MicroRNA-409 regulates the proliferation and invasion of breast cancer cell lines by targeting special AT-rich sequence-binding protein 1 (SATB1).

This study aims to determine the cellular functions and clinical significance of microRNA-409 (miR-409) in breast cancer by targeting special AT-rich sequence-binding protein 1 (SATB1). Breast cancer tissues and adjacent normal tissues, breast cancer cell lines (MDA-MB-453, MDA-MB-231, BT-549, BR3, and MCF-7) were used. miR-409 mimics, miR-409 inhibitor, SATB1, and siSATB1 were transiently transduced into cancer cells independently or together. RT-qPCR, Western blot, Cell Counting Kit-8 (CCK8), and Transwell assays were carried out to analyze the expression, cellular proliferation, and invasion. The results showed that the expression of miR-409 in breast cancer tissues is lower than that in adjacent tissues. The application of a target prediction algorithm predicts that the candidate gene regulated by miR-409 may be SATB1. The expression level of miR-409 in MDA-MB-453 cells is lower, while in BT-549 cells it is higher, when compared with MDA-MB-231, BR3, and MCF-7. The proliferation rate and invasive ability of MDA-MB-453 cells transfected with the miR-409 mimic was significantly lower than that of the miRNA negative control (miR-NC) cells, while the proliferation rate and invasive ability of BT-549 cells transfected with the miR-409 inhibitor were significantly increased. Cell proliferation and invasion of miR-409 mimic and SATB1 co-transfected MDA-MB-453 cells increased compared with that of miR-409 mimic-transfected cells, while miR-409 inhibitor and siSATB1 co-transfected BT-549 cells showed the opposite result. All these results indicated that miR-409 regulates breast cancer proliferation and invasion by targeting SATB1 and might be a potential therapeutic target for the treatment of breast cancer.

Highlights

miR-409 is downregulated in human breast cancer tissues.

SATB1 is the target of miR-409.

miR-409 regulates breast cancer cell proliferation and invasion by targeting SATB1.

miR-409 can affect the proliferation and invasion of breast cancer cells by regulating the expression of SATB1.

Introduction

Breast cancer is a very common cancer among women. Although treatments have improved in recent years, it is still a major challenge. A growing body of research has shown that the dynamic expression of microRNAs is closely associated with tumor cell proliferation and invasion. Such researches include that MiRNA-409-5p being lowly expressed in children with chronic myeloid leukemia (CML), which inhibits proliferative potential and cell cycle progression, [1] and that miR-409-3p is playing roles in inhibiting invasion, migration, and EMT, and promoting apoptosis of gastric cancer cells. [2] It is also reported that miR-409-3p can ameliorate cisplatin-sensitivity of ovarian cancer cells. [3] At present, inhibiting the proliferation and invasion capacity of breast cancer cells through regulating cancer genes in cancer cells is a strategy that has received much attention [4].

The special AT-rich sequence-binding protein 1 (SATB1) is a kind of cancer gene that is highly expressed in multiple cancer cells. The invasion and metastasis capacity of breast cancer is closely related to the SATB1 gene expression [5,6]. SATB1 organizes chromatin into spatial loops, providing a ‘docking site’ necessary for the binding of further transcription factors and chromatin modifying enzymes. SATB1 has the ability to regulate whole sets of genes, even those located on distant chromosomes. SATB1 was found to be overexpressed in numerous malignancies, including lymphomas, breast, colorectal, prostate, liver, bladder, and ovarian cancers. In the solid tumors, an elevated SATB1 level was observed to be associated with an aggressive phenotype, presence of lymph node, distant metastases, and a poor prognosis [7]. But the mechanism of SATB1 gene regulation is still unclear. Considering that the microRNA family is a system with multiple regulatory abilities that can inhibit tumor invasion and metastasis by downregulating the expression of multiple cancer genes [8], in this study, we aim to clarify whether miR-409 regulates the proliferation and invasion of breast cancer cell lines through the interaction with SABT1. Expression, proliferation, and invasion are evaluated by RT-qPCR, Western blot, CCK8, and Transwell assays with miR-409 mimics, miR-409 inhibitor, SATB1, and siSATB1 transiently transduced into cancer cells independently or together.

Patients and methods

Ethical approval

The study was approved by the Institutional Ethics Committee of the Fourth Affiliated Hospital of Hebei Medical University, and written informed consent was obtained from all participants.

