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Vol. 18, Issue 12, 5004-5013, December 2007
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*Department of Pharmacology and Institute of Basic Medical Sciences, College of Medicine, and Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, Taiwan 701; and Department of Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan 701
Submitted April 27, 2007;
Revised September 21, 2007;
Accepted October 3, 2007
Monitoring Editor: William Tansey
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Wilusz and Wilusz, 2004). The aberrant control of mRNA turnover has been implicated in disease states, including cancer, chronic inflammatory responses, and coronary disease (Hollams et al., 2002; Audic and Hartley, 2004). For example, in myeloma and human T-cell leukemia, the c-myc gene is mutated due to a translocation or loss of the 3' untranslated regions (UTRs). This renders the c-myc mRNA up to 7 times more stable than the wild type (Hollis et al., 1988; Aghib et al., 1990).
mRNA turnover rate is mainly specified by control elements that are usually located within the 3'UTR of mRNAs and they are recognized by various RNA-binding proteins. The most well studied specific cis-acting element controlling the half-life of mRNA is the adenylate- and uridylate-rich (AU-rich) element (ARE) found in 3'UTR of a variety of short-lived mRNAs, including those encoding cytokines, lymphokines, proto-oncogenes, and growth factors (Chen and Shyu, 1995; Wilusz et al., 2001). A plethora of ARE-binding proteins have been identified, characterized, and cloned, including AUF1, HuR, TIA-1, and tristetraprolin, which exerted either negative or positive effects on mRNA stability (Brennan and Steitz, 2001; Bevilacqua et al., 2003). C-rich element has also been reportedly involved in the regulation of mRNA stability. Three discontinuous C-rich elements within the 3'UTR of 
-globin mRNA are linked to its high-level accumulation in erythroid cells (Weiss and Liebhaber, 1995). The poly(C) binding proteins, including PCBP1 (CP-1), and PCBP2 (CP-2), were proved to bind with these motifs to prevent mRNA degradation (Wang et al., 1995).
Gastrin is a classical gut peptide hormone, which was identified originally as a stimulant of gastric acid secretion. It is normally produced at high levels by endocrine (G) cells located in the gastric antrum and proximal duodenal mucosa, although lower levels have been detected in the colon and pancreas (Dockray et al., 2005). Besides, gastrin has been proven to play a crucial role in the development of gastric carcinoids. Up-regulation of gastrin gene expression is reported in numerous cancers, including colorectal, pancreatic, and lung cancers (Rehfeld et al., 1989; Aly et al., 2004; Ferrand and Wang, 2006). Overexpression of gastrin in transgenic animals induces increased colonic proliferation and accelerates the development of colon cancer (Wang et al., 1996; Cobb et al., 2004). The higher expression level of gastrin by human lung cancers was associated with a significantly decreased survival in patients (Koh et al., 2004). Recent studies further highlight that the Ras, transforming growth factor-β, and Wnt/β-catenin signaling pathway involved in the gastrin gene expression, indicate that gastrin is in fact a downstream target of multiple oncogenic pathways (Lei et al., 2004; Chakladar et al., 2005).
Although the phenomenon of higher gastrin mRNA expression exists in different kinds of cancers, the detailed mechanism involved in the up-regulation of gastrin is not very clear. It has been reported that epidermal growth factor (EGF) receptor activation is a major regulator of gastrin gene expression and that the transcription factor specificity protein 1 is the most likely target of the EGF receptor signaling cascade that in turn binds to and transactivates the gastrin promoter (Ford et al., 1997). However, no information is known about the posttranscriptional regulation mechanism involved in gastrin gene expression. Here, we found that gastrin mRNA stability increased under EGF treatment. This indicates that mRNA turnover regulation mechanism plays a role in regulating the gastrin mRNA expression. Moreover, we identified heterogeneous nuclear ribonucleoprotein K (hnRNP K), poly(C) binding protein 1 (PCBP1), and nucleolin were involved in the regulation of gastrin mRNA turnover and contributed to the increased gastrin mRNA expression.
