Human CRC-derived cell lines, including HT-29, Lovo, SW-480, Caco2, and control human umbilical vein endothelial cells (HUVECs), provided by Shanghai Institutes For Biological Science, CAS, were resuscitated routinely, resuspended with RPMI-1640 supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, UT, United States), 100 kU/mL penicillin G and 100 g/L streptomycin, and then planted in a 25-cm² culture bottle and incubated in a 5% CO₂ humidified atmosphere at 37 °C. The media were changed every 3 d and the cells were trypsinized using trypsin/edetic acid when they reached 80%-90% confluence. Cells aged at passages 4-8 were used for the experiments. The day before transfection, cells were seeded in antibiotic-free medium. Cells (1 × 10⁴/well) were seeded in a 96-well plate, incubated for 24 h to allow them attach to the bottom of the well, and then transfected with 50 nmol/L negative control, pre-miR-221 or anti-miR-221 oligonucleotides, respectively (Shanghai GenePharma, Shanghai, China). Transfection of miRNAs was carried out using Lipofectamine 2000 in accordance with the manufacturer’s procedure (Invitrogen, Carlsbad, CA, United States). The above experiment was repeated at least three times.

The status of cell proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl trozolium bromide (MTT; Amresco, Solon, OH, United States) assay. In short, exponentially growing CRC cells were adjusted to 1.5 × 10⁴ cells/mL with RPMI-1640, planted in 96-well plates (Corning, Corning, NY, United States) at 200 μL/well, and incubated for 12 h. After transfection with 50 nmol/L pre-miR-221 or anti-miR-221 and incubation for 48 h (five duplicate wells for each sample), 20 μL/well MTT (5 g/L) was added to each well. The medium was removed after 4 h incubation and 100 μL/well dimethylsulfoxide was added to dissolve the reduced formazan product. Finally, the plate was read in an enzyme-linked immunity implement (Bio-Rad 2550, Hercules, CA, United States) at 490 nm. Cellular proliferation inhibition rate (CPIR) was calculated using the following formula: CPIR = (1 - average A value of experimental group/average A value of control group) × 100%. The above experiment was repeated at least three times.

The analysis of miR-221-predicted targets was performed using the algorithms TargetScan (http://targetscan.org/), PicTar (http://pictar.mdc-berlin.de/) and MiRanda (http://www.microrna.org/microrna/home.do).

The human 3′-untranslated region (UTR) of the PTEN gene was amplified by PCR using the primers 5’-CGATTCTAGAAATCATGTTCTGGTGG-3’ for PTEN-3’-UTR-Forward and 5’-GCATTCTAGAATTCTGCACAGTAAGCATA-3’ for PTEN-3’-UTR-Reverse and cloned into the XbaI site of the pGL3-control vector (Promega, Madison, WI, United States), downstream of the luciferase gene, to generate the vector pGL3-PTEN. For luciferase assay, the CRC cells were cultured in 24-well plates and transfected with 500 ng of either pGL3-PTEN or pGL3-control vector and 50 pmol pre-miR-221, anti-miR-221 or negative control. Transfection was performed using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. At 24 h after transfection, firefly luciferase activity was measured using the Dual Luciferase Reporter Assay (Promega, Madison, WI, United States). The above experiment was repeated at least three times.

Irradiation was performed at room temperature in a linear accelerator (Varian 600; Palo Alto, CA, United States) at a dose rate of 3.2 Gy/min. Monolayer cells were plated into six-well plates, placed 100 cm from the source, and exposed to the specified dose (0, 2, 4, 6 and 8 Gy) of X-rays[14].

Total RNA was extracted with routine Trizol reagent (Invitrogen). The precipitation was dissolved in diethylpyrocarbonate-treated water. Nucleic acid protein analyzer (Beckman Coulter, Fullerton, CA, United States) was used to determine RNA concentration. The purity and integrity of RNA were identified by two aspects: A_260nm/A_280nm≥ 1.8, and a band ratio of 28S to 18S RNA ≥ 1.5 in formaldehyde denaturing gel electrophoresis. miR-221 and PTEN mRNA was quantified as described previously[10]. The comparative 2^-ΔΔCT method was used for relative quantification and statistical analysis. The above experiment was repeated at least three times.

