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Supplementary Figure Legends
Figure S1. RRBS methylation results of full miR-148a-associated CpG island. Top panel,
map of miR-148a CpG island (chr7:25990013-25991319), position of pre-miR-148a coding
region (black box, chr7: 25989539-25989606), predicted putative transcription start sites (TSS1,
TSS2) and PCR products used for bisulfite sequencing of Regions 1 and 2 (black arrows);
bottom panel, CpG site methylation pattern of IDH1/2WT glioma or IDH1MUT gliomas (circle
shading proportional to extent of methylation).
Figure S2. The methylation status of IDH1 mutation is prognostic in GBM and Anaplastic
Astrocytoma (Grade III). A, Kaplan-Meier overall survival analysis of GBM patients in the
validation cohort (n=224). Survival among IDH1MUT (n=20) and IDH1WT (n=204) is shown. B,
Kaplan–Meier overall survival analysis of Grade III glioma patients in the validation cohort
(n=42). Survival among IDH1MUT (n=29) and IDH1WT (n=13) is shown. Hash marks indicate
censored patients.
Figure S3. miR-148a promotor CpG island was demethylated and expression was
upregulated after demethylating agent 5-aza-CdR treatment. U251 cells were treated with
0.625 µM 5-aza-CdR for 9 days. DNA and RNA were isolated for bisulfite sequencing,
methylation specific PCR and TaqMan qPCR assay. A, representative CpG sites of miR-148a
promoter CpG island was sequenced by using bisulfite sequencing method in U251 cells with or
without 5-aza-CdR treatment. B, Methylation specific PCR was performed in in U251 cells with
or without 5-aza-CdR treatment by using MSP primers shown in Supplementary table S11. C,
miR-148a expression level was determined in in U251 cells with or without 5-aza-CdR treatment
by TaqMan qPCR. U, Unmethylated band; M, Methylated band.
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Figure S4. Knockdown of DNMT1 in U251 cells by using siRNA. DNMT1 siRNA or
negative control (NC) siRNA (10nM) were transfected into U251 cells by using Lipofectamine®
RNAiMAX at day 1, day 3 and day 5 after seeded into 24-well plates. Total RNA was isolated
48h after the last transfection. DNMT1 mRNA level was determined by qPCR using β-actin as an
internal control and standardized to NC which was set as 100%. Results were presented as mean
± s.e.m. (n=3). ***, P<0.001 relative to NC.
Figure S5. Introduction of mutant IDH1 into HEK293T. A, representative Western blot
shows the expression of IDH1 mutant protein expression. B, D-2-HG level in HEK293T cells is
determined by the enzymatic assay (1).
Figure S6. Overexpression of miR-148a in 293T-IDH1MUT doesn’t block the methylation of
miR-148a and RBP1. A. miR-148a overexpression level was determined by TaqMan qPCR in
vector control HEK293T cells (293T-EV), IDH1WT expressing HEK293T cells (293T-IDH1WT)
or IDH1MUT expressing 293T cells (293T-IDH1MUT) with or without miR-148a overexpression at
passage 26. *** , P<0.001 relative to cells transfected with control empty vector pMIF-GFPzeo. B and C, total number of methylated CpG sites determined by targeted BiSEQ of region 1
of miR-148a CpG island (B) or RBP1 CpG island (C) in vector control HEK293T cells (293TEV), IDH1WT expressing HEK293T cells (293T-IDH1WT) or IDH1MUT expressing 293T cells
(293T-IDH1MUT) with or without miR-148a overexpression. Data shown represents mean value
from 2 independent clones for each construct.
Figure S7. miR-148a expression level in miR-148a stably transfected U251 and T98G cells.
Total RNA was isolated from hNSC cells, 293T cells, U251 and T98G cells with or without
stably overexpressing miR-148a. miR-148a expression level was determined by TaqMan qPCR
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using RNU6B as internal control and standardized to the expression level in hNSC which is set
as 100% . Results were presented as mean ± s.e.m. (n=3). ***, P<0.001 relative to hNSC.
Figure S8. Cell cycle analysis of T98G-miR-148a stable cells.
Cell cycle fraction and
apoptosis index were determined by FACS assay after fixing cells with 70% ethanol and PI
staining. Results are presented as mean ± s.e.m. (n=3) from 3 independent experiments. *,
P<0.05; **, P<0.01 relative to EV.
Figure S9. The expression of G-CIMP genes DLC1 and CIDEB are downregulated in
IDH1MUT GBM patient tissue samples and glioma cell lines. A and B, the gene expression of
DLC1 and CIDEB are downregulated in IDH1MUT GBM from TCGA data set. C and D, the
mRNA expression level of DLC1 and CIDEB in HEK293T, hNSC, U251 and T98G cells are
determined by qPCR using β-actin as an internal control and standardized to hNSC which was
set as 100%. Results were presented as mean ± s.e.m. (n=3). Data for RBP1 has been published
in previous study (2). *, P<0.05; **, P<0.01; *** , P<0.001 relative to hNSC.
Figure S10. Model summarizing the role of miR-148a in IDH1 mutant gliomas. miR-148a
expression level is down-regulated in IDH1 mutant gliomas due to DNA hypermethylation of the
miR-148a promoter CpG island induced by IDH1 mutation. Hypermethylation of the miR-148a
promoter region silences miR-148a expression, subsequently upregulates its target gene DNMT1.
Highly expressed DNMT1 maintains the CpG Island Methylator Phenotype (CIMP) including
hypermethylated potential tumor suppressor genes. As a result, tumor development is initiated
and promoted. This is presumably an early event in gliomagenesis with of IDH1 mutant tumors.
Re-expression of miR-148a in turn inhibits DNMT1 expression by targeting its 3’-UTR, and
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subsequently up-regulates G-CIMP genes (RBP1, CIDEB and DLC1) with tumor suppressor
features.
1.
Balss J, Pusch S, Beck AC, Herold-Mende C, Kramer A, Thiede C, et al. Enzymatic assay for
quantitative analysis of (D)-2-hydroxyglutarate. Acta neuropathologica. 2012;124:883-91.
2.
Chou AP, Chowdhury R, Li S, Chen W, Kim AJ, Piccioni DE, et al. Identification of retinol binding
protein 1 promoter hypermethylation in isocitrate dehydrogenase 1 and 2 mutant gliomas. Journal of
the National Cancer Institute. 2012;104:1458-69.
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