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MATERIALS AND METHODS |
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Cell Culture
The experiments were performed using the human gastric adenocarcinoma AGS cell line purchased from American Type Culture Collection (Manassas, VA). AGS cells were cultured in F-12K culture medium (Biowest, Loire Valley, France) supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin. The cultures were incubated at 37°C in a humidified 5% CO2 incubator.
Subcellular Fractionation of AGS Cells
AGS cells were scraped, rinsed with phosphate-buffered saline, and lysed with cytoplasm lysis buffer (10 mM HEPES, pH 8.0, 40 mM KCl, 3 mM MgCl2, 5% glycerol, 2 mM dithiothreitol [DTT], and 0.5% NP-40) supplemented with protease inhibitors (1 mg/ml leupeptin, 1 mg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride) on ice for 10 min. The cytosolic fraction was prepared by performing high-speed centrifugation (6000 rpm for 5 min at 4°C), and the supernatant was transferred to new Eppendorf and stored at –80°C. The nuclei fraction was prepared by suspended the pellet with nuclei lysis buffer (10 mM HEPES, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, and 0.5 mM DTT) on ice for 10 min. After centrifugation (6000 rpm for 5 min at 4°C), the supernatant was collected as nuclear fraction and stored at –80°C.
mRNA Turnover Analysis
Cells were grown to 70% confluence and treated with or without 10 nM EGF for 4 h, followed by addition of 5 mg/ml actinomycin D. Total RNAs were isolated from the cells at different time points after addition of actinomycin D and analyzed by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The mRNA half-life was determined by linear regression analysis.
Quantitative RT–PCR
The primers used are listed as follows: for gastrin mRNA, forward: 5' GCAGC GACTATGTGTGTATGTG 3' and reverse: 5' TCCATCCATAGGCTTCTTCTTC 3'; and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward: 5' CCCACTCCTCCA CCTTTGAC 3' and reverse: 5' TCTCTCTTCCTCTTGTGCTCTTG 3'. Total RNAs were isolated, and the mRNA levels were measured using TITANIUM one-step RT-PCR reagent (Clontech, Mountain View, CA) and analyzed by LightCycler 480 real-time PCR system (LightCycler Instrument; Roche Molecular Biochemicals, Mannheim, Germany).
RNA Electrophoretic Mobility Shift Assay (EMSA)
To perform gel shift analysis, 20–30 µg of proteins were incubated with 50 pM biotin-labeled RNA probes in RNA-EMSA buffer [50 mM KCl, 5% (vol/vol) glycerol, 0.1% (vol/vol) NP-40, 1 mM MgCl2, 1 mM dithiothreitol, 10 mM Tris-HCl, pH 8.0, 0.5 mg/ml heparin, and 0.5 mg/ml yeast tRNA] for 30 min at room temperature. Samples were subjected to electrophoresis on a 6% native acrylamide gel (acrylamide/bisacrylamide ratio of 37.5:1), and transferred to positive-charge nylon membrane. The signals of RNA-EMSA reaction were detected by LightShift chemiluminescent kit (Pierce Chemical, Rockford, IL). For competition assays, RNA homopolymers [20 ng; poly(rU), poly(rC), and poly(rA); GE Healthcare, Chalfont, St. Giles, United Kingdom) were added to the reaction mixture for 10 min at 25°C before incubation with labeled RNA probe.
Biotin Pull-Down Assay and Mass Spectrometry Analysis
AGS cytoplasmic extract (500 µg) was incubated with 50 pM biotin-labeled RNA and rotated for 2 h at 4°C. The sample was placed on ice and irradiated with UV (240-nm UV bulb; Stratagene, La Jolla, CA) for 15 min at 4°C. The biotin-labeled RNA was isolated by streptavidin-conjugated agarose beads (Sigma-Aldrich), and washed with wash buffer (10 mM HEPES, pH 8.0, 40 mM KCl, 3 mM MgCl2, 5% glycerol, 2 mM DTT, and 2% NP-40) supplemented with protease inhibitors (1 mg/ml leupeptin, 1 mg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride) for three times. The RNA–protein complex was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), and the gel was fixed for silver staining. The protein bands of interest on the gel were excised, ground manually, and transferred into a siliconized 0.5-ml microcentrifuge tube for further identification by mass spectrometry analysis. The in-gel digestion, mass spectrometry analysis, and database search were performed as described previously (Tyan et al., 2005).