Tissue specimens

Pathological data of tumor tissues were collected from patients with breast cancer from January 2016 to April 2019. Breast cancer tissue specimens were obtained from patients in the Pathology Department of the Fourth Affiliated Hospital of Hebei Medical University. The tumor and adjacent tissues were frozen in liquid nitrogen and kept in an ultralow-temperature refrigerator. Patients agreed to the collection of all specimens, and they signed letters of consent.

Cell culture and transfection

These breast cancer cell lines stored in the research center (Research Center and Tumor Research Institute, the Fourth Affiliated Hospital of Hebei Medical University, Shijiazhuang, Hebei, China): MDA-MB-453, MDA-MB-231, BT-549, BR3, and MCF-7 were selected and cultured in a RPMI-1640 culture medium containing 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). The conditions of the incubator were 37°C and 5% CO2. When cells were confluent, 0.25% trypsin was used for digestion and passaging. The medium was changed on alternate days, with one passage needed every 3–4 d. The cells in the logarithmic growth phase were collected for the experiment.

MDA-MB-453 and BT-549 cells were cultured in a DMEM culture medium containing 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. They were placed in the incubator at 37°C and 5% CO2. One day before the miRNA transfection, MDA-MB-453 and BT-549 cells were inoculated into the 6-pore plate. When the cell confluence was 60%–70%, the experiment was conducted according to the specifications of the HiPerFect Transfection Reagent. MDA-MB-453 cell transfection was divided into miR-NC, miR-409-mimic, and miR-409 mimic+ SATB1 groups. BT-549 cell transfection was divided into miR-NC, miR-409-inhibitor, and miR-409-inhibitor + siSATB1 groups. During transfection, the final concentration of each group treatment was 100 nmol/L. After transfection for 6 h, the normal complete culture medium was used to continuously culture the cells for 48 h or 72 h for subsequent experiments.

Reverse transcription

In this experiment, Promega’s reverse transcription kit was employed to prepare cDNA from RNA. In a small Eppendorf (EP) tube, 2 μg total RNA was added as the template, and the proper amount of diethylpyrocarbonate-treated H2O was added to achieve a total volume of 10 μl. Random primer (1 μl, 0.5 µg/reaction) and Oligo(dT)15 primer (1 μl) were added to the reagent mixture. The mixture was heated for 5 min at 70°C and was immediately added to the EP tube on ice for 2 min. Finally, the reverse transcription reaction system was established.

Real-time quantification of the polymerase chain reaction

The amplification reaction followed the instructions for the TransStart Top Green qPCR Supermix. The QuantStudio™ Dx instrument was set to calculate the 2− ΔΔCT value of the SYBR Green signal, and a cDNA template was adopted. The control group was set, and the reference genes and target genes were set. The solubility curve adopted the default setting of the system. The amplification procedure was set to a two-step method:

95°C 10 min

95°C 15 sec

60°C 1 min42 cycles

After setting up the instrument, the protocol was run. After the program ended, the CT value was read. The amplification and solubility curves were observed. According to the amplification curve, the fluorescence values of 3 repeated curves were comparable, and the CT values were close, indicating that the polymerase chain reaction (PCR) system was stable with good repeatability and reliable results. The solubility curve had a main peak, and the temperature at the peak was stable, indicating that the amplification was specific. The 2− ΔΔCT value was calculated, and a statistical analysis was conducted.

Western blot

Breast cancer cells transfected with the indicated plasmids were collected and centrifuged to obtain cell pellets. The total protein was extracted using RIPA lysis buffer containing 1% PMSF, and the denatured protein was separated by 10% SDS-PAGE. The protein bands were transferred from the gel to PVDF membrane (Millipore, USA). Rabbit monoclonal antibodies against SATB1 (abcam, UK) were used to detect protein immunoreactivity. The immunoreactivity on PVDF membranes was developed using ECL Plus solution (Solarbio, China).

CCK-8 assay

Cells were seeded at a cell density of 5 × 103 cells/well in 96-well plates. After the attachment overnight, CCK8 assays were performed at the indicated time periods. In brief, 10 μl of CCK8 solution was added to each well. After 1 h of incubation at 37°C, the absorbance readings for each well were carried out at 450 nm using the microplate reader (Tecan, USA).

