Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics
Aim: To analyze expression of miRNA in human breast cancer cells, sensitive and resistant to cisplatin and doxorubicin, and to explore possible modification of drug sensitivity via treatment of cells with 5-azacytidine (5-aza), a demethylating agent. Materials and Methods: The study was performed on...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2016
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| Цитувати: | Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics / V.F. Chekhun, T.V. Borikun, N.Y. Lukianova // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 26–30. — Бібліогр.: 32 назв. — англ. |
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Chekhun, V.F. Borikun, T.V. Lukianova, N.Y. 2018-06-17T20:13:56Z 2018-06-17T20:13:56Z 2016 Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics / V.F. Chekhun, T.V. Borikun, N.Y. Lukianova // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 26–30. — Бібліогр.: 32 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/137982 Aim: To analyze expression of miRNA in human breast cancer cells, sensitive and resistant to cisplatin and doxorubicin, and to explore possible modification of drug sensitivity via treatment of cells with 5-azacytidine (5-aza), a demethylating agent. Materials and Methods: The study was performed on wild-type MCF-7 cell line (MCF-7/S) and its two sublines MCF-7/Dox and MCF-7/DDP resistant to doxorubicin and cisplatin, respectively. Cells were treated with 5-aza, cisplatin, doxorubicin and their combinations. Relative expression levels of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were examined, using qRT-PCR. The MTT assay was used to monitor cell viability. Results: We compared miRNA expression profiles in MCF-7/S and drug resistant MCF-7/Dox and MCF-7/DDP cells. Changes of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were observed in both resistant cell lines. The most significant differences were found for miRNA-200b (decreased in 50.0 ± 2.6 and 63.0 ± 3.1 times for MCF-7/Dox and MCF7/DDP cells, respectively) and for oncogenic miRNA-221 levels (increase in 62.0 ± 5.7 times for MCF-7/Dox and 83.8 ± 7.2 times for MCF-7/DDP cells). 5-aza treatment caused an increase of miRNA-10b, -122, -200b levels in MCF-7/S cells, miRNA-34a, -10b, -122, -200b and -320a levels in MCF-7/Dox cells and miRNA-34a, -10b, -200b and -320a levels in MCF-7/DDP cells. Pretreatment of all studied lines with 5-aza resulted in the increase of their sensitivity to studied cytostatics. In particular, the IC50 of doxorubicin decreased by 2-, 4- and 3-fold for cell lines MCF-7/S, MCF-7/Dox and MCF-7/DDP cells, respectively, and IC50 of cisplatin in studied cultures decreased by 3-, 2- and 1.5-fold, respectively. Conclusions: It was shown that use of 5-aza can modify sensitivity of breast cancer cells to cytotoxic drugs not only by it’s demetylation effect, but also by changes in expression of miRNAs, involved in cell proliferation, migration and drug resistance development. The study was supported by scientific grant of the NAS of Ukraine 2015–2019 “Molecular and Cell Biotechnologies for the needs of medicine, industry and agriculture”. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics |
| spellingShingle |
Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics Chekhun, V.F. Borikun, T.V. Lukianova, N.Y. Original contributions |
| title_short |
Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics |
| title_full |
Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics |
| title_fullStr |
Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics |
| title_full_unstemmed |
Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics |
| title_sort |
effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics |
| author |
Chekhun, V.F. Borikun, T.V. Lukianova, N.Y. |
| author_facet |
Chekhun, V.F. Borikun, T.V. Lukianova, N.Y. |
| topic |
Original contributions |
| topic_facet |
Original contributions |
| publishDate |
2016 |
| language |
English |
| container_title |
Experimental Oncology |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| format |
Article |
| description |