Protein Immunoprecipitation Assay
In total, 100–300 µg of AGS cytosolic extract was incubated with a specific antibody and rotated at 4°C overnight, and then 50 µl of protein A/G-agarose beads was added for 2 h at 4°C. The complexes were washed with wash buffer (10 mM HEPES, pH 8.0, 40 mM KCl, 3 mM MgCl2, 5% glycerol, 2 mM DTT, and 0.5% NP-40) twice, and then they were resuspended in 50 µl of 1x SDS sample buffer for analysis by SDS-PAGE and Western blotting.
RNA Immunoprecipitation Assay
AGS cells were grown to 70% confluence and lysed with lysis buffer (10 mM HEPES, pH 8.0, 40 mM KCl, 3 mM MgCl2, 5% glycerol, 0.5% NP-40, and 1 U/µl RNaseOUT) for 20 min on ice. The cell extract was incubated with nucleolin antibodies (H-250) or hnRNP K (D-6) antibodies, and protein A/G agarose beads at 4°C overnight. Immunoprecipitated complexes were washed three times with lysis buffer, and bound RNAs were extracted by TRIzol reagent (Invitrogen, Carlsbad, CA). Gastrin mRNA was detected by RT-PCR or quantitative real-time PCR.
Short Interfering RNA
The short interfering RNA against hnRNP K and PCBP1 were purchased from Ambion (Austin, TX). The short interfering RNA sequence of hnRNP K was sense: 5'-GCGCAUAUUGAGUAUCAGU-3' and antisense: 5'ACUGAUACUCAAUAUGCGC-3'. The short interfering RNA sequence of PCBP1 was sense: 5'-CCUCUAGAUGCCUACUCGA-3' and antisense: 5'-UCGAGUAGGCAUCUAGAGC-3'. The short interfering RNA against nucleolin was purchased from Invitrogen. The sequence was sense: 5'-GGAAGGUCAGCAGUCUUCCAUGAGA-3' and antisense: 5'-UCUCAUGGAAGACUGCUGACCUUCC-3'. To deliver the siRNA, the AGS cells were plated at 
40% confluence in a six-well plate, and 50 nM siRNA was transfected to cells using Lipofectamine 2000 (Invitrogen) at 20 h after plating the cells. The cells were changed to serum starvation medium 20 h later, and they continued to incubate for 24 h. After plating the cells for 64 h, the cells were treated with 10 nM EGF for 4 or 8 h, and then cells were lysed and RNAs or proteins were collected for analysis.