The cells were rinsed twice with cold phosphate buffered solution (PBS) buffer, and were then lysed in an ice-cold lysis buffer containing 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.6), 0.1% SDS, 1% Nonidet P-40, and protease inhibitor cocktail (Boehringer Mannheim, Lewes, Sussex, United Kingdom). The samples were cleared by centrifugation at 13000 g for 10 min. Fifty micrograms of protein from the tissue was subjected to SDS-PAGE and electrotransferred to polyvinylidine fluoride membranes (Immobilon, Bedford, MA). After blocking in 20 mmol/L Tris-HCl, pH 7.6 (containing 150 mmol/L NaCl, 0.1% Tween-20, and 5% nonfat dry milk), membranes were incubated with primary antibodies against target protein or β-actin (used as a sample loading control) overnight at 4 °C and then incubated with horseradish-peroxidase-conjugated secondary antibody. The blot was developed using the ECL detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, United States) according to the manufacturer’s instructions and the protein imprinting band was obtained. The above experiment was repeated at least three times.

At 24 h after irradiation, all cells were trypsinized and counted. Corresponding numbers of cells were seeded into 10-cm dishes containing RPMI-1640 supplemented with 10% FBS, in triplicate, incubated for 14 d to allow colony growth, and colonies were stained with crystal violet. Colonies containing ≥ 50 cells were counted. The plating efficiency was calculated by dividing the average number of colonies per dish by the number of cells plated. Survival fractions were calculated by normalization to the plating efficiency of appropriate control groups. The above experiment was repeated at least three times.

The effects of miR-221 and irradiation on CRC cell death were examined by flow cytometry. Pretreated CRC cells were harvested and washed twice with PBS, fixed with 70% ethanol at -20 °C for 30 min, and stored at 4 °C overnight, then washed with PBS again, treated with 100 mL 100 mg/L RNase at 37 °C for 30 min, and stained with 100 mL 50 mg/L propidium iodide at 4 °C for 30 min in the dark. The multiplication cycle and apoptotic rate were assayed using flow cytometry, and the data were analyzed using CellQuest software. The percentages of necrotic and apoptotic cells were measured by calculating the ratio of the number of corresponding cells to the number of total cells. For each sample, 10000 cells were measured.

All data in the experiment are presented as mean ± SD. Comparisons between groups were analyzed with one-way ANOVA and Student-Newman-Keuls Q test using SPSS version 15.0 (SPSS, Chicago, IL, United States). P < 0.05 was considered to be statistically significant.

To study the expression pattern of miR-221 in CRC cells, we performed real-time RT-PCR to detect miR-221 expression in four CRC-derived cell lines. The expression values of miR-221 in HT-29, Lovo, SW-480 and Caco2 were 4.094 ± 0.208, 1.122 ± 0.138, 3.927 ± 0.232 and 1.831 ± 0.149, respectively. A significant overexpression of miR-221 was observed in all four CRC cell lines relative to HUVEC (0.223 ± 0.047, P < 0.01). The Caco2 cell line was chosen for both pre-miR-221 and anti-miR-221 transfection in the successive experiment because it exhibits, among the four cell lines tested, an intermediate miR-221 expression level.

To determine the biological impact of miR-221 in the CRC-derived cell line, Caco2 cells were transfected with pre-miR-221 or anti-miR-221 to increase or reduce miR-221 level, respectively. Real-time RT-PCR analysis revealed that introduction of pre-miR-221 caused a significant increase of miR-221 value; conversely, anti-miR-221 caused a significant decrease of miR-221 value (Figure 1A, P < 0.01). These strategies were then used as the basis of the remaining experiments.

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Figure 1 Modulation of miR-221 expression and cell proliferation in Caco2 cells. A: Expression of miR-221 was detected by real-time RT-PCR. Expression of U6 snRNA was used as internal control; B: The status of cell proliferation was determined by MTT assay. CPIR in the presence of pre-miR-221 or anti-miR-221 was compared with those of controls. n = 6, mean ± SD; ^bP < 0.01 vs control group.

We further tested whether the cell proliferation potential of the transfected CRC cells was modified and the status of cell proliferation was determined by MTT assay. We observed a significant increase in proliferation after transfection of pre-miR-221. In contrast, anti-miR-221 significantly decreased cell proliferation (Figure 1B, P < 0.01). These data indicate that cell proliferation can be significantly enhanced by increase of miR-221 expression, a result which strongly supports the potential oncogenic activity of miR-221 in CRC.

