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MicroRNA-1285 inhibits the expression of p53 by directly targeting its 3′ untranslated region
Biochemical and Biophysical Research Communications 396 (2010) 435–439
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
MicroRNA-1285 inhibits the expression of p53 by directly targeting its 30 untranslated region Shu Tian a,b,1, Shenglin Huang b,1, Shunquan Wu b,c, Weijian Guo a, Jin Li a,*, Xianghuo He a,b,** a
Department of Medical Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, PR China c Department of Hematology, Fujian Medical University Union Hospital, Fujian Institute of Hematology, Fuzhou, PR China b
a r t i c l e
i n f o
Article history: Received 11 April 2010 Available online 24 April 2010 Keywords: p53 microRNA miR-1285 30 Untranslated region
a b s t r a c t The well-known tumor suppressor p53 plays critical roles in the modulation of multiple cellular processes. The regulation of p53 is complicated and remains elusive. In this study, we used a high-throughput luciferase reporter screen to demonstrate that p53 can be regulated by microRNA-1285 (miR-1285). Notably, miR-612, which has the same seed sequence as miR-1285, cannot bind to the 30 untranslated region (30 UTR) of p53. Mutational analyses confirmed that the 30 UTR of p53 mRNA contains two miR1285 target sites, which are nearly perfectly complementary to the mature miR-1285 sequence. Ectopic expression of miR-1285 inhibits expression of p53 mRNA and protein. In contrast, silencing of miR-1285 increases p53 expression. Furthermore, miR-1285 inhibits the transcription of p21, a master gene downstream of p53. In conclusion, our findings provide the first evidence that miR-1285 directly regulates the expression of p53 by directly targeting its 30 UTR. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The p53 protein was first described in 1979 and is one of the most studied cellular proteins of the last three decades [1]. In humans, p53 is encoded by the TP53 gene, which contains 11 exons and is located on the short arm of chromosome 17 (17p13.1). The p53 protein, regarded as the ‘‘guardian of the genome”, plays a central role in the regulation of multiple cellular processes and prevents tumorigenesis [1]. The literature indicates that p53 functions as a transcription factor by binding specific DNA sequences and activating or repressing numerous target genes, including not only protein-coding genes but also miRNA-coding genes [2–6]. Although p53 mRNA is constitutively expressed, p53 activity is tightly regulated by protein–protein interactions, post-translational modifications and subcellular localization [7]. The post-translational modifications of p53 include phosphorylation, acetylation and sumoylation [8]. However, the detailed mechanisms regulating p53 expression are still unclear. MicroRNAs (miRNAs) are a class of endogenously expressed small non-coding RNAs that regulate gene expression post-transcriptionally [9]. miRNAs are initially transcribed as primary precursor molecules, then undergo a series of cleavage events to become a
* Corresponding author. Fax: +86 21 64170366. ** Corresponding author at: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, PR China. Fax: +86 21 64436539. E-mail addresses: [email protected] (J. Li), [email protected] (X. He). 1 These authors contributed equally to this study. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.112
mature 22 nt miRNA. miRNAs bind to complementary sequences in the 30 UTRs of target mRNAs and inhibit protein synthesis by repressing translation or promoting mRNA degradation [9,10]. Computational analyses suggest that 30% of protein-coding genes may be directly regulated by multiple miRNAs [11]. miRNAs are involved in diverse physiological processes, including cell differentiation, proliferation and apoptosis [12]. Numerous studies have demonstrated that miRNAs can function as oncogenes or tumor suppressors in the multistep processes of carcinogenesis [13]. In this study, we investigated whether p53 could be regulated by miRNAs. We used a miRNA target-prediction tool to identify miRNAs that might target p53. A high-throughput luciferase assay was used to identify miRNAs that target the 30 UTR of p53. We found that miR-1285 can downregulate the expression of p53 by directly binding its 30 UTR. Further, mutational analyses confirmed that the 30 UTR of p53 mRNA contains two miR-1285 target sites, which are nearly perfectly complementary to the mature miR1285 sequence. Moreover, miR-1285 inhibits the transcription of p21, a master gene downstream of p53.
2. Materials and methods 2.1. Cell culture Human HEK 293T cells, human neuroblastoma SH-SY5Y cells, human hepatoblastoma HepG2 cells and human breast cancer MCF-7 cells were maintained in Dulbecco’s Modified Eagle’s Medium
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S. Tian et al. / Biochemical and Biophysical Research Communications 396 (2010) 435–439
(DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37 °C with 5% carbon dioxide. 2.2. Luciferase reporter constructs with wild-type or mutant p53 30 UTR We used PCR to amplify the p53 30 UTR from human genomic DNA. The PCR product was cloned downstream of a cytomegalovirus (CMV) promoter-driven firefly luciferase cassette in the pCDNA3.0 vector. To construct the luciferase reporters with mutated versions of the p53 30 UTR, we first used two primer sets to PCR amplify the p53 30 UTR as two fragments with overlapping mutation sites. These two fragments were then annealed and used as templates to amplify a mutant p53 30 UTR. We then processed the resulting amplicons as described above to generate luciferase reporters with mutant p53 30 UTRs. The primer sequences are shown in Supplementary Table S2.