Aim: To analyze expression of miRNA in human breast cancer cells, sensitive and resistant to cisplatin and doxorubicin, and to explore possible modification of drug sensitivity via treatment of cells with 5-azacytidine (5-aza), a demethylating agent. Materials and Methods: The study was performed on wild-type MCF-7 cell line (MCF-7/S) and its two sublines MCF-7/Dox and MCF-7/DDP resistant to doxorubicin and cisplatin, respectively. Cells were treated with 5-aza, cisplatin, doxorubicin and their combinations. Relative expression levels of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were examined, using qRT-PCR. The MTT assay was used to monitor cell viability. Results: We compared miRNA expression profiles in MCF-7/S and drug resistant MCF-7/Dox and MCF-7/DDP cells. Changes of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were observed in both resistant cell lines. The most significant differences were found for miRNA-200b (decreased in 50.0 ± 2.6 and 63.0 ± 3.1 times for MCF-7/Dox and MCF7/DDP cells, respectively) and for oncogenic miRNA-221 levels (increase in 62.0 ± 5.7 times for MCF-7/Dox and 83.8 ± 7.2 times for MCF-7/DDP cells). 5-aza treatment caused an increase of miRNA-10b, -122, -200b levels in MCF-7/S cells, miRNA-34a, -10b, -122, -200b and -320a levels in MCF-7/Dox cells and miRNA-34a, -10b, -200b and -320a levels in MCF-7/DDP cells. Pretreatment of all studied lines with 5-aza resulted in the increase of their sensitivity to studied cytostatics. In particular, the IC50 of doxorubicin decreased by 2-, 4- and 3-fold for cell lines MCF-7/S, MCF-7/Dox and MCF-7/DDP cells, respectively, and IC50 of cisplatin in studied cultures decreased by 3-, 2- and 1.5-fold, respectively. Conclusions: It was shown that use of 5-aza can modify sensitivity of breast cancer cells to cytotoxic drugs not only by it’s demetylation effect, but also by changes in expression of miRNAs, involved in cell proliferation, migration and drug resistance development.
|
| issn |
1812-9269 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/137982 |
| citation_txt |
Effect of 5-azacytidine on mirna expression in human breast cancer cells with different sensitivity to cytostatics / V.F. Chekhun, T.V. Borikun, N.Y. Lukianova // Experimental Oncology. — 2016 — Т. 38, № 1. — С. 26–30. — Бібліогр.: 32 назв. — англ. |
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| first_indexed |
2025-11-25T20:54:31Z |
| last_indexed |
2025-11-25T20:54:31Z |
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1850543472296067072 |
| fulltext |
26 Experimental Oncology 38, 26–30, 2016 (March)
EFFECT OF 5-AZACYTIDINE ON miRNA EXPRESSION IN HUMAN
BREAST CANCER CELLS WITH DIFFERENT SENSITIVITY
TO CYTOSTATICS
V.F. Chekhun, T.V. Borikun, N.Y. Lukianova*
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology,
NAS of Ukraine, Kyiv 03022, Ukraine
Aim: To analyze expression of miRNA in human breast cancer cells, sensitive and resistant to cisplatin and doxorubicin, and to explore
possible modification of drug sensitivity via treatment of cells with 5-azacytidine (5-aza), a demethylating agent. Materials and Me
thods: The study was performed on wild-type MCF-7 cell line (MCF-7/S) and its two sublines MCF-7/Dox and MCF-7/DDP re-
sistant to doxorubicin and cisplatin, respectively. Cells were treated with 5-aza, cisplatin, doxorubicin and their combinations. Relative
expression levels of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were examined, using qRT-PCR. The MTT assay was
used to monitor cell viability. Results: We compared miRNA expression profiles in MCF-7/S and drug resistant MCF-7/Dox and
MCF-7/DDP cells. Changes of miRNA-221, -200b, -320a, -10b, -34a, -122 and -29b were observed in both resistant cell lines. The
most significant differences were found for miRNA-200b (decreased in 50.0 ± 2.6 and 63.0 ± 3.1 times for MCF-7/Dox and MCF-
7/DDP cells, respectively) and for oncogenic miRNA-221 levels (increase in 62.0 ± 5.7 times for MCF-7/Dox and 83.8 ± 7.2 times
for MCF-7/DDP cells). 5-aza treatment caused an increase of miRNA-10b, -122, -200b levels in MCF-7/S cells, miRNA-34a,
-10b, -122, -200b and -320a levels in MCF-7/Dox cells and miRNA-34a, -10b, -200b and -320a levels in MCF-7/DDP cells.
