The Estrogen Responsive Breast Cancer Cells Biology Essay

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Purpose In estrogen responsive breast cancer cells, estradiol is a key regulator of cell proliferation and survival. MiR-155 has emerged as an 'oncomiR', which was the most significantly up-regulated miRNA in breast cancer. TP53INP1 is one of the targets of miR-155. Moreover, miR-155 is higher in ERα (+) breast tumors than ER (-), but no one has examined whether E2 regulates the expression of miR-155 in MCF-7 cells. In this study, the aim was to explore whether miR-155 involved in E2 regulated expression of estrogen responsive genes.

Methods Differential expression of miR-155 was detected between ER-positive MCF-7 cells and ER-negative MDA-MB-231cells by Q-PCR. Transient transfection of MCF-7 cells with miR-155i (or miR-155i NC) using the lipfectamine2000.The effects of E2 on miR-155 expression, TP53INP1 mRNA expression, cell proliferation, cell cycle, cell apoptosis, and protein expression were evaluated by Q-PCR, CCK8 assay, FCM assay, western blotting, and immunofluorescence.

Results MiR-155 was overexpressed in MCF-7 cells compared with MDA-MB-231 cells. E2 promoted miR-155 expression and negatively regulated TP53INP1 mRNA expression. Treatment with E2 increased proliferation and suppressed apoptosis, whereas transfection with miR-155i reduced proliferation and promoted apoptosis of MCF-7 cells in vitro. In addition, E2 negatively regulated the protein expression of TP53INP1, caspase-3, 8, 9 and p21, whereas transfection with miR-155i increased TP53INP1, caspase-3, 8, 9 and p21 protein level.

Conclusions E2 promoted breast cancer development and progression possibly through increasing the expression of miR-155. MiR-155, which was overexpressed in MCF-7 cells compared with MDA-MB-231 cells, contributes to proliferation of MCF-7 cells possibly through down-regulating TP53INP1.

Keywords 17β-Estradiol, miR-155, TP53INP1, MCF-7 breast cells

Introduction

The 17β-estradiol (E2) is a key regulator of proliferation and differentiation in the mammary gland , where it has a significant impact on the development, maintenance, treatment and prognosis of breast cancer. E2 regulates genes directly through binding to estrogen receptors (ERs) or indirectly through activating plasma membrane-associated ER . Therefore, ERs may participate in the genomic and non-genomic actions of E2 . Ligand-activated ERs mediate transcription by interacting directly with specific estrogen response elements (EREs) or interacting indirectly with other transcription factors, such as AP-1 or Sp-1 . This leads to transcriptional activation or repression of target genes involved in the control of cell cycle, proliferation and apoptosis .

MicroRNAs (miRNAs) are a class of small on-coding RNAs that control gene expression at the post-transcriptional level . MiRNAs controlling expression by binding to the 3'-untranslated region (3'UTR) of the target mRNAs, leading to mRNA cleavage and/or translational inhibition, thereby downregulating expression of proteins . MiRNAs encoding genes are mostly transcribed by RNA polymerase Ⅱ by Drosha into short hairpin RNAs, which are then exported from the nucleus, and processed by Dicer to form mature 21-25 nucleotide miRNAs, which are finally transferred to Argonaute proteins in RISC . In fact, almost 30% of the protein-coding genes are under the regulation of miRNAs, and many miRNAs play a crucial role in different biological processes such as proliferation, apoptosis and differentiation . Aberrant miRNA expression, not surprisingly, is a hallmark of several diseases, including cancer . MiRNAs exert their function as oncogenes or tumor suppressor genes depending on their target genes.

Several studies on miRNA analysis have indicated that E2 regulates a variety of miRNAs in ERα positive cells. E2 up-regulates 21 miRNAs and represses 7 miRNAs in MCF-7 cells, E2 induced the expression of Let-7 family members, miR-21 and miR-98 which inhibited c-Myc and E2F2 proteins expression . E2 down-regulates pre-miRNAs and mature miRNAs (miR-16, miR-143, miR-195, miR-145 and miR-125a) expression in both mice and cells . It is reported that miR-155 identified as an 'oncomiR', was the most significantly up-regulated miRNA in breast cancer , and was significantly higher in ERα (+) breast tumors than ERα (-) . MiR-155 down-regulated the translation of human FOXO3a, SOCS1, Rho protein, the tumor suppressors in breast cancer cells . But no one has examined whether E2 regulates miR-155 expression in human breast cancer cells.

