FEBS Letters 589 (2015) 1958–1965
journal homepage: www.FEBSLetters.org
MDM4 regulation by the let-7 miRNA family in the DNA damage response of glioma cells Chen Xie a,d, Wei Chen b,c, Mengdie Zhang a, Qiuxian Cai a, Weiyi Xu a, Xiaodi Li a, Songshan Jiang a,⇑ a
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China Department of Gynecology, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China c Gene Science & Health Company, Shenzhen 518048, China d Shenzhen Weiguang Biological Products Co., Ltd., Shenzhen 518107, China b
a r t i c l e
i n f o
Article history: Received 4 January 2015 Revised 7 May 2015 Accepted 15 May 2015 Available online 27 May 2015 Edited by Angel Nebreda Keywords: MicroRNA let-7 DNA damage MDM4 Coding DNA sequence Glioma
a b s t r a c t Despite extensive investigation into the role of let-7 miRNAs in pathological tumor processes, their involvement in the DNA damage response remains unclear. Here we show that most let-7 family members down-regulate MDM4 expression via binding to MDM4 mRNA at a conserved DNA sequence. Expression of exogenous let-7 miRNA mimics decreased MDM4 protein but not mRNA levels. Several DNA damage reagents increased let-7 expression, thereby decreasing MDM4 protein levels in glioma cells. Inhibition of endogenous let-7 with antisense RNAs rescued MDM4 protein levels with or without MG132, a proteasome-dependent degradation inhibitor. An MDM4 mutation identified in a glioma patient was associated with loss of the putative MDM4 target site. Therefore, let-7 binding to MDM4 is implicated in the DNA damage response. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction MicroRNAs (miRNAs) are small non-coding RNAs that mediate sequence-specific gene expression at the post-transcriptional level by targeting the 30 untranslated region (30 UTR) or the putative coding DNA sequence (CDS) [1]. Recently, many studies have implicated miRNAs in the regulation of genome stability and the response to DNA damage. For instance, miR-24 is upregulated in the terminal differentiation of hematopoietic cell lines and inhibits the histone variant H2AX, which causes the terminal differentiation of hematopoietic cells hypersensitive to gamma-irradiation and genotoxic drugs [2]. miR-138 can target H2AX, affecting genomic stability [3]. The miRNA miR-182 suppresses BRCA1 to impede homologous recombination [4]. In general, let-7 acts as a tumor suppressor in various cancers. Nine let-7 family members exist in human cells, including let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i and miR-98 [5]. Let-7 can inhibit tumor cell
Author contributions: X.C., W.C., Z.M., C.Q., L.X. and W.X. performed research and analyzed data; X.C. and S.J. contributed to the design of experiments and data analysis and wrote the paper. ⇑ Corresponding author at: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, 132 East Waihuan Rd, Rm 312, Higher Education Mega, Guangzhou 510006, China. Fax: +86 (20) 8403 6551. E-mail address: [email protected] (S. Jiang).
proliferation, the efficiency of colony formation [6] and the epithelial-mesenchymal transition [7]. Consequently, the let-7 expression level can indicate the stage of cancer [7]. Let-7 can also influence genomic stability [8,9], and the expression level of let-7 in tumors is related to survival after chemotherapy and radiotherapy [10,11]. It seems that high let-7 expression could induce tumor cell death when DNA damage is irreparable. Several studies have shown that the expression levels of let-7 family members are significantly altered by DNA damage. However, different cell types exhibit different changes in let-7 expression after irradiation. In the human glioma cell line M059K, the expression levels of all let-7 family members were increased after radiation [12]. In the Jurkat p53-negative human T lymphocyte cell line, ionizing radiation can induce increases in all let-7 family members. In the p53-positive TK6 cell line, let-7a, let-7b, let-7c and let-7f were increased after radiation [13]. Let-7a, let-7c, let-7d, and let-7g were shown to be upregulated in normal human thyroid cells by irradiation [14]. Recently, Lee et al. showed that stimulation of p53 by a DNA damage agent can increase let-7 levels by downregulating Lin28A [15]. However, let-7a and let-7b expression levels were reported to be decreased in the human colon cancer cell line HCT116 in a p53-dependent manner after various types of DNA damage including irradiation, ultraviolet radiation, etoposide and hydrogen peroxide [16].
http://dx.doi.org/10.1016/j.febslet.2015.05.030 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
MDM4 is an important p53 regulator [17] that contributes to tumor formation by inhibiting p53 activity [18]. MDM4 is one of the well-characterized molecules that can play an important role in regulating genomic stability [19]. DNA damage could rapidly reduce the half-life of MDM4 [20]. Some experiments support the conclusion that MDM4 is phosphorylated by ATM and Chk2 and then degraded by MDM2 and the proteasome [20]. However, when the proteasome is inhibited, MDM4 levels could still decrease after DNA damage [21], suggesting that another MDM4 repression pathway exists, such as an miRNA pathway. The miRNAs miR-191, miR-10a, miR-15 and miR-34a were reported to target MDM4 and repress the level of MDM4 protein [21–24]. In addition, miR-191 is highly expressed in both normal and tumor tissues. A single nucleotide polymorphism (SNP) in the 30 UTR of MDM4 causes the loss of an miR-191 target site, which is associated with a poor ovarian cancer prognosis [24]. Despite extensive investigation into the roles of let-7 miRNA family members in various pathological tumor processes and a study showing that let-7 could inhibit glioma cell growth and migration [25], the role of let-7 after DNA damage in glioma has not been fully characterized. This study demonstrates that let-7 could suppress MDM4 expression and that MDM4 is downregulated through an increase in let-7 expression in U87 and A172 glioma cells after DNA damage induced by cisplatin, bleomycin and hydroxyurea. Furthermore, the results demonstrate that a mutation in the let-7-MDM4 target site found in a human glioma sample might affect let-7 binding at the MDM4 CDS. 2. Materials and methods 2.1. Bioinformatic analysis DIANA LAB, RegRNA, microRNA, RNAhybrid and PITA software were used to predict the miRNA targets of MDM4. The clustal W algorithm was used for multiple sequence alignment analysis. 2.2. Cell culture Human glioma cell lines (U87 and A172) were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% (V/V) fetal bovine serum (FBS, Invitrogen), 100 U/ml penicillin and 100 lg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) with 5% CO2 at 37 °C. 2.3. Vector construction and transfection To express miRNAs, pre-miRNAs with 80–150 bp of flanking sequences at both ends were cloned into the modified pLL3.7 vector under the control of the human U6 promoter using the primers listed in Table S1. To construct luciferase reporter vectors, MDM4 CDS wild type or mutated sequences corresponding to the predicted miRNA target sites were synthesized using the sequence listed in Table S1 and cloned downstream of Renilla luciferase in the psiCHECK-2 vector (Promega, Madison, WI, USA). Let-7a, let-7c mimics, antisense RNAs and their cognate control RNAs were synthesized and purified by GenePharma, China. Cell transfection was performed using X-tremeGENE siRNA Transfection Reagent (Roche, Rotkreuz, Switzerland) according to the manufacturer’s instructions. Transfections were carried out with 100 nmol/L of miRNA mimics or 200 nmol/L of antisense oligonucleotides. The efficiency of small RNA transfection was estimated to be greater than 95% for U87 and A172 cells using a Cy3 dye-labeled RNA oligonucleotide (Ribobio, Guangzhou, China). FuGENE HD (Roche, Rotkreuz, Switzerland) was used for plasmid
1959
transfection according to the manufacturer’s instructions. DNA (400 ng) was added in a 96-well plate. miRNA expression vectors and reporter constructs were co-transfected at a 3:1 ratio. 2.4. RNA extraction and real-time quantitative RT-PCR Total RNA was extracted from cells using Trizol (Invitrogen, Carlsbad, CA, USA), reverse-transcribed with Rever-Tra-Ace-a-Tra nscriptase (Toyobo, Tokyo, Japan) and then amplified by PCR using the SYBRÒ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Tokyo, Japan). RNAs were quantified and checked for purity by spectrophotometry at 260 and 280 nm. Real-time quantification of microRNAs by stem-loop RT-PCR was carried out using U6 snRNA as an internal standard. The primer sequences are listed in Table S1. Quantitative PCR was performed on a LightCycler 480 Real-Time PCR system (Roche, Rotkreuz, Switzerland). Relative-fold changes in miRNA or mRNA expression in treated cells compared with control cells were calculated using the comparative Ct (2DDCt) method [26]. All reactions were performed in triplicate. 2.5. Western blotting Cells were lysed in RAPI lysis buffer (Bioteke, Beijing, China), and the whole-cell protein concentration was determined by a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Protein samples (20 lg per lane) were denatured and separated by SDS–polyacrylamide gel electrophoresis (PAGE) on a 12% gel and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was incubated overnight at 4 °C with a primary antibody and then incubated for 1 h with secondary antibody. The bound antibody was detected using enhanced chemiluminescence detection reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The band intensities were quantified with Kodak Image Station 4000 MM Pro (Kodak, Tokyo, Japan). Anti-human MDM4 and b-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-Human c-H2AX was purchased from Millipore (Bedford, MA, USA). Goat-anti-mouse and goat-anti-rabbit were purchased from Jackson ImmunoResearch (West Grove, PA, USA). 2.6. Dual-luciferase reporter assays For the luciferase reporter assay, 2.0 104 293T cells were plated in 100 lL growth medium in 96-well plates. The cells were transfected with 100 ng reporter plasmids and 300 ng miRNA expression plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) or FuGENE (Roche, Rotkreuz, Switzerland). The cells were harvested 48 h after transfection and assayed using the dual-luciferase reporter assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Transfections were repeated in triplicate. 2.7. Drug treatment U87 or A172 cells were seeded in 3.5 cm dishes at 2 105 cells per dish, incubated overnight, and then treated with the reagents of interest for various periods of time. Cisplatin (Sigma, St. Louis, MO, USA) was dissolved in 0.9% NaCl before use and was added to the cell culture medium at various final concentrations. Bleomycin (Aladdin, Shanghai, China) and hydroxyurea (Sigma, St. Louis, MO, USA) were dissolved in sterile water and stored at 20 °C.
