Differentially RegulatedMicro-RNAs and ActivelyTranslated Messenger RNATranscripts by Tumor Suppressor p 53 in Colon Cancer

Purpose: The aim of this study was to investigate the role of p53 in regulating micro-RNA (miRNA) expression due to its function as a transcription factor. In addition, p53 may also affect other cellular mRNA gene expression at the translational level either via its mediated miRNAs or due to its RNA-binding function. Experimental Design:The possible interaction between p53 and miRNAs in regulating gene expression was investigated using human colon cancer HCT-116 (wt-p53) and HCT-116 (nullp53) cell lines. The effect of p53 on the expression of miRNAs was investigated using miRNA expression array and real-time quantitative reverse transcription-PCRanalysis. Results: Our investigation indicated that the expression levels of a number of miRNAs were affected by wt-p53. Down-regulation of wt-p53 via small interfering RNA abolished the effect of wt-p53 in regulating miRNAs in HCT-116 (wt-p53) cells. Global sequence analysis revealed thatover46%of the326miRNAputativepromoters containpotentialp53-binding sites, suggesting that some of these miRNAs were potentially regulated directly by wt-p53. In addition, the expression levels of steady-state total mRNAs and actively translated mRNA transcripts were quantified by high-density microarray gene expression analysis. The results indicated that nearly 200 cellular mRNA transcripts were regulated at the posttranscriptional level, and sequence analysis revealed that some of these mRNAs may be potential targets of miRNAs, including translation initiation factor eIF-5A, eIF-4A, and protein phosphatase1. Conclusion:To the best of our knowledge, this is the first report demonstrating that wt-p53 and miRNAs interact in influencing gene expression and providing insights of how p53 regulates genes at multiple levels via uniquemechanisms. The tumor suppressor gene p53 is one of the key regulators of cell cycle control and apoptosis and has been named the guardian of the genome (1). In addition to its function as a transcription factor, p53 also acts as an RNA-binding protein capable of regulating its own mRNA translation (2). As an RNA-binding protein, p53 regulates the expression of other cellular mRNA transcripts at the posttranscriptional level (3). p53 also influences apoptosis by accumulating to mitochondria (4, 5). With the recent discovery of noncoding RNAs [micro-RNAs (miRNA) and small interfering RNAs (siRNA)] and their function as translational regulators, it is clear that miRNAs play important roles in regulating gene expression. The notion that miRNAs regulate gene expression at the translational level is based on the study of the first two miRNAs, lin-4 and let-7, in Caenorhabditis elegans. Lin-4 attenuates the translation, but not the mRNA level, of two target genes, lin-14 and lin-28, by imperfect base pairing to complementary sequences in the 3V untranslated region of the target mRNAs (6, 7). Translational regulation has been extensively studied in plant biology (8). In plants, translational regulation provides acute responses due to sudden environmental changes and this process is highly reversible and energy efficient. Translational control also provides the same advantage for mammalian systems, in particular during genotoxic stress (9). The central concept of translational regulation is that gene expression may be controlled by the efficiency of translation of a given mRNA in the absence of a corresponding change in the steady-state level of that mRNA. Translational regulation provides the cell with a more precise, immediate, and energy-efficient way of controlling expression of proteins, and can induce rapid changes in protein synthesis without the need for transcriptional activation and subsequent mRNA processing steps. In addition, translational control also has the advantage of being readily reversible, providing the cell with great flexibility in responding to various cytotoxic stresses. Human Cancer Biology Authors’Affiliations: University of South Alabama-Cancer Research Institute, Mobile, Alabama and Weizmann Institute of Science, Rehovot, Israel Received 8/23/05; revised1/18/06; accepted 2/1/06. Grant support: University of South Alabama-Cancer Research Institute start-up fund (J. Ju) and Israel Science Foundation (Y. Pilpel). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Jingfang Ju, Cancer Genomics Laboratory, University of South Alabama-Cancer Research Institute, MSB2316, 307 North University Boulevard, Mobile, AL 36688. Phone: 251-460-7393; Fax: 251-460-6994; E-mail: jju@usouthal.edu. