Artemisia absinthium (AA) modifies the expression profile of microRNAs in
human breast cancer cells
Gowhar Shafi1,2,3 and Anjana Munshi1,2
1
Dept. of Molecular Biology, Institute of genetics and Hospital for Genetic Diseases,
Osmania University Hyderabad India
2
Dr NTR University of Health Sciences Vijayawada AP India
3
Unit of Computational Medicine, Dept. of Medicine, Karolinska Institutet Stockholm
Sweden
Abstract
Increasing evidences in recent years reveal that several biological processes and
disease pathogenesis are regulated by microRNAs (miRNAs) and restoration of
normal miRNA activity can be novel way of treating cancers. Side effects of
conventional chemotherapeutic drugs are well known. Therefore, from therapeutic
point of view, the best strategy is to induce apoptosis in cancer cells without affecting
the normal cells of the body. In this context, phytochemicals are a potential alternative
source of safer chemicals and have several other physiological synergistic benefits. In
this study we determined if the methanolic extract of Artemisia absinthium (AA)
could modify the expression pattern of miRNAs. Microarray analysis was used to
profile miRNA expressions in human MCF-7 and MDA-MB-231 breast cancer cells
treated with AA. Transcripts with regulated expression patterns on the microarrays
were validated by SYBR Green based real time PCR.
AA modifies miRNA
expression in human MCF-7 and MDA-MB-231 breast cancer cells. It up-regulates
miRNA-22 and down-regulate miRNA-199a*, as confirmed by SYBR Green Realtime PCR. These observations suggest that modulation of miRNA expression might
be an important mechanism underlying the biological effects of AA in containing
cancer. Results also suggest that these miRNAs might be the future therapeutic agents
in breast cancer with lesser side effects.
Keywords: Artemisia absinthium, MicroRNA, Cancer, Chemotherapy
Introduction
The chemotherapeutic agents long used in oncologic treatment produce deleterious
side effects that augment the mortality and morbidity caused by cancer. Safer
treatments are thus desperately needed, some of which can be found in natural
compounds such as phytochemicals. Although chemopreventive activities and
preclinical antitumor effects are established, phytochemicals provide a novel
therapeutic approach that merits further exploration. Artemisia absinthium (AA) is
commonly called wormwood and is locally known as ‘Tethwen’ in Kashmir valley,
India. It is used in indigenous system of medicine as a vermifuge, an insecticide, an
antispasmodic, an antiseptic and in the treatment of chronic fevers and for
inflammation of the liver [Koul 1997]. Its essential oil has antimicrobial [Juteau et al
2003] and antifungal activity [Saban et al 2005]. Chemical analysis of AA plant has
shown that its volatile oil is rich in thujone, which has been earlier reported as an
anthelmintic [Meschler and Howlett 1999]. In Turkish folk medicine, AA has been
used as an antipyretic, antiseptic, anthelmintic, tonic, and diuretic and for the
treatment of stomach aches [Baytop 1984].
miRNAs are tiny (19–23 nucleotide) non-coding RNA molecules that are currently
being recognized as endogenous physiological regulators of gene expression. These
small RNAs are capable of controlling gene expression either by repression of
translation/transcription [Bartel 2009] or by activation of transcription [Li 2008].
miRNAs are also known to play important roles in many physiological and
pathological processes, including tumorigenesis [Mocellin et al 2009], proliferation
[Johnnidis et al 2008], hematopoiesis [Merkerova et al 2008], metabolism [Aumiller
and Forstemann 2008], immune function [Carissimi et al 2009], epigenetics and
neurodegenerative diseases [Shafi et al 2010, Bushati and Cohen 2008]. miRNAs
have also been found to be useful in identifying the etiology of lymphoma [Lawrie et
al 2007] and progression of certain neurological diseases [Nelson et al 2008].