1960
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
2.8. Statistical analysis Data analysis was performed using Origin 8.0 (ADITIVE, Friedrichsdorf, Germany) and Excel software (Microsoft, Redmond, WA, USA), and results are presented as the mean ± standard deviation (S.D.) of at least three separate experiments. Statistical analyses were performed using analysis of variance or Student’s t test. P values < 0.05 were considered statistically significant (⁄, P < 0.05; ⁄⁄, P < 0.01). 3. Results 3.1. Let-7 miRNA family members repress MDM4 expression Bioinformatic analysis predicted that all let-7 family members target the human MDM4 CDS (456–474) (Fig. 1A). The putative let-7 target site is conserved when aligning the sequences of mammals and other species (Fig. 1B). To investigate whether the expression of let-7 family members correlates with MDM4 expression, we first constructed expression vectors for let-7 miRNA family members. Each miRNA expression vector and two controls (empty vector (pll3.7-control) and rno-miR-344 (pll3.7-miR-344), a microRNA that does not have a homologue in humans) were co-transfected with an MDM4 CDS reporter vector, and then a dual-luciferase assay was used to determine the decline in the relative luciferase activity resulting from transfection with let-7 family members compared with the control (Fig. 1C). As let-7c and let-7a lead to the greatest inhibition, we analyzed these two microRNAs further. To validate whether MDM4 is directly targeted by let-7 family members, we constructed five different mutations of the putative let-7-binding site in the MDM4 CDS reporter (MT1-MT5) (Fig. 2A). The results of the dual-luciferase assay showed that neither let-7a nor let-7c can suppress the expression of the mutated constructs as effectively as they suppress the wild type constructs (Fig. 2B and C). In addition, each let-7 family member was co-transfected with the MT5 CDS to further characterize the interaction. As expected, the introduction of silent mutations into predicted targets disrupted the ability of the miRNAs let-7a, let-7b, let-7c, let-7e and let-7g to repress the expression of the luciferase reporter, with the MT5 reporter expressing higher levels of luciferase than the wild type reporter (Fig. 2D). Taken together, these results indicate that these miRNAs could directly target the MDM4 CDS. To determine whether let-7 can disrupt endogenous MDM4 expression in glioma cells, let-7a and let-7c mimics were transfected into U87 cells. qRT-PCR results showed that the expression levels of let-7a and let-7c were approximately six to nine times higher compared to the control (Fig. 2E). A Western blot assay showed that both let-7a and let-7c consistently and substantially downregulated the expression level of the MDM4 protein without affecting its mRNA level in U87 cells (Fig. 2E), indicating that let-7a and let-7c mainly impair MDM4 expression through translational inhibition. Furthermore, similar experiments were performed using let-7a and let-7c antisense treatment, and decreases in let-7a and let-7c increased the MDM4 protein level without changing the mRNA level (Fig. 2F). 3.2. DNA damage decreased MDM4 expression, partially via let-7 The MDM4 protein is associated with the DNA damage response [20]. We hypothesized that the reduction of MDM4 after DNA damage was mediated by let-7 family members. A Western blot showed that the level of the MDM4 protein was significantly decreased following treatment with different doses of cisplatin (CDDP) for 24 h in U87 cells (Fig. S1), where phosphorylated
Fig. 1. Let-7 miRNA family members can target the MDM4 CDS. (A) The predicted target sequence of let-7 family members within the human MDM4 CDS. (B) Alignment of human and other species MDM4 DNA binding sites to let-7 family members. (C) The effect of different let-7 family members on MDM4 CDS reporter constructs in HEK293T cells was tested by a luciferase reporter assay. The data were normalized to the ratio of Firefly and Renilla luciferase activities measured at 48 h post-transfection. Rno-miR-344 (pll3.7-miR-344) is an miRNA that does not have homologue in humans. The results were presented as relative luciferase activity, with the control assigned a value of 1. Values represent the mean ± S.D. from three independent transfection experiments. Significant differences from the control value are indicated by ⁄, P < 0.05, ⁄⁄, P < 0.01.
H2AX (p-H2AX) was used as a marker of DNA damage. In addition, qRT-PCR was used to determine the expression level of let-7. The miRNA expression level of most let-7 family members increased significantly after U87 cells were treated with 2.5 lM CDDP (Fig. 3A). Interestingly, we found that the changes in the let-7 and MDM4 protein levels were inversely correlated (Fig. 3A and B). To confirm these results, A172 glioma cells were used in the same experiment. The results showed that the expression levels of let-7a and let-7c were increased, whereas the level of MDM4 protein was decreased when the A172 glioma cells were
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
1961
Fig. 2. The let-7 miRNA family members directly correlate with MDM4. (A) Sequences of MDM4 CDS (WT) and mutations (MT1-MT5). Several nucleotides within the seed region as well as other predicted binding regions were mutated in the MDM4 sequence. (B and C) Empty vector (pll3.7-control) or miRNA expression vector (pll3.7-let-7a or pll3.7-let-7c) were co-transfected with MDM4 or mutants MT1-MT5. Luciferase activity was measured at 48 h post-transfection. Each data point was measured in triplicate. (D) Let-7 family members were co-transfected with MDM4 CDS mutant expression vectors (MT5) or wild type MDM4 CDS, respectively. (E and F) miRNA (let-7a or let-7c) mimics or antisense RNAs were transfected into U87 cells. Relative expression levels of let-7a, let-7c and MDM4 were determined by qRT-PCR 48 h after transfection. Western blot was used to analyze the MDM4 protein level. b-actin served as the internal control. Values represent the means ± S.D. Significant differences from the control value are indicated by ⁄, P < 0.05, ⁄⁄, P < 0.01.
