Cancer Res 69(20): 8157-8165

PMC: PMC2763324

PMID: 19826040

MicroRNA-21 negatively regulates Cdc25A and cell cycle progression in colon cancer cells

Introduction

MicroRNAs(miRNAs) are evolutionarily conserved, 20–25 nucleotide long, non-coding RNAs which bind to their targets through partial complementary sequence recognition. This results in either degradation of mRNA or inhibition of translation, thus modulating expression of miRNA targets (1). Several hundred miRNAs have been identified in human cells (2). It is estimated that a single miRNA can regulate hundreds of targets, and 30% or more of human mRNAs are regulated by miRNAs (1, 2). Therefore, it is not surprising that miRNAs are involved in diverse biological processes, including cell differentiation, proliferation, and apoptosis presumably through a myriad of targets (2).

Deregulation of miRNAs contributes to human pathogenesis including cancer (2). For example, aberrant expression of miRNAs, including miR-21, miR-17-92, miR-15, miR-16, and let-7, has been reported in cancer (3). Furthermore, a substantial number of miRNA genes are located in the fragile sites in the genomic regions that are frequently amplified, deleted, or rearranged in cancer, providing plausible mechanisms of deregulated expression (4, 5). A theme is emerging that a miRNA can be considered either a tumor suppressor or oncogene depending on its targets in different tissues and cell types (6–8). Identification of relevant targets or pathways controlled by miRNAs will ultimately provide insights into their biological functions.

Altered expression of miR-21 has been reported in cancer. For example, miR-21 was reported to have substantially higher expression in normal tissues than in colon cancers or in NCI-60 tumor cell lines (8). On the other hand, miR-21 is overexpressed in cancers of the breast, lung, pancreas, prostate, stomach and brain (9, 10). Higher expression of miR-21 was found in colon adenocarcinomas than in the normal mucosa, and was associated with decreased overall survival (11). A limited number of genes, including PTEN, TPM1, Pdcd4, Spry 1 and Spry 2 have been reported to be targets of miR-21, suggesting potential functions in regulating cell proliferation, apoptosis, and invasion (12–16). However, the precise role of miR-21 in cancer remains to be defined.

The cell division cycle 25 (Cdc25) family of proteins are highly conserved dual specificity phosphatases that dephosphorylate and activate cyclin-dependent kinase (CDK) complexes. Three isoforms have been identified in mammalian cells, Cdc25A, Cdc25B and Cdc25C (17). Overexpression of Cdc25 family proteins, mostly Cdc25A and Cdc25B, correlates with more aggressive disease and poor prognosis in some cancers, and leads to genetic instability in mice (18, 19). Cdc25A positively regulates G1-S and G2-M transitions by activating distinct cyclin/Cdk complexes (18, 19). Moreover, timely inactivation of Cdc25A facilitates checkpoint activation upon DNA damage. Cdc25A activities are tightly regulated by multiple mechanisms during the cell cycle, and ubiquitin-mediated proteolysis is the major mechanism of Cdc25A turnover (17). For example, hyperphosphorylation of Cdc25A by the ATR-Chk1 signaling leads to its degradation and contributes to a delay in the cell cycle, which allows either DNA repair or apoptosis, depending on the extent of DNA damage (17, 19, 20).

In the current study, we reported a novel role of miR-21 in modulating cell cycle progression and DNA damage checkpoint activation via Cdc25A. Cdc25A was identified and validated as a miR-21 target using miR-21 knockout colon cancer cell lines. miR-21 was found to be induced by serum starvation, negatively regulate G1-S transition, and participate in DNA damage checkpoint activation in response to γ-irradiation. Our data provide a novel mechanism of Cdc25A mRNA turnover, and a potential role of miR-21 deregulation in tumorigenesis.

Materials and Methods

Targeting the miR-21 gene

Gene targeting vectors were constructed using a rAAV system as described (21–23) with minor modifications. Briefly, two homologous arms flanking the miR-21 locus, which are 1.17 kb and 1.15 kb, respectively, along with the neomycin-resistant gene cassette (Neo), were inserted between two NotI sites in the AAV shuttle vector pAAV-MCS (Stratagene, La Jolla, CA) by a 4-way ligation reaction. Packaging of rAAV was performed by using the AAV Helper-Free System (Stratagene) according to the manufacturer’s instructions. RKO and DLD1 cells were infected with rAAV and selected by G418 (0.4 mg/ml) for 3 weeks. G418-resistant clones were screened by PCR for targeting events with primer pairs listed in Table S1 using pooled genomic DNA (24). The same targeting construct was used in the second round of gene targeting following the excision of Neo gene flanked by Lox P sites in a heterozygous clone with an adenovirus expressing Cre recombinase (Ad-Cre) (24). After the second round of gene targeting, Neo was excised by Ad-Cre infection again, and gene targeting was verified by genomic PCR, RT-PCR, and Northern blot. The detailed procedures of gene targeting and PCR screening are available upon request, and the primers used are listed in Table S1.

Microarray analysis

Total RNA was isolated 48 h following transfection from cells cultured in T25 flasks. Microarray analysis was performed and relative gene expression was analyzed as described previously by the Core facility at the University of Pittsburgh School of Medicine (25).

