Introduction
Breast cancer is the most commonly diagnosed cancer in women, and the second leading cause of cancer deaths in the developed world. Although many advanced treatments have emerged following improvement in clinical instruments and methods, metastasis still leads to cancer mortality and poor prognosis (1). Almost 30% of early breast cancers eventually develop recurrence and metastasis (2). As a result, research and development of treatment targeting breast cancer are of great importance.
miRNAs are endogenous, noncoding small RNAs with 20–25 nucleotides in length (3). They play an important regulatory role through complimentary binding of the 3′ untranslated regions (UTRs) of target genes thus resulting in the degradation of the target mRNA and inhibition of translation (4). Since the initial discovery of miRNAs in 1993 (5), they have been shown to affect multiple cellular processes (6), and in particular, have been shown to play significant roles in cancer development and progression (7,8). Abnormal patterns of miRNA expression have been observed in various cancer types, including breast cancers (6,9–11), colon (12), lymphomas and leukemias (13), head and neck (14) and hepatocellular carcinoma (15). Functionally, abnormal miRNA expression can affect tumor cell proliferation (16), apoptosis (8), development of metastases (17), invasion (16,17), chemo- and radiation-sensitivity (18). Previous studies have indicated that miRNAs can be useful for cancer diagnosis and therapy (19). Let-7 was the first identified miRNA and its downregulation has a prognostic impact on the survival of surgically treated lung cancer patients (20). Let-7a expression increases after differentiation and in mature tissue, but is nearly undetectable in the embryonic stage (21). Although let-7 family members can all function as tumor suppressors (22–24), let-7a is the one that is the most reported to downregulate c-myc (24–26).
In this study, we investigated function of let-7a in human breast cancer. First, total RNAs of breast cancer cells (MDA-MB-231, MCF-7), breast cancer tissues and corresponding adjacent normal tissues were extracted and used to detect let-7a expression by qRT-PCR. Secondly, the effects of let-7a on proliferation, colony formation, migration and invasion of breast cancer cells were assessed by in vitro cell culture experiments, which further clarified the role of let-7a in breast cancer development. Finally, western blotting demonstrated that let-7a negatively regulated HMGA1 protein expression which in turn contributed to tumor formation.
Materials and methods
Breast cancer tissues and normal tissues
Documented informed consent was obtained from all subjects and the Ethics Committee of Jiangsu University approved all aspects of the study. Breast cancer tissues and corresponding adjacent normal tissues were obtained at the Department of Surgery, the Second People’s Hospital of Kunshan, China. Both tumor tissues and corresponding adjacent normal tissues were histologically confirmed. The tissues obtained were immediately stored at −80°C.
Cell culture
The human breast cancer cell lines MCF-7, and MDA-MB-231 were provided from Nanjing University and cultured in Dulbecco’s modified Eagle’s medium with low glucose (L-DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, ExCell Biology, China) at 37°C in humidified 5% CO2.
miRNA transfection
Let-7a mimics or mimics negative control (mimics NC) and let-7a inhibitor or inhibitor negative control (inhibitor NC) were synthesized and purified by GenePharma (Shanghai, China). All of the sequences are listed in Table I. Transfection was performed with lipofectamine 2000 (Invitrogen). Let-7a mimics and let-7a inhibitors were used at a concentration of 100 nM. The same concentrations of mimics and inhibitor negative controls were used.