treated with 10 lM CDDP for 24 h, which is consistent with the results for U87 glioma cells (Fig. 3C). To determine whether let-7 repression of MDM4 could also mediate DNA damage induced by other reagents, bleomycin and hydroxyurea were used to induce DNA damage instead of CDDP. The results in U87 and A172 glioma cells showed that the
expression levels of let-7a and let-7c were increased after treatment with bleomycin or hydroxyurea, and MDM4 protein level was reduced (Fig. 4A–D). To better understand the role of let-7 in the repression of MDM4 in response to DNA damage, we transfected let-7a and let-7c antisense RNAs to inhibit these miRNAs in U87 and A172
1962
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
3.3. A mutation found in glioma may disrupt the relationship between let-7 and MDM4 The Cancer Genome Atlas Research Network has shown that the Thr160Ser missense mutation of MDM4 is found in human glioma [27]. As Thr and Ser have a similar structure and this missense mutation exists in the seed region of the let-7-MDM4 target site, we proposed that this mutation might contribute to the disruption of the let-7 repression of MDM4. To test this hypothesis, we disrupted the target site by mutating 160Thr (ACC) to 160Ser with the AGC (observed in a glioma patient), TCG, AGT or TCC codons (Fig. 5A). As all these mutations code for Ser, the latter three mutations were used to test whether a change in one or two bases could disrupt the let-7 repression of MDM4. Then, the let-7 expression vector was co-transected with the MDM4 CDS reporter construct and a dual-luciferase assay was followed. The results showed that the repressive effect of let-7 family members on luciferase activity was inhibited by a single C to G transition, the mutation observed in a glioma patient (Fig. 5B). The repressive effect of let-7a and let-7c on luciferase activity was also inhibited by all other missense mutations TCG (S1), AGT (S2) and TCC (S3) (Fig. 5C and D). These results suggest that these mutations may disrupt the let-7 repression of MDM4. 4. Discussion
Fig. 3. Cisplatin treatment results in a change in let-7 and MDM4 expression. (A) Expression level of let-7 family members was measured by qRT-PCR after cisplatin (CDDP) treatment. miR-34a served as a positive control, and miR-125b and miR135b served as negative controls. (B) Western blot analysis of the MDM4 protein level after CDDP treatment. p-H2AX acted as a DNA damage marker. b-actin served as the internal control. (C) A172 cells were treated with 10 lM CDDP for 24 h, and then the MDM4 protein level was measured by Western blot and the relative expression levels of let-7a and let-7c were determined by qRT-PCR. Natural saline (NS) served as a negative control.
cells treated with CDDP. A Western blot assay showed that the relative MDM4 protein level increased (Fig. 4E and F), indicating that decreasing let-7a or let-7c can eliminate the effect of CDDP treatment on the MDM4 protein. Furthermore, inhibition of proteasome degradation by pretreatment of U87 cells with 5 lM of MG132 does not completely block the reduction in the level of the MDM4 protein following CDDP treatment (Fig. 4G). Taken together, these results suggest that rather than proteasome degradation, let-7 family members may play a key role in mediating the decrease in the level of the MDM4 protein after DNA damage.
Cells overexpressing let-7 have been reported to be hypersensitive to DNA damage [28]. Several studies have shown that let-7 family members are upregulated after DNA damage [12–14,29], although in some cells the let-7 family members are downregulated by DNA damage [16]. In this study, we found that the let-7 expression level in glioma cells was upregulated after DNA damage, which, in turn, suppressed MDM4 expression. Although the alterations in the let-7 expression level after DNA damage have been widely observed, the mechanisms of this progress remain unclear. To explain this phenomenon, Chaudhry et al. proposed that the increase in let-7 after DNA damage may be mediated by a DNA-dependent serine/threonine protein kinase in glioma cell lines [12]. Lin28 is a well-known gene that can inhibit the maturity of let-7 [30]. Recently, Lee et al. showed that DNA damage increased let-7 levels through downregulation of Lin28A [15]. KSRP was also reported to interact with Drosha and Dicer to promote the maturity of let-7a [31]. KSRP accumulates in the nucleus when Hela cells are cultured with cisplatin [32], which may suggest that KSRP mediates the change in let-7 expression. Further study should focus on the role of KSRP and Lin28A in the process of let-7 maturation in glioma cells. Approximately 40% of gliomas exhibit a p53 mutation or deletion, and approximately 80% of gliomas have a p53 pathway defect [33]. In response to DNA damage, MDM4 expression is downregulated and contributes to p53 activation [20]. Post-translational modifications are considered to play an important role in MDM4 regulation after DNA damage. For example, MDM4 is first phosphorylated by ATM and Chk2 and then degraded by MDM2 and the proteasome after DNA damage [20]. However, even after suppressing MDM4 degradation by adding MDM2 siRNA or the proteasome inhibitor MG132, the amount of endogenous MDM4 remains largely reduced after DNA damage [21]. We also demonstrated that blockade of proteasome-dependent degradation with MG132 in U87 and A172 glioma cells cannot suppress the decrease in the endogenous MDM4 protein level after cisplatin treatment (Fig. 4G). All of these points suggest that there could be exist another mechanism besides MDM2-mediated proteasome-dependent degradation of MDM4 after DNA damage. In this study, we show that several members of let-7 family including let-7a, -7c, -7e, -7g and -7i can repress MDM4 at the protein level. Moreover, knockdown of let-7a and
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
1963
Fig. 4. DNA damage could interfere with MDM4 expression through let-7. The MDM4 protein level and the relative expression levels of let-7a and let-7c were determined by Western blot and qRT-PCR, respectively, after treatment with 100 lM bleomycin or 1 mM hydroxyurea for 24 h in U87 cells (A and B) and A172 cells (C and D). PBS served as the negative control. (E and F) Western blot was used to compare MDM4 protein levels among cells transfected with antisense let-7a, antisense let-7c or a negative control with or without CDDP in U87 and A172 cells. (G) MG132 was used to block proteasome-mediated MDM4 degradation. Whole cell extracts from the indicated treatment conditions were subjected to Western blot analysis for MDM4 and b-actin. b-actin served as the internal control.
let-7c with antisense RNAs can increase the MDM4 protein level (Fig. 2F). This result can be explained as follows: (1) let-7a and let-7b are the most abundant species in glioma cells [34], knockdown of let-7a could reduce the total let-7 level significantly; (2) there is only one nucleotide difference between let-7c and let-7a or let-7b (Fig. 1A), antisense let-7c may exert an effect through let-7a and let-7b. We also found that the MDM4 protein level became more sensitive to antisense let-7a and let-7c after U87 cells were treated with MG132 alone or in combination with CDDP than the same cells without MG132 treatment (Fig. 4G). This may be because investigating the proteasome degradation inhibited by MG132 made the role of let-7 in MDM4 regulation more clear. These genetic traits are always linked to tumorigenesis, tumor progression and prognosis and chemotherapy. For example, a let-7 binding site polymorphism in the KRAS 30 UTR is associated with reduced survival in oral cancers and metastatic colorectal cancers
[35,36]. An SNP in the miR-191 target site within the 30 UTR of MDM4 also affects ovarian cancer progression and chemosensitivity [24]. Consequently, more studies should be carried out to understand the function of mutations or SNPs at miRNA target sites. In our studies, we demonstrated that a single base mutation identified in a glioma patient can disrupt the interaction between let-7 and MDM4 mRNA. Coincidentally, asynonymous somatic mutation (COSM1646071 (C/A), Thr(ACC)160Thr(ACA)) also located in this seed region of the let-7 binding site, was identified in both human lung cancer and large intestine cancer samples in the COSMIC project (release 71). These results suggest that this binding site is important for tumor formation or progression. In summary, this study reports MDM4 as a new target of let-7,which can bind to the MDM4 CDS and then downregulate the level of the MDM4 protein. The let-7 expression level was upregulated after DNA damage induced by cisplatin, bleomycin or
1964
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
Fig. 5. A mutation found in glioma, Thr160Ser, may disrupt the relationship between let-7 and MDM4. (A) Sequences of MDM4 and its missense mutations. One or two nucleotides underlined within the seed region of the MDM4 sequence were mutated (MT-G, S1, S2 and S3), where MT-G was found in a tumor sample. (B) let-7 family members were co-transfected with wild type MDM4 or the MT-G mutant. Luciferase activity was measured at 48 h post-transfection. (C and D) Empty vector (pll3.7-control) or miRNA expression vector (pll3.7-let-7a or pll3.7-let-7c) were co-transfected with MDM4 or mutants S1, S2 or S3. Luciferase activity was measured at 48 h posttransfection. Each data point was measured in triplicate. Values represent the means ± S.D. Significant differences from the control value are indicated by ⁄, P < 0.05, ⁄⁄, P < 0.01.
