Background
Breast cancer is the most common cause of cancer-related death in women worldwide [
1‐
3]. Similar to other solid tumors, distant metastasis (especially lung metastasis) is the leading cause of breast cancer-associated death and resistance to various treatments [
4]. Therefore, it is essential for us to elucidate the molecular mechanism underlying breast cancer progression.
MicroRNAs are around 22-nucleotide-long non-coding RNAs [
5]. They modulate gene expression through targeting 3′-UTR of mRNAs, leading to mRNA degradation or translational block [
6,
7]. Many studies have revealed that miRNAs are involved in cell growth, migration, apoptosis and cancer metastasis [
8‐
10]. Arora et al. [
11] reported that
miR-
506 had a role in regulating EMT in breast cancer cell lines. As a validation, Yu et al. [
12] has shown that
miR-
506 overexpression inhibits proliferation and metastasis of breast cancer cells. However, the mechanism of
miR-
506 inhibition of breast cancer metastasis remains elusive.
MEG3 is identified as an imprinted gene with maternal expression and encodes a long non-coding RNA [
13]. Dysregulation of
MEG3 has been found in various human tumors, including bladder cancer, hepatocellular carcinoma, lung cancer and ovarian cancer [
14‐
16]. More interestingly,
MEG3 has been implicated into tumorigenesis and progression of breast cancer [
17,
18]. Previous studies have revealed that overexpression of
MEG3 could induce cell growth arrest and increase cell apoptosis in human breast cancer cells. In addition, downregulated
MEG3 regulates proliferation, migration and invasion of breast cancer in a p53-dependent manner [
17]. Whether
miR-
506 cooperates with
MEG3 to regulate the metastasis of breast cancer remains unclear. In pituitary tumors, hypermethylation of the
MEG3 regulatory region is identified as an important mechanism associated with the loss of
MEG3 expression [
19].
miR-
29a was shown to regulate methylation of
MEG3 via DNA methyltransferase (DNMT) 1 and 3b, thus contributing to hepatocellular carcinoma (HCC) growth [
20]. Likewise, Li et al. [
18] demonstrated that
MEG3 was epigenetically repressed by DNMT1 to suppress the p53 pathway in glioma. Based on these findings, we hypothesized that
MEG3 may be regulated in a DNA methylation-dependent manner in breast cancer cells.
SP1 and SP3 transcription factors are expressed in almost all mammalian cells. They belong to the specificity protein/Kruppel-like factor (SP/KLF) transcription factor family and are involved in regulation of DNMTs [
21]. Davie et al. [
22] showed SP1 and SP3 could either enhance or repress the activity of promoters of genes implicated in differentiation, cell cycle progression, and oncogenesis. Although SP1 and SP3 has been investigated in breast cancer, the detailed mechanism by which SP1 and SP3 regulate progression of breast cancer requires to be further investigated [
23].
Here, we show that miR-506 inhibits migration and invasion of breast cancer cell lines through the SP3/DNMT1/MEG3 axis. Our findings reveal the detailed mechanism by which miR-506 regulates metastasis of breast cancer, which facilitates the development of therapeutical strategies for treating breast cancer.
Materials and methods
Patients and samples
The present study was approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University. A total of 20 breast tumor samples and 20 adjacent normal tissue samples were obtained from patients aged 20–70 in 2016–2017. No patients had received chemotherapy or radiotherapy prior to surgery. Breast cancer was validated by histological examination in all cases according to World Health Organization criteria. Breast tumors and normal tissue specimens excised surgically from patients were immediately snap-frozen and stored in liquid nitrogen until use.
Cell lines
Human breast cancer cells (MCF-7, MDA-MB-231, SKBR3) and Human Embryonic Kidney (HEK) 293T cells were purchased from ATCC and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin at 37 °C, 5% CO2. Human breast epithelial MCF10A cells were grown in the base medium for this cell line (MEBM) along with the appropriate additives (MEGM, Lonza/Clonetics Corporation, CC-3150). HEK 293T cells were employed in lentiviruses packaging.
Plasmid generation and lentivirus package
SP3 cDNA was cloned into pcDNA4 vector. The short hairpin RNA (shRNAs) targeting SP3 (target sequence showed blow) were purchased from GenePharma, Shanghai, China and cloned into PLKO.1 vector. To generate lentiviruses, the packaging vectors (pPAX2 and pVSVG) and transfer vector were co-transfected into 293T cells. The supernatant was harvested at 24 h and 48 h after transfection and filtered through 0.45 μm membrane. For virus infection, the virus supernatant was added to medium at 1:3 ratio, 24 h later, 2ug/ml puromycin was used to select stable cell lines. scramble shRNA and empty pcDNA4 vector was used as negative control respectively.
shRNA targeting sequences of SP3: shSP3#1: GCAAGAACTGTGGTGTCTTGG; shSP3#2: CCTTCTGCTAACATCCAGAAT; shSP3#3: CGCGAGATGATACTTTGATTA; shSP3#4: GTGGTGATTCTACCTTGAATA.