Most miRs are thought to control gene expression by base-pairing with the miR-recognizing elements found in their messenger target. We utilized all three currently available major prediction programs, TargetScan, Miranda and PicTar, to analyze the potential interactions between miR-221 and PTEN. All these algorithms reveal a potential miR-221 target site in the PTEN mRNA 3’-UTR region (Figure 2A). To demonstrate the direct interaction between miR-221 and PTEN mRNA, we cloned PTEN-3’-UTR segment, which includes a potential target site for miR-221, downstream of the pGL3 luciferase reporter gene, to generate the pGL3-PTEN vector. This vector was cotransfected into Caco2 cells together with pre-miR-221 or anti-miR-221. Luciferase activity in Caco2 cells cotransfected with pGL3-PTEN vector and miR-221 was decreased markedly compared with negative controls. On the contrary, luciferase activity in Caco2 cells transfected with anti-miR-221 was increased significantly compared with negative controls (Figure 2B). These results support the bioinformatic prediction indicating the 3’-UTR of PTEN mRNA as a target for miR-221.

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Figure 2 miR-221 regulated expression of phosphatase and tensin homolog deleted on chromosome 10 in colorectal carcinoma-derived cells. A: Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) 3’-UTR site potentially targeted by miR-221 as predicted by Miranda; B: Luciferase activity assay showing direct interaction between miR-221 and PTEN 3’-UTR site. Firefly luciferase reporter activity in the presence of both pGL3-PTEN vector and pre-miR-221 or anti-miR-221 was compared with that of the controls. Luciferase activity in Caco2 cells cotransfected with pGL3-PTEN vector and pre-miR-221 was decreased markedly compared with the negative control. Luciferase activity in Caco2 cells transfected with anti-miR-221 was increased significantly compared with the negative control, ^bP < 0.01 vs control group; C: Western blotting showing PTEN protein expression in Caco2 cells transfected with pre-miR-221 or anti-miR-221. β-actin was used as a housekeeping gene to normalize PTEN protein expression. The relative PTEN protein levels normalized against β-actin are shown at the top of each panel; D: Real-time RT-PCR analysis showing PTEN mRNA expression in Caco2 cells transfected with pre-miR-221 or anti-miR-221. All the results are representative of three independent experiments.

To check whether miR-221 actually affects PTEN expression in CRC cells, we analyzed the consequence of the ectopic expression of miR-221. We transfected the pre-miR-221 or anti-miR-221 into Caco2 cells, and we searched for changes in PTEN protein levels by Western blotting. Introduction of miR-221 caused a significant increase of miR-221 value and decreased PTEN protein levels. Conversely, anti-miR-221 caused a significant decrease of miR-221 value and increased PTEN protein amounts (Figure 2C). No significant changes in the PTEN mRNA levels were observed in the cells either transfected with miR-221 or anti-miR-221 (Figure 2D). This result strongly validates a post-transcriptional regulation of PTEN protein by miR-221, and also excludes its role in PTEN mRNA degradation.

To determine whether miR-221 and PTEN were involved in the cellular response to radiotherapy in CRC, Caco2 cells were exposed to different doses of X-rays to observe the regular pattern of miR-221 and PTEN. The radiation dose had a significant effect on the expression of miR-221 and PTEN protein in human Caco2 cells in a dose-dependent manner. The miR-221 expression level improved gradually with the increase in radiation dose, while the PTEN protein expression level reduced gradually (Figure 3A and B). However, no significant changes in the PTEN mRNA levels were observed in the cells exposed to irradiation (Figure 3C). All these results gave us a first hint that the expression of miR-221 might be one of the mechanisms acting to regulate radiosensitivity in CRC cells negatively.

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Figure 3 Effects of X-ray dose on miR-221 and phosphatase and tensin homolog deleted on chromosome 10 expression in colorectal carcinoma-derived cells. A: Expression of miR-221 was detected by real-time RT-PCR. The miR-221 expression level improved in a dose-dependent manner with the increase in radiation dose, ^bP < 0.01 vs control group; B: Expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein was detected by Western blotting, β-actin was used as a housekeeping gene to normalize PTEN protein expression. The relative PTEN protein levels normalized against β-actin are shown at the top of each panel. The expression of PTEN protein was decreased in a dose-dependent manner with the increase in radiation dose; C: Expression of PTEN mRNA was detected by real-time RT-PCR. No significant changes in the PTEN mRNA levels were observed in the cells exposed to irradiation. The results are representative of three independent experiments.