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-1853 www.aacrjournals.org Clin Cancer Res 2006;12(7) April 1, 2006 2014 Research. on April 5, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Little is known, however, how miRNAs are regulated at the transcriptional level. After transcription, pre-miRNAs are processed by Dicer complex to their corresponding mature miRNAs. We hypothesize that p53 may also mediate certain miRNAs expression due to its function as a transcription factor. In addition, p53 may also affect other cellular mRNA gene expression at the translational level either via its mediated miRNAs or due to its own RNA-binding function. This hypothesis is partially supported by a recent report from O’Donnell et al. (10) showing that c-Myc regulated a number of miRNAs, and two of the miRNAs regulated E2F expression. c-Myc is a helix-loop-helix leucine zipper transcription factor that regulates an estimated 10% to 15% of genes in the human genome. Translational control has been shown to play a key role in oncogenesis (9). One of the examples is thymidylate synthase, one of the important targets for fluoropyrimidine-based anticancer therapy (11). Another example is vascular endothelial growth factor, which was shown to be regulated, at least in part, at the translational level (12). More importantly, p53 , the critical tumor suppressor gene, was also regulated at the translational level (2). However, the RNA-binding function of p53 and its potential for regulating other downstream genes has not been fully elucidated. The main function of miRNAs is to regulate gene expression at the translational level. Although the exact function of most of the newly discovered miRNAs and siRNAs are just emerging, their ability to regulate cell proliferation and cell death has been recently shown (13). Recent reports have shown that expression of miRNAs can be altered in cancer (14). With the recent discovery of the function of miRNA as translational attenuators, we have reasoned that there might be a potential interaction between miRNAs and p53 because of the dual function of p53 as a transcription factor and RNA-binding protein, and the roles of both in the translational regulation process. Therefore, we chose to explore the potential relationship between the transcription factor function of p53 and miRNA expression in a colon cancer–related context, as p53 is one of the most frequently altered tumor suppressor genes in colon cancer due to mutations and deletions. The human HCT-116 (wt-p53) and HCT-116 (null-p53) colon cancer cell lines were chosen as model systems to investigate the role of p53 on the expression of miRNAs. HCT-116 (null-p53) cell line was developed via targeted deletion using homologous recombination using HCT-116 (wt-p53) cells (15). This model has been used extensively for the investigation of p53 functions in cell cycle control and apoptosis (15–18). We expect that the functional miRNAs are localized in the actively translated polyribosome complexes (19). Hence, we have investigated the effect of wt-p53 on miRNAs and their translationally regulated mRNA targets by isolating both actively translated mRNA transcripts and miRNAs from polyribosome complexes from these two colon cell lines. The effect of p53 on miRNA expression and on the expression levels of both steady-state and actively translated mRNA transcripts were analyzed. Our study indicated that the expression levels of a number of miRNAs were affected by wtp53. Down-regulation of wt-p53 via siRNA abolished the effect of wt-p53 in regulating miRNAs in HCT-116 (wt-p53) cells. Global sequence analysis revealed that >46% of the 326 miRNA putative promoters contain potential p53-binding sites, suggesting that some of these miRNAs were potentially regulated directly by wt-p53. Nearly 200 cellular mRNA transcripts were regulated at the posttranscriptional level, and sequence analysis revealed that some of these mRNAs may be potential targets of miRNAs. Materials and Methods Cell lines and reagents. The HCT-116 (wt-p53) and HCT-116 (nullp53) cell lines were a gift from Dr. Bert Vogelstein (The Johns Hopkins University, Baltimore, MD) and were described in detail previously (15, 16). Both cell lines were maintained in McCoy’s medium supplemented with 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 2 mmol/L L-Glutamine, and antibiotics. All cell lines were grown at 37jC in a humidified incubator with 5% CO2. 5-Fluorouracil (5-FU) was purchased from Sigma (St. Louis, MO). Isolation of steady-state total mRNA and actively translated mRNA transcripts. The procedures for isolating steady-state total mRNA and actively translated mRNA transcripts were described in detail previously via sucrose gradient ultracentrifugation (20). The activated translated mRNA transcripts were isolated from pooled polysome fractions (fractions 7-13) using Trizol-LS Reagent (Invitrogen, Carls-