MicroRNA 218 acts as a tumor suppressor by targeting multiple cancer phenotype-associated genes in medulloblastoma

Abstract

Aberrant expression of microRNAs has been implicated in many cancers. We recently demonstrated differential expression of several microRNAs in medulloblastoma. In this study, the regulation and function of microRNA 218 (miR-218), which is significantly underexpressed in medulloblastoma, was evaluated. Re-expression of miR-218 resulted in a significant decrease in medulloblastoma cell growth, cell colony formation, cell migration, invasion, and tumor sphere size. We used C17.2 neural stem cells as a model to show that increased miR-218 expression results in increased cell differentiation and also decreased malignant transformation when transfected with the oncogene REST. These results suggest that miR-218 acts as a tumor suppressor in medulloblastoma. MicroRNAs function by down-regulating translation of target mRNAs. Targets are determined by imperfect base pairing of the microRNA to the 3'-UTR of the mRNA. To comprehensively identify actual miR-218 targets, medulloblastoma cells overexpressing miR-218 and control cells were subjected to high throughput sequencing of RNA isolated by cross-linking immunoprecipitation, a technique that identifies the mRNAs bound to the RNA-induced silencing complex component protein Argonaute 2. High throughput sequencing of mRNAs identified 618 genes as targets of miR-218 and included both previously validated targets and many targets not predicted computationally. Additional work further confirmed CDK6, RICTOR, and CTSB (cathepsin B) as targets of miR-218 and examined the functional role of one of these targets, CDK6, in medulloblastoma.

FIGURE 1.

Differential expression of miR-218 in medulloblastoma. A, relative expression of miR-218 mRNA, normalized to U6b expression, shows significant down-regulation in pediatric medulloblastoma patient samples compared with normal cerebellum by quantitative PCR analysis (***, p < 0.0001). B, miR-218 expression as measured by microRNA microarray analysis in 90 medulloblastoma tumor specimens that are characterized and divided into four subgroups (WNT, SHH, Group 3, and Group 4). miR-218 is significantly down in SHH (*, p < 0.02) and Group 3 (**, p < 0.01) compared with adult cerebellum. C, in all well characterized medulloblastoma cell lines, the miR-218 expression was significantly lower than that of the normal cerebellum by quantitative PCR analysis. p < 0.05 normal versus all medulloblastoma cell lines shown above by analysis of variance. Error bars, S.E.

FIGURE 2.

Effect of re-expression of miR-218 in the medulloblastoma cell lines. A, Daoy cells transfected with miR-218 showed a significant decrease in cell growth compared with that of both non-transfected (no trt) cells and cells transfected with empty vector pSIF as measured in real time using xCELLigence. By analysis of variance, p < 0.05 for miR-218 versus empty vector pSIF for the indicated time period. B, Daoy cells transfected with miR-218 resulted in a significant inhibition in cell colony-forming ability compared with that of non-transfected and empty vector. **, p < 0.01, miR-218-transfected cells versus pSIF. C and D, Daoy cells transfected with miR-218 resulted in the formation of tumor spheres with smaller diameters compared with that of non-transfected and empty vector. Representative images of tumor spheres formed from each of the treatments, cells treated either with empty vector pSIF or miR-218, or cells with no treatments are shown. *, p < 0.02 miR-218 versus pSIF. E and F, real-time measurement of cell migration and invasion shows that ectopic expression of miR-218 in Daoy cells inhibits cell migration (E) and invasion (F) compared with that of control cells. Error bars, S.E.

FIGURE 3.

