Introduction

MicroRNAs (miRNAs) are small (19-25 ribonucleotides), single-stranded non-coding RNAs found in plant, invertebrate and vertebrate genomes (1). They regulate gene expression at the post-transcriptional level by inducing mRNA degradation or translational inhibition of specific messenger RNA (mRNA) targets (2,3). miRNAs are involved in regulating cell differentiation, cell cycle progression and apoptosis. More than 900 miRNAs have been identified in humans to date, and each miRNA is assigned a numerical identifier (4).

Deregulation of miRNA expression has been demonstrated in many types of cancers (5-7). Many miRNAs are located at fragile sites or cancer-associated regions of the genome, which may explain why there is a correlation between tumorigenesis and aberrant expression of certain miRNAs (8). Although a large number of miRNAs have been identified to date, the role for many of them in tumorigenesis and their underlying mechanisms remain to be determined. miR-205, located in 1q32.2, was shown to be overexpressed in head and neck cancer compared with lung, breast, colorectal, prostate, and pancreatic cancer cell lines (9). Yanaihara et al. (10) have shown that miRNA expression profiles are diagnostic and prognostic markers for lung cancer. Upregulation of miR-205 was observed in bladder cancer (11) as well as in adult mouse corneal and footpad epithelium (12).

Taylor and Gercel-Taylor (13) found that miRNA from both ovarian tumor cells and exosomes from the same patients were positive for 218 of 467 mature microRNAs analyzed. miRNA profiling of circulating tumor exosomes could thus potentially be used as a surrogate diagnostic marker for biopsy profiling, which could allow its utility to be extended to screening asymptomatic populations. Chen et al. (14) sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. They also identified specific expression patterns of serum miRNAs from lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints of various diseases. The expression of miR-205 in the serum of breast cancer has not been reported. In this study, we report that miR-205 is downregulated in breast tumor tissues, which is in good agreement with a previous report (15). Importantly, ectopic expression of miR-205 significantly enhance apoptosis and anchorage-independent growth of MCF-7 breast cancer cells. We further demonstrated that miR-205 is down-regulated and miR-155 is up-regulated in breast cancer serum. This observation may be a new biomarker for breast cancer diagnosis and screening.

Materials and methods

Main reagents

Penicillin-streptomycin solution, ficoll-hypaque solution, trypsin-EDTA solution, RPMI-1640 medium, Dulbecco’s modified Eagle’s medium (DMEM/F-12), and 1% antibiotic-antimycotic solution were obtained from Life Technologies GIBCO, Grand Island, NY, USA. LiporeetamineTMZ000 was purchased from Invitrogen. OPTI-MEM was obtained from GIBCO. AnllexinV-FITC apoptosis detection kit was obtained from KeyGEN BioTECH, Nanjin, China. hsa-miR-205 mimics and cy3 dye-labeled miR negative control was purchased from RiboBio Co. Ltd., Guangzhou, Guangdong. 10% fetal bovine serum was obtained from HyClone, Logan, UT.

Samples and cases

For the miRNA microarray analysis, fresh samples from four cases of human BC and paired normal adjacent tissues (NATs, >2 cm from cancer tissue) were obtained from Tianjin cancer hospital between 2007 and 2009. The patients aged 50-55, two samples with ER(+)PR(+)Her-2(+) were collected and the others with ER(-)PR(-)Her-2(-).The fresh specimens were stored at 4 °C for 24 h in RNA Later (Ambion Inc.) and then at −80 °C liquid nitrogen until further use. The samples used were not subjected to preoperative radiotherapy and/or chemotherapy and were diagnosed as infiltrating carcinoma by pathology. Before RNA extraction, sections were stained with H&E for histological diagnosis and tumor cell evaluation. Only those cases with >70% tumor cell population in the section were used in this study. Two independent sets of plasma samples were used. Plasma samples from age- and gender-matched healthy donors and breast cancer patients (Stage I-IV) were obtained at the Tianjin Medical University Cancer Institute and Hospital, Tianjin, China, between 2007 and 2009. Twenty of these individuals were breast cancer patients, and 10 were normal healthy individuals with no evidence of any disease. All blood samples were collected prior to any therapeutic procedures, including surgery. The plasma was isolated within an hour by centrifugation at 1,500× g at room temperature for 20 minutes and stored as multiple aliquots in fresh tubes at −80 °C.

