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Microrna-22 Suppresses DNA Repair and Promotes Genomic Published OnlineFirst January 27, 2015; DOI: 10.1158/0008-5472.CAN-14-2783 Cancer Molecular and Cellular Pathobiology Research MicroRNA-22 Suppresses DNA Repair and Promotes Genomic Instability through Targeting of MDC1 Jung-Hee Lee1,2, Seon-Joo Park1,3, Seo-Yeon Jeong1, Min-Ji Kim1, Semo Jun1,4, Hyun-Seo Lee1, In-Youb Chang1,5, Sung-Chul Lim6, Sang Pil Yoon7, Jeongsik Yong8, and Ho Jin You1,4 Abstract MDC1 is critical component of the DNA damage response overexpression of constitutively active Akt1, homologous recom- (DDR) machinery and orchestrates the ensuring assembly of the bination was inhibited by miR-22–mediated MDC1 repression. DDR protein at the DNA damage sites, and therefore loss of In addition, during replicative senescence and stress-induced MDC1 results in genomic instability and tumorigenicity. How- premature senescence, MDC1 was downregulated by upregulat- ever, the molecular mechanisms controlling MDC1 expression are ing miR-22 and thereby accumulating DNA damage. Our results currently unknown. Here, we show that miR-22 inhibits MDC1 demonstrate a central role of miR-22 in the physiologic regulation translation via direct binding to its 30 untranslated region, leading of MDC1-dependent DDR and suggest a molecular mechanism to impaired DNA damage repair and genomic instability. We for how aberrant Akt1 activation and senescence lead to increased demonstrated that activated Akt1 and senescence hinder DDR genomic instability, fostering an environment that promotes function of MDC1 by upregulating endogenous miR-22. After tumorigenesis. Cancer Res; 75(7); 1–13. Ó2015 AACR. Introduction DNA double-strand breaks (DSB) activate the DDR by trigger- ing the kinase activity of ataxia telangiectasia mutated (ATM), Repeated exposure to both exogenous and endogenous insults thereby initiating a signaling cascade in which the histone variant challenges the integrity of cellular genomic material. Eukaryotes H2AX (g-H2AX), located at DSB sites, becomes phosphorylated, have evolved a system called the DNA damage response (DDR), and other DDR factors, including the adaptor protein mediator of which allows cells to sense DNA damage and orchestrate the DNA damage checkpoint 1 (MDC1), are recruited. MDC1 ampli- appropriate cell-cycle checkpoints and DNA repair mechanisms fies the ATM signaling activity, leading to a higher percentage of (1). The failure to respond to DNA damage is a characteristic phosphorylated H2AX proteins and contributing to the recruit- associated with genomic instability and with the onset of diseases, ment and retention of additional DDR factors at the sites of DNA including neurodegenerative diseases, immune deficiency, can- damage (3). Thus, MDC1 has been termed a master regulator, cer, and premature aging (2). modulating the specific chromatin microenvironment required to maintain genomic stability. MDC1 knockout mice show chromo- somal instability, defective DNA repair, and radiation sensitivity 1 DNA Damage Response Network Center,Chosun University School of (4). Furthermore, loss of MDC1 is associated with an increased Medicine, Gwangju, Republic of Korea. 2Department of Cellular and Molecular Medicine, Chosun University School of Medicine, Gwangju, occurrence of tumors in mice (5), and reduction or lack of MDC1 Republic of Korea. 3Division of Natural Medical Sciences, Chosun has been observed in breast and lung carcinoma cells in humans 4 University School of Medicine, Gwangju, Republic of Korea. Depart- (6). Therefore, cellular levels of MDC1 appear to impact genomic ment of Pharmacology, Chosun University School of Medicine, Gwangju, Republic of Korea. 5Department of Anatomy, Chosun Uni- instability and tumorigenicity directly. Although posttranslational versity School of Medicine, Gwangju, Republic of Korea. 6Department modification via small ubiquitin-like modifiers affects the stability of Pathology,Chosun University School of Medicine, Gwangju, Repub- of MDC1 and its function in DDR (7–9), little is known about how lic of Korea. 7Department of Anatomy, School of Medicine, Jeju National University, Jeju-Do, Republic of Korea. 