Pretreatment of all studied lines with 5-aza resulted in the increase of their sensitivity to studied cytostatics. In particular, the
IC50 of doxorubicin decreased by 2-, 4- and 3-fold for cell lines MCF-7/S, MCF-7/Dox and MCF-7/DDP cells, respectively, and
IC50 of cisplatin in studied cultures decreased by 3-, 2- and 1.5-fold, respectively. Conclusions: It was shown that use of 5-aza can
modify sensitivity of breast cancer cells to cytotoxic drugs not only by it’s demetylation effect, but also by changes in expression
of miRNAs, involved in cell proliferation, migration and drug resistance development.
Key Words: MCF-7, drug sensitivity, epigenetics, miRNA, methylation.
Oncological diseases remain one of the most com-
mon causes of death. Genetics studies accelerated
cancer research significantly. Moreover, identifica-
tion of oncogenes and tumor suppressor genes was
a breakthrough for the development of specific drugs
to treat cancer, but has not lead to creation of a pana-
cea. Recent studies showed that in addition to ge-
netic disorders, the development of cancer is triggered
by epigenetic changes. Changes in DNA methylation
and miRNA expression are important factors, controlling
oncogenesis and drug resistance development [1, 2].
DNA methylation in oncogenesis. DNA methyla-
tion is a key epigenetic modification that often leads
to silencing on the gene transcription. Cancer cells are
characterized by abnormal DNA methylation patterns,
accompanied by global hypomethylation and gene-
specific hypermethylation, as a rule [2].
DNA methylation occurs when DNA methyltransfe-
rases (DNMT1, DNMT3a and DNMT3b) add the
methyl group to the cytosine residue of DNA, leading
to the 5-methylcytosine synthesis. DNA methylation
occurs mainly in areas rich of so called CpG-islands.
CpG-islands are found in promoter regions of majority
of genes, and negative correlation between promoter
methylation and gene expression is already well known.
Hypermethy lation of CpG-islands in promoter region
almost always causes the gene silencing, regardless
on gene mutations. During breast cancer (BC) develop-
ment, changes of DNA methylation are responsible for
inactivation of large number of genes, including genes
that control the cell cycle, steroid receptor genes, genes
associated with metastasis, resistance to drugs, etc [3].
Changes in DNA methylation lead to genetic instabi-
lity: 5-methylcytosine serve as a transition mutations
site — through hydrolytic deamination of 5-methylcy-
tosine becomes thymine. This mutation is frequently
observed in such genes as TP53, RB1 and HRAS. Also,
epigenetic inactivation of critical genes through pro-
moter hypermethylation is a prerequisite for genetic
instability — for example, methylation of MLH1, which
participates in mismatch reparation.
Furthermore, methylation of promoter CpG-islands
and inactivation of glutathione-S-transferase leads
to the accumulation of free radicals and further DNA
damage. Besides some regional gene hypermethy-
lation, genome hypomethylation is one of the important
characteristics of the tumor, which also contributes
to genetic instability [4].
It has been suggested that aberrant DNA methyla-
tion can affect the sensitivity of tumor cells to anticancer
agents altering the expression of genes that are involved
in the response to the impact of xenobiotics. DNA hyper-
methylation may play an important role in development
of drug resistance phenotype by inactivation of genes
that are involved in the cytotoxicity reactions [5].
Significant reduction in the accumulation of metho-
trexate and other drugs in human hepatoma and
Submitted: January 20, 2016.
*Correspondence: E-mail: lu_na_u@rambler.ru
Abbreviations used: 5-aza — 5-azacytidine; BC — breast cancer; DDP —
cisplatin; DNMT — DNA methyltransfe rases; Dox — doxorubicin; EMT —
epithelial-mesenchymal transition; MDR — multidrug resistance.