Biological effects of differential expression of miR-218 in murine neural progenitor stem cells. A, C17.2 cells transfected with miR-218 showed significant inhibition in their ability to form colonies compared with empty vector pSIF-transfected and non-transfected cells. #, p < 0.05 miR-218 versus pSIF. B, C17.2 cells form a negligible number of colonies in soft agar. C17.2 cells, when co-transfected with REST and pSIF plasmids, formed a robust number of colonies in soft agar that is partially inhibited by the overexpression of miR-218 with REST (**, p < 0.01). Representative wells from each treatment are shown (left). The colonies were counted using the Qpro software program and graphed (right). C, microRNA 218 promotes differentiation of neural stem cells in vitro. C17.2 cells were transfected with miR-218 or anti-miR-218 or with their corresponding control vectors and grown in differentiation medium. Tuj1 and NESTIN proteins were detected by immunofluorescence. i, representative merged immunofluorescence images of C17.2 cells stained for Tuj1 (green) that include the signal detected from DAPI staining of nuclear cells (blue). ii, representative merged immunofluorescence images of C17.2 cells treated as indicated and stained for NESTIN (green); DNA was fluorescently labeled with DAPI (blue). D, percentage of cells with either Tuj1 or NESTIN positive in DAPI-positive cells were counted and normalized to the total DAPI-stained nuclei in different fields in n = 4 wells. p values were calculated by Student's t test. The increase in miR-218 showed more cells expressing the neuronal differentiation marker Tuj1 and a concomitant decrease in cells expressing the neural progenitor marker NESTIN. #, p < 0.05; **, p < 0.01, comparisons as denoted in the figure. Error bars, S.E.

FIGURE 4.

Venn diagram showing the number of overlapping genes targeted by miR-218 that were identified by the HITS-CLIP method and predicted by TargetScan. Shown below are the genes that were selected for validation in this study.

FIGURE 5.

Validation of targets. A, targets identified by HITS-CLIP. miR-218 down-regulates the expression of CTSB, CDK6, and RICTOR protein levels in both the Daoy and ONS76 cell lines. B, verification of HITS-CLIP-identified target protein levels. Shown is Western blot analysis of CTSB, CDK6, and RICTOR protein levels in different well characterized medulloblastoma cell lines compared with pediatric and adult normal cerebellum. UPN 514 and UPN 605 are normal pediatric cerebellum samples. CDK6 and RICTOR are consistently up-regulated in all low miR-218-expressing cell lines, and CTSB is differently up-regulated in different medulloblastoma cell lines. C, validation of targets predicted by TargetScan alone by Western blot. miR-218 overexpression in Daoy cells did not down-regulate the protein expression of JARID1A, BMI1, or MEL-18. D, validation by luciferase assay. Top, base pairing between miR-218 and the mRNA seed sequence of CDK6 and RICTOR. The seed sequences of the CDK6 and RICTOR shown are the ones that were pulled down with AGO2 using HITS-CLIP. The mutated nucleotides are underlined. Bottom, siCHECK plasmid containing the plasmid sequences of either CDK6 or RICTOR (as indicated) downstream of Renilla luciferase was co-transfected with control miR (N.C.) or miR-218 oligonucleotide in HEK293T cells. There was a significant decrease in the ratio of Renilla/firefly luciferase activity in cells transfected with either CDK6 or RICTOR 3′-UTR compared with N.C. #, p < 0.05; **, p < 0.01. Mutated sequence of both CDK6 and RICTOR showed no regulation. Error bars, S.E.

FIGURE 6.

Rescue effect of CDK6 on colony formation and tumor sphere size in Daoy cells overexpressing miR-218. Co-transfection with miR-218 and CDK6 that lacks the 3′-UTR rescued the cells from the inhibitory effect of miR-218 alone on the cell colony-forming ability (A) and on the tumor sphere diameter (B). Cells co-transfected with CDK6 that lacks the 3′-UTR and miR-218 increased the number of colonies and increased the tumor sphere size significantly (**, p < 0.01 miR-218 versus CDK6 + miR-218). CV, control vector for CDK6 that lacks the 3′-UTR. Error bars, S.E.

FIGURE 7.

A working model of miR-218 interactions with its target genes in medulloblastoma. Down-regulation of miR-218 contributes to increased expression of CDK6, CTSB, and RICTOR, which promotes a malignant phenotype in medulloblastoma cells.