Knockdown of SETDB1 inhibits breast cancer progression by miR-381-3p-related regulation
SET domain bifurcated 1 (SETDB1) has been widely considered as an oncogene playing a critical role in many human cancers, including breast cancer. Nevertheless, the molecular mechanism by which SETDB1 regulates breast cancer tumorigenesis is still unknown.
qRT-PCR assay or western blot analysis was performed to assess the expression level of SETDB1 mRNA or protein, respectively. siSETDB1, pCMV6-XL5-SETDB1, miR-381-3p mimic, or miR-381-3p inhibitor was transfected into cells to regulate the expression of SETDB1 or miR-381-3p. MiRNA directly interacted with SETDB1 was verified by luciferase reporter assay and RNA immunoprecipitation. CCK-8 assay, colony formation assay, flow cytometric analysis, and transwell assay were used to detect the abilities of cell proliferation, cell cycle progression and migration, respectively. Animal model of xenograft tumor was used to observe the regulatory effect of SETDB1 on tumor growth in vivo.
We verified that SETDB1 mRNA level was upregulated in breast cancer tissues and cell lines, and SETDB1 depletion led to a suppression of cell proliferation, cell cycle progression and migration in vitro, as well as tumor growth in vivo. SETDB1 was verified to be a target of miR-381-3p. Moreover, miR-381-3p overexpression suppressed cell proliferation, cell cycle progression and migration, whereas SETDB1 abated miR-381-3p-mediated regulatory function on breast cancer cells.
This study revealed that SETDB1 knockdown might suppress breast cancer progression at least partly by miR-381-3p-related regulation, providing a novel prospect in breast cancer therapy.
KeywordsSET domain bifurcated 1 miR-381-3p Breast cancer Proliferation Cell cycle progression Migration
SET domain bifurcated 1
H3 lysine 9
3′ untranslated regions
small interference RNA targeting SETDB1
quantitative real-time PCR
Breast cancer, one of the most common malignancy in women, is a leading cause of cancer death all over the world . Although the developments of diagnosis and therapy techniques have highly improved the survival rate of breast cancer patients, up to 30% of patients died from palindromia and metastasis after having the standard-of-care therapy . Therefore, it is of importance to explore the molecular mechanism of breast cancer pathogenesis for the effective therapy.
SET domain bifurcated 1 (SETDB1), also known as KMET1 or ESET, is a novel SET domain protein with histone H3 lysine 9 (H3K9)-specific methyltransferase activity which plays a pivotal role in early embryonic development . It is recruited to the chromatin for silence promoters via the methyl-CpG-binding protein MBD1  or some tumor inhibitor genes such as P53BP2 and RASSF1A . Accumulating evidences suggest that SETDB1 might function as a novel oncogene to be involved in multiple human cancers, such as hepatocellular carcinoma , lung cancer , and sporadic cutaneous melanoma . Recently, a research document demonstrated that SETDB1 was regulated by miR-7 played a critical role in the metastasis of breast cancer . Moreover, the abnormal expression of SETDB1 protein was found in human breast cancer cell lines by SILAC-based proteomic analysis . Nevertheless, the molecular mechanism by which SETDB1 regulates breast cancer tumorigenesis is still unknown.
MicroRNAs (miRNAs), a type of short non-protein-coding RNAs with 20–22 nucleotides, are negative regulators of gene expression by base-pairing to the 3′ untranslated regions (3′-UTR) of mRNAs . Following binding to partially complementary sites, miRNAs lead to a repression of translation and degradation of transcript . Growing amount of evidences have suggested that miRNAs implicate in multiple physiological and developmental cellular processes, such as cell growth, differentiation, autophagy and apoptosis . Dysregulation of some miRNAs has been widely acknowledged to be involved in a multitude of human cancers, including breast cancer . Of these miRNAs, miR-126 was found to inhibit breast tumor proliferation and growth, and miR-335 repressed tumor metastasis . Whereas, upregulated miR-10b was positively correlated with cell migration and invasion through targeting homeobox D10 (HOXD10) in breast cancer .
Here, we asked whether SETDB1 played a certain role partly by miRNAs regulation in breast cancer. We found that SETDB1 level was upregulated in breast cancer, and SETDB1 knockdown repressed tumor growth in vitro and in vivo. Moreover, SETDB1 was verified to be a functional target of miR-381-3p. Therefore, this study hinted that SETDB1 knockdown might repress breast cancer progression at least partly by miR-381-3p-related regulation, highlighting a novel therapeutic target for breast cancer treatment.