In this study, we tested the assumption that miR-155, an 'oncomiR', is regulated by E2 in MCF-7 breast cancer cells. We focused on the effects of E2 on miR-155 expression in human breast cancer cells with two goals: the one is to determine whether E2 regulates the expression of miR-155, which expressed at higher levels in ERα positive breast cancer; and the second is to determine whether E2-regulated miR-155 subsequently control the expression of cell cycle and apoptosis-related gene at the post-transcriptional level. In this study, we indicated that E2 significantly represses TP53INP1, caspase-3, 8, 9 and p21expression by up-regulates the expression of miR-155 in ERα dependent manner in MCF-7 cells. These results revealed that E2-induced cell proliferation and survival by coordinately up-regulated miR-155, which may serve as a therapeutic option in breast cancer treatment.

Methods

Reagents

Two synthetic, chemically modified short single-or double-stranded RNA oligon- ucleotides: miR-155 inhibitors (miR-155i) and miR-155 inhibitors negative control (miR-55i NC) were synthesized from Shanghai GenePharma Co.Ltd.17β-Estradiol (E2) was purchased from Cayman Chemical Company.

Cell culture and transfection

Human MCF-7, MDA-MB-231 cells were provided by Staff Room of Pathophysiology, Chongqing Medical University and were maintained DMEM (Gibco) or RPMI1640 (Gibco) media supplemented with 10% NBCS (Gibco, New Zealand), 100 units/ml penicillin, and 100µg/ ml streptomycin. All cells were grown in sterile conditions at 37℃ in a humidified atmosphere of 5% CO2. Cells were seeded in 6-well plates at 30-50% confluence and grown overnight before transfection. Transfection of MCF-7 cells with miR-155i (or miR-155i NC) using the lipfectamine2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. The total experiments were classified into three groups, which were E2+miR-155i group, E2 group, and untransfected group. The sequences of oligonucleotides used are shown in Table 1.

Removal of sex hormones by charcoal-dextran

Charcoal was washed twice with cold sterile water immediately before use.5 g charcoal suspension in 500 mg dextran T40 of the same volume as serum was centrifuged at 1000-g for 10min. Supernatants were aspirated, and the serum was mixed with the charcoal precipitation. This charcoal-serum mixture was maintained in suspension by mixing at 56 °C for 30min. This suspension was centrifuged at 1000-g for 20min. This procedure was repeated twice, and the supernatants were filtered through a 0.22 µm cellulose acetate-filter. The charcoal dextran-treated NBCS was stored at -20 °C until needed.

Choose appropriate concentration of 17β-Estradiol (E2)

MCF-7 cells were seeded in 96-well plates (2000 cells/well) with phenol red-free DMEM medium containing 10% charcoal dextran-treated NBCS. After 24 h, cells were stimulated with E2, which was used in concentrations of 0.1 nM, 1nM, 10nM, 100nM, 500nM, 1000nM, followed by a further incubation for 24, 48, or 72 h. Afterwards 10 µl of CCK8 (Beyotime Inst Biotech, China) solution was added to the culture medium, and incubated for additional 3 h. The absorbance was determined at 450 nm wavelength, choose appropriate concentration of E2.

RNA isolation and Q- PCR

Total miRNA and total RNA were isolated from cultured cells with MiRNA rapid extraction kit and RNApure rapid extraction kit (Bioteke Corporation, Beijing, China) according to manufacturer's instructions. Q-PCR analyses for miRNA and mRNA were performed using TaqMan miRNA assays. U6 snRNA and β-actin served as an endogenous control for normalization. Reverse transcriptase reactions and real-time PCR were performed according to the manufacturer's protocols. Q-PCR reactions were performed on a CFX96 real-time PCR detection system from Bio-Rad Co. LTD (America), with cycle threshold values determined using the manufacturer's software. Relative expression of miR-155, TP53INP1 mRNA, relative to U6 snRNA, β-actin, was calculated using the 2-ΔΔCT method.

Cell proliferation assay

Cells at 12 h post-transfection were seeded in 96-well plates at 4000 cells/well. After that, cell proliferation was evaluated using the CCK8 according to manufacturer's instructions. Briefly, 10 µl of CCK8 solution was added to the culture medium, and incubated for additional 3 h. The absorbance was determined at 450 nm wavelength.

Cell cycle assay

Cell cycle analysis was performed using the standard propidium iodide method. In brief, cells were trypsinized, washed with cold PBS, fixed in 70% ethanol for 24 h, and stained with propidium iodide for 30 min. Finally, the cells were analyzed by flow cytometer (FCM).