Cell culture

Human colorectal cancer cell lines RKO and DLD1 were obtained from American Type Cell Collection (ATCC, Manassas, VA) and cultured in McCoy’s 5A modified media (Invitrogen, Carlsbad, CA) supplemented with 10% defined FBS (HyClone, Logan, UT), 100 units/ml penicillin and 1% streptomycin (Invitrogen). Cells were maintained at 37°C with 5% CO₂. In some experiments, cells were grown in medium containing 0.5% serum. Details on serum-stimulated G1-S transition, radiation-induced transient G2/M checkpoint and clonogenic survival are described in the supplemental material.

miRNA target prediction

The miRNA targets were predicted using the algorithms TargetScan (http://genes.mit.edu/tscan/targetscanS2005.html) and PicTar (http://pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi).

Isolation of microRNAs, Real-Time Polymerase Chain Reaction (PCR) assays, and Northern blotting for mature miRNAs

The expression of mature miRNAs was determined by real-time PCR (26) and Northern blot. The expression of protein coding mRNAs was quantitated by real-time PCR. Details are described in the supplemental material.

Transfections

Transfection with 100 nM pre-miR-21, 100 nM anti-miR-21 (Ambion) or 200 nM Cdc25A siRNA (Dharmacon, Lafayette, CO) was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturers’ instructions. The target sequence for Cdc25A is GGAAAAUGAAGCCUUUGAG (27). Cells plated at 20–30% confluence in 6-well plates were transfected twice in 48 h, and split into T25 flask 10 h after the second transfection. The next day, the cells in T25 were either subjected to serum starvation and stimulation or irradiated as described above.

Luciferase reporter constructs

The reporter constructs containing the 3′UTR of Cdc25A were cloned into the pMIR-REPORT^{vector (Ambion) using PCR generated fragment. Site-directed mutagenesis with the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) was used to introduce mutations in the}miR-21 binding site. Reporter assays were carried out as previously described with a transfection control (28). All the experiments were performed in triplicate and repeated at least three times on different days. Details were described in the supplemental material (Table S2).

BrdU incorporation and mitotic index

BrdU incorporation was analyzed by microscopy or flow cytometry following staining with anti-BrdU, Alexa Fluor 488 conjugated antibody. Mitotic index was measured by phosphorylated histone 3 (H3) staining. Detailed methods are described in supplemental material.

Western blotting

Western blotting was performed as previously described (29). The antibodies used for Western blotting included those against Cdc25A, Cdc25C, Cdc2, Cyclin B1, Chk1 (Santa Cruz, Santa Cruz, CA), phospho-histone H3 (Millipore, Bellerica, MA), α-tubulin (EMD Biosciences, San Diego, CA), phospho-Cdc2 (Cell Signaling, Danvers, MA), Cdc25B (BD Biosciences, Sparks, MD), and β-Trcp (Invitrogen). Quantification of relative expression was determined by densitometry as described (30).

Statistical analysis

Statistical analysis was performed using GraphPad Prism IV software. P values were calculated by student’s (t) test. P-values less than 0.05 were considered significant. The means +/− one standard deviation (SD) were displayed in the figures.

Targeting the miR-21 gene

Microarray analysis

Cell culture

miRNA target prediction

The miRNA targets were predicted using the algorithms TargetScan (http://genes.mit.edu/tscan/targetscanS2005.html) and PicTar (http://pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi).

Isolation of microRNAs, Real-Time Polymerase Chain Reaction (PCR) assays, and Northern blotting for mature miRNAs

Transfections

Luciferase reporter constructs

BrdU incorporation and mitotic index

Western blotting

Statistical analysis

Results

Targeted deletion of miR-21 in colon cancer cells

Aberrant expression of miR-21 has been reported in colon cancer (8). We were interested in determining its potential role in tumorigenesis by identifying miR-21 targets. To avoid the limitations of downregulating miRNA expression with antisense oligos (31), we knocked out the miR-21 precursor sequence in RKO and DLD1 colorectal cancer cells, using the recombinant adeno-associated virus (rAAV) system (Fig. 1A) (21, 22). Both of these lines express relatively high levels of miR-21 (8, 32). After two rounds of homologous recombination, miR-21 knockout clones were identified by PCR amplification of the corresponding genomic regions (Fig. 1B, S1A). RT-PCR and Northern blot confirmed that the mature miR-21 was not expressed in these knockout clones (Figs. 1C and D).

An external file that holds a picture, illustration, etc.
Object name is nihms143917f1.jpg

Figure 1

Targeted deletion of the primary miR-21 locus in RKO and DLD1 colon cancer cells

A. Schematic diagram of miR-21 targeting strategy. The targeting construct consists of two homologous arms and the neomycin-resistance gene (Neo) flanked by 2 LoxP sites. Homologous recombination resulted in a deletion of 750 base pairs, including the sequence encoding mature miR-21. The same construct was used in the second round of gene targeting after the excision of Neo gene by Cre recombinase. The positions of the primers (P1 and P2) for PCR screening were indicated. B. Identification of miR-21-KO clones by genomic PCR. C. Mature miR-21 expression was measured by RT-PCR (left panel) or northern blot (right panel) in indicated RKO cell lines. D. Mature miR-21 expression was measured as in C in indicated DLD1 cell lines.