hydroxyurea in U87 and A172 glioma cell lines. Further, we found that the reduction of MDM4 after DNA damage can be recovered by inhibiting the increase in let-7. In addition, a mutation found in glioma can damage the interaction between let-7 and MDM4. Furthermore, additional abnormal targets of let-7 in glioma must be identified to determine the contributions of let-7 to glioma tumorigenesis and tumor progress. This result suggests that let-7 directly and functionally targets MDM4, which may relate to the activation of p53 to maintain genome stability. Therefore, let-7-based therapy may be beneficial for the clinical treatment of cancers, including gliomas. Funding This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81272773 and 81101960), the Natural Science Foundation of Guangdong Province (S2013010012170), the Scientific and Technological Planning of Guangzhou (Grant No. 2012J4100082) and the Shenzhen Municipal Government of China (Grant No. LXRY20121106142947958). Conflict of interest statement None declared. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.05. 030.
References [1] Tay, Y., Zhang, J.Q., Thomson, A.M., Lim, B. and Rigoutsos, I. (2008) MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature 455. 1124-U12. [2] Lal, A. et al. (2009) MiR-24-mediated downregulation of H2AX suppresses DNA repair in terminally differentiated blood cells. Nat. Struct. Mol. Biol. 16, 492–498. [3] Wang, Y.M. et al. (2011) MicroRNA-138 modulates DNA damage response by repressing histone H2AX expression. Mol. Cancer Res. 9, 1100–1111. [4] Moskwa, P. et al. (2011) MiR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors. Mol. Cell 41, 210–220. [5] Wang, S., Tang, Y., Cui, H., Zhao, X., Luo, X., Pan, W., Huang, X. and Shen, N. (2011) Let-7/miR-98 regulate Fas and Fas-mediated apoptosis. Genes Immun. 12, 149–154. [6] Kong, D.J. et al. (2012) Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM. PLoS One 7. [7] Shell, S. et al. (2007) Let-7 expression defines two differentiation stages of cancer. Proc. Natl. Acad. Sci. USA 104, 11400–11405. [8] Johnson, S.M. et al. (2005) RAS is regulated by the let-7 MicroRNA family. Cell 120, 635–647. [9] Saavedra, H.I., Knauf, J.A., Shirokawa, J.M., Wang, J.W., Bin, O.Y., Elisei, R., Stambrook, P.J. and Fagin, J.A. (2000) The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway. Oncogene 19, 3948– 3954. [10] Lu, L., Schwartz, P., Scarampi, L., Rutherford, T., Canuto, E.M., Yu, H. and Katsaros, D. (2011) MicroRNA let-7a: a potential marker for selection of paclitaxel in ovarian cancer management. Gynecol. Oncol. 122, 366–371. [11] Weidhaas, J.B., Babar, I., Nallur, S.M., Trang, P., Roush, S., Boehm, M., Gillespie, E. and Slack, F.J. (2007) MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy. Cancer Res. 67, 11111–11116. [12] Chaudhry, M.A., Sachdeva, H. and Omaruddin, R.A. (2010) Radiation-induced micro-RNA modulation in glioblastoma cells differing in DNA-repair pathways. DNA Cell Biol. 29, 553–561. [13] Chaudhry, M.A. (2009) Real-time PCR analysis of micro-RNA expression in ionizing radiation-treated cells. Cancer Biother. Radiopharm. 24, 49–56.
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965 [14] Nikiforova, M.N., Gandhi, M., Kelly, L. and Nikiforov, Y.E. (2011) MicroRNA dysregulation in human thyroid cells following exposure to ionizing radiation. Thyroid 21, 261–266. [15] Lee, J.Y. et al. (2013) Tumor suppressor p53 plays a key role in induction of both tristetraprolin and let-7 in human cancer cells. Nucleic Acids Res. 41, 5614–5625. [16] Saleh, A.D. et al. (2011) Cellular stress induced alterations in microRNA let-7a and let-7b expression are dependent on p53. PLoS One 6, e24429. [17] Shvarts, A. et al. (1997) Isolation and identification of the human homolog of a new p53-binding protein, Mdmx. Genomics 43, 34–42. [18] Danovi, D. et al. (2004) Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity. Mol. Cell. Biol. 24, 5835–5843. [19] Matijasevic, Z., Krzywicka-Racka, A., Sluder, G. and Jones, S.N. (2008) MdmX regulates transformation and chromosomal stability in p53-deficient cells. Cell Cycle 7, 2967–2973. [20] Chen, L., Gilkes, D.M., Pan, Y., Lane, W.S. and Chen, J. (2005) ATM and Chk2dependent phosphorylation of MDMX contribute to p53 activation after DNA damage. EMBO J. 24, 3411–3422. [21] Markey, M. and Berberich, S.J. (2008) Full-length hdmX transcripts decrease following genotoxic stress. Oncogene 27, 6657–6666. [22] Gatt, M.E. et al. (2010) MicroRNAs 15a/16-1 function as tumor suppressor genes in multiple myeloma. Blood. [23] Ovcharenko, D. et al. (2011) MiR-10a overexpression is associated with NPM1 mutations and MDM4 downregulation in intermediate-risk acute myeloid leukemia. Exp. Hematol. 39, 1030–1042. [24] Wynendaele, J. et al. (2010) An illegitimate microRNA target site within the 30 UTR of MDM4 affects ovarian cancer progression and chemosensitivity. Cancer Res. 70, 9641–9649. [25] Lee, S.T. et al. (2011) Let-7 microRNA inhibits the proliferation of human glioblastoma cells. J. Neurooncol. 102, 19–24.
1965
[26] Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. [27] Chin, L. et al. (2008) Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455, 1061–1068. [28] Metheetrairut, C. and Slack, F.J. (2013) MicroRNAs in the ionizing radiation response and in radiotherapy. Curr. Opin. Genet. Dev. 23, 12–19. [29] Wagner-Ecker, M., Schwager, C., Wirkner, U., Abdollahi, A. and Huber, P.E. (2010) MicroRNA expression after ionizing radiation in human endothelial cells. Radiat. Oncol. 5, 25. [30] Jeong, S.H., Wu, H.G. and Park, W.Y. (2009) LIN28B confers radio-resistance through the posttranscriptional control of KRAS. Exp. Mol. Med. 41, 912–918. [31] Trabucchi, M., Briata, P., Garcia-Mayoral, M., Haase, A.D., Filipowicz, W., Ramos, A., Gherzi, R. and Rosenfeld, M.G. (2009) The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. Nature 459. 1010U144. [32] Drayton, R.M. (2012) The role of microRNA in the response to cisplatin treatment. Biochem. Soc. Trans. 40, 821–825. [33] Dunn, G.P. et al. (2012) Emerging insights into the molecular and cellular basis of glioblastoma. Genes Dev. 26, 756–784. [34] Landgraf, P. et al. (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414. [35] Christensen, B.C., Moyer, B.J., Avissar, M., Ouellet, L.G., Plaza, S.L., McClean, M.D., Marsit, C.J. and Kelsey, K.T. (2009) A let-7 microRNA-binding site polymorphism in the KRAS 30 UTR is associated with reduced survival in oral cancers. Carcinogenesis 30, 1003–1007. [36] Zhang, W. et al. (2011) A let-7 microRNA-binding site polymorphism in 30 untranslated region of KRAS gene predicts response in wild-type KRAS patients with metastatic colorectal cancer treated with cetuximab monotherapy. Ann. Oncol. 22, 104–109.