probed with the following primary antibodies: mouse anti-p53 monoclonal (Thermo Fisher scientific Inc., Pittsburgh, PA, USA), mouse anti-p21 monoclonal (Cell Signaling Technology, Massachusetts, USA) or mouse anti-beta-actin monoclonal (Sigma). Blots were then probed with a horseradish peroxidase (HRP) conjugated goat anti-mouse secondary antibody (Pierce, Rockford, IL, USA). Proteins were detected using ECL reagents (Pierce). 2.7. Statistical analysis Data are presented as mean ± SEM. Differences between groups were calculated using the Student’s t-test. p Values less than 0.05 were defined as statistically significant. 3. Results 3.1. Screening for miRNAs that directly target the 30 untranslated region of p53
2.3. Dual-luciferase reporter assays For the miRNA screen, HEK 293T cells were seeded in 96-well plates at a density of 5000 cells per well. After 24 h, the cells were transiently transfected with 5 ng of pRL-CMV (Renilla luciferase reporter), 50 ng of either p-LUC or p-LUC-p53UTR (firefly luciferase reporter), and 5 pmol of miRNA mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Firefly and Renilla luciferase activities were measured 36 h after transfection using a dual-luciferase reporter assay kit (Promega, Madison, WI, USA). Firefly luciferase activity was normalized using Renilla luciferase activity. For p21 promoter assays, the WWP-Luc plasmid (kindly provided by Dr. B Vogelstein, John Hopkins University), the pRL-CMV Renilla luciferase reporter and small RNAs were simultaneously introduced into SH-SY5Y cells. The cells were harvested 36 h after transfection and luciferase activity was assayed as above. 2.4. Oligonucleotide transfection All miRNA mimics and p53 siRNAs were synthesized by Genepharma, Shanghai, China. miR-1285 inhibitors were synthesized by RiboBio, Guangzhou, China. The sequences of p53 siRNAs are as follows: sense, 50 -CUACUUCCUGAAAACAACGdTdT-30 ; anti-sense, 50 -CGUUGUUUUCAGGAAGUAGdTdT-30 . Transfections were performed using Lipofectamine 2000 according to the manufacturer’s instructions. The final concentration of small RNAs was 100 nM. 2.5. RNA extraction and quantitative real-time PCR Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. We used the PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan) with random primers to synthesize cDNA from 500 ng of RNA. For mature miRNAs, we used the Taqman MicroRNA RT kit (Applied Biosystems, CA, USA) to synthesize cDNA from 20 ng of RNA. We then conducted SYBR Green-based Bulge-loop quantitative real-time PCR (Ribobio). Quantitative real-time PCR was performed with the 7300 real-time PCR system (Applied Biosystems). The relative expression of mRNAs and miRNAs were calculated using the comparative 2DDC t method and were normalized using beta-actin mRNA and RNU6B mature miRNA, respectively. The primer sequences are shown in Supplementary Table S2. 2.6. Western blotting Proteins were separated on 12% SDS–PAGE gels and blotted on nitrocellulose membranes (Bio-Rad, Hercules, USA). Non-specific binding was blocked with 5% non-fat milk in PBS. Blots were
To identify miRNAs that regulate the expression of p53 through its 30 UTR, we constructed a vector containing a cytomegalovirus (CMV) promoter-driven firefly luciferase cassette upstream of the p53 30 UTR (p-LUC-p53UTR), and a control vector containing only the luciferase cassette (p-LUC). TargetScan, a miRNA target-prediction tool, was used to identify potential p53-targeting miRNAs. A pool of 107 potential p53-targeting miRNAs was selected for further analysis (Supplementary Table S1). miRNA mimics were individually transiently co-transfected with the Renilla luciferase reporter (the internal control) and either p-LUC-p53UTR or p-LUC into HEK 293T cells. Luciferase activities were assayed 36 h after transfection using a dual luciferase assay (Fig. 1A and Supplementary Table S1). miR-1285 was found to have the most robust inhibitory effect, causing a 40% reduction in normalized luciferase activity compared to the control (Fig. 1B and C). Notably, miR612, which has the same seed sequence as miR-1285, did not inhibit luciferase activity of the p-LUC-p53UTR reporter (Fig. 1C). 3.2. miR-1285 regulates p53 by binding its 30 UTR According to the TargetScan prediction, the p53 mRNA 30 UTR contains two sequence motifs, which are nearly perfectly complementary to the miR-1285 sequence (Fig. 2A). To determine if p53 is regulated by miR-1285 binding these putative target sites, we constructed full-length fragments of the p53 mRNA 30 UTR (wild-type and miR-1285 binding site mutants) and inserted them immediately downstream of the luciferase reporter gene (Fig. 2B). The miR-1285 mimic or control RNA was co-transfected with different luciferase-30 UTR constructs into HEK 293T cells. The results showed that luciferase activity with p53 30 UTR mutant constructs decreased less than with the wild-type construct, especially in mutants of the first target site (Fig. 2C). Furthermore, the inhibitory effect of miR-1285 on luciferase activity was abrogated when a p53 30 UTR containing the two mutant binding sites was used (Fig. 2C). These findings suggest that miR-1285 can regulate p53 expression by directly binding two target sites in the 30 UTR. 3.3. miR-1285 decreased the endogenous p53 mRNA and protein expression To determine if endogenous p53 can be modulated by miR1285, we introduced miR-1285 mimics or control RNA into several p53 wild-type tumor cell lines, including MCF-7, SH-SY5Y and HepG2. Next, we determined the levels of p53 mRNA and protein. As shown in Fig. 3A, p53 mRNA levels were reduced in miR-1285 transfected cells compared to negative controls. In accordance with the changes of mRNA level, the p53 protein level was also substan-
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Fig. 1. Screen for candidate miRNAs that target the p53 30 UTR. (A) Schematic diagram of the screening strategy. HEK 293T cells were seeded in 96-well plates and after 24 h were transiently transfected with individual miRNA mimics and the Renilla luciferase reporter (p-LUC or p-LUC-p53UTR). The dual-luciferase reporter assay was performed 36 h after transfection. Raw data were normalized using the ratio of firefly to Renilla luciferase activity. (B) Results of the luciferase screen. Vertical axis: ratio of luciferase activity in cells transfected with p-LUC; horizontal axis: ratio of luciferase activity in cells transfected with p-LUC-p53UTR. (C) Luciferase activity assays of miR-1285 and miR-612. Results are representatives of three independent experiments. Statistical analysis was performed using the Student’s t-test. Error bars represent the SEM. indicates p < 0.01.
