The oncogenic circular RNA circ_63706 is a potential therapeutic target in sonic hedgehog-subtype childhood medulloblastomas

Abstract

Medulloblastoma (MB) develops through various genetic, epigenetic, and non-coding (nc) RNA-related mechanisms, but the roles played by ncRNAs, particularly circular RNAs (circRNAs), remain poorly defined. CircRNAs are increasingly recognized as stable non-coding RNA therapeutic targets in many cancers, but little is known about their function in MBs. To determine medulloblastoma subgroup-specific circRNAs, publicly available RNA sequencing (RNA-seq) data from 175 MB patients were interrogated to identify circRNAs that differentiate between MB subgroups. circ_63706 was identified as sonic hedgehog (SHH) group-specific, with its expression confirmed by RNA-FISH analysis in clinical tissue samples. The oncogenic function of circ_63706 was characterized in vitro and in vivo. Further, circ_63706-depleted cells were subjected to RNA-seq and lipid profiling to identify its molecular function. Finally, we mapped the circ_63706 secondary structure using an advanced random forest classification model and modeled a 3D structure to identify its interacting miRNA partner molecules. Circ_63706 regulates independently of the host coding gene pericentrin (PCNT), and its expression is specific to the SHH subgroup. circ_63706-deleted cells implanted into mice produced smaller tumors, and mice lived longer than parental cell implants. At the molecular level, circ_63706-deleted cells elevated total ceramide and oxidized lipids and reduced total triglyceride. Our study implicates a novel oncogenic circular RNA in the SHH medulloblastoma subgroup and establishes its molecular function and potential as a future therapeutic target.

Keywords: Circular RNA; Global lipidome; Medulloblastoma; Sonic hedgehog.

Fig. 1

Differential expression of circRNAs in medulloblastoma subgroups. A Circular RNA identification pipeline for the 126 medulloblastoma patients in four subgroups. B Raw total circular RNA counts across all 126 medulloblastoma patient samples. C Principal component clustering of 8925 highly expressed circRNAs. D–G The red and blue points and shades show significant circular RNAs for a group versus others with an adjusted p value < 0.05 and |log2 fold change|> 2. The box plot shows normalized expression for two significant upregulated circRNAs in WNT (D), SHH (E), G3 (F), and G4 (G) across all 126 medulloblastoma patients

Fig. 2

CircRNAs validated by qRT-PCR and RNA-FISH in cell lines, PDXs, and clinical MB patient samples. A and B Fold change for normal cerebellum (CB) in medulloblastoma cell line samples (A) and PDX samples (B). Values indicate fold change relative to cerebellum. *p < 0.05, **p < 0.01, Kruskal–Wallis analysis. C and D RNA-FISH confirms that circ_63706 expression is specific to SHH medulloblastoma patients. C Representative RNA-FISH analysis of circ_63706 in medulloblastoma tissues. RNA-FISH analysis of circ_63706 in SHH medulloblastoma patients (top panels) and G3 and G4 medulloblastoma patients (lower panels). D Spot numbers relate to circ_63706 per cell in SHH, G3, and G4 medulloblastoma patients. n = 20, *p < 0.01, Student’s t-test. E Kaplan–Meier survival curves of SHH medulloblastoma patients according to circ_63706 expression. SHH medulloblastoma patient samples were divided into circ_63706 high (average spot number per cell > 1.0) and circ_63706 low (average spot number per cell < 1.0)

Fig. 3

Inhibition of circ_63706 suppresses SHH MB cell proliferation, migration, and invasion in vitro. A circ_63706 expression was detected in DAOY, ONS76, UW228, DMB012, icb1712, and RCMB32 cells with si-circ_63706 or NC. B MTS assays were performed to assess proliferation in DAOY, ONS76, UW228, DMB012, icb1712, and RCMB32 cells with si-circ_63706 or NC. C Cell migration was assessed by a wound healing assay in DAOY, ONS76, and UW228 cells with si-circ_63706 or NC. D Cell invasion was assessed by transwell assays and DAOY, ONS76, and UW228 cells with si-circ_63706 or NC. Scale bar, 200 μm. Data shown are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Circ_63706 promotes tumorigenesis and growth of SHH MB cell in vivo. A Expression of circ_63706 in DAOY and ONS76 control (sh-NC) and DAOY and ONS76 with circ_63706-knockdown (sh-circ_63706 #1 and #2) cells. Relative expression to sh-NC is indicated on the y-axis. B Cell viability assays performed with DAOY and ONS76 control (sh-NC) and DAOY and ONS76 with circ_63706-knockdown (sh-circ_63706 #1 and #2) cells. Points represent the mean ± SD of three biological replicates. C DAOY and ONS76 control (sh-NC) and DAOY and ONS76 with circ_63706-knockdown (sh-circ_63706 #1 and #2) cells expressing luciferase were implanted into the cerebellums of NOD-SCID mice, and tumor formation was assessed by bioluminescence imaging. Changes in bioluminescent signal were examined weekly after tumor implantation. D Quantification of total photon counts from mice implanted with DAOY and ONS76 control (sh-NC) and DAOY and ONS76 with circ_63706-knockdown (sh-circ_63706 #1 and #2) cells. n = 5. E Ki67 staining of xenograft tumor sections. Nuclei are stained with DAPI. Scale bars, 50 μm. Quantification of Ki67-positive cells are shown in (F). *p < 0.05, Student’s t-test. G Overall survival was determined by Kaplan–Meier analysis, and the log-rank test was applied to assess the differences between groups. *p < 0.05, Mantel–Cox log-rank test

Fig. 5

Circ_63706 suppresses lipid oxidation and membrane components. The box plot presents total oxidized lipids in circ_63706 knockdown cells (A) and the heatmap represents the top 40 oxidized lipid molecules in circ_63706 knockdown cells (B). The box plot presents total TG (C) and DG (D) lipids in circ_63706 knockdown cells, and the heatmap represents the top 40 TG molecules in circ_63706 knockdown cells (E). The box plot presents total SMs (F) and the heatmap represents the top 40 SM molecules in circ_63706 knockdown cells (G). The box plot presents total ceramide and its subclasses in circ_63706 knockdown cells (H) and the heatmap represent the top 40 ceramides and its subclasses in circ_63706 knockdown cells compared with control (I)

Fig. 6

Mapping the secondary structure and modeling the 3D structure of circ_63706. The structure of circ_63706 A after the minimization of the modeled structure and B after 500 ns of MD simulation. Potential miRNA binding sites are highlighted in red and numbered according to the list in Additional file 4: table S6