journal homepage: www.FEBSLetters.org
MDM4 regulation by the let-7 miRNA family in the DNA damage response of glioma cells Chen Xie a,d, Wei Chen b,c, Mengdie Zhang a, Qiuxian Cai a, Weiyi Xu a, Xiaodi Li a, Songshan Jiang a,⇑ a
State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China Department of Gynecology, the Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China c Gene Science & Health Company, Shenzhen 518048, China d Shenzhen Weiguang Biological Products Co., Ltd., Shenzhen 518107, China b
a r t i c l e
i n f o
Article history: Received 4 January 2015 Revised 7 May 2015 Accepted 15 May 2015 Available online 27 May 2015 Edited by Angel Nebreda Keywords: MicroRNA let-7 DNA damage MDM4 Coding DNA sequence Glioma
a b s t r a c t Despite extensive investigation into the role of let-7 miRNAs in pathological tumor processes, their involvement in the DNA damage response remains unclear. Here we show that most let-7 family members down-regulate MDM4 expression via binding to MDM4 mRNA at a conserved DNA sequence. Expression of exogenous let-7 miRNA mimics decreased MDM4 protein but not mRNA levels. Several DNA damage reagents increased let-7 expression, thereby decreasing MDM4 protein levels in glioma cells. Inhibition of endogenous let-7 with antisense RNAs rescued MDM4 protein levels with or without MG132, a proteasome-dependent degradation inhibitor. An MDM4 mutation identified in a glioma patient was associated with loss of the putative MDM4 target site. Therefore, let-7 binding to MDM4 is implicated in the DNA damage response. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction MicroRNAs (miRNAs) are small non-coding RNAs that mediate sequence-specific gene expression at the post-transcriptional level by targeting the 30 untranslated region (30 UTR) or the putative coding DNA sequence (CDS) [1]. Recently, many studies have implicated miRNAs in the regulation of genome stability and the response to DNA damage. For instance, miR-24 is upregulated in the terminal differentiation of hematopoietic cell lines and inhibits the histone variant H2AX, which causes the terminal differentiation of hematopoietic cells hypersensitive to gamma-irradiation and genotoxic drugs [2]. miR-138 can target H2AX, affecting genomic stability [3]. The miRNA miR-182 suppresses BRCA1 to impede homologous recombination [4]. In general, let-7 acts as a tumor suppressor in various cancers. Nine let-7 family members exist in human cells, including let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i and miR-98 [5]. Let-7 can inhibit tumor cell
Author contributions: X.C., W.C., Z.M., C.Q., L.X. and W.X. performed research and analyzed data; X.C. and S.J. contributed to the design of experiments and data analysis and wrote the paper. ⇑ Corresponding author at: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, 132 East Waihuan Rd, Rm 312, Higher Education Mega, Guangzhou 510006, China. Fax: +86 (20) 8403 6551. E-mail address: [email protected] (S. Jiang).
proliferation, the efficiency of colony formation [6] and the epithelial-mesenchymal transition [7]. Consequently, the let-7 expression level can indicate the stage of cancer [7]. Let-7 can also influence genomic stability [8,9], and the expression level of let-7 in tumors is related to survival after chemotherapy and radiotherapy [10,11]. It seems that high let-7 expression could induce tumor cell death when DNA damage is irreparable. Several studies have shown that the expression levels of let-7 family members are significantly altered by DNA damage. However, different cell types exhibit different changes in let-7 expression after irradiation. In the human glioma cell line M059K, the expression levels of all let-7 family members were increased after radiation [12]. In the Jurkat p53-negative human T lymphocyte cell line, ionizing radiation can induce increases in all let-7 family members. In the p53-positive TK6 cell line, let-7a, let-7b, let-7c and let-7f were increased after radiation [13]. Let-7a, let-7c, let-7d, and let-7g were shown to be upregulated in normal human thyroid cells by irradiation [14]. Recently, Lee et al. showed that stimulation of p53 by a DNA damage agent can increase let-7 levels by downregulating Lin28A [15]. However, let-7a and let-7b expression levels were reported to be decreased in the human colon cancer cell line HCT116 in a p53-dependent manner after various types of DNA damage including irradiation, ultraviolet radiation, etoposide and hydrogen peroxide [16].
http://dx.doi.org/10.1016/j.febslet.2015.05.030 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
MDM4 is an important p53 regulator [17] that contributes to tumor formation by inhibiting p53 activity [18]. MDM4 is one of the well-characterized molecules that can play an important role in regulating genomic stability [19]. DNA damage could rapidly reduce the half-life of MDM4 [20]. Some experiments support the conclusion that MDM4 is phosphorylated by ATM and Chk2 and then degraded by MDM2 and the proteasome [20]. However, when the proteasome is inhibited, MDM4 levels could still decrease after DNA damage [21], suggesting that another MDM4 repression pathway exists, such as an miRNA pathway. The miRNAs miR-191, miR-10a, miR-15 and miR-34a were reported to target MDM4 and repress the level of MDM4 protein [21–24]. In addition, miR-191 is highly expressed in both normal and tumor tissues. A single nucleotide polymorphism (SNP) in the 30 UTR of MDM4 causes the loss of an miR-191 target site, which is associated with a poor ovarian cancer prognosis [24]. Despite extensive investigation into the roles of let-7 miRNA family members in various pathological tumor processes and a study showing that let-7 could inhibit glioma cell growth and migration [25], the role of let-7 after DNA damage in glioma has not been fully characterized. This study demonstrates that let-7 could suppress MDM4 expression and that MDM4 is downregulated through an increase in let-7 expression in U87 and A172 glioma cells after DNA damage induced by cisplatin, bleomycin and hydroxyurea. Furthermore, the results demonstrate that a mutation in the let-7-MDM4 target site found in a human glioma sample might affect let-7 binding at the MDM4 CDS. 2. Materials and methods 2.1. Bioinformatic analysis DIANA LAB, RegRNA, microRNA, RNAhybrid and PITA software were used to predict the miRNA targets of MDM4. The clustal W algorithm was used for multiple sequence alignment analysis. 2.2. Cell culture Human glioma cell lines (U87 and A172) were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% (V/V) fetal bovine serum (FBS, Invitrogen), 100 U/ml penicillin and 100 lg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) with 5% CO2 at 37 °C. 2.3. Vector construction and transfection To express miRNAs, pre-miRNAs with 80–150 bp of flanking sequences at both ends were cloned into the modified pLL3.7 vector under the control of the human U6 promoter using the primers listed in Table S1. To construct luciferase reporter vectors, MDM4 CDS wild type or mutated sequences corresponding to the predicted miRNA target sites were synthesized using the sequence listed in Table S1 and cloned downstream of Renilla luciferase in the psiCHECK-2 vector (Promega, Madison, WI, USA). Let-7a, let-7c mimics, antisense RNAs and their cognate control RNAs were synthesized and purified by GenePharma, China. Cell transfection was performed using X-tremeGENE siRNA Transfection Reagent (Roche, Rotkreuz, Switzerland) according to the manufacturer’s instructions. Transfections were carried out with 100 nmol/L of miRNA mimics or 200 nmol/L of antisense oligonucleotides. The efficiency of small RNA transfection was estimated to be greater than 95% for U87 and A172 cells using a Cy3 dye-labeled RNA oligonucleotide (Ribobio, Guangzhou, China). FuGENE HD (Roche, Rotkreuz, Switzerland) was used for plasmid
1959
transfection according to the manufacturer’s instructions. DNA (400 ng) was added in a 96-well plate. miRNA expression vectors and reporter constructs were co-transfected at a 3:1 ratio. 2.4. RNA extraction and real-time quantitative RT-PCR Total RNA was extracted from cells using Trizol (Invitrogen, Carlsbad, CA, USA), reverse-transcribed with Rever-Tra-Ace-a-Tra nscriptase (Toyobo, Tokyo, Japan) and then amplified by PCR using the SYBRÒ Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Tokyo, Japan). RNAs were quantified and checked for purity by spectrophotometry at 260 and 280 nm. Real-time quantification of microRNAs by stem-loop RT-PCR was carried out using U6 snRNA as an internal standard. The primer sequences are listed in Table S1. Quantitative PCR was performed on a LightCycler 480 Real-Time PCR system (Roche, Rotkreuz, Switzerland). Relative-fold changes in miRNA or mRNA expression in treated cells compared with control cells were calculated using the comparative Ct (2DDCt) method [26]. All reactions were performed in triplicate. 2.5. Western blotting Cells were lysed in RAPI lysis buffer (Bioteke, Beijing, China), and the whole-cell protein concentration was determined by a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Protein samples (20 lg per lane) were denatured and separated by SDS–polyacrylamide gel electrophoresis (PAGE) on a 12% gel and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was incubated overnight at 4 °C with a primary antibody and then incubated for 1 h with secondary antibody. The bound antibody was detected using enhanced chemiluminescence detection reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The band intensities were quantified with Kodak Image Station 4000 MM Pro (Kodak, Tokyo, Japan). Anti-human MDM4 and b-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-Human c-H2AX was purchased from Millipore (Bedford, MA, USA). Goat-anti-mouse and goat-anti-rabbit were purchased from Jackson ImmunoResearch (West Grove, PA, USA). 2.6. Dual-luciferase reporter assays For the luciferase reporter assay, 2.0 104 293T cells were plated in 100 lL growth medium in 96-well plates. The cells were transfected with 100 ng reporter plasmids and 300 ng miRNA expression plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) or FuGENE (Roche, Rotkreuz, Switzerland). The cells were harvested 48 h after transfection and assayed using the dual-luciferase reporter assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Transfections were repeated in triplicate. 2.7. Drug treatment U87 or A172 cells were seeded in 3.5 cm dishes at 2 105 cells per dish, incubated overnight, and then treated with the reagents of interest for various periods of time. Cisplatin (Sigma, St. Louis, MO, USA) was dissolved in 0.9% NaCl before use and was added to the cell culture medium at various final concentrations. Bleomycin (Aladdin, Shanghai, China) and hydroxyurea (Sigma, St. Louis, MO, USA) were dissolved in sterile water and stored at 20 °C.