Clinical tissues collection
Forty five pairs of breast cancer tissues and adjacent normal breast tissues were obtained from patients who underwent radical mammectomy at the Affiliated Hospital of Qinghai University. No systemic or local treatment was performed in these patients before surgery. Tissue samples were immersed in RNAlater (Qiagen, Hilden, Germany) and snap frozen immediately, followed by stored at − 80 °C until used. Written informed consent were obtained from all patients prior to the study, and the study was approved by the Institutional Review Broad and Ethical Committee of Affiliated Hospital of Qinghai University.
Human mammary epithelial cell line (MCF-10A) and breast cancer cell lines (MCF-7, MDA-MB-231) that were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Rockville, MD, USA) with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco) in a humidified incubator (5% CO2) at 37 °C.
Small interference RNA targeting SETDB1 (siSETDB1) and the homologous negative control (siNC) were purchased from Applied Biosystems (Foster city, CA, USA). Modified miRNA mimic for hsa-miR-381-3p (miR-381-3p mimic) and its cognate negative control (miR-NC), miR-381-3p inhibitor and its negative control (inhibitor-NC), SETDB1 overexpression plasmid (pCMV6-XL5-SETDB1) were commercially synthesized by GenePhama (Shanghai, China). As previously described , 10 nM of oligonucleotides or 2 µg of plasmids were transfected into breast cancer cells using Lipofectamine® RNAiMAX™ transfection reagent (Invitrogen, Waltham, MA, USA) referring to the manual of application.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from tissues and cells using TRIzol™ plus RNA Purification Kit (Invitrogen) according to the manual of application. RNA quality was evaluated by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then, a total of 500 ng RNA was reverse-transcribed into cDNA using the M-MLV reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR analysis was performed in triplicates with the SsoFast™ EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). The expression level of SETDB1 mRNA was calculated using 2−ΔΔCt algorithm and normalized to that of GAPDH. The following primers were used: SETDB1 mRNA: 5′-GACTCTCTGAGACAACTTCCAAGGA-3′ (forward) and 5′-CAGGGATTGAGGGAGGAACA-3′ (reverse); GAPDH: 5′-TGCACCACCAACTGCTTAGC-3′ (forward) and 5′-GGCATGCACTGTGGTCATGAG-3′ (reverse).
Cell proliferation assay
Cell proliferation capacity was detected by Cell Counting Kit-8 Detection Kit (CCK-8, Dojindo Molecular Technologies, Shanghai, China) referring to the protocol of manufacturer. In brief, at 0, 24, 48, and 72 h after transfection, 10 µl of CCK-8 solution was added to each well and incubated at 37 °C for 3 h. Absorbance value at 450 nm was measured by a microplate reader (Bio-Rad).
Colony formation assay
Transfected cells were trypsinized into a single-cell suspension and were cultured in growth medium at 37 °C for 14 days to form natural colony. Then, the colonies were fixed with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and stained with 0.5% crystal violet (Sigma-Aldrich).
Flow cytometric analysis of cell cycle progression
Cell cycle progression was assessed by flow cytometry with Cell Cycle and Apoptosis Analysis Kit (Beyotime Institution of Biotechnology, Shanghai, China). Briefly, at 48 h after transfection, cells were harvested and fixed in pre-cooling 70% ethanol overnight. Following PBS-washing three times, the cells were stained with 100 µg/ml propidium iodide (PI) at 37 °C for 30 min. The evaluation of cell cycle was performed using the Modfit 3.0 software (Verity Software House, Topsham, ME, USA) on a FACScalibur flow cytometer (Becton–Dickinson, Franklin Lakes, NJ, USA).
Cell migration assay
Transfected cells were seeded on the upper chamber of an 8-µm pore size Transwell (Corning, Toledo, NY, USA) to detect the cell migration ability. Upper medium was replaced by serum-free medium, while the lower chamber contained growth medium with 10% FBS. After incubation for 24 h, migrated cells were fixed in methanol and stained with 0.5% crystal violet (Sigma-Aldrich). At last, the number of migrated cells was counter with a microscope (Leica, Wetzlar, Germany).