We hypothesize the therapeutic mechanisms of AA could rely, at least in part, on their
influence on miRNA expression. If such an association is confirmed, it might provide
a potential opportunity for miRNAs acting as novel targets for treatment by
interference and biomarkers for selection of chemotherapeutic agents. To test this
hypothesis, we used microarray analysis to identify the differential expression profiles
of miRNA in MCF-7 and MDA-MB-231 breast cancer cells following exposure to the
AA. Microarray data was confirmed by Real Time PCR analysis.
Methods
Preparation of Plant Material
Plant was selected on the basis of ethnopharmacology and 1/2 Kg of plant material
was collected locally. It was identified by botanist, Department of Botany, Osmania
University, where a voucher specimen is deposited at Department of Botany
herbarium. The aerial parts of AA were air dried in shade then powdered using a
milling machine. The powdered plant material was extracted with methanol as
described previously [Shafi et al 2009]. Briefly, powdered plant material was soaked
into methanol for extraction. The quantity of methanol was taken 10 times the
quantity of plant material. Extraction was performed three times and each extraction
was done for 24 h. Methanolic filtrate was then evaporated under reduced pressure to
obtain a residue (500g of AA yielded 29.15g of residue). The residue was dried using
a rotary evaporator to obtain the powder/paste. The required quantity of the dry
powder/paste was dissolved in dimethyl sulfoxide (DMSO).
Preparation of drug
A stock of plant extract was prepared with concentration of 1mg/mL of DMSO and
sterilized by autoclaving at 121°C and 15 lb for 15 min. Then stock concentrations of
test compound were prepared by diluting stock with DMEM.
Maintenance of Cell culture
MDA-MB-231 and MCF-7 cells were procured from National Centre for Cell Science
(Pune, India). The cell lines were maintained and propagated in 90% Dulbecco's
Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and
1% penicillin/streptomycin. Cells were cultured as adherent monolayers (i.e., cultured
at approximately 70% to 80% confluence) and maintained at 37°C in a humidified
atmosphere of 5% CO2. Cells were harvested after brief trypsinization. All chemicals
used were of research grade.
Extraction of Micro RNA
Total RNA including small RNA was extracted from the both cell lines using RNA
isolation kit (Qiagen, Hilden, Germany) as per the instructions of manufacturer. The
concentration of RNA was quantified by a NanoDrop ND-1000 Spectrophotometer
(Thermo Fisher Scientific, U.S.A). The quality of RNAs was determined using 2%
agarose gel.
Microarray analysis
MiRNA expression was investigated using the Agilent Human miRNA microarray v.2
(#G4470B; Agilent Technologies, Santa Clara, CA, USA). This microarray consists
of 60-mer DNA probes synthesized in situ and contains 15000 features which
represent 923 human miRNAs, sourced from the Sanger miRBASE database (Release
14.1). RNA labelling and hybridization were performed in accordance with the
manufacturer’s instructions. An Agilent scanner and Feature Extraction 10.5 software
(Agilent Technologies) were used to obtain the microarray raw data.
cDNA Synthesis from MicroRNA
Quantitation of miRNAs was carried out using SYBR Green PCR. Briefly, 100 ng of
template RNA was reverse transcribed in 20µL using universal stem-loop primer
(Qiagen, Hilden, Germany). For the PCR reaction, 20ng of RT-product was used.
MicroRNA quantification
SYBR green qRT-PCR assay was used for miRNA quantification. One microgram of
total RNA was polyadenylated and reverse transcribed to cDNA using miScript
Reverse Transcription kit (Qiagen). Quantitative PCR assays were carried out in ABI
PRISM 7500 Fast real-time PCR system (Applied Biosystems) using miScript SYBR
Green PCR kit (Qiagen). Each reaction was performed in a final volume of 20µL
containing 2 µL of the cDNA, 0.5 mM of each primer and 1x QuantiTect SYBR
Green PCR Master mix (Qiagen). The amplification profile was denaturation at 950C
for 15 min, followed by 40 cycles of 940 C for 15 s, 550 C for 30 s, and 700C for 30 s.