Exp Oncol 2016
38, 1, 26–30
Experimental Oncology 38, 26–30, 2016 (March) 27
epidermal carcinoma cells resistant to cisplatin (DDP)
was demonstrated as the result of silencing of folate-
binding gene FBP. Restoration of this gene expression
after treatment with a demethylating agent 2-deoxy-
5-azacytidine (5-aza) proved the hypermethylation
of the FBP promoter in drug resistant cells.
Similar processes occur in testicular tumors; in vivo
DDP induces promoter hypermethylation de novo.
In vitro acquired resistance to DDP causes changes
in the expression of a number of genes, but even in high-
resistant cells the expression of tumor suppressor
genes is restored after treatment with demethylating
agents [6].
It was found that expression of several hundred
genes decreased in DDP resistant cell lines and it can
be restored by DNMT inhibitors [6–8].
Concluding, the aberrant DNA methylation is a com-
mon event in development of drug resistance in cancer
cells. Moreover, CpG-methylation leading to gene
silencing may play an important role in resistance to cy-
tostatic chemotherapy.
miRNAs in cancer development. miRNAs are
small (18–25 nucleotides) noncoding RNAs, negatively
regulating expression of target genes through initiation
of cleavage of their mRNA. miRNA binds to the target
complementary mRNA, resulting in cleavage and/
or de-adenylation of the latter, or obstructs ribosome
movement [2].
Usually miRNA genes are transcribed by RNA poly-
merase II. Formed large transcript consists of seve ral
hundred base pairs, named pri-miRNA, containing
5՛Cap and poly-A-tail [9].
Then double stranded structures in pri-miRNA are
recognized by protein DGCR8 (also called “Pasha”),
which forms microprocessor protein complex with
an enzyme Drosha that possesses RNAse III domain.
In the result, approximately 70 bp-length pre-miRNA
is formed with the shape of a stem-loop-structure (hair-
pin). This pre-miRNA is recognized by Exportin-5 and
with the participation of Ran-GTPase is transported
to the cytoplasm [10, 11].
In the cytoplasm, pre-miRNA is digested by enzyme
Dicer (RNAse III) into a duplex of 22 nucleotides length.
Dicer initiates the formation of RNA-induced silencing
complex (RISC). RISC protein family consists of the
Argonautes (Ago), with endonuclease activity towards
mRNA that is complementary to microRNA, incorpo-
rated in RISC [12].
RISC is responsible for gene silencing, due to binding
to 3՛-UTR of the mRNA and repressing its translation [13].
Thus, miRNAs can inhibit the expression of target
genes through a variety of mechanisms by preven-
ting protein translation without changing of the mRNA
level or by mRNA degradation. miRNA genes are often
located in so-called “fragile sites” of chromosomes,
where often occur deletions, insertions, DNA strand
breaks (single and double), translocations, transitions,
amplification etc., making them weak link in the forma-
tion of mutated phenotype [2, 15].
Epigenetic mechanisms in development of drug
resistance. As was mentioned above, altered expres-
sion of genes, leading to drug resistance development
can be regulated not only by genetic alterations, but also
by epigenetic mechanisms. Thus, correction of methy-
lation disturbances and microRNAs expression can
be used for correction of cancer cells sensitivity [1, 15].
While miRNAs are involved in regulation of many cel-
lular processes, from zygote polarization to cell death,
upon development of drug resistance only miRNAs,
responsible for cell proliferation, migration, etc. showed
altered expression.
Many miRNAs are described as oncogenic — they
negatively regulate expression of tumor suppressor
genes, for example, RB1, a cell cycle regulator and
PTEN (phosphatase and tensin homolog) an inhibitor
of cell survival/growth. PTEN is the target of several dif-
ferent miRNA — miRNA-21, -216, -221, and -144. Such
variety of regulatory pathways can be used to overcome
the drug resistance by maintenance of expression
of tumor suppressor [1, 16].
Also, there are tumor suppressor miRNAs. Mutations
in their genes and/or epigenetic silencing of expression
can cause cancer progression, and also incre asing
in invasive and migration properties. For example,
miRNA-200b negatively regulates family of ZEB pro-
teins, responsible for epithelial-mesenchymal transition
(EMT). Decrease in miRNA-200b expression is one
of EMT triggers in BC cells [1].