RNA isolation and qRT-PCR
The total RNA of tissues was isolated with the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA was also isolated with the TRIzol reagent from cell lines which were transfected with let-7a mimic and negative control or let-7a inhibitor and inhibitor negative control for 24 h. For measurement of let-7a RNA expression, qRT-PCR was performed using a SYBR green-containing PCR kit (GenePharma). For the detection of HMGA1 mRNA, cDNA was synthesized from 1 μg of total RNA using the reverse reaction kit in accordance with the manufacturer’s instructions (Thermo). After that, qRT-PCR was performed using UltraSYBR Mixture (with ROX) Assay kits (CWBio, China) according to the manufacturer’s instructions. The CFX-96 real-time fluorescence thermal cycler (Bio-Rad) was used for quantitative miRNA and mRNA detection. The relative expression levels of miRNA and mRNA were normalized to the expression of U6 snRNA and β-actin mRNA, respectively. The expression of each gene was quantified by measuring cycle threshold (Ct) values and normalized using the 2−ΔCt or 2−ΔΔCt Ct method relative to U6 snRNA or β-actin mRNA. All the primer sequences are listed in Table II.
Cell proliferation assay
Cell proliferation was measured 3 days after transfection with let-7a mimic and negative control or let-7a inhibitor and inhibitor negative control by Thiazolyl Blue Tetrazolium Bromide (MTT, Amresco). The results are represented as proliferating cells quantified at OD 490 nm by FLx800 Fluorescence Microplate Reader (BioTek). The experiment was performed in triplicate.
Colony forming assay
The tumor cells were transfected with let-7a mimic and negative control or let-7a inhibitor and inhibitor negative control for 6 h. Then, 1×103 cells were seeded into 6-well plates and incubated for 10 days, fixed and stained, followed by colony counting. The experiment was performed in triplicate.
Wound healing assay
The tumor cells were seeded into 6-well plates and transfected with let-7a mimic and negative control or let-7a inhibitor and inhibitor negative control and allowed to grow until 100% confluency. Next, the cell layer was scratched through the central axis using a sterile plastic tip and loose cells were washed away by PBS. The wound healing was observed and photographed at three pre-selected time points (0, 24, and 48 h) in three randomly selected microscopic fields for each condition and time point. The experiment was performed in triplicate.
Cell invasion assay
The tumor cell invasion was evaluated using a Transwell insert (8 μm, Corning). Cells were transfected with let-7a mimic and negative control or let-7a inhibitor and inhibitor negative control. After 24 h, the cells were starved in L-DMEM without fetal bovine serum overnight, and then 4×104 cells resuspended in 0.2 ml serum-free L-DMEM were added to the upper chamber and L-DMEM containing 10% fetal bovine serum was added to the lower chamber as a chemoattractant at 37°C in humidified 5% CO2 for 14 h (MDA-MB-231) or 16 h (MCF-7). The invasive cells were fixed and stained with 0.1% crystal violet. Three low-magnification areas (x100) were randomly selected and counted for the cell numbers. The experiment was performed in triplicate.
Protein extraction and western blot analysis
Cells were transfected with let-7a mimic and negative control or let-7a inhibitor and inhibitor negative control. After 48 h, the total cellular protein of the cells was extracted using a modified radioimmunoprecipitation assay (RIPA, Vazyme Biotech, China) lysis buffer and phenylmethanesulfonyl fluoride (PMSF, Beyotime, China). The protein concentration was then determined by NanoDrop 1000 spectrophotometer (Thermo) and equal amounts of protein lysates (100 μg) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime) and then transferred to polyvinylidene fluoride (PVDF) membrane (Beyotime). The membranes were blocked with 5% defatted milk/TBST (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween-20) at room temperature for 1 h and incubated with primary antibodies at 4°C overnight. The next day, the membranes were washed with TBST and then incubated with HRP-linked secondary antibodies (anti-rabbit IgG, diluted at 1:1000; Cell Signaling Technology). The protein bands were developed with chemiluminescence (ECL) reagents (Beyotime). The antibodies were anti-HMGA1 (diluted at 1:500; Abgent), anti-GAPDH (diluted at 1:1000; Cell Signaling Technology).
Statistical analysis
For statistical analyses, mean values with standard deviation are shown in the graphs that were generated from several repeats of biological experiments. P-values were obtained from t-tests with paired or unpaired samples and <0.05 were considered significant.
