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Knockdown of SETDB1 Inhibits Breast Cancer Progression by miR-381-3p-related Regulation

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Knockdown of SETDB1 Inhibits Breast Cancer Progression by miR-381-3p-related Regulation

Milu Wu et al. Biol Res.

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Breast cancer; Cell cycle progression; Migration; Proliferation; SET domain bifurcated 1; miR-381-3p.

Figures

Fig. 1
Fig. 1
SETDB1 mRNA expression was upregulated in breast cancer tissues and cell lines. a SETDB1 mRNA expression was measured in 45 pairs breast cancer tissues and adjacent normal breast tissues by qRT-PCR assay. b qRT-PCR assay of SETDB1 mRNA level in breast cancer cell lines (MCF-7, MDA-MB-231) and human mammary epithelial cell line (MCF-10A). c Kaplan–Meier survival assay and log-rank test were used to evaluate the correlation between SETDB1 mRNA level and breast cancer patient prognosis in 45 breast cancer patients in low- and high-risk groups based on SETDB1 mRNA expression. *P < 0.05 vs. corresponding control
Fig. 2
Fig. 2
SETDB1 knockdown suppressed proliferation, cell cycle progression and migration in MCF-7 and MDA-MB-231 cells. Western blot assay of SETDB1 expression (a), CCK-8 assay (b) and colony formation assay (c) of cell proliferation capacity, flow cytometry analysis of cell cycle progression (d), and transwell assay of cell migration ability (e) in MCF-7 and MDA-MB-231 cells transfected with siNC or siSETDB1. *P < 0.05 vs. siNC
Fig. 3
Fig. 3
SETDB1 depletion repressed tumor growth in vivo. Nude mice were subcutaneously injected with 1.0 × 106 MCF-7 cells transfected with siNC or siSETDB1. 50 days later, mice were killed and tumor masses were removed. a Tumor volume was mearsured by a caliper every 10 days. b Bright-field imaging of the xenograft tumors. c The average weight of the xenograft tumors. d qRT-PCR assay of SETDB1 mRNA expression in removed tumor tissues. *P < 0.05 vs. siNC control
Fig. 4
Fig. 4
SETDB1 was regulated by miR-381-3p in a direct interaction in breast cancer cell lines. a Schematic of potential binding sites for miR-381-3p in the SETDB1 3′-UTR, the seed and the mutated sequences of potential binding sites. Luciferase reporter assays were performed to verify the interaction between miR-381-3p and SETDB1 by cotransfection with SETDB1 3′-UTR-WT or SETDB1 3′-UTR-MUT construct and miR-381-3p mimic into MDA-MB-231 cells (b) and MCF-7 cells (c), or by cotransfection with SETDB1 3′-UTR-WT or SETDB1 3′-UTR-MUT construct and miR-381-3p inhibitor into MDA-MB-231 cells (d) and MCF-7 cells (e). f RIP analysis was used to evaluate the binding between miR-381-3p and SETDB1 using anti-Ago1 in MCF-7 and MDA-MB-231 cells transfected with miR-381-3p mimic, followed by measurement of SETDB1 mRNA by qRT-PCR assay. g Western blot analysis of SETDB1 protein expression in MCF-7 and MDA-MB-231 cells transfected with miR-381-3p mimic or miR-381-3p inhibitor. *P < 0.05 vs. respective negative control
Fig. 5
Fig. 5
SETDB1 abrogated miR-381-3p-mediated inhibition on proliferation, cell cycle progression and migration in breast cancer cell lines. a Western blot analysis of SETDB1 expression, b, c CCK-8 assay and d colony formation assay of cell proliferation capacity, e flow cytometry analysis of cell cycle progression, and f transwell assay of cell migration ability in MCF-7 and MDA-MB-231 cells transfected with miR-381-3p mimic or miR-381-3p mimic + pCMV6-XL5-SETDB1. *P < 0.05 vs. corresponding control (miR-NC or miR-381-3p mimic)

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References

    1. DeSantis C, Ma J, Bryan L, Jemal A. Breast cancer statistics, 2013. CA Cancer J Clin. 2014;64(1):52–62. doi: 10.3322/caac.21203. - DOI - PubMed
    1. Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, et al. Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin. 2016;66(4):271–289. doi: 10.3322/caac.21349. - DOI - PubMed
    1. Matsui T, Leung D, Miyashita H, Maksakova IA, Miyachi H, Kimura H, et al. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature. 2010;464(7290):927–931. doi: 10.1038/nature08858. - DOI - PubMed
    1. Sarraf SA, Stancheva I. Methyl-CpG binding protein MBD1 couples histone H3 methylation at lysine 9 by SETDB1 to DNA replication and chromatin assembly. Mol Cell. 2004;15(4):595–605. doi: 10.1016/j.molcel.2004.06.043. - DOI - PubMed
    1. Li H, Rauch T, Chen ZX, Szabo PE, Riggs AD, Pfeifer GP. The histone methyltransferase SETDB1 and the DNA methyltransferase DNMT3A interact directly and localize to promoters silenced in cancer cells. J Biol Chem. 2006;281(28):19489–19500. doi: 10.1074/jbc.M513249200. - DOI - PubMed
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