MiRNA microarray experiments

For miRNA microarray analysis, total RNA from fresh BC tissue was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA samples were analyzed by CapitalBio (CapitalBio Corp.). Procedures were performed as described in detail on the CapitalBio website (http://www.capitalbio.com). Briefly, miRNA was separated from 30-50 mg total RNA using the Ambion miRNA Isolation Kit. Fluorescein-labeled miRNA (16) were used for hybridization on each miRNA microarray chip. Each chip contained 924 probes in triplicate corresponding to 677 human (including 122 predicted miRNAs), 461 mouse and 292 rat miRNAs found in the miRNA Registry (http://microrna.sanger.ac.uk/sequences/; accessed August 2007). Image intensities were measured as a function of the median foreground minus the background as previously described (17). Raw data were normalized and analyzed in GenePix Pro 4.0 software (Axon Instruments). Expression data were median-centered using the global median normalization function of the Bioconductor package (http://www.bioconductor.org). Using miRNA microarray analysis, we evaluated miRNA expression profiles of four BC samples and the corresponding NAT. To identify differentially expressed miRNAs among the human, rat and mouse miRNAs on the chip.

RNA isolation and quantitative RT-PCR in plasma

Total RNA containing small RNA was isolated from plasma using the Trizol LS reagent (Invitrogen Life Technologies) according to the manufacturer’s protocol with the following modifications. The Trizol LS reagent was mixed with plasma in a 3^:1 ratio and incubated for 5 minutes. After the addition of chloroform, tubes were shaken well and centrifuged to separate the upper aqueous phase, which was carefully transferred to a fresh tube. Isopropanol was then added to the aqueous phase for 30 minutes followed by centrifugation at 12,000× g for 10 minutes. The RNA precipitate was washed with 75% ethanol and centrifuged at 7,500× g for 5 minutes. miRNA-specific stem-loop primers (Table 1) and other primers were obtained from RiboBio Co. Ltd, Guangzhou, Guangdong. Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR) was performed with an ABI Prism 7000 sequence detection system (Applied Biosystems). RT PCR system: (1×) 10 mM dNTPs (0.15 L), Multi-Scribe Reverse Transcriptase (1 L), 10× RT buffer (1.5 L), RNase Inhibitor (0.19 L), nuclease-free water (6.66 L), RNA sample (2.5 L), 5× RT primer (3 L); 16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min, 4 °C forever. Pre-amplification system: (1×) TaqMan PreAmp Master Mix (2×) (10 L), nuclease-free (7.67 L) water, 20× primer (Applied Biosystem 1^:50 dillution) (1 L), miRNA RT product (1.33 L); Hold 95 °C 10 min, Hold 55 °C 2 min, Hold 72 °C 2 min, 95 °C 15 sec, 60 °C 4 min, Repeat step 4 and 5 for 12 cycles, Hold 99.0 °C 10 min. Real time PCR system: 2× TaqMan Universal PCR Master Mix (10 µL), 20× PCR Primer (1 µL), nuclease-free water (7.67 µL) and miRNA RT product (1^:50 dilution) (1.33 L). qRT-PCR was then performed at 95 °C for 10 minutes, 95 °C for 15 seconds and 60 °C for 60 seconds, with the last two steps repeated for a total of 45 cycles. Each reaction was performed in triplicate. miRNA expression was defined based on the threshold cycle (Ct), and relative expression levels were calculated as 2^{-[(Ct of miR-205) – (Ct of U6)]} after normalization with reference to expression of U6.