At the end of the PCR cycles, melting curve analyses were performed as well as
electrophoresis of the products on 2% agarose gels in order to validate the specific
generation of the expected PCR product. The expression levels of miRNAs were
normalized to RNU1A.
Statistical analysis
Microarray results were analysed by using GeneSpring GX 11 software (Agilent
Technologies). Data transformation was applied to set all the negative raw values at
1.0, followed by quantile normalization and a log2 transformation. Filters on gene
expression were used to keep only the miRNAs expressed in at least one sample. A
1.5 fold-change filter and ANOVA (analysis of variance) statistical test were applied.
Differentially expressed genes were employed in cluster analysis, using the
Manhattan correlation as a measure of similarity. The statistical analyses were
performed using the SPSS software package, version 18.0 (SPSS Inc. Chicago, IL,
USA). The cycle threshold (Ct) value for the genes was determined by SDS software
v1.2 (Applied Biosystems, Foster City, CA). Ct is the threshold cycle, the cycle
number at which fluorescence is generated in a reaction crossing the threshold. The
expression level of miRNA was normalized by calculating the ΔCt value based on
subtracting its Ct value from that of the internal control RNU1A. The relative gene
expression was calculated as 2−ΔCt. The amplitude of change of the expression
miRNA observed in patients in relation to control group was analyzed by the
2−ΔΔCt method [Livak and Schmittgen 2001]. Statistical significance was measured
using Student's t-test, difference was considered significant at P < 0.05.
Resuts
AA Treatment Modifies miRNA Expression Profiles
To study the responses of miRNAs to AA, microarray analysis of miRNA expression
was conducted with miRNA-enriched total RNAs extracted from both cell lines
treated with respective IC50 concentration of AA (data not shown). RNA samples
were processed, labelled, and hybridized to Agilent miRNA chips as described in
Methods. Figure 1 and 2 show the effects of AA on miRNA expression in MCF-7 and
MDA-MB-231 breast cancer cells. After 48-h incubation, 11 miRNAs were
significantly upregulated, whereas 18 were significantly down-regulated in MCF-7 by
AA compared with untreated cells (Fig. 1). Among these miRNAs, miRNA-22 was
upregulated by 65.5%, whereas miRNA-199a* was downregulated by 54.2%.
Similarly, in MDA-MB-231 at the IC50 concentration, AA significantly up-regulated
the expression of 5 miRNAs and down-regulated 10 miRNAs (Fig. 2). AA enhanced
miRNA-22 expression (68%) but decreased miRNA-199a* expression (71%). A
greater number of miRNAs were down-regulated than up-regulated in both cases.
Interestingly, miRNA-22 was up-regulated and miRNA-199a* was down-regulated
consistently and substantially in both cell lines by AA.
Differentially Expressed miRNAs by Real-time PCR
Quantitative Real Time PCR was done for the two miRNAs, miRNA-22 and miRNA199a*, which were up-regulated and down-regulated, respectively, in both cell lines
by AA treatment in the microarray experiments (Fig. 3A). miRNA-enriched total
RNAs from the same preparation used for microarray analysis were reversetranscribed and amplified in the ABI Prism 7500 Sequence Detection System for
SYBR Green analysis. As shown in Fig. 3B, miRNA-22 was up-regulated by AA
treatment in
both MCF-7 and MDA-MB-231 breast cancer cells by 60.3% and
68.6%, respectively (Fig. 3B), whereas miRNA-199a* was down-regulated by 25.1%
and 36.40% (Fig. 3C), respectively. The results obtained by quantitative real-time
PCRs were comparable with and confirmed the microarray data (Fig. 3A).
Discussion
Several recent studies have demonstrated that various chemical compounds induce
differential expression of miRNAs in a variety of animals and animal cell lines
(Marsit, Eddy et al. 2006; Saito, Liang et al. 2006; Moffat, Boutros et al. 2007;
Pogribny, Tryndyak et al. 2007; Rossi, Bonmassar et al. 2007; Blower, Chung et al.