Luciferase reporter assay
A fragment of SETDB1 3′-UTR containing the putative binding site of miR-381-3p, was amplified from human genomic DNA and inserted into a modified pGL3 Luciferase Reporter Vector (Promega) to construct wild SETDB1 3′-UTR report vector (SETDB1 3′-UTR-WT). To construct mutated SETDB1 3′-UTR report vector (SETDB1 3′-UTR-MUT), the residues that base-pairs with miR-381-3p were mutated by site-directed mutagenesis with Q5 Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). 20 µg of SETDB1 3′-UTR-WT or SETDB1 3′-UTR-MUT construct was cotransfected with 200 ng of miR-381-3p mimic or miR-381-3p inhibitor into MDA-MB-231 or MCF-7 cells. 48 h later, the luciferase activity was measured using a Lumat LB9508 luminometer (Berthold, Bad Wildbad, Germany).
RNA immunoprecipitation (RIP)
Coimmunoprecipitation (co-IP) experiment of miRNA with anti-Argonaute1 (anti-Ago1, Abcam, Cambridge, UK) was performed as previously reported . Briefly, cells were transfected with miR-NC or miR-381-3p mimic for 48 h, and were lysed with cell lysis buffer (25 mM Tris–HCl, pH = 7.5, 150 mM KCl, 2 mM EDTA, 0.5% NP-40, 1 mM DTT, 100 U/ml RNasin). Then, the complex of anti-Ago1 and Protein A magnetic beads was added to cell lysates, and incubated at 4 °C overnight to get the immunoprecipitation complex. Lastly, the enrichment of SETDB1 mRNA was measured by qRT-PCR assay and anti-IgG (Abcam) was as negative control.
Western blot analysis
Protein fractions were obtained from cells using ice-cold RIPA buffer (50 mM Tris–HCl, pH = 7.5, 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate) supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics, Mannheim, Germany). About 50 µg of protein extractive was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (10% SDS-PAGE) and transferred to polyvinyldene fluoride (PVDF) membrance (Bio-Rad). Followed by blocking with 5% non-fat milk at room temperature for 1 h, the membrances were incubated with SETDB1 (1:2000, Abcam) and β-actin (1:5000, Abcam) antibodies at 4 °C overnight. Then, the membrances were further probed with horseradish peroxidase-conjugated secondary antibodies (1:5000, Cell Signaing Technology, Danvers, MA, USA) for 1 h at room temperature. All protein bands were analyzed using an Image-Pro plus 4.5 software (Media Cybernetics, Silver Spring, MD, USA).
In vivo animal model
All animals used were under an approved protocol of the Institutional Animal Care and Use Committee of Affiliated Hospital of Qinghai University. 6–8 weeks male BALB/c mice were purchased from Qinghai Research Center of Laboratory Animal (Xining, China) and were housed in a specific-pathogen-free environment. For xenograft tumors formation, 1.0 × 106 MCF-7 cells transfected with siNC or siSETDB1 were subcutaneously injected into the nude mice (n = 8 per group). Tumor volume was determined with a caliper every 10 days. On 50 days after cell implantation, mice were euthanized and tumors were excised for weight assessment and qRT-PCR assay of SETDB1 expression.
All data were presented as mean ± standard deviation (SD) from three independent experiments. Differences between two groups were compared using Student’s t-test. Kaplan–Meier method was used to evaluate overall survival, and log-rank test was performed to analyze the difference in survival between two groups. P value of < 0.05 was considered statistically significant.
Upregulation of SETDB1 mRNA in breast cancer tissues and cell lines
Subsequently, Kaplan–Meier survival assay and log-rank test using patient post-operative survival were performed to further determine the correlation between the expression level of SETDB1 mRNA and the prognosis of breast cancer patients. According to the median ratio of SETDB1 mRNA expression, the 45 breast cancer patients were classified into two groups: high SETDB1 mRNA expression group (n = 24) and low SETDB1 mRNA expression group (n = 21). Kaplan–Meier survival curve presented that low SETDB1 mRNA expression group had markedly longer survival times compared to those with high SETDB1 mRNA expression group (P < 0.01, Fig. 1c). Taken together, these data hinted that abnormal expression of SETDB1 might be associated with the progression of breast cancer.
SETDB1 knockdown inhibited proliferation, cell cycle progression and migration in breast cancer cells in vitro
Knockdown of SETDB1 repressed tumor growth in vivo
Regulation of SETDB1 by miR-381-3p in a direct interaction
Restoration of SETDB1 expression abrogated the regulatory function of miR-381-3p in breast cancer cell lines
Further, to explore whether the suppression function of miR-381-3p was mediated by SETDB1, MCF-7 and MDA-MB-231 cells were cotransfected with miR-381-3p mimic and SETDB1 overexpression vector (pCMV6-XL5-SETDB1). The data illustrated that miR-381-3p-induced the suppression of SETDB1 expression was drastically abated by cotransfection with pCMV6-XL5-SETDB1 (Fig. 5a). Moreover, the restoration of SETDB1 expression remarkably abrogated miR-381-3p-mediated inhibition effect on cell proliferation capacity, colony formation ability, cell cycle progression and migration ability in MCF-7 and MDA-MB-231 cells (Fig. 5b–f). All these findings hinted that the restoration of SETDB1 expression undermined miR-381-3p-mediated retardation on proliferation, cell cycle progression and migration in breast cancer cell lines.