To determine whether miR-221 affected Caco2 cell radiosensitivity, cells were transfected with pre-miR-221 or anti-miR-221 and colony formation was assessed following 0-8 Gy radiation. Transfection of Caco2 cells with pre-miR-221 significantly increased survival following 0-8 Gy radiation compared to blank and negative controls. Conversely, transfection of Caco2 cells with anti-miR-221 significantly decreased survival following radiation exposure (Figure 4). The D₀ value, the radiation dose required to reduce the level of cell survival from 100% to 37%, which is considered a measure of the intrinsic radiosensitivity of the cell, was calculated following genetic manipulation of miR-221. The quasi-threshold dose (D_q) value represents the sublethal damage repair capacity of the cells, which is also a sensitive indicator for the evaluation of cell radiosensitivity. Blank control cells, cells transfected with negative control or pre-miR-221 or anti-miR-221 oligonucleotides exhibited D₀ values of 1.681, 1.666, 2.208 and 1.068 Gy, respectively. The sensitization enhancement ratio (SER), calculated by determining the ratio of the D₀ of the control group vs treated cells, was 1.009, 0.761 and 1.574 for negative control-, pre-miR-221-, or anti-miR-221-treated cells, respectively (Table 1), indicating a radiosensitization potential for targeting miR-221.

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Figure 4 miR-221 modulates colorectal carcinoma cell radiosensitivity. Caco2 cells transfected with negative control, pre-miR-221 or anti-miR-221 oligonucleotides were exposed to 0-8 Gy radiation and incubated for 14 d prior to fixation, staining and assessment of colony formation. The colony formation assays were performed in triplicate.

To study the mechanism of miR-221 knockdown-induced radiosensitization, we measured irradiation-induced cell death in cells transfected with negative control or anti-miR-221 by flow cytometry. We found that in unirradiated cells transfected with anti-miR-221, there was an increase in necrosis and apoptosis compared to that in the controls. This is consistent with our previous observations that miR-221 knockdown leads to inhibition of cell growth[10]. More interestingly, in irradiated cells, anti-miR-221 transfection enhanced cell death (Figure 5), demonstrating a synergistic effect of miR-221 knockdown with irradiation. Collectively, these results provide strong evidence that miR-221 regulates the radiosensitivity of Caco2 cells.

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Figure 5 Effects of anti-miR-221 on irradiation-induced death in Caco2 cells. The percentages of necrotic and apoptotic cells were measured by calculating the ratio of the number of corresponding cells to the number of total cells. A: Blank control; B: Caco2 cells transfected with negative control; C: Caco2 cells transfected with anti-miR-221; D: Caco2 cells under irradiation; E: Caco2 cells transfected with negative control under irradiation; F: Caco2 cells transfected with anti-miR-221 under irradiation. The right upper quadrant (FITC⁺/PI⁺) shown as necrotic cells. The right lower quadrant (FITC⁺/PI^-) shown as apoptotic cells; G: The status of cell death was determined by flow cytometry. Groups A-F are described as above. n = 3, mean ± SD; ^bP < 0.01 vs blank or negative control group. ^dP < 0.01 vs sole anti-miR-221 or irradiation group.

PTEN is a tumor suppressor protein, thus, we hypothesized that anti-miR-221 might sensitize Caco2 cells to radiotherapy by upregulating PTEN protein expression. To study further downstream pathways of miR-221, we conducted Western blotting to look at phosphorylation of AKT, a downstream target of PTEN. In Caco2 cells transfected with anti-miR-221, there was a significant increase in PTEN expression accompanied by downregulation of AKT phosphorylation (Figure 6A).