Transwell invasion assay

The cells (2 × 105 cells suspended in 200 μl of serum-free medium) were seeded in the upper chamber that was pre-coated with Matrigel (BD Biosciences). The lower chamber was filled with a serum-contained medium. After incubation for 24 h, cells were fixed with paraformaldehyde and stained with crystal violet. The invaded cells were observed and calculated under a microscope (Olympus, Tokyo, Japan).

Statistical analysis

All statistical analyses were performed with SPSS™ Statistics 22.0 software (SPSS Inc., Chicago, IL, USA). The data were expressed as the mean ± standard deviation (SD). The Student's t-test was used to compare the mean values between different groups. A one-way analysis of variance was used for comparison between multiple groups. All statistical tests were two-sided, and P value less than or equal to 0.05 was considered statistically significant.

Results

The expression of miR-409 in breast cancer tissues is lower than that in adjacent tissues. The application of a target prediction algorithm predicts that the candidate gene regulated by miR-409 may be SATB1. We detected the expression level of miR-409 in breast cancer cells MDA-MB-453, MDA-MB-231, BT-549, BR3, and MCF-7. The expression level of miR-409 in MDA-MB-453 cells is lower, while in BT-549 cells it is higher. CCK8 experiments showed that the proliferation rate of MDA-MB-453 cells transfected with the miR-409 mimic was significantly lower than that of miR-NC cells, and the proliferation rate of BT-549 cells transfected with the miR-409 inhibitor was significantly increased. Similarly, the invasive ability of MDA-MB-453 cells transfected with the miR-409 mimic was significantly lower than that of miR-NC, and the invasive ability of BT-549 cells transfected with the miR-409 inhibitor was enhanced. And cell proliferation and invasion of miR-409 mimic and SATB1 co-transfected MDA-MB-453 cells had increased compared with that of miR-409 mimic-transfected cells. However, in BT-549 breast cancer cells, cell proliferation and invasion of miR-409 inhibitor and siSATB1 co-transfected BT-549 cells had decreased compared with that of miR-409 inhibitor-transfected cells.

miR-409 is downregulated in human breast cancer tissues

First, we detected the expression of miR-409 in breast cancer tissues and adjacent tissues. According to the results of RT-qPCR, the expression of miR-409 is downregulated in human breast cancer tissues as compared with the adjacent tissues (Figure 1a). In addition, we detected miR-409 expression levels in the breast cancer cell lines MDA-MB-453, MDA-MB-231, BT-549, BR3, and MCF-7 (Figure 1b). Experiments detected that, among these five breast cancer cells, the miR-409 expression was lower in the MDA-MB-453 cell line and higher in the BT-549 cell line. These two breast cancer cell lines were screened for subsequent experiments.

Figure 1.

The expression of miR-409 is downregulated in human breast cancer tissues (Ca) as compared with the adjacent tissues (Adj) (a). The MDA-MB-453 cell line has a lower miR-409 expression, and the BT-549 cell line has a higher miR-409 expression, among five breast cancer cell lines (b). Data represent the mean ± SD. ** p < 0.01 compared with adj group.

SATB1 is the target of miR-409

To explore the biological mechanism of miR-409 in breast cancer, we first utilized the target prediction algorithms TargetScan and PicTar to predict the candidate target of miR-409. Based on the results of the software, SATB1 was determined as the candidate. To further explore the expression of miR-409 and SATB1 in breast cancer cells and their relationship, we used the breast cancer cell line BT-549 that had a high expression of miR-409 and the breast cancer cell line MDA-MB-453 that had a low expression of miR-409. MDA-MB-453 cells that stably overexpressed miR-409 were established by transfection with synthetic miR-409 mimics. Meanwhile, BT-549 cells with a low miR-409 expression were established by transfection with a synthetic miR-409 inhibitor. MDA-MB-453 cells with a low miR-409 expression could express SATB1 at a high level, while BT-549 breast cancer cells with a high miR-409 expression had clearly decreased expression of SATB1. These cell lines were verified by Western blotting and RT-qPCR. In addition, miR-409 and SATB1 were negatively correlated (R = −0.6911, p< 0.01), as shown in Figure 2.

Figure 2.