Tet-Off mRNA Turnover Assay
The 1.7 kb of luciferase gene from pGL-3 vector (Promega, Madison, WI) was subcloned into pTRE2-hyg vector (Clontech) and fused with gastrin 3'UTR to produce pTRE-luc-gastrin 3'UTR (pTRE-luc-3'UTR) construct. To establish pTet-Off stable AGS cell line, the pTet-Off plasmid was transiently transfected into AGS cells and cultured through 400 µg/ml Geneticin (G-418; Invitrogen) for selection. The stable pTet-Off AGS cells were transfected with 20 µM scramble si-control or si-nucleolin oligonucleotides with 0.2 µg of pTRE-luc-3'UTR by using Lipofectamine 2000. After 36 h of transfection, cells were treated with or without 10 nM EGF for 3 h, and then luciferase mRNA expression was turned off by adding 50 µg/ml doxycycline. To analyze gastrin mRNA turnover rate under specific knockdown condition, the cells were harvested at different time points, and luciferase activity was measured. Results were the mean ± SD of three independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To confirm this phenomenon existed in our study system, AGS cells were treated with EGF, and gastrin mRNA levels were detected at different time points using RT-PCR. Consistent with previous results, we observed EGF increased gastrin mRNA expression in a time-dependent manner (Figure 1A). The mRNA expression levels were also quantified by quantitative RT-PCR. As shown in Figure 1B, gastrin mRNA expression levels are elevated to 3.1- and 14.5-fold after EGF treatment for 4 and 16 h, respectively. To check whether the mRNA turnover regulation mechanism was involved in the up-regulation of gastrin mRNA under EGF treatment, AGS cells were incubated with or without EGF for 4 h, and then actinomycin D was added to block the transcriptional activities. The cells were harvested at different time intervals, and the levels of gastrin mRNA were analyzed by quantitative RT-PCR. The half-life of gastrin mRNA was 1.8 h in normal conditions. In contrast, when AGS cells were pre-treated with EGF for 4 h, the half-life of gastrin mRNA was extended to 3.7 h (Figure 1C). This result indicated that EGF increases the gastrin mRNA stability, and it implies an mRNA turnover regulation mechanism is involved in the gastrin mRNA expression.
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). Therefore, we tried to identify the cis-acting elements in gastrin 3'UTR and trans-acting proteins. Figure 2A illustrates the structure of human gastrin mRNA. The length of 3'UTR is only 71 nucleotides, and the structure predicted by Vienna RNA Secondary Structure Prediction program is shown in Figure 2B. It forms two stem-loop regions connected by a spacer region. Three RNA probes (A, B, and C) based on these structures were synthesized (Figure 2C). To identify which probes interacted with proteins, RNA- EMSAs were performed. Only probes B and C interacted with proteins to form observable RNA–protein complexes. (Figure 2D, lanes 7 and 12). The RNA–protein complex disappeared after incubation with poly(rC) homopolymer. This indicates that the binding proteins belong to the family of poly(C) binding proteins (Figure 2D, lanes 9 and 14). To identify whether the same protein complex interacted with probes B and C, we synthesized nonbiotin-labeled probe B to compete in the reaction of protein binding with biotin-labeled probes B and C. We found that it could inhibit the RNA–protein complex formation in a dose-dependent manner (Figure 2E, lanes 2–4 and 6–8). These results suggested that probe B and probe C interact with the same RNA-binding proteins in AGS cells.
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hnRNP K and PCBP1 Bind with Gastrin mRNA 3'UTR
To identify the trans-acting factors that may interact with the gastrin mRNA 3'UTR, the biotin pull-down assay was performed to analyze the potential protein associations with this region. Two major proteins were observed to interact with probe B and probe C (Figure 3A). Using mass spectrometry analysis, hnRNP K and PCBP1, belonging to the poly(C) binding protein family, were identified to be the target proteins interacted with the gastrin mRNA 3'UTR (Figure 3B). To further confirm this finding, UV cross-linking/biotin-labeled probe pull-down combined with Western blot analysis using hnRNP K and PCBP1 antibodies was performed. The data revealed that both hnRNP K and PCBP bound with probe B and C, but not probe A (Figure 3C, lanes 3–5). Also, both hnRNP K and PCBP1 could not bind with probe D, which had a mutated C-rich region (Figure 3C, lane 6).
