on chromosome 10 (an
important tumorsuppressor gene) expression [6–13,29]. miR-21 was identified as significantly overexpressed in pancreatic cancer versus NP in our study,
suggesting that miR-21 plays an important role in preventing apoptosis, thus
functioning as a proto-oncogene in pancreatic cancer. Most known miRNA genes have the same type of promoters, within the
500-bp upstream proximal regions of pre-miRNA
hairpins, as protein-coding genes have [22]. Mutations that affect the activity
of the promoters of miRNA genes may also be important
for miRNA expression levels. But promoter assays
showed that the two identified mutations of miR-21 did not significantly alter
the activity of the promoter of miR-21 gene in our study. Hypothetically,
mutations in the pri– and pre-miRNA
regions of miRNA genes could affect processing of the
precursor to the mature form of miRNA, resulting in
aberrant expression of miRNA. Using the RNAfold program, we found that the A t In summary, we presented evidence that miRNA sequences vary in pancreatic cancer, and miR-21 was highly
expressed in pancreatic carcinoma. Most of the mutations so far identified in
cancer did not exert any apparent effect on miRNA
function. However, one identified sequence variant disrupted the base-pairing
close to the stem region and relatively reduced the expression of the miRNA. Our study indicated that miRNAs
might play an important role in pancreatic tumorigenesis,
but the molecular mechanism underlying the particular sequence variations in miRNA that can cause aberrant expression remains to be determined.
Acknowledgements
We greatly appreciate the technical support of the
functional assays from Dr. Li Wu (Laboratory of the Department of Gerontology
of the First Affiliated Hospital of
Funding
This work was supported in part by the grants from the
National Natural Science Foundation of China (No. 30500492) and by the Natural
Science Foundation of
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