Results
Let-7a is decreased in breast cancer tissues and breast cancer cells
We performed qRT-PCR to determine let-7a levels in breast cancer cells, 27 breast cancer tissues and adjacent normal breast tissues. As shown in Fig. 1, the expression levels of the let-7a were downregulated in cancer tissues compared to the adjacent normal tissues. We compared let-7a expression in breast cancer cell lines MDA-MB-231 and MCF-7 which was in the similar range of that in breast cancer tissues, thus we used these two breast cancer cell lines for further experiments in this study. To confirm the function of let-7a, we transfected let-7a mimics and inhibitor into breast cancer cell lines (MDA-MB-231 and MCF-7). At 6 h after transfection of let-7a mimics and inhibitor into breast cancer cells, the transfection efficiency were estimated by fluorescence microscopy and the let-7a expression level was verified by real-time PCR (Fig. 2). We found that let-7a mimics significantly increased let-7a RNA expression while let-7a inhibitor significantly decreased let-7a RNA expression in both breast cancer cell lines.
Let-7a inhibits breast cancer cell proliferation
Breast cancer cells were treated with let-7a mimics or mimics NC and let-7a inhibitor or inhibitor NC for 24, 48, 72 h and measured the absorbance at 490 nm. Compared with the treatment with the mimics NC, cell proliferation was inhibited when cells were treated with let-7a mimics. Compared with the inhibitor NC treatment, cell proliferation was promoted when cells were treated with let-7a inhibitor (Fig. 3).
Let-7a decreases breast cancer cell colony formation
Colonies formed from let-7a mimic-transfected cells were significantly less than that of mimics NC transfected cells. The let-7a inhibitor transfected cells formed significantly more colonies than that of inhibitor NC transfected cells (Fig. 4). These data demonstrated that let-7a inhibited breast cancer cell colony formation.
Let-7a inhibits breast cancer cells migration
Cell scratch assay showed that cells transfected with let-7a mimics migrated slowly. The scratch in mimics NC treated cells was almost healed 48 h after the scratch had been made, but not in let-7a mimics treated cells. The cells transfected with let-7a inhibitor migrated more rapidly than cells transfected with inhibitor NC (Fig. 5). These data demonstrated that let-7a inhibited breast cancer cell migration.
Let-7a inhibited breast cancer cell invasion
Transwell invasion assays showed that the number of tumor cells invading out of the chamber after treatment with let-7a mimics was significantly less than that after treatment with mimics NC. The number of tumor cells invading from the chamber after treatment of let-7a inhibitor was significantly more than that after treatment with inhibitor NC, demonstrating that let-7a inhibited tumor cell invasion (Fig. 6).
HMGA1 is a target gene of let-7a
In silico analyses of potential let-7a targets (www.microrna.org and www.targetscan.org) indicated that the proteins of the high mobility group A1 (HMGA1) is a possible target of let-7a. HMGA1 mRNA has one potential complimentary binding site with let-7a within its 3′ UTR (Fig. 7A). Based on these results, we performed qRT-PCR assays and western blot analysis to assess the impact of let-7a on HMGA1 expression. Although we did not find apparent effects on HMGA1 mRNA expression in the breast cancer cells after treatment of let-7a mimics or mimics NC and let-7a inhibitor or inhibitor NC (Fig. 7B–E), western blot analysis showed that HMGA1 protein expression was significantly decreased or increased in let-7a mimics or let-7a inhibitor transfected cells (Fig. 7F). These data suggest that let-7a may target HMGA1 mRNA, and inhibits its translation into proteins.
Discussion
Breast cancer is the most common type of malignant tumor and its metastatic progression is a complex process (27–30). At present, surgery and chemotherapy are the primary treatments for breast cancer, but many patients under chemotherapy have early tumor recurrence and metastasis, which results in poor prognosis. Consequently, it is especially important to explore targeted treatment for breast cancer. Over the last decades, miRNA has become a hotspot for research. Previous studies suggest that dysregulation of miRNAs is a common event in breast cancer (31) and that they may thus act as key regulators of tumorigenesis of breast cancer. Based on these findings, it has been proposed that more effective targeted drugs for treatment of breast cancer may involve miRNAs.