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EGF Signaling Regulates Nucleolin Interacted with Gastrin mRNA and hnRNP K
Increased binding between trans-factors with cis-acting elements in mRNA 3'UTR was reported to enhance the mRNA stability under stimulator treatment (Wilusz et al., 2001; Bevilacqua et al., 2003). From Figure 3D, we found HnRNP K and PCBP1 interacted with gastrin mRNA in vivo; however, the level of gastrin mRNA associated with hnRNP K and PCBP1 was unchanged under EGF treatment (compare lanes 5–7). This raises the possibility that other factors are involved in the increase of gastrin mRNA stability induced by EGF. To identify this regulation factor, we examined the possible RNA-binding motifs existing in the gastrin mRNA 3'UTR. Interestingly, we found a motif sequence AGCCCU located between the C-rich region similar to the reverse orientation of nucleolin binding sequence (U/G)CCCG(A/G). Then, the biotin-labeled probe pull-down combined with Western blot analysis was conducted to confirm the interaction between nucleolin and gastrin mRNA. We found nucleolin, a RNA-binding protein involved in many cellular processes, interacted with probes B and C, but not with probe A (Figure 4A, lanes 3–5). This result indicated that the nucleolin associates with gastrin mRNA 3'UTR in vitro.
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It is also interesting to know whether nucleolin binds with gastrin mRNA and also whether it interacts with hnRNP K and PCBP1. To address this question, a protein immunoprecipitation assay was performed. As shown in Figure 4D, we found the hnRNP K-immunoprecipitated complex contained the nucleolin protein, and vice versa. To further demonstrate the binding between nucleolin and hnRNP K was through protein–protein interaction, and not due to binding with the same RNA, RNase was added to the reaction mixture to destroy the binding ability through protein–RNA–protein interaction. We found the binding pattern did not change under RNase treatment (Figure 4D, right), which indicated nucleolin and hnRNP K form a protein complex to interact with gastrin mRNA. Furthermore, we observed the interaction between hnRNP K and nucleolin increased under EGF treatment (Figure 4E, compare lanes 3 and 4, immunoblot [IB]: hnRNP K, and lanes 7 and 8, IB: nucleolin). Combining these findings, the increase in binding between nucleolin and hnRNP K seems to contribute to the increased association of nucleolin and gastrin mRNA under EGF treatment.
Nucleolin Binds with AGCCCU Motif in the Gastrin mRNA and This Binding Ability Is Enhanced by hnRNP K/PCBP1 Complex
To identify the exact binding site of nucleolin, the putative nucleolin binding site AGCCCU was mutated to AGAAAU to test the binding activity of nucleolin (Figure 5A, probe E). As shown in Figure 5B, we found that nucleolin could not bind with probe E, which lacked the putative nucleolin binding site. These results indicated the AGCCCU motif was the target site for nucleolin to bind with gastrin mRNA. Interestingly, the binding activity decreased with probe D-N. The binding ability of hnRNP K/PCBP1 complex with this probe was abolished due to lack of C-rich region. This result indicated that nucleolin binds with gastrin mRNA not only dependent on the binding sequence but also regulated by hnRNP K/PCBP1 complex interacting with the C-rich region. Besides, we also found the probe B bound with lower level nucleolin protein than probe C. Mutation of the C-rich region of probe B abolished the binding activity of nucleolin (Figure 5B, probe F). It indicated the C-rich region of probe B may contain a minor binding site for nucleolin. But, the putative nucleolin binding site in probe C may be the major interaction element with nucleolin.
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60% under the reduced expression of hnRNP K and PCBP1 (Figure 6B). Knocked down nucleolin protein expression only reduced the gastrin mRNA expression level 15%. Simultaneous silencing of these three genes had the serious inhibitory effect on gastrin mRNA expression. These results indicated that these factors contribute to stabilization of gastrin mRNA. Meanwhile, hnRNP K and PCBP1 play a more important role than nucleolin in prevention of gastrin mRNA degradation under normal conditions.
