Genistein-induced mir-23b expression inhibits the growth of breast cancer cells.

AIM OF THE STUDY: Genistein, an isoflavonoid, plays roles in the inhibition of protein tyrosine kinase phosphorylation, induction of apoptosis, and cell differentiation in breast cancer. This study aims to induce cellular stress by exposing genistein to determine alterations of miRNA expression profiles in MCF-7 cells.
MATERIAL AND METHODS: XTT assay and trypan blue dye exclusion assays were performed to examine the cytotoxic effects of genistein treatment. Expressions of miRNAs were quantified using Real-Time Online RT-PCR.
RESULTS: The IC50 dose of genistein was 175 μM in MCF-7 cell, line and the cytotoxic effect of genistein was detected after 48 hours. miR-23b was found to be up-regulated 56.69 fold following the treatment of genistein. It was found that miR-23b was upregulated for MCF-7 breast cancer cells after genistein treatment.
CONCLUSIONS: Up-regulated ex-expression of miR-23b might be a putative biomarker for use in the therapy of breast cancer patients. miR-23b up-regulation might be important in terms of response to genistein.

Introduction

Breast cancer is the most common cancer type among women. After respiratory cancers, including lung and respiratory tract cancers, breast cancer is the second leading cause of death among women [1]. Incidence and mortality rates of breast cancer may differ depending on the age, ethnicity, and socioeconomic status of patients [2]. The presence or absence of oestrogen receptor (ER), progesterone receptor (PR), and HER2/Neu receptor determine the sub-molecular classification and progression of breast cancer [3]. miRNAs are non-coding small RNA molecules (17–24 nucleotide) that repress mRNA transcription when they bind to their target region (3’ untranslated region). Reregulation of expression profiles of miRNAs has a putative role in cancer development [4]. Decreasing expression of miRNA that suppress mRNA of oncogene, or increasing of expression of miRNA that suppress mRNA of tumour suppressor gene, can trigger the cancer process [4, 5]. It has been demonstrated that oncomirs such as miR-21, miR-27a, miR-155, and miR-145 exhibit different expression patterns between breast cancer cells and non-cancerous breast cells [6-8]. In addition to this difference, altered miRNA expression profile is detected differently among molecular sub-types of breast cancer [9]. Abnormal miRNA expression pattern can induce angiogenesis and metastasis in breast cancer tumours [10]. miRNA dysregulation in breast cancer is also associated with poor survival and poor therapeutic outcome [11, 12].

Genistein, an isoflavonoid, is a prime anti-cancer component of soybean, and it plays roles in the inhibition of protein tyrosine kinase phosphorylation, induction of apoptosis, and cell differentiation in breast cancer [13]. The interaction between genistein and oestrogen receptor signalling pathway in breast cancer has been well characterised. Genistein induces oestrogen-dependent cell growth and up-regulation of ER expression, thus it has a potential impact for hormone therapy [14, 15]. Genistein-mediated ERα expression is associated with histone modification changes and genistein re-sensitises ERα-negative breast cancer cells to tamoxifen [16]. Although it has been known that genistein affects chemotherapy agent efficiency and apoptosis, the effect of genistein on miRNA profiles is still unknown for breast cancer. This study aims to induce cellular stress by exposing genistein to determine the IC50 doses of treatment conditions in MCF-7 cells. This study also aims to evaluate the single effect in terms of miRNA expression levels.

Material and methods

Tumour cell line

Breast cancer cell line (MCF-7), which was purchased from ATCC, was used as a breast cancer model.

Cell culture

Breast cancer cell line (MCF-7) was cultured in RPMI-1640 medium supplemented with 100 IU/ml penicillin, 10 mg/ml streptomycin, 1% L-glutamine, and 10% heat-inactivated foetal bovine serum, at 37°C in a humidified 95% air 5% CO2 atmosphere.

Treatment of genistein and cytotoxicity assay

MCF-7 cells were incubated at a density of 2 × 105 cells/ml of medium using 96-well plates for 24, 48, and 72 hours. Studied concentrations of genistein were 75 µM, 100 µM, 125 µM, 150 µM, 175 µM, and 200 µM. XTT assay and trypan blue dye exclusion assays were performed to examine the cytotoxic effect of IC50 dose of genistein in the MCF-7 cell line. Formazan formation was quantified spectrophotometrically at 450 nM (reference wavelength 620 nM) with a microplate reader. Viability was calculated using the background-corrected absorbance. Cells without any treatment were taken as a control group.