Identification of potential miR-21 targets through microarray analysis

miRNAs regulate their target genes via mRNA degradation and/or inhibition of translation (33). Their potential targets can be identified using high-throughput methods such as microarray analysis (34). To identify potential miR-21 targets predicted to have elevated expression in miR-21 KO cells, we performed microarray analysis on RKO parental cells transfected with pre-miR-21 and miR-21 KO cells transfected with a control siRNA for 48 hours. Over a hundred candidates showed at least a 2-fold increase in their expression in miR-21 KO cells (Table S3) (P ≤ 0.02). Interestingly, several proteins involved in cell cycle and DNA damage responses were among them. We then chose 12 candidates and validated 7 of them by quantitative RT-PCR analysis (Fig. S2). Cdc25A was chosen for further analysis due to its well established role in cell cycle regulation and cancer.

Cdc25A expression is regulated by miR-21 in colon cancer cells

To validate Cdc25A as a miR-21 target, its mRNA and protein levels were compared in parental and miR-21 KO RKO cells. Consistent with the results obtained in microarray analysis, Cdc25A levels were significantly up-regulated in miR-21 KO cells (Fig. 2A and B). Transfection of precursor (pre)-miR-21 decreased the levels of Cdc25A transcript and protein (Figs. 2C, D, and S1B and S3). These findings were confirmed in DLD1 parental and miR-21 KO cells (Fig. S3A–D). miR-21 deletion did not affect the expression of the other two Cdc25 family members, Cdc25B and Cdc25C, or its established regulators Chk1 or Beta-TrCP (Fig. S4A). Transient transfection of anti-miR-21 also elevated Cdc25A expression in RKO cells as did miR-21 targeting (Fig. 2D, right panel). To examine the expression of miR-21 in relation to Cdc25A in cancer, we analyzed the expression of miR-21 in 12 colon cancers that overexpress Cdc25A using matched normal and tumor tissues (35). miR-21 was found to be underexpressed in 6 of 12 (50%) tumors (range from 2–7 fold) (Fig. 3).

An external file that holds a picture, illustration, etc.
Object name is nihms143917f2.jpg

Figure 2

Cdc25A is regulated by miR-21 in colon cancer cells

A. Relative Cdc25A mRNA expression was measured by real-time RT-PCR in RKO wild-type (WT) and miR-21 knockout (KO) cells. Levels were standardized to Cdc25A mRNA in the WT cells normalized to GAPDH. Values are means ± SD, n=3. *P<0.02. B. Expression of Cdc25A protein was determined by Western blotting. C. The effect of precursor miR-21 on CDC25A mRNA levels. Cells were transfected with precursor miR-21 or control siRNA for 48 hrs and analyzed for CDC25A expression by RT-PCR. Values are means ± SD, n=3. *P<0.04. Expression levels were normalized to those in control siRNA transfected cells. The values are mean of three experiments +/− SD. D. The effects of anti-miR-21, or pre-miR-21 on Cdc25A expression. RKO cells were transfected with control siRNA, anti-miR-21, pre-miR-21. Cdc25A levels were analyzed by Western blotting at 48 h after transfection.

An external file that holds a picture, illustration, etc.
Object name is nihms143917f3.jpg

Figure 3

The miR-21 levels in Cdc25A overexpressing colon cancers

The levels of miR-21 were analyzed by Real-Time RT-PCR in 12 pairs of matched adjacent normal and tumor tissues that overexpress Cdc25A. The miR-21 levels were normalized to that of U6. The Cdc25A levels were normalized to that of GAPDH. Values are means ± SD, n=3.

Cdc25A is a miR-21 target

Upon a closer inspection, a putative miR-21 binding site located in the 3′ untranslated region (3′UTR) of Cdc25A gene was predicted by two algorithms (TargetScan and PicTar) (Fig. 4A). Importantly, this putative miR-21 binding site is 100% conserved in five species in the region that pairs with the seed sequence (Fig. 4A). The 3′-UTR region of Cdc25A containing this site was cloned into pMIR-REPORT^{miRNA reporter vector. The luciferase activities of this reporter in}miR-21 KO cells were about 60% higher than that in parental RKO cells, but were suppressed by pre-miR-21 transfection (Fig. 4B), suggesting a regulatory element in its 3′-UTR. We then mutated the miR-21 binding site in the reporter construct Luc-Cdc25A-Mut-UTR and found its activities were similar in parental and miR-21 KO RKO cells (Fig. 4). Transfection of pre-miR-21 did not decrease the activities of the mutant reporter in either parental or miR-21 KO cells (Fig. 4B), suggesting specificity of this sequence. We also examined the expression of several reported miR-21 targets in the microarray data or by Western blotting, including PTEN, Pdcd4, Bcl-2, TMP1, Spry 1 and Spry 2 (Table S4 and Fig. S4B). Only Spry 1 and Spry 2 appear to be significantly upregulated (1.8- and 1.44-fold) in miR-21 KO cells, but not the other 3 genes (Table S4 and Fig. S4B). Together, these results indicate that miR-21 regulates Cdc25A through the miR-21 binding site in its 3′UTR, and establish Cdc25A as a direct target of miR-21.

An external file that holds a picture, illustration, etc.
Object name is nihms143917f4.jpg

Figure 4

The conserved miR-21 binding site in the 3′UTR of Cdc25A.