The mRNA expression and protein of SATB1 after over-expression of miR-409 in MDA-MB-453 cells (a). The mRNA expression and protein of SATB1 after low-expression of miR-409 in BT549 cells (b). Western blot analysis was performed to test the expression of SATB1. β-actin was used as internal control. miR-409 and SATB1 were negatively correlated (c). ** p < 0.01 compared with the miR-NC group.

miR-409 regulates breast cancer cell proliferation and invasion by targeting SATB1

To further explore the biological effect of miR-409 in breast cancer cells, the miR-409 mimic was used to transfect MDA-MB-453 cells, and the miR-409 inhibitor was used to transfect BT-549 cells. A transwell invasion assay showed that overexpression of miR-409 repressed the invasive ability of MDA-MB-453 cells and knockdown of miR-409 increased the invasive ability of BT-549 cells (Figures 3A & 3b). Meanwhile, the CCK8 experiment showed that the proliferation rate of MDA-MB-453 cells transfected with the miR-409 mimic was obviously lower than that of the cells transfected with miR-NC. Moreover, the proliferation rate of BT-549 cells transfected with miR-409 inhibitor clearly increased (Figure 3c).

Figure 3.

The transfection of miR-409 mimic suppressed MDA-MB-453 cell invasion (a). The transfection of miR-409 inhibitor enhanced BT-549 cell invasion (b). Overexpression of miR-409 inhibited the proliferation of MDA-MB-453 cells. Knockdown of miR-409 can enhance the proliferation of BT-549 cells (c). ** P < 0.01 compared with miR-NC group.

In order to verify whether miR-409 affects the proliferation and invasion of breast cancer cells by regulating SATB1 in breast cancer cell lines in vitro, the SATB1 expression vector plasmid was designed and constructed to confirm that its expression process was not regulated by miR-409. The SATB1 expression was rescued in breast cancer cells after transfection. The results showed that SATB1 expression increased in MDA-MB-453 breast cancer cells with stable miR-409 expression, and cancer cell proliferation was obvious. In BT-549 breast cancer cells, after co-transfection with the miR-409 inhibitor and siSATB1, the cell proliferation was lower than that with the miR-409 inhibitor transfection alone (Figure 4).

Figure 4.

After co-transfection of SATB1 and the miR-409 mimic into MDA-MB-453 cells, the inhibition of miR-409 on breast proliferation was interrupted. (a). ** p < 0.01 compared with the miR-409 mimic group. After co-transfection with the miR-409 inhibitor and siSATB1, the cell proliferation was lower than that of cells transfected with the miR-409 inhibitor alone (b). ** p < 0.01 compared with the miR-409 inhibitor group.

Furthermore, miR-409 overexpression significantly inhibited invasion of MDA-MB-453 cells compared with untreated cells, whereas the number of invaded cells was clearly increased following transfection with the SATB1 expression vector. To further explore whether SATB1 was involved in the invasion function of miR-409, rescue experiments were performed by co-transferring siSATB1 and the miR-409 inhibitor into BT-549 cells. The results showed that SATB1 knockdown partially abrogated the effects of miR-409 on the invasion behavior of BT-549 cells (Figure 5). The above results indicate that the SATB1 gene is a downstream effector of miR-409 and that miR-409 can regulate the biological behaviors of breast cancer cells in vitro through the target gene SATB1.

Figure 5.

The number of invaded cells was clearly increased following transfection with the SATB1 expression vector (a). Knockdown of SATB1 eliminated the effect of miR-409 on the invasion behavior of BT-549 cells (b). ** p < 0.01 compared with the miR-NC group.

Discussion

SATB1 is a kind of binding protein with tissue specificity in the matrix attachment regions (MARs). SATB1 is located at chromosome 3p23 and contains 11 exons [9]. SATB1 plays the role of genetic organizer and changes gene expression through recombining the spatial structure of chromosomes so that breast cancer cells can gain invasion capacity, thus leading to cancer cell infiltration and metastasis [10-14]. SATB1 participates in the formation of the high-level structure of chromatin, anchors relevant chromosomes to their cage structure through recognizing and bonding base pairing regions, remodels chromatin structure and recruits genes, and uniformly regulates expression [15]. SATB1 is closely related to the proliferation and invasion abilities of breast cancer cells, thus influencing the prognosis of breast cancer patients. Breast cancer cells with high SATB1 expression have strong proliferation and distant metastasis capacities [16,17]. Recurrence and metastasis occur more easily in such cancer patients, and the prognosis is poor. Increasing SATB1 expression in breast cancer cells with low invasion ability can induce the development of cancer cells with high invasion ability. In contrast, reducing the SATB1 gene expression in breast cancer cells with high invasion ability can eliminate tumor growth and metastasis and facilitate cell recovery to a normal epithelial morphology [18].