It is well documented that resistance of MCF-7 cells
to doxorubicin (Dox) is mediated by increase of MDR1 (mul-
tidrug resistance) gene copy numbers. Transfection
of miRNA-451 causes increase in sensitivity to this cyto-
static, as result of inhibition of MDR1 protein translation
by degradation of mRNA [17].
As was mentioned already, development of drug
resistance is often accompanied by disturbances
in DNA methylation. For example, total DNA methy-
lation is lower in MCF-7 cells, compared with normal
breast cells. It was proposed that resistance of cancer
cells to Dox and DDP was developed due to alterations
in methylation pattern of DNA in the specific regions,
leading to differentially methylated genes. Such chan-
ges in methylation of promoter CpG-islands, respon-
sible for the specificity of transcription factors are
essential not only for expression of genes, but also for
genome stability [18].
Therefore, the aim of the present work was to ana-
lyze expression of miRNA in human BC cells sensitive
and resistant to DDP and Dox, and explore possible
modification of drug sensitivity via treatment of the cells
with 5-aza, a demethylating agent.
MATERIALS AND METHODS
Three sub-lines of breast carcinoma MCF-7 cells
(obtained from invasive breast ductal carcinoma),
namely MCF-7/S, sensitive to cytostatics; MCF-7/
Dox — resistant to Dox; and MCF-7/DDP — resis-
tant to DDP were used in our study. Cells were pro-
vided by the Bank of human and animal tissues lines
28 Experimental Oncology 38, 26–30, 2016 (March)
at R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology of NAS of Ukraine and
described earlier in [19].
All cells were grown in Dulbecco Modified Eagles
Medium (DMEM, Sigma), supplemented with re-
combinant human insulin (0.01 mg/ml) and 10% fetal
bovine serum. All cultures were grown in humidified
atmosphere with 5% CO2 at 37 °C.
Measurement of cell viability, using the MTT as-
say. Ability of living cells to reduce tetrazole (MTT, solu-
tion of the yellow color) into purple crystalline formazan
by the mitochondrial enzymes allow us to estimate cell
viability. The resulting intracellular formazan can be so-
lubilized and quantified, using spectrophotometry [20].
For this purpose, cells were cultivated in 96-well
plates in DMEM, supplemented with insulin (see above)
for 24 h. The 5-aza, Dox, and DDP were added to media
at different concentrations.
After 24 h, 10 µl of the MTT dye solution (Sigma,
USA) (5 mg/ml in phosphate buffer saline) was added
to the cells; the cells were incubated at the same condi-
tions for 3 h. After centrifugation (1500 rpm, 5 min) the
supernatant was removed. 100 µl of dimethyl sulfoxide
(Serva, Germany) was added to each well, to dis-
solve formazan. The absorption was measured, using
a multiwell spectrophotometer (STAT FAX 2100, USA)
at a wavelength of 540 nm.
Cytotoxicity of studied agents was studied using
IC50 and IC30 values [21].
Total RNA isolation. Total RNA extraction was
performed, using Ribozol RNA Isolation Kit (Amplisens,
Russia). Concentration of RNA was measured, using
NanoDrop 2000c Spectrophotometer (Thermo Scien-
tific, USA). The purity of isolated RNA was controlled,
analyzing the ratio of OD at 260/280 nm. RNA was
dissolved in TE buffer and stored at −20 °С.
Single-stranded cDNA was synthesized from
100 ng of total RNA, using TaqMan® MicroRNA Kit for
reverse transcription.
Real-time quantitative reverse transcription
polymerase chain reaction (qRT-PCR). Preparation
of reverse transcription reaction mix was performed ac-
cording to manufacturer’s protocol. Reverse transcrip-
tion was performed at a “Tertsik” (“DNA Tehnologіya”,
Russian Federation) thermal cycler. qRT-PCR was
performed on Applied Biosystems 7900HT Fast Real-
Time PCR System using TaqMan® MicroRNA primers
and manufacturer’s protocol.