Isolation of miRNA

miRNA was isolated from cells exposed to IC50 dose of genistein and the control group. Isolation of miRNA and cDNA synthesis was performed using RT2 qPCR-Grade miRNA Isolation Kit and RT2 first Strand Kit, respectively, according to the manufacturers’ instructions.

Relative quantification of miRNAs

Relative quantitation of 88 microRNAs (Table 1) was measured by using real-time online RT-PCR (LightCycler 480). SNORD48, SNORD47, SNORD44, U6 were used as human endogenous controls. Alterations in the miRNAs expressions of genistein were compared to the control group. Data analysis was evaluated by ΔΔCT method, “Light Cycler® 480 Quantification Software” program, and statistical analysis was evaluated with web-based RT2 Profiler PCR Array Data Analysis.

Table 1

Target and housekeeping miRNAs that were analysed for the genistein group. Expression analyses for 88 target miRNAs were performed by real-time PCR. Table 1 describes the sequences of miRNAs

miRNA	Sequence	miRNA	Sequence	miRNA	Sequence
hsa-miR-142-5p	CAUAAAGUAGAAAGCACUACü	hsa-miR-191	CAAC GGAAUC C CAAAAGCAGCUG	hsa-miR-320	AAAAGCUGGGUDGAGAGGGCGA
hsa-miR-16	UAGCAGCACGÜAAAUAUÜGGCG	hsa-miR-17	CAAAGUGCUUACAGÜGCAGGUAG	hsa-miR-374a	UUAUAAUACAACCUGAUAAGUG
hsa-miR-142-3p	UGUAGUGUUDCCÜACUÜUADGGA	hsa-miR-130a	CAGUGCAAUGUUAAAAGGGCAÜ	hsa-let-7e	UGAGGUAGGAGGUUGUAUAGUÜ
hsa-miR-21	UAGCUUAUCAGACUGAUGUUGA	hsa-miR-20a	UAAAGUGCUUAUAGUGCAGGUAG	hsa-miR-151-5p	UCGAGGAGCUCACAGUCUAGU
hsa-miR-15a	UAGCAGCACAÜAAüGGüUUGüG	hsa-miR-27b	UUCACAGUGGCUAAGUUCUGC	hsa-miR-374b	AUAUAAUACAAC CUGCUAAGUG
hsa-miR-29b	UAGCAC CAUUUGAAAUCAGDGUU	hsa-miR-26b	UUCAAGUAAUUCAGGAUAGGU	hsa-miR-196b	UAGGUAGUUUC C UGUUGUUGGG
hsa-let-7a	UGAGGUAGUAGGUUGUAUAGUU	hsa-miR-146a	UGAGAACUGAAUUCCAUGGGUU	hsa-miR-140-3p	UACCACAGGGUAGAACCACGG
hsa-miR-126	UCGüACCGUGAGUAAUAAUGCG	hsa-miR-200c	UAAUACUGCCGGGUAAUGAUGGA	hsa-miR-100	AACCCGUAGAUCCGAACUUGUG
hsa-miR-143	UGAGAUGAAGCACUGUAGCUC	hsa-miR-99a	AACCCGUAGAÜCCGAUCUUGÜG	hsa-miR-103	AGCAGCAÜUGUACAGGGCÜAUGA
hsa-let-7b	UGAGGUAGUAGGUUGU GUGGUU	hsa-miR-19a	UGUGCAAAUCUAUGCAAAACUGA	hsa-miR-96	UUUGGCACUAGCACAUUUUUGCU
hsa-miR-27a	UUCACAGUGGCUAAGUUCCGC	hsa-miR-23a	AUCACAUUGCCAGGGAUÜÜCC	hsa-miR-302b	UAAGUGCUUCCAUGÜUUÜAGUAG
hsa-let-7f	UGAGGUAGUAGAÜUGUAÜAGÜU	hsa-miR-30a	UGUAAACAUCCUCGACUGGAAG	hsa-miR-194	UGUAACAGCAAC UC CAUGUGGA
hsa-miR-9	UCUUUGGÜUAÜCUAGCUGUAÜGA	hsa-let-7i	UGAGGUAGUAGUUUGUGCUGUÜ	hsa-miR-125a-5p	UCCC UGAGACC C UUUAACCUGüGA