Cell apoptosis rate assay

Apoptotic cells were differentiated from viable or necrotic ones by combined application of annexin V-FITC and propidium iodide (PI). The samples were washed twice and adjusted to a concentration of 1-106 cells/ ml with cold PBS. 10 µL of annexin V-FITC and 10 µL PI were added into 100 µl of cell suspension, incubated for 15 min at room temperature in the dark. Finally, 400µL of binding buffer was added to each sample without washing and analyzed using FCM.

Immunofluorescence

Caspase-3 and p21 proteins expression of tumor cells were detected using standard immunofluorescence techniques. Cells were fixed in precooked methanol (-20℃) for 10 min and blocked with 10% goat serum for 30 min, and incubated with an anti-caspase-3 activated (1:500, Beyotime, China) and p21 antibody(1:100, Beyotime, China) at 4℃ overnight. The cell were washed and incubated with FITC anti-rabbit IgG and anti-mouse IgG for 50 min at room temperature in dark, washed extensively, and examined under a confocal laser scanning microscope.

Western blotting analysis

Total proteins extracts of each group cells were resolved by12% SDS-PAGE and transferred on PVDF (Millipore) membranes. After blocking, the PVDF membranes were washed 3 times for 15 min with TBST at room temperature and incubated with TP53INP1 (rabbit anti-human polyclonal antibody; 1:100, Santa Cruz), caspase-3 (rabbit anti-human monoclonal antibody; 1:1000, epitomics), caspase-3 activated (rabbit anti-human monoclonal antibody; 1:1000, Beyotime), caspase-8 (rabbit anti-human monoclonal antibody; zymogen1:1000, fragment1:500, Beyotime), caspase-9 (mouse anti- human monoclonal antibody; 1:1000, Beyotime), p21(mouse anti- human monoclonal antibody; 1:200, Beyotime) for overnight at 4℃. Following extensive washing, membranes were incubated with secondary peroxidase-linked goat anti-rabbit IgG (1:5000, Beijing 4A Biotech Co.Ltd) and goat anti-mouse IgG (1:5000, Beijing 4A Biotech Co.Ltd) for 2 h. After washing 3 times for 15 min with TBST at room temperature once more, the immunoreactivity was visualized by enhanced chemiluminescence (BeyoECL Plus kit, Beyotime), and membranes were exposed to Kodak XBT-1 films.

Statistical analysis

Each experiment was performed as least three times, and data are shown as the mean ± SD (`x±s) where applicable, and differences were evaluated using one-way ANOVA for 3-group comparisons and t tests for 2-group comparisons. The probability of P < 0.05 was considered to be statistically significant.

Results

Differential expression of miR-155 between MCF-7 and MDA-MB-231 cells

The ERα positive breast cancer cell line MCF-7 was used in vitro model of E2, ERα expression was monitored by western blot analysis, using as control extracts from ERα negative MDA-MB-231 cells (Fig. 1A). In our study, we found that miR-155 was significantly overexpressed in MCF-7cells with ERα positive compared with MDA-MB-231 cells with ERα negative using Q-PCR analysis. As shown in Figure1B, the expression of miR-155 in MCF-7 cells was 4.86 fold (p=0.024) that of MDA-MB-231cells, and the difference has statistical significance.

The appropriate concentration of E2

The growth stimulatory effects of E2 on MCF-7 cells in a range of concentrations were determined by CCK8 assay. The percentage survival of MCF-7 cells increased upon incubation with 0.01 nM to 500 nM E2 (Fig. 2) compared with the untreated control. Within 24 h incubation, no obvious survival stimulation was observed. It was not until 48 h incubation that estrogen induced an increase in percentage survival from 100% in untreated controls to a maximum of 142% in 100 nM E2- treated cells. Increasing the incubation time to 72 h further increased the percentage survival. At this time period, 100 nM E2 attained the maximum survival stimulation of 171% with respect to controls. On the other hand, 1000 nM E2 reduced cell survival from 99% to 84% after 24 and 72 h treatment respectively. So, we choose 100 nM E2 for the future experiment.

E2 promotes the expression of miR-155 and negatively regulated the expression of TP53INP1 mRNA

To examine the identity of E2-regulated miR-155 in estrogen-responsive breast cancer cells, ER-positive MCF-7 breast cancer cells were treated with 100 nM E2 or 100 nM E2+miR-155i for 48 h. Transfection efficiency was detected at 24 h post-transfection (Fig. 3A) by confocal laser scanning microscopy. Q-PCR indicated a 93% increase in miR-155 by E2 (Fig. 3B) and a 88% reduction in TP53INP1 mRNA by E2 (Fig. 3C).