Fig. 2. miR-1285 regulates p53 by binding to its 30 UTR. (A) Location of putative target sites for miR-1285 in the p53 30 UTR. TargetScan software was used to predict target sites. Sequence inspection indicated that the p53 30 UTR contains two elements nearly perfectly complementary to miR-1285 sequence. (B) Schematic diagram of the p53 30 UTR luciferase reporters with one or two target sites mutated. Constructs were generated using overlap-extension PCR. (C) Luciferase activity assays of wild-type and mutant p53 30 UTR luciferase reporters after co-transfection with miR-1285. Normalized luciferase activity was presented as the mean ± SEM of triplicate experiments.
tially reduced when miR-1285 was expressed in these cells (Fig. 3B). We used p53 specific siRNA (si-p53) as a positive control.
Furthermore, miR-1285 anti-sense oligonucleotides were synthesized and transfected into SH-SY5Y cells, which display relatively
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Fig. 3. miR-1285 inhibits expression of endogenous p53 mRNA and protein. (A) Quantitative real-time PCR analysis of p53 after transfection with miR-1285 mimics or siRNA against p53 in tumor cell lines MCF-7, SH-SY5Y and HepG2. (B) Western blot analysis of p53 expression after transfection with miR-1285 mimics or siRNA against p53 in tumor cell lines MCF-7, SH-SY5Y and HepG2. Beta-actin was used as a loading control. (C) Quantitative real-time PCR analysis of p53 mRNA expression in SH-SY5Y cells transfected with miR-1285 inhibitors. (D) Western blot analysis of p53 expression in SH-SY5Y cells transfected with miR-1285 inhibitors. Beta-actin was used as a loading control. (A, C) Beta-actin was used for normalization. The relative mRNA levels were presented as fold differences based on calculations of 2DDC t . Results are representative of three independent experiments. Statistical analysis was performed using the Student’s t-test. Error bars represent the SEM. indicates p < 0.05. indicates p < 0.01.
high expression of miR-1285 (Supplementary Fig. S1). As shown in Fig. 3C and D, miR-1285 silencing led to increased expression levels of p53 mRNA and protein in SH-SY5Y cells. Taken together, these results indicate that miR-1285 can reduce the expression level of p53. 3.4. miR-1285 suppresses the expression of p21, a master gene downstream of p53 To further evaluate the potential function of miR-1285, we determined the effect of miR-1285 on the expression of p21, one of the most important genes downstream of p53. p53 primarily activates p21 expression at transcriptional level by binding to the p21 promoter. As such, we used a p21 promoter based luciferase reporter WWP-Luc [14], which contains p53 binding sites, to assay the effect of miR-1285. As shown in Fig. 4A, the luciferase activity of WWP-Luc in SH-SY5Y cells was significantly decreased when cotransfected with miR-1285 or siRNA against p53. This result indicates that miR-1285 could inhibit the activity of the p21 promoter, thus suppressing the transcription of p21. Consistent with this, in SH-SY5Y cells both p21 mRNA and protein levels were suppressed by miR-1285 or siRNA against p53 (Fig. 4B and C). 4. Discussion In the present study, we performed a luciferase reporter screen to determine if p53 could be targeted by miRNAs. We selected a pool of