hsa-miR-26a	UUCAAGUAAUCCAGGAUAGGCU	hsa-miR-93	CAAAGUGCUGUUCGUGCAGGUAG	hsa-miR-423-5p	UGAGGGGCAGAGAGCGAGACUUU
hsa-miR-24	UGGCUCAGUÜCAGCAGGAACAG	hsa-let-7c	UGAGGUAGUAGGUUGUAÜGGUÜ	hsa-miR-376c	AACAUAGAGGAAAUUCCACGU
hsa-miR-30e	UGUAAACAUCCUUGACUGGAAG	hsa-miR-106b	UAAAGUGCUGACAGUGCAGAU	hsa-miR-195	UAGCAGCACAGAAAUAUUGGC
hsa-miR-181a	AACAUUCAACGCUGUCGGUGAGU	hsa-miR-101	UACAGUACUGUGAUAACUGAA	hsa-miR-222	AGCUACAUCUGGCUACUGGGU
hsa-miR-29a	UAGCAC CAUCUGAAAU CGGUUA	hsa-let-7g	UGAGGUAGUAGUUUGUACAGUU	hsa-miR-28-3p	CAC UAGAUUGUGAGCÜC CUGGA
hsa-miR-124	UAAGGCACGCGGUGAAUGCC	hsa-miR-425	AAUGACACGAUCACUCCCGUUGA	hsa-miR-128a	UCACAGUGAAC C GGUCUCUUU
hsa-miR-144	UACAGUAÜAGAÜGAÜGUACU	hsa-miR-15b	UAGCAGCACAÜCAUGGUÜÜACA	hsa-miR-302c	UAAGUGCUUCCAUGUÜUCAGUGG
hsa-miR-30d	UGUAAACAUCCCCGACÜGGAAG	hsa-miR-28-5p	AAGGAGCUCACAGUCUAÜÜGAG	hsa-miR-423-3p	AGCUCGGUCUGAGGCCCCÜCAGU
hsa-miR-19b	UGÜGCAAAUCCAUGCAAAACÜGA	hsa-miR-18a	UAAGGUGCAUCUAGUGCAGAUAG	hsa-miR-185	UGGAGAGAAAGGCAGUUCCUGA
hsa-miR-22	AAGCUGCCAGUUGAAGAACUGU	hsa-miR-25	CAUUGCACUUGUCUCGGÜCUGA	hsa-miR-30b	UGUAAACAUCCUACACUCAGCU
hsa-miR-122	UGGAGUGÜGACAAUGGUGUUÜG	hsa-miR-23b	AUCACAUUGC CAGGGAUÜAC C	hsa-miR-210	CUGUGCGUGUGACAGCGGCUGA
hsa-miR-150	UCUCCCAACCCUUGUACCAGUG	hsa-miR-302a	UAAGUGCUUCCAUGUUUUGGUGA	SNORD48	TAACTCTGAGTGTGTCGCTGA
hsa-miR-32	UAÜUGCACAUUACUAAGUUGCA	hsa-miR-186	CAAAGAAUUCUCCUUUUGGGCU	SNORD47	CCGTTCCATTTTGATTCTGAG
hsa-miR-155	UUAAUGCUAAUCGüGAÜAGGGGU	hsa-miR-29c	UAGCACCAUUUGAAAUC GGUUA	SNORD44	GGTCTTAATTAGCTCTAACTGAC
hsa-miR-140-5p	CAGUGGUÜUUACCCUAUGGÜAG	hsa-miR-7	UGGAAGACUAGUGAUUUUGUUGU	U6	ATT GGAAC GATACAGAGAAGAT TAG
hsa-miR-125b	UCCCUGAGACCCUAACDUGüGA	hsa-let-7d	AGAGGUAGUAGGUUGCAUAGUÜ	miRTC	ACAC TAAGTAC GT C GTAT TAC
hsa-miR-141	UAACACUGUCUGGUAAAGAUGG	hsa-miR-30c	UGUAAACAUCCUACACUCUCAGC	miRTC	ACAC TAAGTAC GT C GTATTAC
hsa-miR-92a	UAUÜGCACUÜGUCCCGGCCUGU	hsa-miR-181b	AACAUUCAUUGCUGUCGGUGGGU
hsa-miR-424	CAGCAGCAAUUCAUGUUUUGAA	hsa-miR-223	UGUCAGUUUGUCAAAUACCCCA

Results

Cytotoxic effect of genistein on MCF-7 cells

Cells were incubated at a density of 2 × 105 cells/ml of medium using 96-well plates for 24, 48, and 72 hours. Studied concentrations of genistein were 75 µM, 100 µM, 125 µM, 150 µM, 175 µM, and 200 µM (Fig. 1). Untreated MCF-7 cells were considered as a control group. The IC50 dose of genistein was 175 µM and the cytotoxic effect of genistein was detected after 48 hours.