A. Schematic representation of CDC25A transcript with its 3′ UTR. The predicted miR-21 binding sites in the Cdc25A gene of 5 species are shown with miR-21 targeting sequences aligned (GenBank accession numbers are {"type":"entrez-nucleotide","attrs":{"text":"NM_007658","term_id":"92373435","term_text":"NM_007658"}}NM_007658, {"type":"entrez-nucleotide","attrs":{"text":"NM_133571","term_id":"19424257","term_text":"NM_133571"}}NM_133571, {"type":"entrez-nucleotide","attrs":{"text":"XR_014086","term_id":"109040929","term_text":"XR_014086"}}XR_014086, {"type":"entrez-nucleotide","attrs":{"text":"XM_001155610","term_id":"1034156438","term_text":"XM_001155610"}}XM_001155610, and {"type":"entrez-nucleotide","attrs":{"text":"NM_201567","term_id":"1675177798","term_text":"NM_201567"}}NM_201567, respectively). The base pairing nucleotides are in bold. B. Activities of the Luc-Cdc25A-UTR reporter or miR-21 binding site mutated Luc-Cdc25A-MUT-UTR reporter in RKO wild-type and miR-21 KO cells with or without precursor miR-21 transfection. Values are means ± SD, n=3. *P<0.05, **P>0.1. The mutated nucleotides in the Luc-Cdc25A-MUT-UTR reporter are underlined in A.

miR-21 inhibits cell proliferation following serum starvation and delays G1-S transition through Cdc25A

Cdc25A is an important regulator of cell cycle progression during G1–S transition (36, 37). To evaluate whether miR-21 affects cell cycle progression, we compared the growth rate of parental and miR-21 KO cells under normal serum (10%) and low-serum condition (0.5%) over a course of 7 days. The growth rate of parental and miR-21 KO cells was indistinguishable under the normal serum condition in the entire 7 days (Fig. S4C). However, RKO miR-21 KO cells exhibited enhanced proliferation over WT cells in the low serum condition (Fig. 5A, top panel). Under these conditions, no significant levels of apoptosis were detected in either WT or miR-21 KO cells (data not shown). Using quantitative RT-PCR, we found that miR-21 levels were induced 2–10 fold by serum starvation in WT cells within 24 hours (Fig. 5A, middle panel). Serum starvation also caused an apparent reduction in Cdc25A levels, which was significantly blunted in miR-21 KO cells (Fig. 5A, bottom panel). The induction of miR-21 in parental cells, and elevated Cdc25A and enhanced proliferation in miR-21 KO cells were also observed following complete serum starvation (0% serum) (data now shown).

An external file that holds a picture, illustration, etc.
Object name is nihms143917f5.jpg

Figure 5

miR-21 modulates cell proliferation under low serum conditions and G1-S transition through Cdc25A

A. The indicated cells were cultured in medium containing 0.5% serum medium for 7 days. The cell numbers were determined by counting. The levels of miR-21 and Cdc25A were determined by realtime RT-PCR and Western blotting, respectively. The levels of mature miR-21 expression were normalized to those of U6. The cells cultured in 10% serum for one day were used as controls. Values are means ± SD, n=3. B. The indicated RKO cells were cultured in serum free medium for 48 h and then stimulated with 10% fetal calf serum. Cell cycle analysis was performed using flow cytometry. The increases in the fraction of S-phase cells at the indicated time points compared with 0 h were quantified. C. BrdU incorporation in parental and miR-21 KO cells with or without precursor miR-21 transfection. Cells were subjected to treatment as in B and pulse labeled with BrdU for 15 minutes. BrdU/DAPI staining was performed at 15 h following 10% serum stimulation. The percentage of BrdU positive cells were scored by fluorescence microscopy (top panel). Values are means ± SD, n=3. *P<0.05. BrdU incorporation was also determined by flow cytometry (low panel). D. The effects of pre-miR-21 on BrdU incorporation were analyzed as in C. Values are means ± SD, n=3. *P<0.05. The effects of Pre-miR-21 on Cdc25A levels at 16 h following serum stimulation were analyzed by Western blotting in the indicated cell lines.

Since serum has been well documented to stimulate the G1-S transition, we therefore specifically evaluated a potential role of miR-21 in this process (17, 27, 38). Parental and miR-21 KO RKO cells were serum starved for 48 hours and subsequently stimulated with 10% fetal calf serum. The cell cycle profiles were followed by flow cytometry in a time course experiment. Serum addition induced a higher degree of S phase entry in miR-21 KO cells compared with parental RKO cells. The effects were most pronounced at 15 (17 % vs. 7%) and 16 (16% vs. 10%) hours, and gradually diminished (Fig. 5B). BrdU staining indicated increased DNA synthesis in miR-21 KO cells compared to parental cells (Figs. 5C and S5A). Consistent with an accelerated entry into S phase, high levels of Cdc25A were detected in miR-21 KO cells as early as 15 hours (Fig. S5B). Transfection of pre-miR-21 significantly reduced DNA synthesis and Cdc25A levels in miR-21 KO cells (Fig. 5D). No significant difference in the cell cycle distribution was found between WT or miR-21 KO RKO or DLD1 cells growing in log phase with 10% serum (Fig. S5C). Taken together, these results indicate that miR-21 induction inhibits the G1-S transition by suppressing Cdc25A expression.