8Department of Bio- the expression of MDC1 is regulated and which pathophysiologic chemistry, Molecular Biology and Biophysics, University of Minnesota conditions are associated with this regulation. Twin Cities, Minneapolis, Minnesota. miRNAs are small noncoding RNAs that suppress protein 0 Note: Supplementary data for this article are available at Cancer Research synthesis, usually by interacting with the 3 -untranslated region Online (http://cancerres.aacrjournals.org/). (30-UTR) of target mRNAs (10). Several lines of evidence suggest Corresponding Authors: Ho Jin You, Chosun University Medical School, 375 that miRNAs negatively regulate the expression of DDR proteins Seosuk-dong, Gwang-ju 501-759, Republic of Korea. Phone: 82-62-230-6337; (11–14). Therefore, miRNAs may play an important role in the Fax: 82-62-233-3720; E-mail: [email protected]; Jung-Hee Lee, Phone: 82-62- regulation of DDR and may contribute to the maintenance of 230-6399; Fax: 82-62-230-6586; E-mail: [email protected]; and Jeongsik genomic integrity. Yong, Phone: 612-626-2420; Fax: 612-625-2163; E-mail: [email protected] To investigate the possibility that some specific miRNA might doi: 10.1158/0008-5472.CAN-14-2783 directly regulate MDC1 expression and its function, we screened Ó2015 American Association for Cancer Research. for miRNAs that could potentially regulate MDC1 expression and www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst January 27, 2015; DOI: 10.1158/0008-5472.CAN-14-2783 Lee et al. A B 1.5 1.2 0.9 0.6 MDC1 0.3 α-Tubulin 0.0 Normalized luciferase luciferase Normalized activity C HeLa U2OS HEK293T D miR-22 –+ –+ –+ MDC1 HeLa U2OS HEK293T 53BP1 1.2 1.2 1.2 BRCA1 0.9 0.9 0.9 α-Tubulin 0.6 ∗∗ 0.6 ∗∗ 0.6 ∗∗ 30 ∗∗ 15 150 Relative MDC1 0.3 0.3 0.3 ∗∗ ∗∗ mRNA expression 20 10 100 0 0 0 miR-22–+ –+ –+ 10 5 50 expression Relative miR-22 0 0 0 miR-Ctrl E F miR-22 1.6 ∗∗ ns 1.2 0.8 0.4 Normalized luciferase Normalized activity 0 Vector MDC1 MDC1 3′-UTR-wt 3′-UTR-mt Figure 1. miR-22 directly affects MDC1 expression. A, HEK293T cells were cotransfected with the MDC1 30-UTR luciferase reporter vector along with the candidate miRNAs, which were predicted by at least five bioinformatics algorithms, or the miRNA-negative control (miR-Ctrl). Results are shown as mean Æ SD (n ¼ 3). B, the levels of MDC1 protein were measured using Western blotting in HEK293T cells transfected with the indicated miRNAs. C, indicated cells were transfected with control miRNA or miR-22. The levels of indicated proteins were determined using Western blotting (top). miR-22 levels in the indicated cells were determined using real-time qPCR analysis (bottom). Results are shown as mean Æ SD (n ¼ 3). ÃÃ, P < 0.01. D, expression of MDC1 mRNA in miR-22–transfected HeLa, U2OS, and HEK293T cells was quantitated using real-time qPCR. Results are shown as mean Æ SD (n ¼ 3). ÃÃ, P < 0.01. E, a schematic representation of MDC1 30-UTR. Red, the seed sequence of miR-22. F, MDC1 30-UTR-wt and MDC1 30-UTR-mt were cotransfected with miR-22 in HEK293T cells. Luciferase activity was measured 24 hours after the transfection. Data represent mean Æ SD; n ¼ 3; ns, not significant; ÃÃ, P < 0.01. fi fi identi ed miR-22 as an miRNA that could speci cally suppress Materials and Methods MDC1 expression. We show that miR-22–mediated downregula- tion of MDC1 induces impaired DDR activation and genomic Antibodies instability. Further, we demonstrated that this new pathway plays a Polyclonal MDC1 antibody (R2) was raised in rabbit against a crucial role in the regulation of DNA repair in sustained activation glutathione S-transferase fusion protein containing the BRCT of Akt1 and senescence and may represent a new therapeutic target. domain of MDC1 (residues 1882-2089). Anti-MDC1 polyclonal OF2 Cancer Res; 75(7) April 1, 2015 Cancer Research Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst January 27, 2015; DOI: 10.1158/0008-5472.CAN-14-2783 miR-22 Impedes DDR Function of MDC1 MDC1 cDNA – – + A miR-22 ++– B MDC1 HA-MDC1 MDC1 cDNA – – + α-Tubulin miR-22 –++ 1.2 ∗∗ miR-Ctrl+control vector MDC1 ∗∗ ∗∗ ∗∗ miR-22+control vector 80 0.9 miR-22+MDC1 cDNA ∗∗ 60 ∗∗ γ-H2AX -H2AX 0.6 ∗∗ γ 40 ∗∗ ∗∗ ∗∗ (%)staining 20 merge Relative 0.3 ∗∗ ∗∗ 0 Relative BrdUrd incoporation (%) incoporation Relative BrdUrd MDC1 cDNA – – + 0.0 DAPI miR-22 –++ 0 2 5 10 20 50 Gy 2 hours after-IR treatment C MDC1 cDNA – – + D MDC1 cDNA –– + E MDC1 cDNA –– + miR-22 –+ + miR-22 –+ + miR-22 –+ + MDC1 MDC1 MDC1 HA-MDC1 HA-MDC1 HA-MDC1 α-Tubulin α-Tubulin α-Tubulin miR-22 –– + miR-control Control vector MDC1 cDNA MDC1 cDNA miR-Ctrl+control vector miR-22 –+ + miR-22+control vector Comet miR-22+MDC1 cDNA tail ∗∗ 100 P = 0.0008 P = 0.0001 15 ∗∗ ∗∗ ∗∗ ∗∗ 75 ∗∗ ∗∗ 10 60 10 45 ∗∗ 30 5 Relative cell survival (%)survival Relative cell 1 15 02510 Gy Comet tail moment (%) Comet tail 2 hours after-IR treatment 0 Number of chromosomal breaks 0 MDC1 cDNA – – + Control-miR Control vector MDC1 cDNA miR-22 –++ miR-22 F Figure 2. miR-22–mediated MDC1 downregulation leads to impaired DNA damage response. A, BrdUrd incorporation was measured using a colorimetric assay after indicated doses of IR, using U2OS cells transfected with indicated combinations of miRNAs and HA-MDC1 constructs. Results are shown as mean Æ SD (n ¼ 3). ÃÃ, P < 0.01. B and C, miR-22–expressing U2OS cells were transfected with miR-22–insensitive MDC1 and irradiated with 10 Gy of IR. Cells were then analyzed by g-H2AX and MDC1 staining 16 hours after IR (B) and by comet assay 3 hours after IR (C).
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