Among human cancer-related miRNAs, the let-7 family has attracted significant attention because its family members are expressed aberrantly in many cancers, such as lung carcinoma, and colon carcinoma (20,22). As a member of the let-7 miRNA family, let-7a has been reported to be expressed at lower than normal levels in a variety of cancer (32–34). However, the suppressive role of let-7a in tumorigenesis is still poorly understood. In this study, we examined the expression levels of let-7a in breast cancer cells (MDA-MB-231 and MCF-7), breast cancer tissues and corresponding adjacent normal tissues. We found that the expression of let-7a was significantly lower in breast cancer cells and breast cancer tissues than corresponding adjacent normal tissues, which suggested that downregulation of let-7a was associated with the development of breast cancer. Thus, we hypothesized that let-7a may function as a tumor suppressor. To prove the role of let-7a in breast cancer, we transfected let-7a mimics or let-7a inhibitor into breast cancer cells to induce overexpression or low expression of let-7a. Exogenous overexpression of let-7a significantly inhibited the cell growth as indicated by MTT and colony formation assays whereas low expression of let-7a significantly promote cell growth. Moreover, cell migration and invasion were also significantly decreased or increased by overexpression or low expression of let-7a in breast cancer cells as shown by wound healing and Transwell assays. Recent evidence indicated that let-7a plays a role in the progression of human tumors such as renal cell carcinoma, gastric carcinoma and hepatocellular carcinoma (35–37). In addition, it has been shown that let-7a is downregulated in Burkitt’s lymphoma and acted as an anticancer miRNA repressing C-MYC expression at the translational level (38). These previous studies are consistent with our current data, suggesting that let-7a plays a role as a tumor suppressor in regulation of breast cancer progression.
In this study, we first predicted by online biological software that HMGA1 is a potential target gene of let-7a. HMGA family members have previously been reported to be involved in breast carcinogenesis (39). They are non-histone and DNA-binding proteins, which are often referred to as architectural transcription factors. They contain basic A-T hook domains which mediate binding to the minor groove of AT-rich regions of chromosomal DNA. Upon binding to DNA, HMGA proteins regulate gene expression by forming the transcriptional complex through protein-protein and protein-DNA interactions (40–42). The HMGA family includes the products of the HMGA1 and HMGA2 genes. HMGA1 has been found to be abnormally expressed in several types of malignant tumors, including breast (43–45), ovarian (46), leukemia (47), colon (48), pancreatic (49), thyroid (50), lung (51), prostate (52), endometrial (53), and head and neck malignant tumor (54).
Next, we tested whether HMGA1 is a target of let-7a by qRT-PCR and western blotting. Noteworthy, we found that the mRNA expression of HMGA1 did not alter in let-7a mimics or let-7a inhibitor transfected breast cancer cells, but protein expression was significantly decreased or increased in let-7a mimics or let-7a inhibitor transfected cells. Previous studies indicated that one miRNA might have multiple mRNA targets and that one mRNA might be targeted by multiple miRNAs. When a miRNA is perfectly complementary to its target, it can specifically degrade the target mRNA (55). However, if it is not perfectly complementary to its target, the miRNA will inhibit mRNA translation (56). Most miRNAs are involved in the regulation of the expression of target genes through the two pathways discussed above (57). Therefore, the result indicates that let-7a is not perfectly complementary to its target. In other words, let-7a regulates HMGA1 only at protein level, but not at the genetic level. The result also confirmed the prediction from bioinformatics.
Collectively, these data strongly suggested that let-7a might act as a tumor suppressor in breast cancer by targeting HMGA1. The upregulation of let-7a targeting HMGA1 shows promise as new strategy to treat breast cancer.