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.