METHODS
Invasive ductal carcinoma from tumoral paraffin tissues and normal breast tissues taken from breast
reduction surgeries was used in the study. The study examines the expressions of the PIWIL-2, PIWIL-1,
DICER-1, and DDX-4 genes in the invasive ductal carcinoma of the breast.
RESULTS
As predicted, PIWIL-2 and DDX4 expressions were found to be significantly higher compared to normal
tissues. PIWIL-1 expression, on the other hand, was recorded at borderline levels of significance.
The expression of DICER-1, a gene responsible for the formation and maturation of microRNAs (miRNAs),
revealed no significant difference.
CONCLUSION
It was concluded that the increased expression of PIWIL-2 and DDX-4 in breast cancer cases that are
not detectable in normal tissue enables its use as a marker in the diagnosis and treatment of mamarian
cancer and the post-therapy follow-up of the disease. It appears to be more informative to study the
specific miRNAs of the gene rather than its expression to discern the function of the DICER-1 gene in
breast cancer.
Keywords: Breast cancer; DDX-4; DICER-1; invasive ductal carcinoma; PIWIL-2; real-time polymerase chain reaction
At the cellular level, the development of cancer is viewed as a multistep process. At every step, important genetic processes are thought to occur that characterizes the cell as a cancer cell. As a result of these changes, alterations may occur in the expression of genes or in the function of gene products that cause tumor formation or progression.
Mutations or overproduction of receptors or proteins
in signaling pathways cause cells to become independent
from growth factors and other mitotic factors,
thereby causing uncontrolled cell division. It is known
that the histologically similar tumors have clinically
different outcomes. Moreover, tumors with no established
differences based on histological analysis can
respond to treatments differently.[
Breast cancer is a heterogeneous disease, resulting
from the progressive accumulation of genetic abnormalities,
including point mutations, chromosomal
instability, aneuploidies, deletions, rearrangements
(chromothripsis), translocations, and duplications. These
situations of DNA cause damage of DNA repair and
telomere dysfunction.[
Piwi genes have been preserved during evolution,
and stem cells of different organisms ranging from the
Arabidopsis plant to human are known to play a role
in the regeneration, gametogenesis, and RNA interference.
Piwi germline is involved as an autonomic organizer
in stem cell division. PIWIL-2 is a member of the
Argonaute protein family. This gene family has been
revealed in studies to play a key role in gene silencing
through small RNAs. The Piwi gene family contains two
domains known as PAZ and Piwi, which are responsible
for "stem-cell self-renewal, RNA silencing and translational regulation." PIWIL-2, a member of the Piwi gene
family, was demonstrated to be first expressed only in
testicular tissue among normal tissues. In spermatogenesis,
it plays a fundamental role in differentiation of spermatocytes,
germ cells, and spermatogonia stem cells. It
is thought to have a similar function during oogenesis in
women. PIWIL-2 has a key role in regulation of proliferation
and apoptosis in signal transduction pathways in
cancer stem cells. Overexpression of this gene in germ
cells leads to germ cell malignancies. This finding suggests
that PIWIL-2 may cause the same effect in other
stem cells including cancer development.
To understand the role of PIWIL-2 in different organs,
northern blot analysis was performed in major
adult organs and among the normal adult human tissues
(spleen thymus, prostate, testis, ovary, small intestine,
colon, peripheral blood, and leukocytes) analyzed,
the expression of PIWIL-2 protein was observed
only in the testis. In immunohistochemical studies,
PIWIL-2 expression was detected in benign and malignant
lesions of the breast. No difference in expression
was noted between different breast cancer types. However,
no expression was detected in benign lesions of
the breast including fibroadenoma and fibrocystic disease.
It is not clear why and how PIWIL-2 is activated
in cancer stem cells while it is silent in somatic cells.
Since this gene serves at a critical point at the beginning
of DNA repair mechanisms, the primary reason
for activation of the gene may be the initiation of DNA
repair to stop oncogenesis. To confirm this hypothesis
in invasive ductal carcinomas (IDC) of the breast, the
molecular pathogenesis of IDCs will be elucidated by
examining the expressions in the four genes as compared
to the normal tissue and with each other.[
DICER is a key ribonuclease in microRNA (miRNA)
and short interfering RNAs (siRNA) biosynthesis during
mammalian development and cell differentiation.
Recent studies have revealed that DICER-1 may also
be involved in oncogenesis. Death occurred as a result
of depletion of stem cells in DICER gene-silenced
mice. Embryonic stem cells with DICER deficiency can
survive but lose their differentiation and proliferation
ability. Therefore, DICER-1 is the basis for stem cells"
regeneration and proliferation. DICER is involved in
the metabolic processes of Piwi-interacting RNAs
(piRNAs), which are 24-30 nucleotide RNA fragments
that are formed by an independent mechanism and are
primarily composed of transposon and other repeating
sequence elements. Besides suppressing transposons,
piRNAs are also involved in some functional positions
and translational regulation, possibly during meiosis. It also plays a role in the stabilization of miRNA and the
formation of the chromatid body. Small RNAs such as
siRNAs, miRNAs, or piRNAs bind to specific binding
sites, leading to silencing and destruction in the target
mRNA. Small RNAs and Argonaute proteins have been
found in high eukaryotes and have very different functions
such as transposon silencing, cell differentiation,
and embryonic development.[
All participants were females aged 35-60 years. The study group was selected from patients operated for breast cancer and the control group from those who underwent mammoplasty (breast reduction). Patients diagnosed as "Invasive Ductal Carcinoma" in the pathology report were included in the study group and those identified as "Normal breast tissue" in the pathology report served as the control group. Males and patients with any pathological diagnosis other than or in addition to "Invasive Ductal Carcinoma and Normal Breast Tissue" in fine-needle aspiration biopsy materials were excluded from the study.
