Molecular mechanism of chemoresistance by miR-215 in osteosarcoma and colon cancer cells

The human osteosarcoma cell lines U-2 OS, MG63 were obtained from the American Type Culture Collection (ATCC). The human colon cancer cell lines HCT 116 (wt-p53) and HCT 116 (null-p53) were a gift from Professor Bert Vogelstein (The Johns Hopkins University). U-2 OS, HCT 116 (wt-p53) and HCT 116 (null-p53) cells were maintained in McCoy's 5A medium (Gibco Laboratories), and MG63 cells were maintained in Eagle's Minimum Essential Medium (ATCC) respectively. All the media were supplemented with 10% dialyzed fetal bovine serum (HyClone Laboratories). MTX, TDX, cisplatin and doxorubicin were purchased from Sigma-Aldrich.

Microscopically confirmed tumor samples and paired adjacent normal tissues were obtained from 24 patients undergoing surgical resection of primary colorectal adenocarcinoma at the Department of General, Visceral and Transplantation Surgery, University of Ulm, Germany. Following surgery, samples from tumor and adjacent normal tissues were frozen in liquid nitrogen and stored at -80°C for subsequent RNA extraction. Patient consent forms were obtained from all patients according to the institutional regulations. The characteristics of these patients are shown in Additional file 1.

HCT 116 (wt-p53) cells were sorted with multiparametric flow cytometry using BD FACS Aria cell sorter (Becton Dickinson) under sterile conditions. Cells were prepared and labeled with conjugated anti-human CD133-PE (clone 105902; R&D Systems) and CD44-FITC (clone F10-44-2; R&D Systems). Antibodies were diluted in MACS buffer containing 5% BSA, 1 mM EDTA and 15-20% blocking reagent (Miltenyi Biotec) to inhibit non-specific binding to non-target cells. After 15 min incubation at 4°C, staining cells were washed, resuspended in 500 μl of MACS buffer, and sorted.

U-2 OS, MG63, HCT 116 (wt-p53) and HCT 116 (null-p53) cells (2 × 10⁵) were plated in six-well plates and transfected with 100 nM of either miR-215 mimics or non-specific miRNA (Ambion) after 24 h by Oligofectamine (Invitrogen) according to the manufacturer's protocols. siRNA against DHFR (ON-TARGET plus SMARTpool L-008799-00-0010, human DHFR, NM_000791), siRNA against TS [30], and siRNA against DTL (ON-TARGET plus SMARTpool L-020543-00-0005, human DTL, NM_016448) were purchased from Dharmacon and transfected with Oligofectamine at a final concentration of 100 nM.

To knock down endogenous miR-215, HCT 116 (wt-p53) cells were transfected with 100 nM of scramble-miR locked nucleic acid (LNA-control) or LNA anti-miR215 (LNA-miR215) oligonucleotides by Lipofectamine 2000 (Invitrogen) in six-well plates (2 × 10⁵ cells/well). LNA-control and LNA-miR215 were purchased from Exiqon. To mimic the stress situation, HCT 116 (wt-p53) and HCT 116 (null-p53) cells were first transfected with 100 nM of miR-215 in six-well plates (2 × 10⁵ cells/well), 24 h later, 100 nM of LNA-control or LNA-miR215 were transfected into the cells respectively.

Total RNAs, including miRNAs, were isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions (see Additional file 2).

The relative quantity of miR-215 was evaluated by real time qRT-PCR (details see Additional file 2).

The levels of DHFR and TS mRNAs were determined as described in Additional file 2.

Cell proliferation and cell cycle analyses were performed, for details see Additional file 2.

Western immunoblot was performed, details and information for antibodies see Additional file 2.

pMIR-REPORT Luciferase miRNA Expression Reporter Vector (Ambion) was used to determine the targets of miR-215. Double stranded DNA oligonucleotides containing the miR-215 binding sequence (wild-type, underlined) or a mismatch sequence (mutant, italic) of the 3'UTR of DHFR or TS mRNAs and the HindIII and SpeI restriction site overhangs were synthesized (IDT). After annealing, double strand oligonucleotides were inserted into the pMIR-REPORT plasmid, downstream of the firefly luciferase reporter. The sequences of these synthesized oligonucleotides are listed in Additional file 3. The 3'UTR of TS includes 2 binding sites of miR-215 at 84-104 bp (wild type-1) and 216-236 bp (wild type-2) respectively. Transfection and luciferase assays were performed as described in Additional file 2.