Fig. 1

Dose-dependent cytotoxicity of genistein. MCF-7 cells were treated with various concentrations of genistein. The studied concentrations of genistein were 75 μM, 100 μM, 125 μM, 150 μM, 175 μM, and 200 μM. The IC50 dose of genistein was 175 μM

miR-23b is up-regulated miRNA by genistein in MCF-7 cell line

Alterations in the expressions of miRNAs were compared with genistein untreated MCF-7 cells. miRNA expression was detected 48 hours after genistein treatment. SNORD44, SNORD47, SNORD48, and U6 genes were used for housekeeping miRNAs as the endogenous normalisation factor to define miRNA expression profiles of 88 miRNAs. miR-23b was found to be up-regulated 56.69 fold in the treatment of genistein compared to the control group of genistein untreated cells (Fig. 2).

Fig. 2

miRNA expression profiles after treatment. For the genistein group, miR-23b was up-regulated 56.69 fold after treatment. miRNA expression visualization about log2 (Fold Change) associated with genistein, compared with control

Discussion

Several studies have reported that genistein, which is an isoflavonoid and is a prime anti-cancer component of soybean, can affect miRNA expression levels [17-20]. miR-151, which has an oncogenic effect, is up-regulated in prostate cancer cell lines (PCa), and genistein treatment down-regulates the relative expression of miR-151 in PCa [21]. It is known that genistein induces expression of miR-574-3p, which has a tumour suppressor role, and this induction inhibits cell proliferation, migration, and invasion in vitro and in vivo for prostate cancers [22]. Zaman et al. showed that genistein decreases the expression of miR-23b-3p in A-498 renal cancer cell line [23]. Furthermore, suppression of miR-23b-3p increases the number of total apoptotic cells and decreases cell invasion [23]. Although it is known that genistein affects chemotherapy agent efficiency and apoptosis, the effect of genistein on miRNA profiles is still unknown for breast cancer. In this study, it was found that treatment condition, which was genistein, affected miRNA expressions in MCF-7 breast cancer cell line. The cytotoxic effects of the defined group were examined independently. Cells in the genistein group were treated with an IC50 dose of genistein for three days. The cytotoxic effect of treatment group was observed after 48 hours. After the IC50 dose of genistein was determined, miRNA qPCR array method was performed to detect regulation of miRNAs expressions in MCF-7 cell line. In this study, it was found that expression of miR-23b was up-regulated in the genistein treatment group. Majid et al. clearly showed that miR-23b is a methylation-silenced tumour suppressor in prostate cancer, and a high expression level of miR-23b is associated with higher survival rates in prostate cancer patients [24]. Stabile ectopic expression of miR-23b in HCT-116 colon carcinoma cell line reduces migration, invasion, and resistance to anoikis [25]. In vivo tumour models, which are generated from miR-23b-expressing HCT 116 cells, show that miR-23b-expressing tumours are encapsulated, non-invasive, and have low growth rate [25]. miR-23b regulates colony morphology and increases epithelial characteristics in MCF-7 cells. It is observed that miR-23b enhances focal adhesion connections and provides less lamellipodia structure after transfection in MDA-MB-231 breast cancer cells [26]. miR-23b regulates cytoskeletal reorganization and reduces cell motility and invasion via the PAK2 gene, which is a target for miR-23b in MCF-7 and MDA-MB-231 cells [26]. Furthermore, inhibition of miR-23b increases cell migration and metastasis for in vivo breast cancer models [26]. Because it is known that miR-23b has a tumour suppressor role for metastasis of breast cancer cells, miR-23b up-regulation might be important in terms of response to genistein.

In conclusion, up-regulated expression of miR-23b might be a putative biomarker for use in the therapy of breast cancer patients.