In this study, the expressions of PIWIL-2, PIWIL-1, DICER-1, and DDX-4 genes in the IDC of the breast were analyzed by real-time polymerase chain reaction (RT-PCR) method. For the expression analysis, betaactin (ACTB), a housekeeping gene was used as the control gene, and NBT samples obtained from mammoplasty operations were used as the control tissue sample. RNeasy FFPE Kit was used for RNA isolation from the tissues. The c-DNA synthesis and pre-amplification of the RNAs were performed with RT2 Pre- AMP c-DNA Synthesis Kit. RT-PCR method was applied after pre-amplification.
In tissues diagnosed with IDC, the ER positivity
rate was 70% and PR positivity rate was 58%. The
c-erbB-2 was positive in 46% of tumor tissues and
negative in 53%. The ki-67 positivity was very high
including 95% of the cases. Figures
According to the Delta-Delta Ct method, that we
used to compare the expression levels in normal tissues
and the tissues with IDC, the level of expression
in all included genes of the study had increased in
IDC tissues compared to NBT, as presented in Table
In a study by Nickpour et al. on bladder cancers,
PIWIL-2 expression was reported to be at least 240
times lower in all cell series when compared to levels
in the testicular tissue. Such a low level of PIWIL-2
expression in most tissues may enable this gene to be
considered as a tumor marker in the future. However,
the same researchers showed that PIWIL-2 had no role
in carcinogenesis in bladder cancer[
Therefore, the authors suggested that Piwil 2 may be
a novel biomarker for breast cancer.[
Lee et al. found that PIWIL-2 inhibits apoptosis by
activating the STAT3-BCL-XL pathway that has expression
in tumors.[
DICER-1 is a gene involved in the formation
and maturation of miRNAs. At the beginning of our study, we expected the expression of this gene to
be increased in IDC compared to NBT, because the
miRNAs" crucial role in cancer pathogenesis could
not be denied. In our study, we did not find a statistical
difference in the expression of the DICER-1 gene
in IDC group compared to normal individuals. As
our research progressed further, we considered this
as a normal finding, since DICER-1 contributes not
only to carcinogenesis but also to the formation of
miRNAs in hundreds of different mechanisms. Therefore,
the presence of specific miRNAs formed by this
gene and their relation with the breast cancer should
be planned to be investigated by another study, rather
than global expression of this gene in the cell. The
identification of the genes that are involved in tumorigenesis
is possible by genomic and transcriptomic
comparisons of different tumor samples with normal
tissue samples and with each other and by the use of
genomic information in oncology. Data of these studies
will enable to categorize the tumors as subgroups
and to use them as personalized treatments.
1. Among these genes, the expression of PIWIL-2 and DDX-4 was not evident or very low in NBT, while their expression increased in breast cancer tissue. PIWIL-2 and DDX-4 genes work in parallel. Although one of them was increased with the increase in other, the increase in DICER-1 was not found to be significant among the groups. Given that both PIWIL-2 and DDX-4 genes are increasingly expressed in breast cancer despite not expressed in normal tissue, it seems likely that they can be used as markers in the diagnosis and treatment of breast cancer and follow-up of disease after treatment
2. Considering no change in the expression of DICER-1 between normal and IDC tissues, we think that the miRNAs that are involved in the production of the gene, other than the gene expression, should be investigated
3. Until the genomic revolution, the tumors were classified according to two criteria; localization (the region of the tumor originated) and the appearance of the tumor (histological structure). These two criteria are still in use today for the determination of the prognosis and application of the best proper treatment.
Nevertheless, it has been known for long that histologically similar tumors have different clinical outcomes. In addition to this information, the new tumor taxonomy, in which the presence of genetic lesions is the major criteria, will enable the likelihood of better diagnosis and individualized treatment plan by means of the genome-based information. The gene expressions and proteins will be included in the new classification as a discriminating criterion besides to the other two criteria due to contribution of the analysis of gene expression profiles in the diagnosis and treatment.
These results will help to highlight the molecular pathogenesis of breast cancer, while possibly one or more of these genes can be used as molecular markers that can be determined before the oncogenesis in the diagnosis of IDC.
Peer-review: Externally peer-reviewed.
Conflict of Interest: The authors declare that they have no conflict of interest.
Ethics Committee Approval: The study was approved by the Erciyes University Clinical Research Ethics Committee (No: 2012/130, Date:10/02/2012).
Financial Support: The authors declared that this study has received no financial support.
Authorship contributions: Concept - Y. Özkul; Design - Y.Ö.; Supervision - Y.Ö.; Funding - Y.Ö.; Materials - M.Ç., Y. Özkul; Data collection and/or processing - Y.Ö.; Data analysis and/or interpretation - M.Ç., Y. Özkul; Literature search - M.Ç.; Writing - M.Ç., Y. Özkul; Critical review - M.Ç.