miR-21 modulates DNA damage-induced G2/M checkpoint through Cdc25A

Cdc25A has been shown to regulate the G2-M transition and its inactivation is critically involved in establishing a G2/M checkpoint following γ-irradiation (17, 27, 38). We tested whether miR-21 is involved in establishing a transient G2/M checkpoint 1 hour following irradiation by analyzing phospho-histone-H3 (p-H3) positive cells (39). As expected, radiation (12 Gy) induced an over 90% drop in mitotic cells, which was significantly inhibited in miR-21 knockout RKO cells (Fig 6A, S6A). Using quantitative RT-PCR, we found that miR-21 levels were induced in parental RKO cells within one hour of γ-irradiation (Fig. 6A, right panel). miR-21 KO DLD1 cells also exhibited a defective G2/M checkpoint associated with elevated levels of Cdc25A (Fig. S6B and S6C). Transfection of pre-miR-21 or Cdc25A siRNA significantly suppressed the fraction of mitotic cells in both WT and miR-21 KO cells, but the inhibition was more pronounced in miR-21 KO cells (Fig. 6B). Transfection of anti-miR-21 only elevated the fraction of mitotic cells in WT cells but did not further elevated that in miR-21 KO cells (Fig. 6B). Cdc25A levels rapidly decreased following radiation in both parental and KO cells, but were substantially higher in miR-21 KO cells (Fig. 6C). As expected, transfection of pre-miR-21 or Cdc25A siRNA decreased the levels of Cdc25A while anti-miR-21 elevated those in WT cells (Fig. 6C).

An external file that holds a picture, illustration, etc.
Object name is nihms143917f6.jpg

Figure 6

miR-21 modulates irradiation-induced G2/M checkpoint through Cdc25A

A. The RKO cell lines were harvested at 1 h after 12 Gy irradiation (IR). The cells were stained with phospho-histone H3 (Ser10) antibody and the nuclei were counterstained by propidium iodide (PI). The fractions of p-H3 positive cells were analyzed by flow cytometry and plotted. Values are means ± SD, n=3. *P<0.05. The levels of miR-21 normalized to those of U6 at indicated time points after IR were determined by realtime RT-PCR. Values are means ± SD, n=3. B. The indicated RKO cells were transfected with control, pre-miR-21, anti-miR-21 or Cdc25A siRNA twice in 48 h. The cells were replated overnight prior to irradiation at 12Gy, and analyzed as in A. Values are means ± SD, n=3. *P<0.05. C. The levels of Cdc25A were analyzed in indicated cells following indicated treatments as in B by Western blotting, and quantitated by densitometry. D. The indicated cells were irradiated at three doses of IR. The clonogenic survival was quantified using colony formation assay (left panel). Values are means ± SD, n=3. Representative pictures for colony enumeration are shown in the right panel with the number of cells plated noted.

To determine whether miR-21 affects radiosensitivity, we evaluated the clonogenic survival of parental and miR-21 KO RKO cells. miR-21 KO RKO cells were found to have increased clonogenic survival following several doses of irradiation (Fig. 6D). miR-21 has previously been reported to regulate cell proliferation and apoptosis in glioblastoma and breast cancer cells (9, 13, 40). However, miR-21 did not appear to affect apoptosis induced by a variety of anticancer agents in either RKO or DLD1 cells (Fig. S4D and data not shown). The regulation of Cdc25A by miR-21 appears to be independent of the tumor suppressor p53, as it occurs in both p53 wild-type RKO cells and p53 mutant DLD1 cells. The above results suggest that miR-2-mediated downregulation of Cdc25A contributes to the activation of the G2/M checkpoint following radiation.

Targeted deletion of miR-21 in colon cancer cells

Figure 1

Targeted deletion of the primary miR-21 locus in RKO and DLD1 colon cancer cells

Identification of potential miR-21 targets through microarray analysis

Cdc25A expression is regulated by miR-21 in colon cancer cells

Figure 2

Cdc25A is regulated by miR-21 in colon cancer cells

Figure 3

The miR-21 levels in Cdc25A overexpressing colon cancers

Cdc25A is a miR-21 target

Figure 4

The conserved miR-21 binding site in the 3′UTR of Cdc25A.

miR-21 inhibits cell proliferation following serum starvation and delays G1-S transition through Cdc25A

Figure 5

miR-21 modulates cell proliferation under low serum conditions and G1-S transition through Cdc25A

miR-21 modulates DNA damage-induced G2/M checkpoint through Cdc25A

Figure 6

miR-21 modulates irradiation-induced G2/M checkpoint through Cdc25A

Discussion

Our study provides a novel function of miR-21 in regulating cell cycle progression and checkpoint activation through Cdc25A in colon cancer cells. This conclusion is supported by several lines of evidence: increased expression of Cdc25A in miR-21 KO RKO and DLD1 cells that is suppressed by expression of pre-miR-21; a putative miR-21 binding site in the 3′UTR that is subject to miR-21 regulation; the induction of miR-21 by serum starvation and DNA damage, accelerated G1-S transition in miR-21 KO cells; and compromised G2/M checkpoint in response to γ-irradiation, all of which were partially rescued by pre-miR-21 or Cdc25A knockdown.