1960
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
2.8. Statistical analysis Data analysis was performed using Origin 8.0 (ADITIVE, Friedrichsdorf, Germany) and Excel software (Microsoft, Redmond, WA, USA), and results are presented as the mean ± standard deviation (S.D.) of at least three separate experiments. Statistical analyses were performed using analysis of variance or Student’s t test. P values < 0.05 were considered statistically significant (⁄, P < 0.05; ⁄⁄, P < 0.01). 3. Results 3.1. Let-7 miRNA family members repress MDM4 expression Bioinformatic analysis predicted that all let-7 family members target the human MDM4 CDS (456–474) (Fig. 1A). The putative let-7 target site is conserved when aligning the sequences of mammals and other species (Fig. 1B). To investigate whether the expression of let-7 family members correlates with MDM4 expression, we first constructed expression vectors for let-7 miRNA family members. Each miRNA expression vector and two controls (empty vector (pll3.7-control) and rno-miR-344 (pll3.7-miR-344), a microRNA that does not have a homologue in humans) were co-transfected with an MDM4 CDS reporter vector, and then a dual-luciferase assay was used to determine the decline in the relative luciferase activity resulting from transfection with let-7 family members compared with the control (Fig. 1C). As let-7c and let-7a lead to the greatest inhibition, we analyzed these two microRNAs further. To validate whether MDM4 is directly targeted by let-7 family members, we constructed five different mutations of the putative let-7-binding site in the MDM4 CDS reporter (MT1-MT5) (Fig. 2A). The results of the dual-luciferase assay showed that neither let-7a nor let-7c can suppress the expression of the mutated constructs as effectively as they suppress the wild type constructs (Fig. 2B and C). In addition, each let-7 family member was co-transfected with the MT5 CDS to further characterize the interaction. As expected, the introduction of silent mutations into predicted targets disrupted the ability of the miRNAs let-7a, let-7b, let-7c, let-7e and let-7g to repress the expression of the luciferase reporter, with the MT5 reporter expressing higher levels of luciferase than the wild type reporter (Fig. 2D). Taken together, these results indicate that these miRNAs could directly target the MDM4 CDS. To determine whether let-7 can disrupt endogenous MDM4 expression in glioma cells, let-7a and let-7c mimics were transfected into U87 cells. qRT-PCR results showed that the expression levels of let-7a and let-7c were approximately six to nine times higher compared to the control (Fig. 2E). A Western blot assay showed that both let-7a and let-7c consistently and substantially downregulated the expression level of the MDM4 protein without affecting its mRNA level in U87 cells (Fig. 2E), indicating that let-7a and let-7c mainly impair MDM4 expression through translational inhibition. Furthermore, similar experiments were performed using let-7a and let-7c antisense treatment, and decreases in let-7a and let-7c increased the MDM4 protein level without changing the mRNA level (Fig. 2F). 3.2. DNA damage decreased MDM4 expression, partially via let-7 The MDM4 protein is associated with the DNA damage response [20]. We hypothesized that the reduction of MDM4 after DNA damage was mediated by let-7 family members. A Western blot showed that the level of the MDM4 protein was significantly decreased following treatment with different doses of cisplatin (CDDP) for 24 h in U87 cells (Fig. S1), where phosphorylated
Fig. 1. Let-7 miRNA family members can target the MDM4 CDS. (A) The predicted target sequence of let-7 family members within the human MDM4 CDS. (B) Alignment of human and other species MDM4 DNA binding sites to let-7 family members. (C) The effect of different let-7 family members on MDM4 CDS reporter constructs in HEK293T cells was tested by a luciferase reporter assay. The data were normalized to the ratio of Firefly and Renilla luciferase activities measured at 48 h post-transfection. Rno-miR-344 (pll3.7-miR-344) is an miRNA that does not have homologue in humans. The results were presented as relative luciferase activity, with the control assigned a value of 1. Values represent the mean ± S.D. from three independent transfection experiments. Significant differences from the control value are indicated by ⁄, P < 0.05, ⁄⁄, P < 0.01.
H2AX (p-H2AX) was used as a marker of DNA damage. In addition, qRT-PCR was used to determine the expression level of let-7. The miRNA expression level of most let-7 family members increased significantly after U87 cells were treated with 2.5 lM CDDP (Fig. 3A). Interestingly, we found that the changes in the let-7 and MDM4 protein levels were inversely correlated (Fig. 3A and B). To confirm these results, A172 glioma cells were used in the same experiment. The results showed that the expression levels of let-7a and let-7c were increased, whereas the level of MDM4 protein was decreased when the A172 glioma cells were
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
1961
Fig. 2. The let-7 miRNA family members directly correlate with MDM4. (A) Sequences of MDM4 CDS (WT) and mutations (MT1-MT5). Several nucleotides within the seed region as well as other predicted binding regions were mutated in the MDM4 sequence. (B and C) Empty vector (pll3.7-control) or miRNA expression vector (pll3.7-let-7a or pll3.7-let-7c) were co-transfected with MDM4 or mutants MT1-MT5. Luciferase activity was measured at 48 h post-transfection. Each data point was measured in triplicate. (D) Let-7 family members were co-transfected with MDM4 CDS mutant expression vectors (MT5) or wild type MDM4 CDS, respectively. (E and F) miRNA (let-7a or let-7c) mimics or antisense RNAs were transfected into U87 cells. Relative expression levels of let-7a, let-7c and MDM4 were determined by qRT-PCR 48 h after transfection. Western blot was used to analyze the MDM4 protein level. b-actin served as the internal control. Values represent the means ± S.D. Significant differences from the control value are indicated by ⁄, P < 0.05, ⁄⁄, P < 0.01.