2008; Sun, Estrov et al. 2008; Zhang and Pan 2009). These chemicals include anticancer drugs as well as environmental toxicants. However, there are no studies on the
effect of natural products on expression pattern of miRNA in in vivo or in vitro. In
this study, we report, for the first time, the global expression profile of miRNAs in
human MCF-7 and MDA-MB-231 breast cancer cells and the effect on the miRNA
expression profile after 48 hours of treatment with AA extract.
Based on our microarray analysis with 923 currently known human miRNAs, 29 and
15 miRNAs were identified to have differential expression after AA treatment in
MCF-7 and MDA-MB-231 respectively. Although some of the differentially
expressed miRNAs, have been reported previously to have differential expression in
response to chemical treatment in human cell lines or animal models. However,
majority of the AA-regulated miRNAs observed in the present study have not been
reported in previous studies. Therefore, it seems that these miRNAs uniquely respond
to AA treatment. Our microarray experiments showed that AA up-regulated the
expression of some miRNAs (such as miRNA-22) while as down-regulated others
(such as miRNA- 199a*). Real-time PCR experiments with miRNA-22 and miRNA199a* further confirmed the expression patterns we found in our microarray
experiments.
The results of our investigations and others suggest that the spectrum of miRNAs that
respond to drug treatment is unique to specific groups of drugs. Investigating the
molecular regulatory mechanism of the common and drug-specific miRNAs will
allow us to better understand the therapeutic efficacy of different drugs thereby
provide new insight on screening new drugs for cancer treatment.
In conclusion, we used microarray technology to analyse the expression of all known
human miRNAs following treatment with AA. Two AA-responsive miRNAs;
miRNA-22 and miRNA- 199a*, showed major and consistent effects, which were
validated by microarray analysis and real-time PCR. Therefore, we suggest that AA
extract has anti-cancer effect that is mediated via regulating the expression of specific
microRNAs in human breast cancer cell lines. Further, manipulating the expression of
miRNA may modify the protein expression of its target genes. This suggests an
important and novel mechanism by which AA mediates its potent effects on cancer
cell proliferation.
References
Aumiller V, Forstemann K (2008) Roles of microRNAs beyond development–
metabolism and neural plasticity. Biochim Biophys Acta 1779(11): 692–6.
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell
136(2): 215–33.
Baytop, Therapy with Medicinal Plants in Turkey, Istanbul University Press, Istanbul,
Turkey (1984) 166–167.
Blower, P. E., J. H. Chung, et al. (2008). "MicroRNAs modulate the chemosensitivity
of tumor cells." Molecular Cancer Therapeutics 7(1): 1-9.
Bushati N, Cohen SM (2008) MicroRNAs in neurodegeneration. Curr Opin Neurobiol
18(3): 292–6.
Carissimi C, Fulci V, Macino G (2009) MicroRNAs: Novel regulators of immunity.
Autoimmun Rev, 2009 Feb 3. [PMID: 19200459].
Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, et al. (2008)
Regulation of progenitor cell proliferation and granulocyte function by
microRNA-223. Nature 451(7182): 1125–9.
Juteau F., Jerkovic I., Masotti V., Milos M., Mastelic J., Bessiere J.M. and Viano J.,
Composition and anti-microbial activity of essential oil of Artemisia
absinthium from Croatia and France, Planta Med. (2003) 69, 158–161.
Koul M.K., Medicinal Plants of Kashmir and Ladakh, Temperate and Cold Arid
Himalaya, Indus Publishing Company, FS-5, Tagore Garden, New Delhi
(1997) pp. 102, ISBN: 81-7837-061-6.
Lawrie CH, Soneji S, Marafioti T, Cooper CD, Palazzo S, et al. (2007) MicroRNA
expression distinguishes between germinal center B cell-like and activated B
cell-like subtypes of diffuse large B cell lymphoma. Int J Cancer 121(5):
1156–61.