107 miRNAs that could potentially target p53 according to the miRNA target-prediction tool TargetScan, which is reported to have the best performance [15,16]. We found that, in our assay, only miR1285 could target the p53 30 UTR. In comparison, our recent study showed that 28 of 266 miRNAs examined could directly target the p21Cip1/Waf1 gene [17]. This discordance might result from the different sequences or secondary structures and suggests that different genes might be regulated by drastically different numbers of miRNAs. It is noteworthy that although p53 was recently reported to be a target of miR-125b [18], there was no significant effect of this miRNA on the p53 30 UTR in our assay (Supplementary Table S1). Notably, miR-612, which has the same seed region sequence as miR-1285, did not significantly alter expression of the p53 30 UTR reporter. It has been reported that perfect binding of nucleotides 2–7, the miRNA seed sequence, to the target mRNA plays a key role in target recognition [11,19]. Our mutational analyses identified two miR-1285 target sites in the 30 UTR of the p53 mRNA. In particular, these two sites are nearly perfectly complementary to the miR-1285 sequence. Therefore, we propose that the effect of miR-1285 on p53 expression might result from the near perfect complementarity, which leads to degradation of the p53 mRNA. miR-1285 was recently discovered from massively parallel sequencing of human embryonic stem cells [20]. However, there has been little reported about miR-1285 until now. miR-1285 expression profiles, available from the miRZ database [21], indicate that it is preferentially expressed in SHS5Y neuroblastoma cells and HepG2 liver cancer cells (data not shown), suggesting that
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Fig. 4. miR-1285 suppresses expression of p21, a master gene downstream of p53. (A) Luciferase activity assays of SH-SY5Y cells after transfection with the p21 promoter reporter (WWP-Luc) and either miR-1285 mimics, siRNA against p53 or control RNA. (B) Quantitative real-time PCR analysis of p21 mRNA expression in SH-SY5Y cells transfected with miR-1285 mimics, siRNA against p53, or control RNA. Beta-actin was used for normalization. The relative mRNA levels were presented as fold differences based on calculations of 2DDC t . (C) Western blot analysis of p21 protein expression in SH-SY5Y cells transfected with miR-1285 mimics, siRNA against p53, or control RNA. p21 protein expression was normalized using beta-actin expression. (A, B) Results are representatives of three independent experiments. Statistical analysis was performed using the Student’s t-test. Error bars represent the SEM. indicates p < 0.05. indicates p < 0.01.
miR-1285 might function in oncogenesis of these two cancer types. The functional linkage is worthy of further exploration. In summary, our study provides the first evidence that miR-1285 directly regulates expression of p53 by binding its 30 UTR. Acknowledgments We thank Dr. B. Vogelstein for providing the WWP-Luc plasmid. This work was supported by grants from The Ministry of Human Resources and Social Security of China (2007-170); The Science & Technology Commission of Shanghai Municipality (07DJ14006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.04.112. References [1] A.J. Levine, M. Oren, The first 30 years of p53: growing ever more complex, Nat. Rev. Cancer 9 (2009) 749–758. [2] D. Menendez, A. Inga, M.A. Resnick, The expanding universe of p53 targets, Nat. Rev. Cancer 9 (2009) 724–737. [3] L. He, X. He, L.P. Lim, E. de Stanchina, Z. Xuan, Y. Liang, W. Xue, L. Zender, J. Magnus, D. Ridzon, A.L. Jackson, P.S. Linsley, C. Chen, S.W. Lowe, M.A. Cleary, G.J. Hannon, A microRNA component of the p53 tumour suppressor network, Nature 447 (2007) 1130–1134. [4] T.C. Chang, E.A. Wentzel, O.A. Kent, K. Ramachandran, M. Mullendore, K.H. Lee, G. Feldmann, M. Yamakuchi, M. Ferlito, C.J. Lowenstein, D.E. Arking, M.A. Beer, A. Maitra, J.T. Mendell, Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis, Mol. Cell 26 (2007) 745–752. [5] N. Raver-Shapira, E. Marciano, E. Meiri, Y. Spector, N. Rosenfeld, N. Moskovits, Z. Bentwich, M. Oren, Transcriptional activation of miR-34a contributes to p53mediated apoptosis, Mol. Cell 26 (2007) 731–743.