U-2 OS and HCT 116 (wt-p53) cells were re-plated in 96-well plates at 2 × 10³ cells/well in triplicate after being transfected with miR-215 mimics, non-specific miRNA, or siRNAs against DHFR, TS or DTL in 100 μl of medium. After twenty-four hours, 10-200 nM of MTX or TDX in 100 μl medium was added, and the cells were incubated for another 72 h. WST-1 was added to each well (10 μl), and after 2 h incubation, optical absorbance was measured at 450 and 630 nm respectively. HCT 116 (wt-p53) cells were transfected with LNA antisense miRNAs and assayed for MTX sensitivity using the same methods described above.

All experiments were repeated at least twice. Statistical significance between the two groups of data was evaluated by Student's t test (two-tailed). Asterisks indicate significant differences of experimental groups compared with the corresponding control condition. Statistical analysis was performed using GraphPad Prism software 5 (GraphPad, Inc.). The expression ΔC_T value of miR-215 in each clinical sample was calculated by normalizing with its internal control RNU6B and relative quantitation values were plotted using SDS software v1.2 (Applied Biosystems). The statistically significant difference in expression level between tumor and normal tissues was calculated using a paired Wilcoxon signed-rank test, and the statistical analysis was performed by MedCalc^® 10.0.2 (MedCalc software, Belgium). Differences were considered statistically significant at P < 0.05.

Given the significance of DHFR and TS as two of the major targets of anti-cancer chemotherapy, we used a bioinformatics approach to identify miRNAs that were predicted to bind to DHFR and TS mRNAs and therefore function in their regulation. Using TargetScan and PicTar in the miRNAs database (http://​www.​mirbase.​org), we found that miR-215 was predicted to have a potential interaction site at the 3'UTR of DHFR mRNA and two sites at the 3'UTR of the TS mRNA (Figure 1A). Ectopic expression of miR-215 by transient transfection decreased the expression of both DHFR and TS proteins in both osteosarcoma U-2 OS and colon cancer HCT 116 (wt-p53) cells analyzed by Western immunoblot analysis (Figure 1B). However, only a slight reduction of DHFR or TS mRNAs expression was observed in HCT 116 (wt-p53) cells (Additional file 4A-B), and the results in U-2 OS cells were consistent with HCT 116 (wt-p53) cells (data not shown). This indicates that the suppression of DHFR or TS expression by miR-215 is, in a large part, reducing the protein translation. By contrast, the decreased expression of DHFR and TS by siRNAs (Additional file 5A-B) was clearly caused by mRNA degradation (Additional file 4A-B), and similar results were found in U-2 OS cells (data not shown). To provide direct evidence that miR-215 interacts with the 3'UTRs of DHFR or TS mRNAs, luciferase reporter constructs were prepared by inserting the DHFR or TS 3'UTRs containing the putative miR-215 binding sites into the downstream of the firefly luciferase reporter gene. Our results clearly demonstrated a significant decrease of luciferase activity compared to either mutant or empty vector controls upon transient over-expression of miR-215 (Figure 1C). Based on these results, we conclude that DHFR and TS are among some of the direct targets of miR-215.

Interestingly, a recent report describing a microarray expression analysis for miR-215 targets did not identify DHFR or TS [32], in a large part because the approach was dependent on mRNA target degradation. In fact, any analysis based on the examination of steady state total RNA levels would be likely to miss these important targets.

Recent studies have clearly demonstrated that miRNAs play important roles in multiple biological processes, such as development, differentiation, cell proliferation, apoptosis, metabolism, and stress response, many of which are often perturbed in cancer [24, 33]. Some miRNAs have been identified acting as either oncogenes or tumor suppressors, so to investigate the potential impact of miR-215 on cell growth, cell proliferation assays were performed in both U-2 OS and HCT 116 (wt-p53) cells. A significant inhibition of cell proliferation (over 40%) was observed in either U-2 OS or HCT 116 (wt-p53) cells compared with the non-specific miRNA control after 5 days (Figure 2A-B). The inhibitory effect was more profound in cells containing wild-type p53 than cells without functional p53 (Figure 2C-D), suggesting that this reduced cell proliferation may be caused by cell cycle arrest or cell senescence.