Li LC (2008) The multifaceted small RNAs. RNA Biol 5(2): 61–4.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2 (-Delta Delta C (T) Method. Methods 2001; 25:
402–8.
Marsit, C. J., K. Eddy, et al. (2006). "MicroRNA responses to cellular stress." Cancer
Research 66(22): 10843-10848.
Merkerova M, Belickova M, Bruchova H (2008) Differential expression of
microRNAs in hematopoietic cell lineages. Eur J Haematol 81(4): 304–10.
Meschler J.P and Howlett A.C. Thujone exhibits low affinity for cannabinoid
receptors but fails to evoke cannabimimetic responses, Pharm. Biochem.
Behav. (1999), 62, 413–480.
Mocellin S, Pasquali S, Pilati P (2009) Oncomirs: from tumor biology to molecularly
targeted anticancer strategies. Mini Rev Med Chem 9(1): 70–80.
Moffat, I. D., P. C. Boutros, et al. (2007). "MicroRNAs in adult rodent liver are
refractory to dioxin treatment." Toxicological Sciences 99(2): 470-487.
Nelson
PT,
Wang WX,
Rajeev
BW
(2008)
MicroRNAs
(miRNAs)
in
neurodegenerative diseases. Brain Pathol 18(1): 130–8.
Pogribny, I. P., V. P. Tryndyak, et al. (2007). "Induction of microRNAome
deregulation in rat liver by long-term tamoxifen exposure." Mutation
Research-Fundamental and Molecular Mechanisms of Mutagenesis 619(1-2):
30-37.
Rossi, L., E. Bonmassar, et al. (2007). "Modification of miR gene expression pattern
in human colon cancer cells following exposure to 5-fluorouracil in vitro."
Pharmacological Research 56(3): 248-253.
Saban, K. Recep, M. Ahmet, C.A.A. and Ali, Y. Determination of the chemical
composition and antioxidant activity of the essential oil of Artemisia
dracunculus and of the antifungal and antibacterial activities of Turkish
Artemisia absinthium, Artemisia dracunculus, Artemisia santonicum, and
Artemisia spicigera essential oils, J. Agric. Food Chem. (2005) 53, 9452–
9458.
Saito, Y., G. Liang, et al. (2006). "Specific activation of microRNA-127 with
downregulation of the proto-oncogene BCL6 by chromatin-modifying drugs
in human cancer cells." Cancer Cell 9(6): 435-443.
Sun, M., Z. Estrov, et al. (2008). "Curcumin (diferuloylmethane) alters the expression
profiles of microRNAs in human pancreatic cancer cells." Molecular Cancer
Therapeutics 7(3): 464-473.
Zhang, B., X. Pan, et al. (2007). "microRNAs as oncogenes and tumor suppressors."
Dev Biol 302(1): 1-12.
Figure 1: Effects of AA treatment on miRNA expression in MCF-7 cell lines.
Microarray profile of miRNA expression in MCF-7 cells treated with AA for 48 h.
The relative increase or decrease of the 29 miRNAs that showed a significant change
in levels of expression after AA treatment compared with untreated cells.
Figure 2: Effects of AA treatments on miRNA expression in MDA-MB-231 cell
line. Microarray profile of miRNA expression in MDA-MB-231 cells treated with
AA, for 48h. The relative increase or decrease of the 15 miRNAs that showed a
significant change in levels of expression after AA treatment compared with untreated
cells.
Figure 3: Comparison of microarray analysis with real-time PCR on the
expressions of miRNA-22 and miRNA-199a*. MCF-7 and MDA-MB-231 cells
were exposed for 48 h with AA. (A) Microarray analysis; the fold changes of AAregulated miRNA-22 and miRNA-199a* compared with untreated controls. (B) RealTime PCR confirmation of miRNA-22 expression compared with normal control
values (mean fold change ± SE (n = 3), P < 0.05). (C) Real-Time PCR confirmation
of miRNA-199a* expression compared with normal control values (mean fold change
± SE (n = 3), P < 0.05).