[6] H.I. Suzuki, K. Yamagata, K. Sugimoto, T. Iwamoto, S. Kato, K. Miyazono, Modulation of microRNA processing by p53, Nature 460 (2009) 529–533. [7] M.F. Lavin, N. Gueven, The complexity of p53 stabilization and activation, Cell Death Differ. 13 (2006) 941–950. [8] A.M. Bode, Z. Dong, Post-translational modification of p53 in tumorigenesis, Nat. Rev. Cancer 4 (2004) 793–805. [9] D.P. Bartel, MicroRNAs: genomics, biogenesis, mechanism, and function, Cell 116 (2004) 281–297. [10] V. Ambros, The functions of animal microRNAs, Nature 431 (2004) 350–355. [11] B.P. Lewis, C.B. Burge, D.P. Bartel, Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets, Cell 120 (2005) 15–20. [12] L. He, G.J. Hannon, MicroRNAs: small RNAs with a big role in gene regulation, Nat. Rev. Genet. 5 (2004) 522–531. [13] G.A. Calin, C.M. Croce, MicroRNA signatures in human cancers, Nat. Rev. Cancer 6 (2006) 857–866. [14] W.S. el-Deiry, T. Tokino, V.E. Velculescu, D.B. Levy, R. Parsons, J.M. Trent, D. Lin, W.E. Mercer, K.W. Kinzler, B. Vogelstein, WAF1, a potential mediator of p53 tumor suppression, Cell 75 (1993) 817–825. [15] D. Baek, J. Villen, C. Shin, F.D. Camargo, S.P. Gygi, D.P. Bartel, The impact of microRNAs on protein output, Nature 455 (2008) 64–71. [16] M. Selbach, B. Schwanhausser, N. Thierfelder, Z. Fang, R. Khanin, N. Rajewsky, Widespread changes in protein synthesis induced by microRNAs, Nature 455 (2008) 58–63. [17] S. Wu, S. Huang, J. Ding, Y. Zhao, L. Liang, T. Liu, R. Zhan, X. He, Multiple microRNAs modulate p21Cip1/Waf1 expression by directly targeting its 30 untranslated region, Oncogene 29 (2010) 2302–2308. [18] M.T. Le, C. Teh, N. Shyh-Chang, H. Xie, B. Zhou, V. Korzh, H.F. Lodish, B. Lim, MicroRNA-125b is a novel negative regulator of p53, Genes Dev. 23 (2009) 862–876. [19] J. Brennecke, A. Stark, R.B. Russell, S.M. Cohen, Principles of microRNA-target recognition, PLoS Biol. 3 (2005) e85. [20] R.D. Morin, M.D. O’Connor, M. Griffith, F. Kuchenbauer, A. Delaney, A.L. Prabhu, Y. Zhao, H. McDonald, T. Zeng, M. Hirst, C.J. Eaves, M.A. Marra, Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells, Genome Res. 18 (2008) 610–621. [21] J. Hausser, P. Berninger, C. Rodak, Y. Jantscher, S. Wirth, M. Zavolan, MirZ: an integrated microRNA expression atlas and target prediction resource, Nucleic Acids Res. 37 (2009) W266–W272.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
MicroRNA-1285 inhibits the expression of p53 by directly targeting its 30 untranslated region Shu Tian a,b,1, Shenglin Huang b,1, Shunquan Wu b,c, Weijian Guo a, Jin Li a,*, Xianghuo He a,b,** a
Department of Medical Oncology, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, PR China c Department of Hematology, Fujian Medical University Union Hospital, Fujian Institute of Hematology, Fuzhou, PR China b
a r t i c l e
i n f o
Article history: Received 11 April 2010 Available online 24 April 2010 Keywords: p53 microRNA miR-1285 30 Untranslated region
a b s t r a c t The well-known tumor suppressor p53 plays critical roles in the modulation of multiple cellular processes. The regulation of p53 is complicated and remains elusive. In this study, we used a high-throughput luciferase reporter screen to demonstrate that p53 can be regulated by microRNA-1285 (miR-1285). Notably, miR-612, which has the same seed sequence as miR-1285, cannot bind to the 30 untranslated region (30 UTR) of p53. Mutational analyses confirmed that the 30 UTR of p53 mRNA contains two miR1285 target sites, which are nearly perfectly complementary to the mature miR-1285 sequence. Ectopic expression of miR-1285 inhibits expression of p53 mRNA and protein. In contrast, silencing of miR-1285 increases p53 expression. Furthermore, miR-1285 inhibits the transcription of p21, a master gene downstream of p53. In conclusion, our findings provide the first evidence that miR-1285 directly regulates the expression of p53 by directly targeting its 30 UTR. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction The p53 protein was first described in 1979 and is one of the most studied cellular proteins of the last three decades [1]. In humans, p53 is encoded by the TP53 gene, which contains 11 exons and is located on the short arm of chromosome 17 (17p13.1). The p53 protein, regarded as the ‘‘guardian of the genome”, plays a central role in the regulation of multiple cellular processes and prevents tumorigenesis [1]. The literature indicates that p53 functions as a transcription factor by binding specific DNA sequences and activating or repressing numerous target genes, including not only protein-coding genes but also miRNA-coding genes [2–6]. Although p53 mRNA is constitutively expressed, p53 activity is tightly regulated by protein–protein interactions, post-translational modifications and subcellular localization [7]. The post-translational modifications of p53 include phosphorylation, acetylation and sumoylation [8]. However, the detailed mechanisms regulating p53 expression are still unclear. MicroRNAs (miRNAs) are a class of endogenously expressed small non-coding RNAs that regulate gene expression post-transcriptionally [9]. miRNAs are initially transcribed as primary precursor molecules, then undergo a series of cleavage events to become a
* Corresponding author. Fax: +86 21 64170366. ** Corresponding author at: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, PR China. Fax: +86 21 64436539. E-mail addresses: [email protected] (J. Li), [email protected] (X. He). 1 These authors contributed equally to this study. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.112
mature 22 nt miRNA. miRNAs bind to complementary sequences in the 30 UTRs of target mRNAs and inhibit protein synthesis by repressing translation or promoting mRNA degradation [9,10]. Computational analyses suggest that 30% of protein-coding genes may be directly regulated by multiple miRNAs [11]. miRNAs are involved in diverse physiological processes, including cell differentiation, proliferation and apoptosis [12]. Numerous studies have demonstrated that miRNAs can function as oncogenes or tumor suppressors in the multistep processes of carcinogenesis [13]. In this study, we investigated whether p53 could be regulated by miRNAs. We used a miRNA target-prediction tool to identify miRNAs that might target p53. A high-throughput luciferase assay was used to identify miRNAs that target the 30 UTR of p53. We found that miR-1285 can downregulate the expression of p53 by directly binding its 30 UTR. Further, mutational analyses confirmed that the 30 UTR of p53 mRNA contains two miR-1285 target sites, which are nearly perfectly complementary to the mature miR1285 sequence. Moreover, miR-1285 inhibits the transcription of p21, a master gene downstream of p53.