Specific miRNAs have been found to regulate cell cycle progression and apoptosis, which represents a new layer of complexity in the cell cycle regulation [34]. Here, we focused on the cell cycle changes induced by miR-215 through examining its impact on cell cycle control using flow cytometry. The proportion of cells in the G2 phase was higher in miR-215 transfected U-2 OS cells than that in non-specific miRNA control cells, whereas the proportion of cells in the S phase was much lower than that in the negative miRNA control cells, with the relative quantity of G2/S ratio >2-fold (Figure 3A). Similar results were observed in HCT 116 (wt-p53) colon cancer cells transfected with miR-215 (Figure 3C). Our results suggest that the reduced proliferation rate is due to the decreased S phase and increased G2 checkpoint control.

We also noticed that the effect of miR-215 on cell proliferation and cell cycle control were in part dependent on the status of p53, as the effect was much less in cell lines containing mutant or deleted p53 (Figure 2C-D and Figure 3B and 3D). This is consistent with recent reports that miR-215 contributes to cell cycle control and cell proliferation mediated by p53 [32, 35]. It's known that p53 plays an important role in stem cell quiescence process [36]. We believe that we have also added a microRNA component to the p53-regulated network in stem cell quiescence.

p53 and p21, a downstream target of the p53 growth control pathway, are reported to block cells at the G2 checkpoint mainly through inhibition of Cdc2 activity, the cyclin-dependent kinase that normally drives cells into mitosis and which is the ultimate target of pathways that mediate rapid arrest in G2 in response to DNA damage [37]. We found that miR-215 can induce G2-arrest in U-2 OS and HCT 116 (wt-p53) cells, so to further investigate the mechanism of cell proliferation inhibition by miR-215, we transfected miR-215 into U-2 OS and HCT 116 (wt-p53) cells and evaluated the levels of cell cycle control genes p53 and p21 by Western immunoblot analysis (Figure 4). The results showed that over-expression of miR-215 caused a significant increase of the p53 and p21 protein in both U-2 OS and HCT 116 (wt-p53) cells. Thus, our results indicate that miR-215 contributes to the inhibition of cell proliferation at least partially by the induction of G2-arrest in U-2 OS and HCT 116 (wt-p53) cells through over-expression of p53 and p21.

We then asked what possibilities might contribute to the induction of p53 and p21 by miR-215. Georges et al. [32] reported that miR-215 induced cell cycle arrest by targeting a number of G1 and G2 checkpoint regulators. They confirmed that eighteen transcripts were direct downstream targets of miR-215, including denticleless protein homolog (DTL), a cell cycle G2/M checkpoint regulatory protein [28, 38]. Moreover, DTL is thought to interact with both DDB1-CUL4 and MDM2-p53 ligase complexes [39, 40]. Inactivation of DTL has been found to impair these complexes and stabilize p53 by preventing its ubiquitination, increasing the levels of p53 and its target p21 [28]. Here, we hypothesized that miR-215 might suppress DTL, the destabilizing factor of p53, to promote p53 stabilization and subsequently inhibit cell growth and cause cell cycle G2-arrest. First, we confirmed that miR-215 down-regulated DTL protein expression by Western immunoblot, and that knock-down of DTL by siRNA induced p53 and p21 expression to the same extent as miR-215 (Figure 5A). We next investigated the effects of DTL knock-down on cell proliferation and cell cycle in HCT 116 (wt-p53) cells, and found that cell proliferation was reduced significantly (about 43%) compared to negative control after 5 days (Figure 5B). Flow cytometry showed that the proportion of cells in the G2 phase was higher in HCT 116 (wt-p53) cells transfected with siRNA targeting DTL than that in negative control cells, while the proportion of cells in the S phase decreased, with the relative quantity of G2/S ratio >2-fold (Figure 5C). These results were similar to those observed in HCT 116 (wt-p53) cells transfected with miR-215 (Figure 2B and 3C). Taken together, our results indicate that miR-215 inhibits cell proliferation by the induction of G2-arrest through down-regulation of G2 checkpoint regulator DTL and up-regulation of p53 and p21.