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To further confirm the functional role of nucleolin under EGF treatment, luciferase reporter gene assay was conducted. The wild-type 3'UTR and putative nucleolin binding site mutated 3'UTR, which abolished the nucleolin binding ability (3'UTRmNucleolin), were ligated to a luciferase reporter vector. The AGS cells were transiently transfected with these reporter constructs in the presence or absence of EGF, respectively, and then the luciferase mRNA expression levels were measured by quantitative real-time RT-PCR. From Figure 6D, we found the luciferase mRNA levels of 3'UTRmNucleolin decreased under normal conditions, and, more importantly, the EGF inducibility was also inhibited under EGF treatment (compare the control and EGF treatment of the 3'UTR mNucleolin clone). Besides, the Tet-Off mRNA turnover assay was performed to study the gastrin mRNA degradation rate under normal and reduced nucleolin protein expression conditions. As shown in Figure 6E, reduction of nucleolin expression accelerates the degradation rate of reporter gene fused with gastrin 3'UTR under EGF treatment, but not the normal conditions (compare si-control + EGF and si-Nucleolin + EGF). These results suggested nucleolin plays an important role in mediating the increased mRNA stability under EGF signaling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Gastrin can impart antiapoptotic activities through activation of genes associated with cell division, invasion, and angiogenesis, including EGF receptor, regenerating protein, cyclooxygenase-2, and matrix metalloproteinases (Chiba et al., 2000; Wroblewski et al., 2002; Konturek et al., 2003; Clarke et al., 2006). Therefore, to dissect the mechanism that maintains higher expression levels of gastrin in cancer cells is important for understanding the progress of gastrointestinal cancer development. Here, we report a novel mechanism that increases the gastrin expression level through regulating mRNA turnover.
First, we found EGF signal increased the gastrin mRNA stability in AGS cells (Figure 1), indicating that mRNA turnover regulation is involved in the mechanism which maintains the gastrin mRNA expression level. To dissect the molecular mechanism governing gastrin mRNA turnover, we identified the trans-acting factors, hnRNP K and PCBP1, interacting with the C-rich region located at the gastrin mRNA 3'UTR (Figure 3). hnRNP K and PCBP1 belong to the family of poly(C) binding proteins, which are involved in a remarkable array of gene expression from transcription, mRNA processing, mRNA transport, mRNA stability, and translation (Makeyev and Liebhaber, 2002). PCBP1/PCBP2 complex was reported to stabilize -globin and erythropoietin mRNA by binding with the C-rich region existing in their 3'UTR (Weiss and Liebhaber, 1995; Czyzyk-Krzeska and Bendixen, 1999). The major functional role of hnRNP K, which interacted with 3'UTR was focused on translational regulation. hnRNP K formed a complex with PCBP1/2, binding to the differentiation control element located at the 15-lipoxygenase mRNA 3'UTR and mediating translational silencing effect (Ostareck et al., 1997). Some literature indicates that hnRNP K is involved in regulating renin and neurofilament mRNA stability (Skalweit et al., 2003; Thyagarajan and Szaro, 2004). But, these studies only show the interaction between hnRNP K and mRNAs, and no direct evidence is provided to prove hnRNP K really regulates mRNA turnover in vivo. In this study, the functional role of PCBP1 and hnRNP K was proved by reducing these two protein expressions using siRNA. We found the expression level of gastrin mRNA decreased (Figure 6B). Therefore, we propose that hnRNP K and PCBP1 are responsible for stabilizing the gastrin mRNA.
Nucleolin, a multifunctional RNA binding protein, is involved in regulation of chromatin condensation, rRNA processing, and nuclear-cytoplasmic transport (Ginisty et al., 1999; Mongelard and Bouvet, 2007). Recent studies indicate that nucleolin is also involved in regulation of mRNA turnover, for example, GADD45 mRNA, bcl-2 mRNA, and CD154 mRNA (Sengupta et al., 2004; Singh et al., 2004; Zhang et al., 2006). From our results, we identified that nucleolin, but not hnRNP K and PCBP1, increased association with gastrin mRNA under EGF treatment (Figure 4B). The functional role of nucleolin was demonstrated by reducing nucleolin protein expression by using siRNA technology. We observed that decreased nucleolin protein expression significantly exerted an inhibitory effect on the EGF inducibility (Figure 6C). Using a reporter assay system, we found mutation of the putative nucleolin binding site that abolished the nucleolin binding displayed the same inhibitory effect (Figure 6D). Moreover, reduction of nucleolin expression reduced the stabilization effect exerting by EGF in Tet-Off mRNA turnover assay (Figure 6E). Therefore, the functional role of nucleolin was proved to mediate the effect of increasing the gastrin mRNA stability under EGF signaling.