Small nucleolar RNA RNU48 was used as an endo-
genous control for normalization of miRNA expression.
Relative expression of the studied miRNAs was identi-
fied by comparative Ct method [22]. Experiments were
performed in triplicates for each line, and PCR was
performed three times for each sample. Expression
differences between the studied miRNA levels relative
to control was calculated by the formula:
Fold change = 2−ΔΔCt [22],
where ΔCt (target — control) is equal to the differ-
ence between threshold cycles for miRNA (target) and
the threshold cycle for RNU48 (control) (ΔCt (target —
control) = Ct target − Ct control). ΔΔCt = ΔCt (experi-
ment) − ΔCt (control).
Experimental data were analysed using the Stu-
dent’s t-test. P-values less than 0.05 were considered
statistically significant. Statistical analysis of the obtained
data was performed, using the STATISTICA 6.0 software.
RESULTS AND DISCUSSION
The analysis of miRNA expression showed that the
development of resistance to cytotoxic drugs is ac-
companied by a significant decrease in the expression
of tumor suppressive miRNA-200b: in cells resistant
to Dox — 50 fold decrease; in cells resistant to DDP —
63 fold decrease. For the miRNA-320a downregulation
was 8.5 fold and 3.9 folds, respectively. In addition, the
increased expression of oncogenic miRNA-221 was ob-
served — 62 fold and 83.8 folds, respectively. miRNA-
10b and miRNA-34a expression changed differently
in MCF-7/Dox (reduced) and MCF-7/DDP (increased)
cell lines (Fig. 1).
-80
-60
-40
-20
0 * * * *
*
*
* *
*
*
*
*
* *
20
40
60
80
100
m
iR
NA
-3
4a
m
iR
NA
-1
0b
m
iR
NA
-2
9b
m
iR
NA
-1
22
m
iR
NA
-2
00
b
m
iR
NA
-2
21
m
iR
NA
-3
20
а
Fo
ld
c
ha
ng
e
MCF7/Dox / MCF7/S
MCF7/DDP / MCF7/S
Fig. 1. Fold change of miRNAs expression in resistant to Dox and
DDP MCF-7 cells comparatively to sensitive cell line; *p < 0.05.
Average ± SD values are shown
5-aza irreversibly binds to DNMT1 during DNA rep-
lication and prevents methylation, reduces the number
of active DNMT1 molecules in cell and provides a cyto-
toxic DNA adducts formation [23, 24].
The depletion of DNMT1 causes progressive loss
of methylation upon DNA replication. 5-aza-induced
hypomethylation affects the entire DNA, and also CpG-
islands in the gene promoter regions; hypomethylation
of promoters is often associated with induction of tran-
scription [25, 26]. For example, due to 5-aza treatment,
loss of promoter methylation and induction of transcrip-
tion was shown for BRCA1, the tumor suppressor gene
that is critical in the ethiology of hereditary and sporadic
BC [18, 27].
The web resources (at http://refgene.com and
https://genome.ucsc.edu) to analyze DNA segments
at a distance of 1.5 kb in up- and downstream regions
from the transcription start site was used to find CpG-
islands in promoter regions of miRNA-221, -200b,
-320a, -34a, -10b, -122 and-29b. Several CpG-islands
were found in areas, where their methylation can prevent
binding of transcription factors to the promoter.
Summarizing, the promoters of miRNA-200b, -10b
and -320a are characterized by a high CpG density, that
Experimental Oncology 38, 26–30, 2016 (March) 29
makes these genes more sensitive to demethylation
influence by described mechanism.
As shown in Table 1, miRNA-10b, -122, -221, and
-320a showed increased expression in MCF-7/S cells
upon 5-aza treatment. The most considerable changes
were detected in miRNA-200b expression (2.55 fold
increase), and this might lead to inhibition of EMT,
metastasizing, proliferation and increase susceptibility
to xenobiotics due to its annotated targets [1].