To evaluate the impact of miR-215 on chemosensitivity, we used MTX or TDX to treat HCT 116 (wt-p53) cells transfected with either miR-215, non-specific miRNA, or siRNAs against DHFR or TS. Examination revealed that cells transfected with siRNAs specific for DHFR or TS showed a considerably heightened sensitivity to both MTX and TDX (Figure 6A-B). This is consistent with previous reports that tumors with lower expression of DHFR or TS are more sensitive to antifolate treatment [1, 3, 30, 41]. However, the cells transfected with miR-215 were considerably less sensitive to both chemotherapeutic agents than were control cells (Figure 6A-B). Similar results were obtained with U-2 OS cells (data not shown). Van Triest et al. [42] and Peters et al. [43] found there is no significant relationship between the TS levels and TDX sensitivity and suggested that the sensitivity to the TS-directed antifolate is unlikely to be determined by one single determinant. Since miR-215 potentially regulates hundreds of mRNA transcripts, its global impact on genes and pathways has the potential to be more important for the resistance mechanism. Based on the results of cell proliferation and cell cycle between miR-215 and siRNAs against DHFR or TS, we reasoned that the opposite impact of miR-215 on chemosensitivity vs. siRNAs specific to DHFR or TS may be largely due to the reduced cell proliferation rate (Figure 2) through decreased S phase and increased G2-arrest (Figure 3). MTX and TDX are considered to be cell cycle-specific agents and mainly affect cells in the S phase [43‐47]. In general, slowly proliferating cells are far more resistant to chemotherapeutic drug treatment, particularly slowly proliferating tumor stem cells [29]. Since siRNAs specific for DHFR and TS reduced the levels of their targets without affecting the rate of cell proliferation, they greatly enhanced the toxicity of DHFR and TS inhibitors (Additional file 5C-D). To identify the possible target(s) of miR-215 responsible for the reduced cell proliferation and cell cycle G2-arrest, we investigated several miR-215-mediated targets with a role in cell cycle control (data not shown). As mentioned above, we discovered that one of them, DTL, was directly responsible for G2-arrest and the induction of p53 and p21 (Figure 5). We then investigated the impact of DTL expression on MTX and TDX chemosensitivity in HCT 116 (wt-p53) cells by performing siRNA-mediated knock-down of DTL. When cells were transfected with siRNA targeting DTL, they exhibited a significant increase in chemoresistance to both MTX and TDX (Figure 6C-D) similar to miR-215 (Figure 6A-B). Taken together, our results suggest that miR-215 may affect chemoresistance to MTX and TDX by suppressing DTL expression, thereby increasing G2-arrest and reducing the proportion of time spent in S phase, during which MTX and TDX are most effective.

We reasoned that if our conclusion was correct, and that the mechanism of miR-215-mediated resistance lies in its ability to trigger G2-arrest with slow cell proliferation, then altering miR-215 levels should have no effect on the toxicity of agents whose actions are cell cycle-independent. To test this, we repeated the previous drug treatment experiments in HCT 116 (wt-p53) cells, but replacing MTX and TDX with the DNA-targeting agents-cisplatin and doxorubicin. As shown in Additional file 6A-B, no significant difference in chemosensitivity was observed between miR-215 transfected cells and negative control cells. Similar results were also found in cells transfected with siRNA against DTL (Additional file 6C-D). These results further support our conclusion that miR-215-mediated MTX and TDX resistance is due to its effects on the cell cycle and suggest that the resistance mechanism mediated by miR-215 is specific to cell cycle-dependent drugs.