The major mechanism of rapid turnover of Cdc25 family proteins is regulated by ubiquitin-mediated proteolysis (19). Our findings suggest that the full extent of Cdc25A inactivation requires miR-21 in colon cancer cells, which represents a novel mechanism of Cdc25A mRNA turnover. An involvement of miR-21 in cell cycle progression following stress is supported by several recent studies, as it was induced by the chemotherapeutic drug 5-Fluorouracil (5-FU) in colon cancer cells (41), and by UV irradiation in primary fibroblasts (42) or in colon cancer cells (Fig. S6D). Cdc25A contains a large number of phosphorylation sites recognized by CDK1, Chk1/Chk2, and p38 (17, 19). However, extensive effort in the mapping of phosphorylation sites in Cdc25Aand the use of cells deficient in Chk2 or ATM indicate that many such sites are not required for Cdc25A-mediated G2/M checkpoint following DNA damage (20, 43–45). Our data suggest that miR-21-mediated Cdc25A downregulation facilitates the rapid establishment of the G2/M checkpoint following DNA damage. Interestingly, the elevated Cdc25A levels in unstressed miR-21 KO cells do not appear to affect proliferation, but profoundly affect cell cycle checkpoint and progression following stress (ie. DNA damage or serum starvation), suggesting the importance of fully inactivating Cdc25Aunder these conditions. These conditional phenotypes associated with miR-21 might be particularly relevant as growth factor deprivation and DNA damage have been shown to play important roles in tumorigenesis (46). In addition, Cdc25A was recently found to be a target of miR-16 that participates in UV-induced DNA damage response (42). Taken together, these observations suggest that critical cell cycle regulators such as Cdc25A are subject to modulation by microRNAs.

Our data provide a novel mechanism of how miR-21 could potentially contribute to tumorigenesis by compromising cell cycle progression and DNA damage-induced checkpoint function under those conditions, which can lead to chromosomal instability that promotes tumorigenesis (47). The cell cycle is composed of highly regulated machinery; the precise coordination of a timely entry into and exit from various stages during normal cell cycle is crucial for maintaining normal cell division that entails faithful DNA replication and segregation. In addition, most, if not all, of the cells in the human body are constantly encountering endogenous or exogenous insults that can damage DNA, and proper activation of checkpoints and recovery from them is probably just as important in ensuring genome integrity. Altered expression of miR-21 can conceivably cause genomic instability and lead to oncogenesis by relaxing or tightening this engine driving cell cycle through Cdc25A-dependent activation of cyclin/CDK complexes, and may also impact therapeutic responses. Similar to Chk2-deficient cells (48), miR-21 KO cells exhibit compromised checkpoint and radioresistance. Given the complexity of the regulation of miRNA targets, much work remains to define and characterize miR-21 targets to better understand its biology in different tissues and cancer. Therefore, future work will determine whether miR-21 affects chromosomal stability following DNA damage and other aspects of tumor biology through novel targets.

Overexpression of Cdc25A and Cdc25B is correlated with more aggressive disease and poor prognosis in some cancer patients (19). The reasons for Cdc25A overexpression are still not clear. Our data offer reduced miR-21 expression as a plausible explanation of Cdc25A overexpression in perhaps a subset of colon cancers. Other factors such as overexpression of c-Myc and E2F, or inactivation of glycogen synthase kinase-3 beta (GSK-3 beta) are likey to be involved (19, 49). It is established that Cdc25A activities are tightly regulated by multiple mechanisms during cell cycle, including inhibitory and activating phosphorylation, changes in intracellular localization, and interactions with other proteins (17). Given a central role of Cdc25A in regulating cell cycle progression, it is perhaps not surprising that additional mechanisms such as miR-16 (42) can fine tune its activity or levels.

Lastly, miR-21 appears to regulate a distinct set of genes and have a limited role in regulating anticancer drug-induced apoptosis in colon cancer cells. The discrepancies in targets identified by different groups are perhaps not surprising, as miRNAs are known to regulate targets in a tissue- and cell type-specific manner (6). It is also possible that some of these targets are primarily regulated by miR-21 at the level of translation. The miR-21 targeted cells and the targeting vector established in this study should be very useful for further dissecting miR-21 biology.

Supplementary Material

1

Click here to view.^{(732K, pdf)}

1

Click here to view.^{(732K, pdf)}

Acknowledgments

We thank other members of our laboratories and Dr. Edward V. Prochownik at University of Pittsburgh for helpful discussion and comments, and Hongtao Liu for technical assistance. We also thank Dr. Jianhua Luo and the Microarray Core facility at University of Pittsburgh School of Medicine for gene expression analysis. This work was supported by Flight Attendant Medical Research Institute (FAMRI), the Alliance for Cancer Gene Therapy (ACGT) (J. Yu), and NIH grant 1R01CA129829, U19-A1068021 (pilot project) (J. Yu), {"type":"entrez-nucleotide","attrs":{"text":"CA106348","term_id":"34959655","term_text":"CA106348"}}CA106348, {"type":"entrez-nucleotide","attrs":{"text":"CA121105","term_id":"34974413","term_text":"CA121105"}}CA121105 (L. Zhang), {"type":"entrez-nucleotide","attrs":{"text":"CA127590","term_id":"35007624","term_text":"CA127590"}}CA127590, U54 {"type":"entrez-nucleotide","attrs":{"text":"CA116867","term_id":"34970174","term_text":"CA116867"}}CA116867 (Z. Wang), and American Cancer Society grant RSG-07-156-01-CNE (L. Zhang). L. Zhang and Z. Wang are V scholars.