Table 1. Fold change of miRNAs expression after treatment of MCF-7 cells
with 5-aza, sensitive and resistant to cytostatics**
miRNA MCF-7/S +
5-aza MCF-7/Dox MCF-7/Dox
+ 5-aza
MCF-7/
DDP
MCF-7/DDP
+ 5-aza
miRNA-34a 1.2 ± 0.1 −4.0±0.2* 1.3 ± 0.2 4.0 ± 0.1* 9.3 ± 0.7*
miRNA-10b 2.1 ± 0.1* −2.0±0.2* 1.3 ± 0.1 5.0 ± 0.9* 8.3 ± 1.3*
miRNA-29b −1.1 ± 0.2 13.0±0.2* 10.2 ± 0.9* 52.4 ± 5.3* 50.2 ± 7.4*
miRNA-122 2.5 ± 0.1* 6.0 ± 0.4* 7.0 ± 1.0* 1.5 ± 0.3* 1.9 ± 0.4*
miRNA-200b 2.55 ± 0.2* −50.0±2.9* −45.1±5.4* −63.0±3.9* −59.1 ± 5.3*
miRNA-221 1.7 ± 0.1* 62.0±4.1* 63.1 ± 7.1* 83.8±8.9* 70.1 ± 9.6*
miRNA-320а 1.89 ± 0.1* −8.5±1.3* −6.1±0.9* −3.9 ± 1.5* 1.7 ± 0.3*
Note: *p < 0.05 compared with MCF-7/S cell culture; ** normalized to the values
obtained for the MCF-7/S cells without treatment. Average ± SD values are shown.
In Dox-resistant cells upon 5-aza treatment, in-
crease in expression levels of miRNA-34a, -200b and
-320a was observed — 2.4, 2.9 and 3.3 folds, respec-
tively, while the expression of miRNA-10b and -122 in-
creased only by half compared with control culture.
The 5-aza treatment caused enhanced expression
of miRNA-34a and -10b up to levels typical for the
sensitive line. In addition, there was a tendency of diffe-
rences in reduction fold between sensitive and resistant
cultures to Dox; miRNA-122 showed diminished expres-
sion, while and miRNA-200b and -320a expression was
elevated. However, changes showed different levels,
in comparison with sensitive cells.
In MCF-7/Dox cells upon 5-aza treatment, expres-
sion of miRNAs, responsible for apoptosis, sensitivity
to cytotoxic drugs, cell proliferation and metastasizing,
was elevated.
DDP resistant cells were characterized by the follow-
ing changes: the level of miRNA-320a increased 2 fold,
miRNA-10b — 3.32 fold, miRNA-200b — 4 fold, miRNA-
34a — 5 fold, in comparison with the control cells.
In MCF-7/DDP cells, in comparison with the sensi-
tive culture the increase of miRNA-320a, up to the level
in MCF-7/S cells, enhanced miRNA-200b expression,
and reduced oncogenic miRNA-221 level were ob-
served. Noteworthy, miRNA-10b, which is responsible
for invasion and metastasis, was expressed at the higher
level (Fig. 2).
Changes in drug sensitivity in MCF-7/S, MCF-
7/Dox, and MCF-7/DDP cells upon the treatment
with 5-aza. The 5-aza is often used in combination with
DDP, cyclophosphamide, and other drugs used in the
anti-cancer treatment, in particular, in case of neuro-
blastoma, rhabdomyosarcoma in children, and also
certain solid tumors [29]. Chekhun et al. [26] showed
the crucial role of epigenetic regulation in acquisition
of drug resistance by MCF-7 cells. DNA-methylation
disturbances are one of the most frequent causes
of DDP and Dox resistance in these cells.