Transfection
For NC and miR-506 mimic transfection, we used LipofectamineVR LTX with PlusTM Reagent (Life Technologies) according to manufacturer’s instructions. mimic NC and miR-506 mimics were synthesized by GenePharma.
Wound healing assay
Migration of cells was measured by a wound healing assay in vitro. Briefly, 2 × 105 MCF-7 and MDA-MB-231 cells were seeded onto 6-well plates, and incubated in appropriate complete culture medium for 16 h under normal conditions at 37 °C. The monolayer was scratched and incubated in fresh medium without FBS for 24 h. Finally, the wound width was measured. Three different locations were visualized and photographed under inverted microscope.
Invasion assay
Invasion assays was performed using chambers containing 8.0-μm pore membranes (Millipore) with matrigel basement membrane matrix. Breast cancer cells (1 × 105 cells) were resuspended in 200 µl of FBS-free medium, and then seeded into the top chamber with Matrigel-coated membrane. Next, 500 µl medium with 10% FBS was added to the bottom chamber as a chemoattractant. After 48 h of incubation, the non-invaded cells were removed from the upper surface of the membrane with a cotton-tipped swab, and the invaded cells were fixed, stained with crystal violet, finally, 5 fields of the stained cells per sample were counted under the inverted microscope.
Western blot
The cells were harvested and lysed by RIPA buffer. The lysates were boiled at 100 °C for 5 min and centrifuged at 10,000 rpm for 1 min. About 50 ug of total protein were loaded onto SDS-PAGE gel. After that, the proteins were transferred to PVDF membrane at 300 mA for 2.5 h. The membrane was blocked with 5% non-fat milk in 1 × TBST for 1 h at room temperature, then incubated with primary antibodies at 4 °C overnight. Then, the membrane was washed with 1 × TBST for 3 times, 5 min each time, and incubated with secondary antibodies at room temperature for 1 h. Finally, the membrane was incubated with ECL and exposed. The following antibodies were used: anti-SP3 (Santa Cruz, USA), anti-DNMT1 (Cell Signaling Technology, USA), anti-SP1 (Cell Signaling Technology, USA), anti-β-actin (Proteintech).
RT-qPCR
Cells were harvested and RNA was extracted by Trizol method, then chloroform was added to the mixture. The sample was centrifuged at 12,000 rpm for 10 min and transferred to new RNase-free EP tubes, mixed with an equal volume of isopropanol and centrifuged. Supernatant was then removed and 75% ethanol added to wash the pellet. Finally, ethanol was discarded and the pellet dried and resuspended in 20–30 µl Rnase-free H2O.
For reverse transcription, ~ 1 ug of total RNA was used for reverse transcription according to manufacturer’s instruction (TAKARA PrimeScript Kit).
For real time PCR, we used SYBR as a probe dye to detect the signal, with GAPDH used as internal control. The Ct value was calculated using the
ΔΔCt method and normalized to GAPDH levels. The following primers were used:
-
MiR-506-QPCR-F: GCCACCACCATCAGCCATAC
-
MiR-506-QPCR-R: GCACATTACTCTACTCAGAAGGG
-
MEG3-QPCR-F: ATCATCCGTCCACCTCCTTGTCTTC
-
MEG3-QPCR-R: GTATGAGCATAGCAAAGGTCAGGGC
-
DNMT1-QPCR-F: CGGCTTCAGCACCTCATTTG
-
DNMT1-QPCR-R: AGGTCGAGTCGGAATTGCTC
-
SP1-QPCR-F: CTGGTCCCATCATCATCCGG
-
SP1-QPCR-R: TGTTTGGGCTTGTGGGTTCT
-
SP3-QPCR-F: GGTCAAGTCCAGGTTCAGGG
-
SP3-QPCR-R: CTGAGAACTGCCCGAGAGTC
-
GAPDH-QPCR-F: GAGTCAACGGATTTGGTCGT
-
GAPDH-QPCR-R: TTGATTTTGGAGGGATCTCG
Luciferase assay
The 3′-UTRs of SP1 and SP3 were fused to the luciferase gene using the XhoI/NotI restriction sites in the psiCHECK2vector. Mutations in the miR-506 target site in these UTRs were generated using the QuikChange Multi Site-directed Mutagenesis kit (Stratagene, LaJolla, CA). Luciferase assays were performed using the Dual-Luciferase assay (Promega). Renilla expression was normalized to the luciferase gene on the psiCHECK2 vector.