^{University of Pittsburgh Cancer Institute, and Departments of Pathology and Pharmacology and Chemical Biology, University of Pittsburgh, 5117 Centre Ave., Pittsburgh, PA 15213, USA}

^{Department of Genetics and Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106}

Correspondence: Jian Yu, Ph.D. Hillman Cancer Center Research Pavilion, Suite 2.26h, 5117 Centre Ave, Pittsburgh, PA 15213. ude.cmpu@2juy; Phone: 412-623-7786; Fax: 412-623-7778

^{These authors contributed equally to this work.}

^{Current affiliation-Department of Biology, Sichuan University, Chengdu, P. R. China 610064}

Abstract

MicroRNAs are small non-coding RNAs that participate in diverse biological processes by suppressing target gene expression. Altered expression of miR-21 has been reported in cancer. To gain insights in its potential role in tumorigenesis, we generated miR-21 knockout colon cancer cells through gene targeting. Unbiased microarray analysis combined with bioinformatics identified cell cycle regulator Cdc25A as a miR-21 target. miR-21 suppressed Cdc25A expression through a defined sequence in its 3′UTR. We found that miR-21 is induced by serum starvation and DNA damage, negatively regulates G1-S transition, and participates in DNA damage-induced G2-M checkpoint through downregulation of Cdc25A. In contrast, miR-21 deficiency did not affect apoptosis induced by a variety of commonly used anticancer agents or cell proliferation under normal cell culture conditions. Furthermore, miR-21 was found to be underexpressed in a subset of Cdc25A overexpressing colon cancers. Our data demonstrated a role of miR-21 in modulating cell cycle progression following stress, providing a novel mechanism of Cdc25A regulation and a potential explanation of miR-21 in tumorigenesis.