2. Materials and methods 2.1. Cell culture Human HEK 293T cells, human neuroblastoma SH-SY5Y cells, human hepatoblastoma HepG2 cells and human breast cancer MCF-7 cells were maintained in Dulbecco’s Modified Eagle’s Medium
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S. Tian et al. / Biochemical and Biophysical Research Communications 396 (2010) 435–439
(DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37 °C with 5% carbon dioxide. 2.2. Luciferase reporter constructs with wild-type or mutant p53 30 UTR We used PCR to amplify the p53 30 UTR from human genomic DNA. The PCR product was cloned downstream of a cytomegalovirus (CMV) promoter-driven firefly luciferase cassette in the pCDNA3.0 vector. To construct the luciferase reporters with mutated versions of the p53 30 UTR, we first used two primer sets to PCR amplify the p53 30 UTR as two fragments with overlapping mutation sites. These two fragments were then annealed and used as templates to amplify a mutant p53 30 UTR. We then processed the resulting amplicons as described above to generate luciferase reporters with mutant p53 30 UTRs. The primer sequences are shown in Supplementary Table S2.
probed with the following primary antibodies: mouse anti-p53 monoclonal (Thermo Fisher scientific Inc., Pittsburgh, PA, USA), mouse anti-p21 monoclonal (Cell Signaling Technology, Massachusetts, USA) or mouse anti-beta-actin monoclonal (Sigma). Blots were then probed with a horseradish peroxidase (HRP) conjugated goat anti-mouse secondary antibody (Pierce, Rockford, IL, USA). Proteins were detected using ECL reagents (Pierce). 2.7. Statistical analysis Data are presented as mean ± SEM. Differences between groups were calculated using the Student’s t-test. p Values less than 0.05 were defined as statistically significant. 3. Results 3.1. Screening for miRNAs that directly target the 30 untranslated region of p53
2.3. Dual-luciferase reporter assays For the miRNA screen, HEK 293T cells were seeded in 96-well plates at a density of 5000 cells per well. After 24 h, the cells were transiently transfected with 5 ng of pRL-CMV (Renilla luciferase reporter), 50 ng of either p-LUC or p-LUC-p53UTR (firefly luciferase reporter), and 5 pmol of miRNA mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Firefly and Renilla luciferase activities were measured 36 h after transfection using a dual-luciferase reporter assay kit (Promega, Madison, WI, USA). Firefly luciferase activity was normalized using Renilla luciferase activity. For p21 promoter assays, the WWP-Luc plasmid (kindly provided by Dr. B Vogelstein, John Hopkins University), the pRL-CMV Renilla luciferase reporter and small RNAs were simultaneously introduced into SH-SY5Y cells. The cells were harvested 36 h after transfection and luciferase activity was assayed as above. 2.4. Oligonucleotide transfection All miRNA mimics and p53 siRNAs were synthesized by Genepharma, Shanghai, China. miR-1285 inhibitors were synthesized by RiboBio, Guangzhou, China. The sequences of p53 siRNAs are as follows: sense, 50 -CUACUUCCUGAAAACAACGdTdT-30 ; anti-sense, 50 -CGUUGUUUUCAGGAAGUAGdTdT-30 . Transfections were performed using Lipofectamine 2000 according to the manufacturer’s instructions. The final concentration of small RNAs was 100 nM. 2.5. RNA extraction and quantitative real-time PCR Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. We used the PrimeScript RT reagent kit (TaKaRa, Tokyo, Japan) with random primers to synthesize cDNA from 500 ng of RNA. For mature miRNAs, we used the Taqman MicroRNA RT kit (Applied Biosystems, CA, USA) to synthesize cDNA from 20 ng of RNA. We then conducted SYBR Green-based Bulge-loop quantitative real-time PCR (Ribobio). Quantitative real-time PCR was performed with the 7300 real-time PCR system (Applied Biosystems). The relative expression of mRNAs and miRNAs were calculated using the comparative 2DDC t method and were normalized using beta-actin mRNA and RNU6B mature miRNA, respectively. The primer sequences are shown in Supplementary Table S2. 2.6. Western blotting Proteins were separated on 12% SDS–PAGE gels and blotted on nitrocellulose membranes (Bio-Rad, Hercules, USA). Non-specific binding was blocked with 5% non-fat milk in PBS. Blots were