Histone lysine methylation has been reported to be implicated in transcriptional activation or suppression, which was of increasing interest owing to its involved in the neoplastic transformation . H3K9 methylation was reported to play a vital role in multiple human cancer . SETDB1, located at chromosome 1q21, is a member of H3K9 methylation catalyzed by histone lysine methyltransferases . Over the past few years, SETDB1 has been widely considered as an oncogene playing a critical role in many human cancers. It was previously reported that SETDB1 protein level was upregulated in human melanomas tissues and it enhanced the formation of melanoma in a zebrafish model . Silence of SETDB1 was found to inhibit lung tumor growth in vitro and in vivo, while it upregulation promoted the tumor invasiveness, highlighting its role as a novel therapeutic target . SETDB1 also was reported to be overexpressed in hepatocellular carcinoma (HCC), which inactivation repressed the growth of HCC cell lines through regulating p53 methylation . In addition to the cancers mentioned above, SETDB1 was proposed as an oncogene in prostate cancer , gliomas  and colorectal cancer . In this study, SETDB1 level was verified to be elevated in breast cancer tissues and cell lines, which was consistent with the finding of Zhang et al. . From a functional standpoint, SETDB1 knockdown inhibited breast tumor growth in vitro and in vivo. All these data suggested that SETDB1 knockdown might suppress breast cancer progression.
MiRNAs are attractive candidates as upstream regulators of tumor progression because they have been demonstrated to repress a set of target genes expression in a post-transcriptional way . For instance, miR-135 and miR-203 inhibited breast tumor growth and metastasis in vitro and in vivo by targeting runx2 . MiR-155 was found to accelerate tumor angiogenesis by directly suppressing von Hippel–Lindau (VHL) expression in triple-negative breast cancer . It was reported that miR-621 overexpression promoted breast cancer chemosensitivity through targeting FBXO11 . Moreover, miR-21 enhanced epithelial-to-mesenchymal transition (EMT) by inhibiting PTEN protein expression in breast cancer . PTPRN2, MERTK, TNC and SOX4 were identified to be targets of miR-335 , AIB1 and CCND1 were the targets of miR-17-5p , and H-RAS and HMGA2 were verified as targets of let-7  in breast cancer.
Then, software algorithms were performed to search for the miRNAs directly interacted with SETDB1 in breast cancer. Among the predicted miRNAs, miR-381-3p was chose for further research owing to its involvement in the progression of multiple human cancers. For example, in oral squamous cell carcinoma, miR-381-3p suppressed cell proliferation and cell cycle progression while enhanced apoptosis through directly targeting fibroblast growth factor receptor 2 (FGFR2) . In non-small cell lung cancer, miR-381 led to a suppression of tumor growth and chemoresistance by direct downregulation of differentiation 1 (ID1) . Additionally, a recent document reported that miR-381 suppressed proliferation, EMT and metastasis of breast cancer cells through targeting CXCR4 . In the present study, SETDB1 was verified to be a functional target of miR-381-3p in breast cancer cells. Consistent with the findings of Xue et al. , miR-381-3p was manifested to suppress breast cancer cells proliferation, cell cycle progression and migration. Moreover, miR-381-3p-mediated regulatory function was abrogated by the restoration of SETDB1 expression.
In conclusion, our study demonstrated that SETDB1 was upregulated in breast cancer and SETDB1 knockdown suppressed breast cancer progression at least partly by miR-381-3p-related regulation, highlighting SETDB1 as a novel biomarker for breast cancer therapy.
This work was conceived and designed by MW, BF and QG. The experiments were carried out by YL, RC and NL. The manuscript was prepared by MW, YD and YL. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated and analyzed during this study are included in this published article and its additional information files. The gene expression dataset used is available on request.
Consent for publication
Ethics approval and consent to participate
All studies involving animal subjects were approved by the institutional animal care and use committees at the Affiliated Hospital of Qinghai University.
Funding and acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (81760730) and the Scientific Research Project Funds of Qinghai Department (2016-ZJ-785).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.