Keywords: MicroRNA-21, Cdc25A, cell cycle, DNA damage, colon cancer

Abstract

References

1. Bartel DPMicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97.[PubMed][Google Scholar]
2. Kloosterman WP, Plasterk RHThe diverse functions of microRNAs in animal development and disease. Dev Cell. 2006;11:441–50.[PubMed][Google Scholar]
3. Lu J, Getz G, Miska EA, et al MicroRNA expression profiles classify human cancers. Nature. 2005;435:834–8.[PubMed][Google Scholar]
4. Calin GA, Dumitru CD, Shimizu M, et al Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 2002;99:15524–9.[Google Scholar]
5. Nairz K, Rottig C, Rintelen F, Zdobnov E, Moser M, Hafen EOvergrowth caused by misexpression of a microRNA with dispensable wild-type function. Dev Biol. 2006;291:314–24.[PubMed][Google Scholar]
6. Calin GA, Croce CMMicroRNA signatures in human cancers. Nat Rev Cancer. 2006;6:857–66.[PubMed][Google Scholar]
7. Chen CZMicroRNAs as oncogenes and tumor suppressors. N Engl J Med. 2005;353:1768–71.[PubMed][Google Scholar]
8. Gaur A, Jewell DA, Liang Y, et al Characterization of microRNA expression levels and their biological correlates in human cancer cell lines. Cancer Res. 2007;67:2456–68.[PubMed][Google Scholar]
9. Chan JA, Krichevsky AM, Kosik KSMicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res. 2005;65:6029–33.[PubMed][Google Scholar]
10. Volinia S, Calin GA, Liu CG, et al A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A. 2006;103:2257–61.[Google Scholar]
11. Schetter AJ, Leung SY, Sohn JJ, et al MicroRNA expression profiles associated with prognosis and therapeutic outcome in colon adenocarcinoma. Jama. 2008;299:425–36.[Google Scholar]
12. Asangani IA, Rasheed SA, Nikolova DA, et al MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene. 2008;27:2128–36.[PubMed][Google Scholar]
13. Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel TMicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology. 2007;133:647–58.[Google Scholar]
14. Zhu S, Si ML, Wu H, Mo YYMicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1) J Biol Chem. 2007;282:14328–36.[PubMed][Google Scholar]
15. Sayed D, Rane S, Lypowy J, et al MicroRNA-21 targets Sprouty2 and promotes cellular outgrowths. Mol Biol Cell. 2008;19:3272–82.[Google Scholar]
16. Thum T, Gross C, Fiedler J, et al MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature. 2008;456:980–4.[PubMed][Google Scholar]
17. Boutros R, Dozier C, Ducommun BThe when and wheres of CDC25 phosphatases. Curr Opin Cell Biol. 2006;18:185–91.[PubMed][Google Scholar]
18. Ray D, Kiyokawa HCDC25A phosphatase: a rate-limiting oncogene that determines genomic stability. Cancer Res. 2008;68:1251–3.[PubMed][Google Scholar]
19. Boutros R, Lobjois V, Ducommun BCDC25 phosphatases in cancer cells: key players? Good targets? Nat Rev Cancer. 2007;7:495–507.[PubMed][Google Scholar]
20. Jin J, Ang XL, Ye X, Livingstone M, Harper JWDifferential roles for checkpoint kinases in DNA damage-dependent degradation of the Cdc25A protein phosphatase. J Biol Chem. 2008;283:19322–8.[Google Scholar]
21. Rago C, Vogelstein B, Bunz FGenetic knockouts and knockins in human somatic cells. Nat Protoc. 2007;2:2734–46.[PubMed][Google Scholar]
22. Wang P, Yu J, Zhang LThe nuclear function of p53 is required for PUMA-mediated apoptosis induced by DNA damage. Proc Natl Acad Sci U S A. 2007;104:4054–9.[Google Scholar]
23. Zhang X, Guo C, Chen Y, et al Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies. Nat Methods. 2008;5:163–5.[Google Scholar]
24. Yu J, Wang Z, Kinzler KW, Vogelstein B, Zhang LPUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci U S A. 2003;100:1931–6.[Google Scholar]
25. Luo JH, Yu YP, Cieply K, et al Gene expression analysis of prostate cancers. Mol Carcinog. 2002;33:25–35.[PubMed][Google Scholar]
26. Yue W, Sun Q, Dacic S, et al Downregulation of Dkk3 activates beta-catenin/TCF-4 signaling in lung cancer. Carcinogenesis. 2008;29:84–92.[PubMed][Google Scholar]
27. Mailand N, Podtelejnikov AV, Groth A, Mann M, Bartek J, Lukas JRegulation of G(2)/M events by Cdc25A through phosphorylation-dependent modulation of its stability. Embo J. 2002;21:5911–20.[Google Scholar]
28. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein BPUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell. 2001;7:673–82.[PubMed][Google Scholar]
29. Yu J, Wang P, Ming L, Wood MA, Zhang LSMAC/Diablo mediates the proapoptotic function of PUMA by regulating PUMA-induced mitochondrial events. Oncogene. 2007;26:4189–98.[PubMed][Google Scholar]
30. Sun Q, Sakaida T, Yue W, Gollin SM, Yu JChemosensitization of head and neck cancer cells by PUMA. Mol Cancer Ther. 2007;6:3180–8.[PubMed][Google Scholar]
31. Zhang B, Pan X, Cobb GP, Anderson TAPlant microRNA: a small regulatory molecule with big impact. Dev Biol. 2006;289:3–16.[PubMed][Google Scholar]
32. Cummins JM, He Y, Leary RJ, et al The colorectal microRNAome. Proc Natl Acad Sci U S A. 2006;103:3687–92.[Google Scholar]
33. Doench JG, Sharp PASpecificity of microRNA target selection in translational repression. Genes Dev. 2004;18:504–11.[Google Scholar]
34. Wang X, Wang XSystematic identification of microRNA functions by combining target prediction and expression profiling. Nucleic Acids Res. 2006;34:1646–52.[Google Scholar]
35. Zhang L, Zhou W, Velculescu VE, et al Gene Expression Profiles in Normal and Cancer Cells. Science. 1997;276:1268–72.[PubMed][Google Scholar]
36. Blomberg I, Hoffmann IEctopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin E- and cyclin A-dependent kinases. Mol Cell Biol. 1999;19:6183–94.[Google Scholar]
37. Hoffmann I, Draetta G, Karsenti EActivation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. Embo J. 1994;13:4302–10.[Google Scholar]
38. Sexl V, Diehl JA, Sherr CJ, Ashmun R, Beach D, Roussel MFA rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2. Oncogene. 1999;18:573–82.[PubMed][Google Scholar]
39. Xu B, Kim ST, Lim DS, Kastan MBTwo molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol. 2002;22:1049–59.[Google Scholar]
40. Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YYmiR-21-mediated tumor growth. Oncogene. 2007;26:2799–803.[PubMed][Google Scholar]
41. Rossi L, Bonmassar E, Faraoni IModification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro. Pharmacol Res. 2007;56:248–53.[PubMed][Google Scholar]
42. Pothof J, Verkaik NS, van Ijcken W, et al MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response. Embo J. 2009[Google Scholar]
43. Goloudina A, Yamaguchi H, Chervyakova DB, Appella E, Fornace AJ, Jr, Bulavin DVRegulation of human Cdc25A stability by Serine 75 phosphorylation is not sufficient to activate a S phase checkpoint. Cell Cycle. 2003;2:473–8.[PubMed][Google Scholar]
44. Goldstone S, Pavey S, Forrest A, Sinnamon J, Gabrielli BCdc25-dependent activation of cyclin A/cdk2 is blocked in G2 phase arrested cells independently of ATM/ATR. Oncogene. 2001;20:921–32.[PubMed][Google Scholar]
45. Jallepalli PV, Lengauer C, Vogelstein B, Bunz FThe Chk2 tumor suppressor is not required for p53 responses in human cancer cells. J Biol Chem. 2003;278:20475–9.[PubMed][Google Scholar]
46. Hanahan D, Weinberg RAThe hallmarks of cancer. Cell. 2000;100:57–70.[PubMed][Google Scholar]
47. Lengauer C, Kinzler KW, Vogelstein BGenetic instabilities in human cancers. Nature. 1998;396:643–9.[PubMed][Google Scholar]
48. Takai H, Naka K, Okada Y, et al Chk2-deficient mice exhibit radioresistance and defective p53-mediated transcription. Embo J. 2002;21:5195–205.[Google Scholar]
49. Kang T, Wei Y, Honaker Y, et al GSK-3 beta targets Cdc25A for ubiquitin-mediated proteolysis, and GSK-3 beta inactivation correlates with Cdc25A overproduction in human cancers. Cancer Cell. 2008;13:36–47.[Google Scholar]