To identify miRNAs that regulate the expression of p53 through its 30 UTR, we constructed a vector containing a cytomegalovirus (CMV) promoter-driven firefly luciferase cassette upstream of the p53 30 UTR (p-LUC-p53UTR), and a control vector containing only the luciferase cassette (p-LUC). TargetScan, a miRNA target-prediction tool, was used to identify potential p53-targeting miRNAs. A pool of 107 potential p53-targeting miRNAs was selected for further analysis (Supplementary Table S1). miRNA mimics were individually transiently co-transfected with the Renilla luciferase reporter (the internal control) and either p-LUC-p53UTR or p-LUC into HEK 293T cells. Luciferase activities were assayed 36 h after transfection using a dual luciferase assay (Fig. 1A and Supplementary Table S1). miR-1285 was found to have the most robust inhibitory effect, causing a 40% reduction in normalized luciferase activity compared to the control (Fig. 1B and C). Notably, miR612, which has the same seed sequence as miR-1285, did not inhibit luciferase activity of the p-LUC-p53UTR reporter (Fig. 1C). 3.2. miR-1285 regulates p53 by binding its 30 UTR According to the TargetScan prediction, the p53 mRNA 30 UTR contains two sequence motifs, which are nearly perfectly complementary to the miR-1285 sequence (Fig. 2A). To determine if p53 is regulated by miR-1285 binding these putative target sites, we constructed full-length fragments of the p53 mRNA 30 UTR (wild-type and miR-1285 binding site mutants) and inserted them immediately downstream of the luciferase reporter gene (Fig. 2B). The miR-1285 mimic or control RNA was co-transfected with different luciferase-30 UTR constructs into HEK 293T cells. The results showed that luciferase activity with p53 30 UTR mutant constructs decreased less than with the wild-type construct, especially in mutants of the first target site (Fig. 2C). Furthermore, the inhibitory effect of miR-1285 on luciferase activity was abrogated when a p53 30 UTR containing the two mutant binding sites was used (Fig. 2C). These findings suggest that miR-1285 can regulate p53 expression by directly binding two target sites in the 30 UTR. 3.3. miR-1285 decreased the endogenous p53 mRNA and protein expression To determine if endogenous p53 can be modulated by miR1285, we introduced miR-1285 mimics or control RNA into several p53 wild-type tumor cell lines, including MCF-7, SH-SY5Y and HepG2. Next, we determined the levels of p53 mRNA and protein. As shown in Fig. 3A, p53 mRNA levels were reduced in miR-1285 transfected cells compared to negative controls. In accordance with the changes of mRNA level, the p53 protein level was also substan-
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Fig. 1. Screen for candidate miRNAs that target the p53 30 UTR. (A) Schematic diagram of the screening strategy. HEK 293T cells were seeded in 96-well plates and after 24 h were transiently transfected with individual miRNA mimics and the Renilla luciferase reporter (p-LUC or p-LUC-p53UTR). The dual-luciferase reporter assay was performed 36 h after transfection. Raw data were normalized using the ratio of firefly to Renilla luciferase activity. (B) Results of the luciferase screen. Vertical axis: ratio of luciferase activity in cells transfected with p-LUC; horizontal axis: ratio of luciferase activity in cells transfected with p-LUC-p53UTR. (C) Luciferase activity assays of miR-1285 and miR-612. Results are representatives of three independent experiments. Statistical analysis was performed using the Student’s t-test. Error bars represent the SEM. indicates p < 0.01.
Fig. 2. miR-1285 regulates p53 by binding to its 30 UTR. (A) Location of putative target sites for miR-1285 in the p53 30 UTR. TargetScan software was used to predict target sites. Sequence inspection indicated that the p53 30 UTR contains two elements nearly perfectly complementary to miR-1285 sequence. (B) Schematic diagram of the p53 30 UTR luciferase reporters with one or two target sites mutated. Constructs were generated using overlap-extension PCR. (C) Luciferase activity assays of wild-type and mutant p53 30 UTR luciferase reporters after co-transfection with miR-1285. Normalized luciferase activity was presented as the mean ± SEM of triplicate experiments.
tially reduced when miR-1285 was expressed in these cells (Fig. 3B). We used p53 specific siRNA (si-p53) as a positive control.
Furthermore, miR-1285 anti-sense oligonucleotides were synthesized and transfected into SH-SY5Y cells, which display relatively
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Fig. 3. miR-1285 inhibits expression of endogenous p53 mRNA and protein. (A) Quantitative real-time PCR analysis of p53 after transfection with miR-1285 mimics or siRNA against p53 in tumor cell lines MCF-7, SH-SY5Y and HepG2. (B) Western blot analysis of p53 expression after transfection with miR-1285 mimics or siRNA against p53 in tumor cell lines MCF-7, SH-SY5Y and HepG2. Beta-actin was used as a loading control. (C) Quantitative real-time PCR analysis of p53 mRNA expression in SH-SY5Y cells transfected with miR-1285 inhibitors. (D) Western blot analysis of p53 expression in SH-SY5Y cells transfected with miR-1285 inhibitors. Beta-actin was used as a loading control. (A, C) Beta-actin was used for normalization. The relative mRNA levels were presented as fold differences based on calculations of 2DDC t . Results are representative of three independent experiments. Statistical analysis was performed using the Student’s t-test. Error bars represent the SEM. indicates p < 0.05. indicates p < 0.01.