We have so far accessed the functional significance of miR-215 using a knock-in approach. This, to a certain extent, mimics a cellular stress response in which miR-215 is induced. The results showed that exogenous miR-215 reduced cell proliferation with increased cell cycle control and chemoresistance. To further elucidate the impact of endogenous miR-215 on cell proliferation, cell cycle and chemosensitivity, we performed a series of knock-down experiments using locked nucleic acid (LNA) oligonucleotides (a scramble-miR LNA negative control, and a LNA antisense miR-215) to test the biological significance of endogenous miR-215 in HCT 116 (wt-p53) cells. Antagonizing the endogenous miR-215 enhanced the cell proliferation rate by 23% compared to the LNA negative control (Additional file 7A), and increased the sensitivity to MTX treatment (Additional file 7B). The expression of TS and DHFR were increased by knocking down miR-215 using Western immunoblot analysis (Additional file 7C). These observations further demonstrate the important effects of endogenous miR-215 on cell proliferation and chemosensitivity.

To test whether we can reverse the cell cycle impact caused by exogenous miR-215, we antagonized miR-215 by transfecting cells with 100 nM LNA antisense miRNAs. We observed that the percentage of cells in the G2 phase decreased from 37% to 24%, and percentage of cells in the S phase increased from 19% to 34% (Additional file 8A, top panel) in the HCT 116 (wt-p53) cell line, while HCT 116 (null-p53) cells showed no change in the G2 and S phases (Additional file 8A, bottom panel). These results further support the notion that miR-215 is important in regulating the cell cycle in a manner of depending on p53 status. Strikingly, antagonizing miR-215 also attenuated the induction of p53 and p21 (Additional file 8B). These results are highly consistent with the data obtained from exogenous miR-215 over-expression experiments.

Cancer stem cells (CSC), as their name implies, are cancer cells that possess the characteristics associated with normal stem cells, in particular the ability to give rise to all cell types found in a particular cancer sample. In contrast with other more differentiated cancers, however, CSCs exhibit a low rate of division and proliferation that allows them to resist chemotherapies and radiation [29], both of which preferentially affect highly proliferative cells, making CSCs a major reason for the failure of chemotherapy. With this in mind, we analyzed the miR-215 expression levels from isolated CD133+HI/CD44+HI colon cancer stem cells from cultured HCT 116 (wt-p53) cells using real time qRT-PCR analysis (Additional file 9B). We used CD133 and CD44 as two selection markers to isolate colon cancer stem cells from HCT 116 (wt-p53) cells (Additional file 9A) because CD133 and CD44 have been shown to be two of the important markers for the isolation of colon cancer stem cells [48‐51]. The details of characterization of CD133+HI/CD44+HI colon cancer stem cells have been previously reported [51]. Expression of miR-215 in the CD133+HI/CD44+HI colon cancer stem cells was nearly 3-fold higher than that in the control bulk CD133+/CD44+ colon cancer cells (Additional file 9B). These results suggest that colon cancer stem cells may utilize miR-215 to slow cell proliferation and avoid damage caused by chemotherapy until receiving a proliferation and differentiation signal, further verifying the impact of miR-215 on cell proliferation and chemotherapy resistance. To further confirm that DHFR and TS are the targets of miR-215, the expression of both DHFR and TS were quantified in CD133+HI/CD44+HI colon cancer stem cells and the control bulk CD133+/CD44+ colon cancer cells. We found DHFR and TS protein levels were remarkably down-regulated in the CD133+HI/CD44+HI colon cancer stem cells based on the relative higher miR-215 expression level (Additional file 9C). This result, in turn, suggests that miR-215 is more important than the levels of DHFR or TS in the chemoresistance.

Previous studies from our laboratory have shown that certain miRNAs were associated with the development and prognosis in colorectal cancer [52]. To provide potential relevance of miR-215 in colorectal cancer, we profiled the expression of miR-215 in the same set of clinical samples (24 colorectal tumor specimens vs. adjacent normal colorectal tissues) using real time qRT-PCR analysis. The expression of miR-215 was significantly decreased (P < 0.01) in colorectal tumor specimens compared to adjacent normal tissues (Additional file 10). These results indicate that the fast proliferating phenotype in the majority of differentiated colorectal tumor cells are associated with the reduction of miR-215 expression. This further supports our hypothesis that the small fraction of tumor stem cells with a slow proliferation rate is mediated, at least in part, by miR-215. Based on these findings, it is clear that the roles miR-215 plays in regulating cellular behavior are too complex for it to be simply defined as a tumor suppressor based on its ability to slow cell proliferation and cause cell cycle arrest under certain conditions, and it has to be defined in the right cellular context.