E2 stimulated the proliferation and inhibited the apoptosis of MCF-7 cells

To investigate the biological effects of E2, cell proliferation was measured after differential treatment. As shown in Figure 4A, treatment with E2 significantly stimulated the growth of MCF-7 cells compared with control and E2+miR-155i  (Fig. 4A, P<0.05). We detected the effects of E2 on cell cycle and cell apoptosis rate of MCF-7 cells, found that E2 significantly reduced G0/G1 phase population, but had more profound effect on G2 phase population compared with control and E2+miR-155i (Fig. 4B, 4D, P<0.05). As shown in Figure 4C and 4E, E2 significantly reduced cell apoptosis rate of MCF-7 cells compared with control and E2+miR-155i (Fig. 4C, 4E, P<0.05).

E2 negatively regulated the protein expression of TP53INP1, Caspase-3, 8, 9, P21 in MCF-7 cells

To search for potential targets of miR-155 that may influence proliferation, apoptosis of cells, TargetScan, Pictar-Vert and microRNA. Org were employed for this purpose. TP53INP1 was found among potential targets of miR-155 combinational predicted by the three softwares. To confirm the role of up-regulation of miR-155 in the reduce of protein expression of TP53INP1, caspase-3, 8, 9 and p21, MCF-7 cells were transfected with miR-155i followed by treatment with 100 nM E2 for 24h.Treatment with E2 led to nearly 28% down-regulation of TP53INP1 compared with control, whereas transfection with miR-155i induced 100% increased in the TP53INP1 compared with E2 (Fig. 6A ).The cell cycle and apoptosis-related protein caspase-3, 8, 9 and p21 were detected as the potential downstream genes of TP53INP1.Treatment with E2 induced 66%, 52%, 39% and 44% reduced in the caspase-3, 8, 9 and p21 compared with control, whereas transfection with miR-155i led to nearly 214%, 89%, 85% and 64% up-regulation of caspase-3, 8, 9 and p21 compared with E2 (Fig. 6B ~E). The expression tendency of caspase-3 and p21 was supported by the results of immunofluorescence (Fig. 5).

Discussion

MiRNAs represent a class of small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level by targeting mRNAs and thereby triggering either translation repression or RNA degradation. MiR-155 represents a typical multifunctional miRNA , which is over-expressed in a variety of human malignant tumors such as breast cancer , lung cancer , thyroid tumor , pancreatic cancer . Recent data indicate that miR-155 plays a crucial role in tumor development and is closely related to disease diagnosis and prognosis. The available experimental evidences indicating that miR-155 promotes tumor growth, invasion and metastasis through inhibits downstream targets such as SHIP1 , C/EBPβ and SOCS1 . Multiple lines of evidence show that miR-155 relevant to the pathogenesis of breast cancer, where it may represents a molecular mechanism that could impact the onset, development and progression of this hormone-responsive disease . The goal of this study was to investigate the role of E2 in modulating miR-155 expression in MCF-7 breast cancer cells. This is the first, to our knowledge, report that E2 up-regulates miR-155 and thus down-regulates the protein expression of miR-155 target gene TP53INP1 and downstream genes caspase-3, 8, 9, p21. In this study, we compared the MCF-7 cells with ER positive and MDA-MB-231 cells with ER negative miR-155 expression. Using Q-PCR, we found that miR-155 was significantly overexpressed in MCF-7 cells with ER positive. Our data were supported by the recent reports showing that miR-155 expression was significantly higher in ERα (+) than ERα (-) breast tumors .

Considering the biological significance of the results obtained, we focused our attention on E2-regulated miR-155 in MCF-7 cells to explore the role of miR-155 in modulating the responses to oestrogen of hormone-responsive genes in breast cancer cells. Firstly, in our study, we chose the appropriate concentration of E2 with 100 nM for the future experiment (Figure 2). We observed that there was a 93% increase in miR-155 expression with E2 treatment, meanwhile, there was a significative reduction in miR-155 expression with miR-155i transfection (Figure 3B). Our results show that E2 increased the expression of miR-155 in MCF-7 cells. As shown in Figure 4, treatment with E2 significantly stimulated the growth and inhibited the apoptosis rate of MCF-7 cells. Therefore, we conclude that E2 stimulated the growth and inhibited the apoptosis of MCF-7 cells relative to the increase of miR-155 expression. We next asked how miR-155 might function inside cells accounting for the effect of miR-155 on biological behavior of MCF-7 cells. TP53INP1 expression was investigated immunohistochemically in 81 cases of breast carcinoma, compared with normal breast tissue, decreased TP53INP1 expression was found in 45 cases (55.6%) . The expression level of TP53INP1 was inversely linked to high histological grade, tumor size, positive lymph node metastasis and aberrant P53 expression . These suggest that TP53INP1 acts as a suppressor of breast carcinoma progression. Moreover, TP53INP1 expression is repressed by the oncogenic miR-155, which is overexpressed in pancreatic ductal adenocarcinoma (PDAC) cells . The TP53INP1 is one of the targets of miR-155 . Therefore, we focused our attention on TP53INP1 involving p53 signal pathways.