high expression of miR-1285 (Supplementary Fig. S1). As shown in Fig. 3C and D, miR-1285 silencing led to increased expression levels of p53 mRNA and protein in SH-SY5Y cells. Taken together, these results indicate that miR-1285 can reduce the expression level of p53. 3.4. miR-1285 suppresses the expression of p21, a master gene downstream of p53 To further evaluate the potential function of miR-1285, we determined the effect of miR-1285 on the expression of p21, one of the most important genes downstream of p53. p53 primarily activates p21 expression at transcriptional level by binding to the p21 promoter. As such, we used a p21 promoter based luciferase reporter WWP-Luc [14], which contains p53 binding sites, to assay the effect of miR-1285. As shown in Fig. 4A, the luciferase activity of WWP-Luc in SH-SY5Y cells was significantly decreased when cotransfected with miR-1285 or siRNA against p53. This result indicates that miR-1285 could inhibit the activity of the p21 promoter, thus suppressing the transcription of p21. Consistent with this, in SH-SY5Y cells both p21 mRNA and protein levels were suppressed by miR-1285 or siRNA against p53 (Fig. 4B and C). 4. Discussion In the present study, we performed a luciferase reporter screen to determine if p53 could be targeted by miRNAs. We selected a pool of
107 miRNAs that could potentially target p53 according to the miRNA target-prediction tool TargetScan, which is reported to have the best performance [15,16]. We found that, in our assay, only miR1285 could target the p53 30 UTR. In comparison, our recent study showed that 28 of 266 miRNAs examined could directly target the p21Cip1/Waf1 gene [17]. This discordance might result from the different sequences or secondary structures and suggests that different genes might be regulated by drastically different numbers of miRNAs. It is noteworthy that although p53 was recently reported to be a target of miR-125b [18], there was no significant effect of this miRNA on the p53 30 UTR in our assay (Supplementary Table S1). Notably, miR-612, which has the same seed region sequence as miR-1285, did not significantly alter expression of the p53 30 UTR reporter. It has been reported that perfect binding of nucleotides 2–7, the miRNA seed sequence, to the target mRNA plays a key role in target recognition [11,19]. Our mutational analyses identified two miR-1285 target sites in the 30 UTR of the p53 mRNA. In particular, these two sites are nearly perfectly complementary to the miR-1285 sequence. Therefore, we propose that the effect of miR-1285 on p53 expression might result from the near perfect complementarity, which leads to degradation of the p53 mRNA. miR-1285 was recently discovered from massively parallel sequencing of human embryonic stem cells [20]. However, there has been little reported about miR-1285 until now. miR-1285 expression profiles, available from the miRZ database [21], indicate that it is preferentially expressed in SHS5Y neuroblastoma cells and HepG2 liver cancer cells (data not shown), suggesting that
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Fig. 4. miR-1285 suppresses expression of p21, a master gene downstream of p53. (A) Luciferase activity assays of SH-SY5Y cells after transfection with the p21 promoter reporter (WWP-Luc) and either miR-1285 mimics, siRNA against p53 or control RNA. (B) Quantitative real-time PCR analysis of p21 mRNA expression in SH-SY5Y cells transfected with miR-1285 mimics, siRNA against p53, or control RNA. Beta-actin was used for normalization. The relative mRNA levels were presented as fold differences based on calculations of 2DDC t . (C) Western blot analysis of p21 protein expression in SH-SY5Y cells transfected with miR-1285 mimics, siRNA against p53, or control RNA. p21 protein expression was normalized using beta-actin expression. (A, B) Results are representatives of three independent experiments. Statistical analysis was performed using the Student’s t-test. Error bars represent the SEM. indicates p < 0.05. indicates p < 0.01.
miR-1285 might function in oncogenesis of these two cancer types. The functional linkage is worthy of further exploration. In summary, our study provides the first evidence that miR-1285 directly regulates expression of p53 by binding its 30 UTR. Acknowledgments We thank Dr. B. Vogelstein for providing the WWP-Luc plasmid. This work was supported by grants from The Ministry of Human Resources and Social Security of China (2007-170); The Science & Technology Commission of Shanghai Municipality (07DJ14006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.04.112. References [1] A.J. Levine, M. Oren, The first 30 years of p53: growing ever more complex, Nat. Rev. Cancer 9 (2009) 749–758. [2] D. Menendez, A. Inga, M.A. Resnick, The expanding universe of p53 targets, Nat. Rev. Cancer 9 (2009) 724–737. [3] L. He, X. He, L.P. Lim, E. de Stanchina, Z. Xuan, Y. Liang, W. Xue, L. Zender, J. Magnus, D. Ridzon, A.L. Jackson, P.S. Linsley, C. Chen, S.W. Lowe, M.A. Cleary, G.J. Hannon, A microRNA component of the p53 tumour suppressor network, Nature 447 (2007) 1130–1134. [4] T.C. Chang, E.A. Wentzel, O.A. Kent, K. Ramachandran, M. Mullendore, K.H. Lee, G. Feldmann, M. Yamakuchi, M. Ferlito, C.J. Lowenstein, D.E. Arking, M.A. Beer, A. Maitra, J.T. Mendell, Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis, Mol. Cell 26 (2007) 745–752. [5] N. Raver-Shapira, E. Marciano, E. Meiri, Y. Spector, N. Rosenfeld, N. Moskovits, Z. Bentwich, M. Oren, Transcriptional activation of miR-34a contributes to p53mediated apoptosis, Mol. Cell 26 (2007) 731–743.
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