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

Abstract

microRNAs (miRNA) are small noncoding 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 into 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'-untranslated region. We found that miR-21 is induced by serum starvation and DNA damage, negatively regulates G(1)-S transition, and participates in DNA damage-induced G(2)-M checkpoint through down-regulation 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 show 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.

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.

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.

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.

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 NM_007658, NM_133571, XR_014086, XM_001155610, and 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.

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.

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.