The main pathway alterations in human breast cancer involve p53, which as a multifunctional transcription factor, exerts its tumor suppressor function mainly through transcriptional induction of target genes involved in several processes, including cell cycle checkpoints and apoptosis . TP53INP1 (tumor protein 53-induced nuclear protein 1) is a pro-apoptotic stress-induced p53 target gene . It is a tumor suppressor gene with a known role in cell cycle arrest and p53-mediated apoptosis through its anti-proliferative and pro-apoptotic activities via p53-dependent and p53-independent pathways . Moreover recent findings have shown a significant reduction or loss of the expression of TP53INP1 during the development of cancers of the breast , stomach , and pancreas . Besides TP53INP1-positive rate decreased with the progression of gastric cancer; and TP53INP1 protein negativity was significantly associated with aggressive pathological phenotypes of gastric cancer . These suggest that TP53INP1 plays an important role in suppression of tumor progression . Our data show that the up-regulation of miR-155 by E2 correlated with the down-regulation of TP53INP1 RNA and protein (Figure 3C and 6A). The conclusion that E2-inhibited TP53INP1 expression through increased miR-155 expression in MCF-7 cells is further supported by data showing that miR-155i inhibited E2-induced function on TP53INP1 RNA and protein. MiR-155 can directly repress the TP53INP1 protein expression through its binding to the binding sites in 3'UTR of human TP53INP1 gene , thereby negatively regulating TP53INP1 function. Thus, we have reasons to believe that TP53INP1 is possible the target gene of miR-155 in MCF-7 breast cancer cells, but that remain to be verified in future.

During the normal development and somatic maintenance of all multicellular organisms, apoptosis, a programmed cell death mechanism, is a fundamental process and thus is highly conserved and tightly regulated through numerous signaling pathways; and its dysregulation contributes significantly to numerous human diseases, primarily through changes in the expression and activation of key apoptotic regulators . Apoptosis is mediated by the caspase family of cysteine proteases that play a key role in coordinating the stereotypical events that occur during apoptosis. Caspases are classified into the upstream initiator, such as caspase-8, -9 and -10, and the downstream executioner, such as caspase-3, -6 and -7 . Cell cycle arrest is closely linked to apoptosis. P21 is located in p53 gene downstream cyclin-dependent kinase inhibitor, it and p53 together constitute the cell cycle checkpoint G1, that plays a key role in promoting G1cell cycle arrest. Our data showing the down-regulation of TP53INP1 by miR-155 correlated with down-regulation of caspase-3, -8, -9 and p21 (Figure 5 and Figure 6). Therefore, TP53INP1 regulates cell cycle may through down-regulates p21and induces apoptosis by activate caspase way.

What is the mechanism that how E2 to regulate the expression of miR-155. The activating protein 1 (AP-1), a known transcription factor for regulate normal growth and development as well as carcinogenesis, is a potential site for cross-talk with hormones in breast cancer cells . To our knowledge, E2 enhances AP-1 activity that could increase the expression of genes that are critical for tumorigenesis, including influencing cell survival, proliferation, differentiation and invasion. MiR-155 encoded by the BIC gene, functions as a miRNA with oncogentic properties. In a previous study, the upstream promoter region of BIC/miR-155 was reported to contain putative AP-1 binding sites . Recent study indicate AP-1 pathway was required for the up-regulation of miR-155 in response to H. pylori and induction of the BIC promoter by B cell receptor avtivation requires a conserved AP-1 element . Moreover, the AP-1 site plays a critical role in facilitating BIC/miR-155 promoter activity in EBV-infected type â…¢ latency cells and LPS-stimulated HTR-8/SVneo cells . These demonstrated that AP-1 plays a central role in the induction of miR-155. Therefore, we can consider that E2 up-regulate the expression of miR-155 may be by interacting indirectly with AP-1 transcription factor, but this deduction required further to evaluate. We believe that our apparent data of E2 up-regulating miR-155 expression add complexity to understanding of E2 action in breast tumorigenesis.

Acknowledgments We thank Professor Huayu Deng (pathophysiology of Chongqing Medical University) and Professor Jing Zhao (pathophysiology of Chongqing Medical University) for the technical assistance and suggestions.

Conflict of interest The authors declare that they have no conflict of interest.

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