In this study, we found that, through artificial synthesis and transfection, MDA-MB-453 cells with a high SATB1 expression could express SATB1 at a low level, while BT-549 breast cancer cells with a low SATB1 expression had clearly increased expression of SATB1. In addition, miR-409 and SATB1 were negatively correlated (R = −0.6911, p< 0.01). Furthermore, SATB1 is expressed in other tumor cells, such as colorectal cancer [19,20]. SATB1 can also recruit genetic loci to the remodeled chromatin, thus regulating directional differentiation and specific cell functions. For example, SATB1 is required for the growth of thymocytes and the maturation of T cells [10,21].

MicroRNAs are composed of approximately 22–24 nucleotides. They are short-chain non-coding RNAs that are highly conserved in the evolutionary process. MicroRNAs mediate gene expression through post-transcriptional regulation. At present, it is believed that microRNAs are strictly regulated by cell type and differentiation stage and that they are expressed in specific tissues at specific stages [22]. The tissue-specific and time-dependent expression of microRNAs highlights their importance in tissue and cell function and multiple important regulatory functions in cell growth and development. Previous studies found that most tumors presented microRNA expression dysregulation. MicroRNAs with abnormal expression can participate in tumor occurrence, proliferation, invasion, and metastasis [23]. In this study, the miR-409 mimic was used to transfect MDA-MB-453 cells, and the miR-409 inhibitor was used to transfect BT-549 cells. The CCK8 experiment showed that the proliferation rate of MDA-MB-453 cells transfected with the miR-409 mimic clearly decreased, while the proliferation rate of BT-549 cells transfected with the miR-409 inhibitor increased. Meanwhile, a Transwell invasion assay showed that overexpression of miR-409 repressed the invasive ability of MDA-MB-453 cells, and knockdown of miR-409 increased the invasive ability of BT-549 cells. Many microRNAs have been confirmed as important regulatory factors of cancer genes, tumor suppressor genes, and cell signaling pathways [24]. The oncogenic or tumor suppressive role of a microRNA depends on the effect of its target gene. If a microRNA targets a tumor suppressor gene, an increase in microRNA activity leads to the inhibition of tumor suppressor gene activity and promotes cell proliferation and tumor progression; a decrease in microRNA activity results in an increase in oncogene translation and promotes tumor formation. In this study, we identified miR-409, which can regulate the SATB1 gene. The research showed that miR-409 inhibited breast cancer cell proliferation and invasion by directly downregulating the SATB1 gene expression. In this study, it was found via bioinformatics analysis that miR-409 regulated the expression of SATB1 in tumor cells. The experimental results showed that the SATB1 gene is a downstream effector of miR-409, and miR-409 can regulate the biological behavior of breast cancer cells in vitro by targeting the SATB1 gene.

miR-409 is localized on the chromosome and is often deficient in human tumor cells. Some studies also found pathological action of miR-409 in autoimmune diseases [25]. Many studies have reported that SATB1 is highly expressed in many tumors and directly or indirectly influences different tumor cells in multiple ways [26-28]. Relevant reports have verified that miR-409 regulates tumor cells in stomach cancer and kidney cancer through multiple pathways [29]. However, through retrieval, we did not find documents about miR-409 and SATB1 genes in breast cancer tissues or MDA-MB-453 and BT-549 breast cancer cell lines. In this study, we used RT-qPCR to detect the miR-409 expression in tumor tissues and adjacent tissues. We also detected the miR-409 expression in MDA-MB-453 and BT-549 breast cancer cell lines. The miR-409 level was regulated by transfection to further detect changes in the SATB1 expression and tumor cell proliferation and invasion. Further experiments verified that miR-409 inhibited breast cancer cell proliferation and invasion by downregulating the SATB1 expression. This study provides new insight by investigating SATB1-mediated regulation of breast cancer cell proliferation and invasion.

Conclusions

The expression level of miR-409 in breast cancer cells is significantly lower than that in adjacent tissues. Overexpression of miR-409 can inhibit breast cancer cell proliferation and invasion. There is a negative correlation between miR-409 and SATB1 expression levels. Regulating the level of SATB1 can block the effect of miR-409 on breast cancer cell proliferation and invasion. miR-409 can affect the proliferation and invasion of breast cancer cells by regulating the expression of SATB1.