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Figure 6 Ectopic expression of miR-221 affects the radiosensitivity of colorectal carcinoma cells by targeting phosphatase and tensin homolog deleted on chromosome 10. A: miR-221 regulates the phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/AKT pathway. Caco2 cells transfected with negative control or anti-miR-221 were mock treated or irradiated (4 Gy). Total cell lysates were obtained for Western blotting using the indicated antibodies. Caco2 cells were pretreated with or without anti-PTEN-siRNA (80 nmol/L) for 24 h prior to the addition of pre-miR-221 (50 nmol/L) or anti-miR-221 (50 nmol/L); B, C: Real-time RT-PCR and Western blotting showing PTEN mRNA and protein reduced markedly after transfection with anti-PTEN-siRNA (^bP < 0.01 vs blank control and negative control group); D: The status of cell radiosensitivity was determined by colony formation assay. The suppression of Caco2 cell survival fraction at 4 Gy by anti-miR-221 was partially, but not completely, abrogated by anti-PTEN-siRNA (SF₄ from 29.77% to 47.88%). n = 3, mean ± SD; ^bP < 0.01 vs negative control group; ^dP < 0.01 vs sole anti-miR-221 group.

Additionally, if anti-miR-221 enhancement of CRC cell radiosensitivity was indeed mediated by PTEN, we would expect that the PTEN-specific and irreversible antagonist, anti-PTEN-siRNA, would abolish this effect. To test this hypothesis, we measured the cell radiosensitivity variations induced by pre-miR-221 or anti-miR-221 in CRC cells previously transfected with anti-PTEN-siRNA. The aim of this experiment was to study if and how the PTEN-depleted cellular environment responds to pre-miR-221 or anti-miR-221 addition and irradiation. Caco2 cells were pretreated with or without anti-PTEN-siRNA (80 nmol/L) for 24 h prior to the addition of pre-miR-221 (50 nmol/L) or anti-miR-221 (50 nmol/L) and the status of cell radiosensitivity was determined by colony formation assay following different doses of X-ray exposure. The data showed that a reduction of PTEN dosage by means different from miR-221 overexpression led to analogous outcomes: when we transfected Caco2 cells with anti-PTEN-siRNA, which was able to reduce both PTEN mRNA and protein by about 80% (Figure 6B and C), we observed a sharp increase in cell survival following radiation as compared with the negative controls (Figure 6D). Thus, reducing PTEN levels in CRC cells, either by miR-221 overexpression or by anti-PTEN-siRNA transduction, is sufficient to induce a comparable cell survival increase.

When pre-miR-221 was transfected into Caco2 cells previously treated with anti-PTEN-siRNA, we observed that anti-PTEN-siRNA and miR-221 seemed to cooperate to increase the survival rate (Figure 6D). However, when anti-miR-221 was transfected into Caco2 cells previously treated with anti-PTEN-siRNA, we observed that the suppression of cell survival by anti-miR-221 was partially abrogated by anti-PTEN-siRNA (Figure 6D, Table 2). These results indicated that the inhibitory effect of anti-miR-221 on CRC cell survival following irradiation was partially, but not completely, mediated by PTEN, suggesting that anti-miR-221 could also activate some PTEN-independent signaling pathways to repress CRC cell growth in addition to the up-regulation of PTEN.

miRNAs regulate gene expression by mainly binding to the 3’-untranslated region (UTR) of target mRNAs, leading to mRNA degradation or translation inhibition. miRNAs are aberrantly expressed in various cancers, suggesting that they play a vital role as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate.

Colorectal carcinoma (CRC) is one of the most dangerous malignancies in China. Previous studies have shown that miR-221 expression is elevated in radioresistant CRC cell lines; however, it is not known whether and how miR-221 controls the cellular response to irradiation. In this study, the authors investigated the alterations of miR-221 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene expression in CRC cells after X-ray irradiation, and the mechanisms underlying the enhancement of radiosensitivity to irradiation in CRC cells transfected with anti-miR-221.

Some human miRNAs are consistently deregulated in human cancer, suggesting a role for these genes in tumorigenesis. This study showed that knocking down miR-221 by antisense oligonucleotides upregulated PTEN expression and PTEN was identified as a direct target of miR-221 in CRC. Moreover, upregulated PTEN expression suppressed AKT activity and increased radiation-induced cell death, resulting in enhancement of radiosensitivity in CRC cells.

This study indicated that anti-miR-221 enhanced the radiosensitivity of CRC cells by upregulating PTEN, and miR-221 might be a novel potential strategy for CRC treatment.

Anti-miR-221 could enhance the radiosensitivity of CRC cells by upregulating PTEN. This study provides evidence for the antioncogenic activity of anti-miR-221 in the irradiation of CRC and this may be a useful biomarker or therapeutic target in CRC.