treated with 10 lM CDDP for 24 h, which is consistent with the results for U87 glioma cells (Fig. 3C). To determine whether let-7 repression of MDM4 could also mediate DNA damage induced by other reagents, bleomycin and hydroxyurea were used to induce DNA damage instead of CDDP. The results in U87 and A172 glioma cells showed that the
expression levels of let-7a and let-7c were increased after treatment with bleomycin or hydroxyurea, and MDM4 protein level was reduced (Fig. 4A–D). To better understand the role of let-7 in the repression of MDM4 in response to DNA damage, we transfected let-7a and let-7c antisense RNAs to inhibit these miRNAs in U87 and A172
1962
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
3.3. A mutation found in glioma may disrupt the relationship between let-7 and MDM4 The Cancer Genome Atlas Research Network has shown that the Thr160Ser missense mutation of MDM4 is found in human glioma [27]. As Thr and Ser have a similar structure and this missense mutation exists in the seed region of the let-7-MDM4 target site, we proposed that this mutation might contribute to the disruption of the let-7 repression of MDM4. To test this hypothesis, we disrupted the target site by mutating 160Thr (ACC) to 160Ser with the AGC (observed in a glioma patient), TCG, AGT or TCC codons (Fig. 5A). As all these mutations code for Ser, the latter three mutations were used to test whether a change in one or two bases could disrupt the let-7 repression of MDM4. Then, the let-7 expression vector was co-transected with the MDM4 CDS reporter construct and a dual-luciferase assay was followed. The results showed that the repressive effect of let-7 family members on luciferase activity was inhibited by a single C to G transition, the mutation observed in a glioma patient (Fig. 5B). The repressive effect of let-7a and let-7c on luciferase activity was also inhibited by all other missense mutations TCG (S1), AGT (S2) and TCC (S3) (Fig. 5C and D). These results suggest that these mutations may disrupt the let-7 repression of MDM4. 4. Discussion
Fig. 3. Cisplatin treatment results in a change in let-7 and MDM4 expression. (A) Expression level of let-7 family members was measured by qRT-PCR after cisplatin (CDDP) treatment. miR-34a served as a positive control, and miR-125b and miR135b served as negative controls. (B) Western blot analysis of the MDM4 protein level after CDDP treatment. p-H2AX acted as a DNA damage marker. b-actin served as the internal control. (C) A172 cells were treated with 10 lM CDDP for 24 h, and then the MDM4 protein level was measured by Western blot and the relative expression levels of let-7a and let-7c were determined by qRT-PCR. Natural saline (NS) served as a negative control.
cells treated with CDDP. A Western blot assay showed that the relative MDM4 protein level increased (Fig. 4E and F), indicating that decreasing let-7a or let-7c can eliminate the effect of CDDP treatment on the MDM4 protein. Furthermore, inhibition of proteasome degradation by pretreatment of U87 cells with 5 lM of MG132 does not completely block the reduction in the level of the MDM4 protein following CDDP treatment (Fig. 4G). Taken together, these results suggest that rather than proteasome degradation, let-7 family members may play a key role in mediating the decrease in the level of the MDM4 protein after DNA damage.
Cells overexpressing let-7 have been reported to be hypersensitive to DNA damage [28]. Several studies have shown that let-7 family members are upregulated after DNA damage [12–14,29], although in some cells the let-7 family members are downregulated by DNA damage [16]. In this study, we found that the let-7 expression level in glioma cells was upregulated after DNA damage, which, in turn, suppressed MDM4 expression. Although the alterations in the let-7 expression level after DNA damage have been widely observed, the mechanisms of this progress remain unclear. To explain this phenomenon, Chaudhry et al. proposed that the increase in let-7 after DNA damage may be mediated by a DNA-dependent serine/threonine protein kinase in glioma cell lines [12]. Lin28 is a well-known gene that can inhibit the maturity of let-7 [30]. Recently, Lee et al. showed that DNA damage increased let-7 levels through downregulation of Lin28A [15]. KSRP was also reported to interact with Drosha and Dicer to promote the maturity of let-7a [31]. KSRP accumulates in the nucleus when Hela cells are cultured with cisplatin [32], which may suggest that KSRP mediates the change in let-7 expression. Further study should focus on the role of KSRP and Lin28A in the process of let-7 maturation in glioma cells. Approximately 40% of gliomas exhibit a p53 mutation or deletion, and approximately 80% of gliomas have a p53 pathway defect [33]. In response to DNA damage, MDM4 expression is downregulated and contributes to p53 activation [20]. Post-translational modifications are considered to play an important role in MDM4 regulation after DNA damage. For example, MDM4 is first phosphorylated by ATM and Chk2 and then degraded by MDM2 and the proteasome after DNA damage [20]. However, even after suppressing MDM4 degradation by adding MDM2 siRNA or the proteasome inhibitor MG132, the amount of endogenous MDM4 remains largely reduced after DNA damage [21]. We also demonstrated that blockade of proteasome-dependent degradation with MG132 in U87 and A172 glioma cells cannot suppress the decrease in the endogenous MDM4 protein level after cisplatin treatment (Fig. 4G). All of these points suggest that there could be exist another mechanism besides MDM2-mediated proteasome-dependent degradation of MDM4 after DNA damage. In this study, we show that several members of let-7 family including let-7a, -7c, -7e, -7g and -7i can repress MDM4 at the protein level. Moreover, knockdown of let-7a and
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
1963
Fig. 4. DNA damage could interfere with MDM4 expression through let-7. The MDM4 protein level and the relative expression levels of let-7a and let-7c were determined by Western blot and qRT-PCR, respectively, after treatment with 100 lM bleomycin or 1 mM hydroxyurea for 24 h in U87 cells (A and B) and A172 cells (C and D). PBS served as the negative control. (E and F) Western blot was used to compare MDM4 protein levels among cells transfected with antisense let-7a, antisense let-7c or a negative control with or without CDDP in U87 and A172 cells. (G) MG132 was used to block proteasome-mediated MDM4 degradation. Whole cell extracts from the indicated treatment conditions were subjected to Western blot analysis for MDM4 and b-actin. b-actin served as the internal control.
let-7c with antisense RNAs can increase the MDM4 protein level (Fig. 2F). This result can be explained as follows: (1) let-7a and let-7b are the most abundant species in glioma cells [34], knockdown of let-7a could reduce the total let-7 level significantly; (2) there is only one nucleotide difference between let-7c and let-7a or let-7b (Fig. 1A), antisense let-7c may exert an effect through let-7a and let-7b. We also found that the MDM4 protein level became more sensitive to antisense let-7a and let-7c after U87 cells were treated with MG132 alone or in combination with CDDP than the same cells without MG132 treatment (Fig. 4G). This may be because investigating the proteasome degradation inhibited by MG132 made the role of let-7 in MDM4 regulation more clear. These genetic traits are always linked to tumorigenesis, tumor progression and prognosis and chemotherapy. For example, a let-7 binding site polymorphism in the KRAS 30 UTR is associated with reduced survival in oral cancers and metastatic colorectal cancers
[35,36]. An SNP in the miR-191 target site within the 30 UTR of MDM4 also affects ovarian cancer progression and chemosensitivity [24]. Consequently, more studies should be carried out to understand the function of mutations or SNPs at miRNA target sites. In our studies, we demonstrated that a single base mutation identified in a glioma patient can disrupt the interaction between let-7 and MDM4 mRNA. Coincidentally, asynonymous somatic mutation (COSM1646071 (C/A), Thr(ACC)160Thr(ACA)) also located in this seed region of the let-7 binding site, was identified in both human lung cancer and large intestine cancer samples in the COSMIC project (release 71). These results suggest that this binding site is important for tumor formation or progression. In summary, this study reports MDM4 as a new target of let-7,which can bind to the MDM4 CDS and then downregulate the level of the MDM4 protein. The let-7 expression level was upregulated after DNA damage induced by cisplatin, bleomycin or
1964
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965
Fig. 5. A mutation found in glioma, Thr160Ser, may disrupt the relationship between let-7 and MDM4. (A) Sequences of MDM4 and its missense mutations. One or two nucleotides underlined within the seed region of the MDM4 sequence were mutated (MT-G, S1, S2 and S3), where MT-G was found in a tumor sample. (B) let-7 family members were co-transfected with wild type MDM4 or the MT-G mutant. Luciferase activity was measured at 48 h post-transfection. (C and D) Empty vector (pll3.7-control) or miRNA expression vector (pll3.7-let-7a or pll3.7-let-7c) were co-transfected with MDM4 or mutants S1, S2 or S3. Luciferase activity was measured at 48 h posttransfection. Each data point was measured in triplicate. Values represent the means ± S.D. Significant differences from the control value are indicated by ⁄, P < 0.05, ⁄⁄, P < 0.01.
hydroxyurea in U87 and A172 glioma cell lines. Further, we found that the reduction of MDM4 after DNA damage can be recovered by inhibiting the increase in let-7. In addition, a mutation found in glioma can damage the interaction between let-7 and MDM4. Furthermore, additional abnormal targets of let-7 in glioma must be identified to determine the contributions of let-7 to glioma tumorigenesis and tumor progress. This result suggests that let-7 directly and functionally targets MDM4, which may relate to the activation of p53 to maintain genome stability. Therefore, let-7-based therapy may be beneficial for the clinical treatment of cancers, including gliomas. Funding This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81272773 and 81101960), the Natural Science Foundation of Guangdong Province (S2013010012170), the Scientific and Technological Planning of Guangzhou (Grant No. 2012J4100082) and the Shenzhen Municipal Government of China (Grant No. LXRY20121106142947958). Conflict of interest statement None declared. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2015.05. 030.