-2
-1
0
1
2
3
4
5
6
7
m
iR
NA
-
34
a
m
iR
NA
-
10
b
m
iR
NA
-
29
b
m
iR
NA
-
12
2
m
iR
NA
-
20
0b
m
iR
NA
-
22
1
m
iR
NA
-
32
0а
MCF-7/Dox+5-aza / MCF-7/Dox
MCF-7/DDP+5-aza / MCF-7/DDP**
**
*
*
**
**
*
*
*
Fig. 2. miRNAs expression patterns after 5-aza treatment in resis-
tant MCF-7 cell sublines compared with the control cell cultures;
*p < 0.05 compared with the control MCF-7/Dox cell culture;
**p < 0.05 compared with the control MCF-7/DDP cell culture.
Average ± SD values are shown
The results of the present work show that preliminary
cultivation of MCF-7 cells with 5-aza resulted in increase
of cytotoxic effects of DDP and Dox (Table 2). In par-
ticular, the IC50 of Dox decreased by 2, 4 and 3 fold
for cell lines MCF-7/S, MCF-7/Dox and MCF-7 /DDP
cells, respectively.
IC50 of DDP in studied cultures decreased by 3, 2 and
1.5 fold, respectively.
Such modulating effect of 5-aza on cytotoxic effects
of Dox can be explained by promoter demethylation
of MDR1 gene, which is responsible in elimination
of Dox from cells. There are twelve CpG-islands in the
MDR1 gene promoter, 5 of which remain methylated
in transformed cells. Increased expression of this gene
is usually caused by its amplification. It is proposed
that demethylation facilitates binding of regulatory
elements to the promoter, inhibiting the transcription
of this gene [26].
Also, increased miRNA-320a is involved in MDR1 si-
lencing, as well as miRNA-34a does [1, 17, 30].
It was proposed earlier, that increase of sensitivity
to DDP could be associated with the following mecha-
nisms — 5-aza causes DNA demethylation, and this
leads to changes in expression of genes responsible
for the transport of DDP into the cell. DDP is associated
with GG, AG and GNG motifs, forming intra-chain DNA
adducts and extra- and intrastrand G-G DNA crosslinks.
Demethylation of CpG-rich areas increases their vulner-
ability to DDP and, thereby increases sensitivity [19,
26, 31].
Table 2. Increased sensitivity of BC cells to anticancer drugs after treatment with 5-aza
IC50, µM
MCF-7/S MCF-7/DOX MCF-7/DDP
Dox DDP Dox DDP Dox DDP
Control 4.1 ± 0.3 15.3 ± 1.3 23.3 ± 2.1 16.0 ± 1.0 12.4 ± 1.2 93.3 ± 7.0
10 days with 5-aza 3.5 ± 0.2* 10.5 ± 1.9* 13.7 ± 1.3* 11.9 ± 0.8* 10.2 ± 1.2* 87.4 ± 3.0*
20 days with 5-aza 2.9 ± 0.7* 7.2 ± 1.4* 6.3 ± 1.1* 9.7 ±1.5* 6.9 ± 0.9* 55.9 ± 2.1*
30 days with 5-aza 2.4 ± 0.3* 5.4 ± 0.3* 5.2 ± 0.2* 4.4 ± 2.4* 4.6 ± 0.2* 45.2 ± 6.3*
Note: *p < 0.05 compared with the control cell culture. Average ± SD values are shown.
30 Experimental Oncology 38, 26–30, 2016 (March)
Another mechanism to restore the sensitivity to DDP
is increased miRNA-200b expression. This miRNA
modulates iron metabolism, thus, enhancing oxidative
stress in cancer cells [19, 32].
In conclusion, we have shown that miRNA expression
was increased in BC cells upon 5-aza treatment, due
to promoter DNA demethylation. At such conditions res-
toration of expression of miRNA-10b, -34a, -200b, and
-320a was observed in MCF-7 sublines resistant to Dox
and DDP. Importantly, this increased sensitivity of prior
resistant cells to treatment with anti-cancer drugs.
We may speculate that this phenomenon is due
to the enhanced epigenetic control of miRNA expres-
sion upon treatment with 5-aza. The obtained data
create the basis for further in vivo investigations.
ACKNOWLEDGEMENT
The study was supported by scientific grant of the
NAS of Ukraine 2015–2019 “Molecular and Cell Bio-
technologies for the needs of medicine, industry and
agriculture”.
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