Table 1 Primers for miR-205, miR-155 and U6
Full table

Cell culture

The MCF-7 human breast cancer cell line was obtained from the center laboratory of Tianjin cancer hospital and was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution. Cells were grown to confluence at 37 °C in 5% CO₂ atmosphere.

Synthesis and transfection of miRNA mimics

The miR-205 mimics and negative control were purchased from RiboBio (RiboBio Co. Ltd, Guangzhou, Guangdong). Transfection of microRNA mimics and the negative control were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. The third group was treated with only lipo-2000, we define it as blank group.

Apoptosis assay

12, 24, 48, and 72 hours after transfection, 40 µL of MTT (5 mg/mL) was added to each well of a 96-well plate and the cells were cultured for 4 h. The MTT medium mixture was then discarded and 100 µL of dimethyl sulfoxide (DMSO) was added to each well. Absorbance was measured at 490 nm using a multi-well spectrophotometer (Bio-Tek, USA). Each reaction was performed in triplicate. For cell cycle analysis, the cells were resuspended in PBS and then fixed in ethanol at −20 °C overnight. The cells were washed with PBS and resuspended in Staining Solution (50 µg/mL of propidium iodide, 1 mg/mL of RNase A, 0.1% Triton X-100 in PBS). The stained cells (1×10⁶) were then analyzed with a flow cytometer. Cell apoptosis was detected using an AnllexinV-FITC apoptosis detection kit (KeyGEN BioTECH, Nanjin, China) according to the manufacturer’s protocol and FACS analysis.

Statistical analysis

Statistical comparisons were performed with SAM software (SAM version 2.1, http://www-stat.stanford.edu/ztibs/SAM/index.html) (18). SAM analysis generated a list of differentially expressed miRNAs with a false discovery rate of 0% (FDR=0).

The relative miRNA levels were quantified using 2^-△△T; the other data are presented as from at least three separate experiments. Multiple group comparisons were performed with ANOVA (P<0.05).

Results

MiRNA expression profile of BC

SAM analysis generated a list of differentially expressed miRNAs at FDR=0 (0% false discovery rate). We identified one miRNA that was up-regulated greater than two-fold (miR-155) in BC compared with NAT. Nine miRNAs were down-regulated greater than two-fold: let-7b, miR-381, miR-10b, miR-125a-5p, miR-335, miR-205 and miR-145 (Table 2).

Table 2 miRNA fold changes in breast cancer detected by miRNA microarray
Full table

MiR-205 and miR-155 expression in the serum of BC patients versus control

Total RNA was isolated from each sample and miRNA content was determined by qRT-PCR. We profiled miRNA expression in the serum of 20 BC patients and 10 healthy controls. miR-205 expression on average was down-regulated in BC patients compared with the matched healthy controls (Figure 1; P<0.05) while miR-155 expression was up-regulated (Figure 1; P<0.05). We investigated associations between miR-205 and the clinicopathologic features of the BC patients but found no significant correlation.

miR-155 expression had significant difference in the clinical stage, molecular type, Ki-67 and p53 status of BC patients (P<0.05) but no significant difference in age, and histological grade. The relative expression of miR-155 was strongest in phase III, followed by phase II and phase I; it was strongest in “triple negative” subtype, followed by HER-2(+), luminal B, and luminal A; and finally, it was stronger in Ki-67(+) than in Ki-67(-) and stronger in p53(-) than in p53(+) patients (Table 3).