References [1] Tay, Y., Zhang, J.Q., Thomson, A.M., Lim, B. and Rigoutsos, I. (2008) MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature 455. 1124-U12. [2] Lal, A. et al. (2009) MiR-24-mediated downregulation of H2AX suppresses DNA repair in terminally differentiated blood cells. Nat. Struct. Mol. Biol. 16, 492–498. [3] Wang, Y.M. et al. (2011) MicroRNA-138 modulates DNA damage response by repressing histone H2AX expression. Mol. Cancer Res. 9, 1100–1111. [4] Moskwa, P. et al. (2011) MiR-182-mediated downregulation of BRCA1 impacts DNA repair and sensitivity to PARP inhibitors. Mol. Cell 41, 210–220. [5] Wang, S., Tang, Y., Cui, H., Zhao, X., Luo, X., Pan, W., Huang, X. and Shen, N. (2011) Let-7/miR-98 regulate Fas and Fas-mediated apoptosis. Genes Immun. 12, 149–154. [6] Kong, D.J. et al. (2012) Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the acquisition of cancer stem cell signatures that are attenuated by BR-DIM. PLoS One 7. [7] Shell, S. et al. (2007) Let-7 expression defines two differentiation stages of cancer. Proc. Natl. Acad. Sci. USA 104, 11400–11405. [8] Johnson, S.M. et al. (2005) RAS is regulated by the let-7 MicroRNA family. Cell 120, 635–647. [9] Saavedra, H.I., Knauf, J.A., Shirokawa, J.M., Wang, J.W., Bin, O.Y., Elisei, R., Stambrook, P.J. and Fagin, J.A. (2000) The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway. Oncogene 19, 3948– 3954. [10] Lu, L., Schwartz, P., Scarampi, L., Rutherford, T., Canuto, E.M., Yu, H. and Katsaros, D. (2011) MicroRNA let-7a: a potential marker for selection of paclitaxel in ovarian cancer management. Gynecol. Oncol. 122, 366–371. [11] Weidhaas, J.B., Babar, I., Nallur, S.M., Trang, P., Roush, S., Boehm, M., Gillespie, E. and Slack, F.J. (2007) MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy. Cancer Res. 67, 11111–11116. [12] Chaudhry, M.A., Sachdeva, H. and Omaruddin, R.A. (2010) Radiation-induced micro-RNA modulation in glioblastoma cells differing in DNA-repair pathways. DNA Cell Biol. 29, 553–561. [13] Chaudhry, M.A. (2009) Real-time PCR analysis of micro-RNA expression in ionizing radiation-treated cells. Cancer Biother. Radiopharm. 24, 49–56.
C. Xie et al. / FEBS Letters 589 (2015) 1958–1965 [14] Nikiforova, M.N., Gandhi, M., Kelly, L. and Nikiforov, Y.E. (2011) MicroRNA dysregulation in human thyroid cells following exposure to ionizing radiation. Thyroid 21, 261–266. [15] Lee, J.Y. et al. (2013) Tumor suppressor p53 plays a key role in induction of both tristetraprolin and let-7 in human cancer cells. Nucleic Acids Res. 41, 5614–5625. [16] Saleh, A.D. et al. (2011) Cellular stress induced alterations in microRNA let-7a and let-7b expression are dependent on p53. PLoS One 6, e24429. [17] Shvarts, A. et al. (1997) Isolation and identification of the human homolog of a new p53-binding protein, Mdmx. Genomics 43, 34–42. [18] Danovi, D. et al. (2004) Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity. Mol. Cell. Biol. 24, 5835–5843. [19] Matijasevic, Z., Krzywicka-Racka, A., Sluder, G. and Jones, S.N. (2008) MdmX regulates transformation and chromosomal stability in p53-deficient cells. Cell Cycle 7, 2967–2973. [20] Chen, L., Gilkes, D.M., Pan, Y., Lane, W.S. and Chen, J. (2005) ATM and Chk2dependent phosphorylation of MDMX contribute to p53 activation after DNA damage. EMBO J. 24, 3411–3422. [21] Markey, M. and Berberich, S.J. (2008) Full-length hdmX transcripts decrease following genotoxic stress. Oncogene 27, 6657–6666. [22] Gatt, M.E. et al. (2010) MicroRNAs 15a/16-1 function as tumor suppressor genes in multiple myeloma. Blood. [23] Ovcharenko, D. et al. (2011) MiR-10a overexpression is associated with NPM1 mutations and MDM4 downregulation in intermediate-risk acute myeloid leukemia. Exp. Hematol. 39, 1030–1042. [24] Wynendaele, J. et al. (2010) An illegitimate microRNA target site within the 30 UTR of MDM4 affects ovarian cancer progression and chemosensitivity. Cancer Res. 70, 9641–9649. [25] Lee, S.T. et al. (2011) Let-7 microRNA inhibits the proliferation of human glioblastoma cells. J. Neurooncol. 102, 19–24.
1965
[26] Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. [27] Chin, L. et al. (2008) Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455, 1061–1068. [28] Metheetrairut, C. and Slack, F.J. (2013) MicroRNAs in the ionizing radiation response and in radiotherapy. Curr. Opin. Genet. Dev. 23, 12–19. [29] Wagner-Ecker, M., Schwager, C., Wirkner, U., Abdollahi, A. and Huber, P.E. (2010) MicroRNA expression after ionizing radiation in human endothelial cells. Radiat. Oncol. 5, 25. [30] Jeong, S.H., Wu, H.G. and Park, W.Y. (2009) LIN28B confers radio-resistance through the posttranscriptional control of KRAS. Exp. Mol. Med. 41, 912–918. [31] Trabucchi, M., Briata, P., Garcia-Mayoral, M., Haase, A.D., Filipowicz, W., Ramos, A., Gherzi, R. and Rosenfeld, M.G. (2009) The RNA-binding protein KSRP promotes the biogenesis of a subset of microRNAs. Nature 459. 1010U144. [32] Drayton, R.M. (2012) The role of microRNA in the response to cisplatin treatment. Biochem. Soc. Trans. 40, 821–825. [33] Dunn, G.P. et al. (2012) Emerging insights into the molecular and cellular basis of glioblastoma. Genes Dev. 26, 756–784. [34] Landgraf, P. et al. (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414. [35] Christensen, B.C., Moyer, B.J., Avissar, M., Ouellet, L.G., Plaza, S.L., McClean, M.D., Marsit, C.J. and Kelsey, K.T. (2009) A let-7 microRNA-binding site polymorphism in the KRAS 30 UTR is associated with reduced survival in oral cancers. Carcinogenesis 30, 1003–1007. [36] Zhang, W. et al. (2011) A let-7 microRNA-binding site polymorphism in 30 untranslated region of KRAS gene predicts response in wild-type KRAS patients with metastatic colorectal cancer treated with cetuximab monotherapy. Ann. Oncol. 22, 104–109.