Nucleolin has been shown to bind the consensus sequence (U/G)CCCG(A/G) in the loop region of hairpin structure with a 7- to 14-bp stem region (Bouvet et al., 2001). Other sequence motifs were also reported to bind with nucleolin, such as the 5'CUCUCUUUA3' motif in interleukin 2 mRNA and the A/U-rich motif in the Bcl-2 mRNA (Chen et al., 2000; Sengupta et al., 2004). In our study, we demonstrate that the AGCCCU motif is the nucleolin binding site, and the C-rich regions are also important for the nucleolin to associate with this motif (Figure 5B). It is interesting to know how the C-rich regions enhance the nucleolin to bind with the putative nucleolin target sequence. From our studies, we observed hnRNP K and PCBP1 bind with the C-rich regions. Other studies report that hnRNP K and PCBP1 can form both homomeric and heteromeric dimers (Kim et al., 2000), and the binding affinity of nucleolin with the hairpin-like structure is higher than with the linear structure (Ghisolfi-Nieto et al., 1996), Therefore, we propose when hnRNP K and PCBP1 bind with the C-rich regions, the linear structure of mRNA may reform to a hairpin-like structure through protein–protein interaction in the stem region and expose the AGCCCU sequence, which is located between the C-rich regions, in the loop region. Nucleolin has higher binding affinity with hairpin structure gastrin mRNA than the linear structure. The novel nucleolin binding model is shown in Figure 7. This model explains our observations, that both the C-rich region and putative nucleolin binding site are important for nucleolin association with gastrin mRNA.
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). The higher expression level of nucleolin in tumors or other rapidly dividing cells was also documented (Srivastava and Pollard, 1999). Examination of the expression profile of nucleolin on different cancers by analysis of expressed sequence tag (EST) counts, using the National Center for Biotechnology Information EST database, we found colorectal cancer and gastrointestinal cancer expressed higher amounts of nucleolin (801 and 411 transcripts per million) than other cancers and normal tissue (190 transcripts per million). According to our novel findings that both hnRNP K and nucleolin protect gastrin mRNA from degradation, these results may explain the higher expression level of gastrin mRNA in colorectal cancer.
hnRNP K and nucleolin are both multifunctional proteins and associate with different kinds of proteins to execute their functional roles. Here, we first describe that hnRNP K associates with nucleolin to play a functional role in maintaining mRNA stability. EGF signaling increases the association between these two proteins and implies posttranslational modification, such as phosphorylation, may be involved in this regulation. CKII and cdc2 kinase were demonstrated to phosphorylate nucleolin in growing cells (Srivastava and Pollard, 1999). hnRNP K was also reported to phosphorylate by Lck and mitogen-activated protein kinase (Bomsztyk et al., 2004). Now, we are studying what kinds of signal pathways regulate this interaction under EGF treatment. This will give us more information about hnRNP K/nucleolin complex in mediating the stabilization of mRNA under growth conditions.
In conclusion, hnRNP K and PCBP1 bound with the C-rich regions of gastrin mRNA 3'UTR facilitates nucleolin to associate with gastrin mRNA. Under EGF treatment, the interaction of nucleolin with hnRNP K/PCBP1/gastrin mRNA complex increased and contributed to the increase of gastrin mRNA stability. This novel mechanism in regulation of gastrin mRNA may provide us another direction to study the mechanism that maintains high expression levels of gastrin mRNA in gastrointestinal cancer.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Joseph T. Tseng (tctseng{at}mail.ncku.edu.tw) or Wen-Chang Chang (wcchang{at}mail.ncku.edu.tw).
Abbreviations used: 3'UTR, 3' untranslated region; EGF, epidermal growth factor; hnRNP K, heterogeneous nuclear ribonucleoprotein K; PCBP1, poly(C) binding protein 1.
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