Table 3 Analysis of miR-155 expression and the clinicopathologic parameters of breast cancer patients
Full table

Figure 1 A. qRT-PCR result of miR-205 expression; B. melting curve of miR-205; C. qRT-PCR analysis shows that miR-205 expression is lower in the serum of BC patients compared to normal controls (Stem loop RT–PCR analysis of miRNA in BC serum and healthy controls - U6 rRNA was used as an internal control and the 2^−ΔΔCt method was used to analyze the qRT-PCR data); D. qRT-PCR analysis of miR-155 expression; E. melting curve for miR-155; F. qRT-PCR analysis shows that miR-155 expression is higher in the serum of BC patients compared with normal controls

Growth inhibition of MCF-7 cells by miR-205

The cellular activity in each group was determined using the MTT method 12, 24, 48, and 72 hours after transfection, from which growth curves were established and the relative cellular proliferation activities were calculated (Figure 2). It was found that the relative proliferation activity was 2.28±0.25%, 4.35±0.28%, and 4.62±0.31% in the 72-hour transfection group, negative control group, and blank group, respectively. Compared with the negative control group and blank group, the growth of MCF-7 cells was significantly inhibited in the transfection groups (P<0.01); however, no such difference was found between the negative control group and the blank group (P>0.05).

Figure 2 Growth curve of MCF-7 cells transfected with miR-205 mimics after 12 h, 24 h, 48 h and 72 h

MiR-205 induces apoptosis of MCF-7 cells

We conducted an MCF-7 cell proliferation assay to determine whether miR-205 suppresses MCF-7 cell entry into S phase. A comparison of treatments between the three groups (Figure 3) indicated that the G0/G1 ratio was increased and S phase was lower, but there was no significant difference between the groups (P>0.05). Moreover, we examined cell apoptosis in MCF-7 cells using an AnllexinV-FITC apoptosis detection kit and found that levels of cell apoptosis were increased in cells transfected with miR-205 mimics versus other groups (Figure 4). Taken together, our data indicate that miR-205 suppresses MCF-7 cell growth via enhancement of apoptosis.

Figure 3 MCF-7 cells were stably transfected with either miR-205 mimics, a negative control, or were untreated (blank). A. Percentage of MCF-7 cells transfected with miR-205 mimics at different phases of the cell cycle; B. Percentage of MCF-7 cells transfected with negative control at different phases of the cell cycle; C. Percentage of blank MCF-7 cells at different phases of the cell cycle. Data were obtained by densitometry measurement and are represented as the mean of three experiments

Figure 4 A. Cell apoptosis of the transfected group was 11.76±1.72%; B. Cell apoptosis of the negative control was 3.03±1.63%; C. Cell apoptosis of the blank control was 2.58±1.49%. Cell apoptosis of the transfected group was higher than the other two groups (P<0.01, but there was no significant difference between the other two groups (P>0.05)

Discussion

miRNAs were first linked with cancer in 2002 initially in chronic lymphocytic leukemia (CLL) and subsequently in many other types of cancer, including BC (19,20). miR-155 was first found as an oncogene and was highly expressed in many types of tumors (21). BC, the second-leading cause of cancer-related deaths in women, is expected to account for 26% of new cancer diagnoses in 2008 (22). Several miRNAs are associated with BC. Patients with low miR-335 and miR-126 have been observed to have shorter median times to metastatic relapse (23). Iorio et al. (24) used a miRNA microarray to evaluate miRNA expression profiles of 10 normal and 76 neoplastic breast tissues. They found that miR-10b, miR125b and miR-145 were down-regulated in breast cancer while miR-21 and miR-155 were up-regulated. Based on microarray expression experiments, we found that miR-155 was highly expressed in BC while miR-145, miR-335, miR-10b, miR-125a and miR-205 were down-regulated. These results were similar to another study (24). miR-205 was found to be higher in ER⁺PR⁺Her2⁺ BC relative to other subtypes (25,26); however, other studies observed high miR-205 expression in ER⁻PR⁻Her2⁻ tumors, but low expression in other tumor types, and low miR-205 expression in metastatic BC cell lines (5). Likewise, other studies found low miR-205 expression in clinical samples of metastatic BC compared to non-metastatic cancers (24). In our study, however, there are only four paired tissues; thus, we cannot analyze specific subsets.