Methylation-specific PCR
DNA methylation status was examined by the methylation-specific PCR with genomic DNA treated with sodium bisulfite using the EZ DNA MethylationDirect kit (Zymo Research). Two primer sets were used to amplify the promoter region of the MEG3 gene containing a number of CpG sites, one for the methylated sequence (forward, 5′-TATGAGTTGTAAGCGGTAGAGTTC-3′; reverse, 5′-TACGAACTTAACGAAAAAAAATCAT-3′) and the other for the unmethylated sequence (forward, 5′-GAATATGAGTTGTAAGTGGTAGAGTTT-3′; reverse, 5′-TACAAACTTAACAAAAAAAAATCATACT-3′).
Statistical analysis
Each experiment was performed for three times independently. All values were presented as mean ± SD. Comparisons were performed using student’s t-test for two groups or one-way ANOVA for multiple groups. *P < 0.05 was considered statistically significant.
Discussion
The major finding of the present study is that miR-506 inhibits migration and invasion of breast cancer cell lines through an undescribed pathway SP1/SP3/DNMT1/MEG3. We revealed a novel epigenetic mechanism of how miR-506 and SP3 play a role in breast cancer progression.
Many studies have shown that
miR-
506 functions as tumor suppressor in different types of malignant tumors [
25,
26]. For instance, Chen et al. [
27] reported that
miR-
506 inhibits colorectal cancer progression by targeting DNMT1 and DNMT3b. In the present study, we further uncover SP1 and SP3 as novel targets by which
miR-
506 regulates DNMT1 expression. A previous meta-analysis revealed that
miR-
506 is associated with the survival of breast cancer patients [
28]. Recently,
miR-
506 has been shown to regulate TGFβ1-induced EMT of breast cancer cells through targeting EMT-related gene expression [
11]. Although it has been found that
miR-
506 has the ability to repress IQGAP1 and MAPK signaling pathway to influence breast cancer metastasis, other downstream targets may exist, which prompted us to search by bioinformatic prediction and for the first time, find out SP1 and SP3 as a direct target of
miR-
506 [
29], thereby regulating breast cancer metastasis via DNMT1/
MEG3 axis. SP1 and SP3 expression level are often greater in cancer cells than in normal cells [
23]. Compared to SP3, SP1 has been extensively studied in breast cancer, thyroid cancer, hepatocellular cancer, pancreatic cancer, colorectal cancer, gastric cancer and lung cancer [
30‐
32]. Hence, we primarily focused on the role of SP3 in present study, bridging
miR-
506 and DNMT1/
MEG3.
It was found that the demethylation of
MEG3 promoter and the change of gene region are the main reasons for the abnormal expression of
MEG3 in tumors [
33]. Consistently, another study has shown that
MEG3 expression is closely regulated by DNA methylation with the treatment of DNA methylation inhibitor (5′-Aza-2′-deoxycytidine) [
18]. The further work can be focused on assessing whether other members of DNMT (e.g., DNMT3a and DNMT3b) or DNA demethylases (e.g., TET1-3) are implicated in regulating the expression of
MEG3 mediated by miR-506. Besides, another interesting question is to search for other miRNAs responsible for upregulating
MEG3 through targeting DNMT1.
A number of previous studies have identified
MEG3 as a classical tumor suppressor [
34,
35]. The mechanism of how
MEG3 exerts its effects on tumorigenesis is almost fully understood. To date, two groups have confirmed that
MEG3 suppresses tumorigenesis and progression of breast cancer and gliomas by p53 pathway [
17,
18]. In addition, by comparing gene expression profiles in embryonic brains between
Meg3 KO mice and wild-type mice using microarray techniques, researchers found that
Meg3 deletion could lead to elevated VEGFA and VEGFR1 [
36]. Based on these findings, we do not draw much attention on the downstream signaling regulated by
MEG3 or function of
MEG3.
Conclusion
In conclusion, our study highlighted a novel regulation axis responsible for miR-506-attenuated migration and invasion of breast cancer cell lines. Of which, for the first time, we reveal miR-506 has a role in targeting and regulating SP1 and SP3 expression, down-regulating methylation level of MEG3 promoter in a DMNT1 dependent manner. Our findings provide new mechanism for explaining the breast cancer progression as well as potential candidate for treating breast cancer in future.
Authors’ contributions
XXW designed the study, prepared and edited the manuscript. GCG and XKQ did literature research and clinical studies. DWD, ZZ and XDX did data analysis and statistical analysis. XD and XHP edited and revised the manuscript.