Although tumor tissue miRNA have been demonstrated to be associated with the development and prognosis of tumors in many studies, the complex maneuver and large trauma to patients during its determination make it difficult to be applied to clinical practice. On the contrary, the levels of tumor associated microRNAs in the serum of the peripheral blood are much easier to determine, and therefore are more feasible for clinical application. Many articles have reported the association between serum miRNA and tumors, which provides a solid foundation for the application of serum miRNA as a molecular marker in the early diagnosis and prognosis of tumors (13,14). miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity (27,28). Plasma miR-92 was used to diagnosis the colorectal cancer (CRC), with a sensitivity of 89% and specificity of 70% in discriminating CRC from control subjects. It can be a potential non-invasive molecular marker for CRC screening (29). Based on the differentially expressed miRNA obtained by chip screening, we further determined the expression of miR-205 and miR-155 in the serum of 20 patients with BC and in control samples using real time technology. It was found that these two miRNAs were differentially expressed within the serum of BC patients; furthermore, miR-155 was associated with the clinical stages of BC and with ki-67, p53 and molecular type. Its relative expression in serum was highest in triple negative BC, followed by HER-2(+), luminal B, and luminal A. In a multi-factor correlation analysis among 49 BC patients who had received adriamycin monotherapy, Sorlie et al. found that the survival rates significantly differed among patients with different molecular types: luminal A > luminal B > triple-negative and HER-2+/ER-group (30). Therefore, serum miR-155 levels are correlated with malignancy and survival rates of BC. Ki-67 is a nuclear antigen expressed in proliferative cells. Ki-67 expression represents the proliferative activity of tumor cells and therefore is often used as a reliable marker of proliferation. BC positive for Ki-67 tends to be more malignant. In such cases, tumor cells had high proliferative activity, strong invasion potential, and a high probability for metastasis; therefore, such patients usually have poor prognosis. Patients with high Ki-67 label index had significantly decreased disease-free survival and overall survival (31). Wild-type p53 gene is a key tumor suppressor gene. It can trigger cell cycle arrest, induce apoptosis, and promote differentiation (32). After genetic mutation, p53 will become mutant p53, which can induce the inhibition of cell apoptosis, uncontrolled cellular proliferation and ultimately cause the cancer development. In our current study, serum miR-155 significantly differed in terms of Ki-67 and p53 (P<0.01), with the relative expression of Ki-67(+) larger than Ki-67(-) and that that of P53 (-) larger than P53 (+), indicating that serum miR-155 levels were associated with tumor cell proliferation, invasion, apoptosis and prognosis. High miR-155 expression was detected when tumor cells had high proliferative activity and decreased apoptotic capability.

Studies have shown that many miRNAs can promote or inhibit tumor cell growth (33). For example, transfection of anti-miR-199a oligonucleotides into cervical cancer cells suppressed cell growth in vitro (34), and inhibition of miR-141 decreased cell growth in human cholangiocarcinoma cell lines (35). Our miRNA profiling suggests that miR-205 is specifically down-regulated in BC. Moreover, miR-205 expression is also lower in BC serum compared to healthy controls, suggesting that miR-205 is a potential tumor suppressor in BC. In support of this notion, we demonstrate that ectopic expression of miR-205 suppresses proliferation and increases breast tumor cell apoptosis, similar to the result of Wu (36). One study found that inhibition of MCF-7 cell growth was related to miR-205 loss in fragile sites (8).

The efficient extraction and accurate identification of miRNAs from serum represents a key first step toward the development of a noninvasive blood-based detection test for BC. Screening serum for miRNAs that predict the presence of breast cancer is feasible and may be useful for BC detection. Furthermore, it will provide useful information for evaluating BC staging and prognosis.

Acknowledgements

This study was supported by National Natural Science Foundation of China (No. 30872955).

Disclosure: The authors declare no conflict of interest.

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