Contribution of microRNA-1275 to Claudin11 protein suppression via a polycomb-mediated silencing mechanism in human glioma stem-like cells

Abstract

Glioblastomas show heterogeneous histological features, and tumor cells show distinct phenotypic states that confer different functional attributes and an aggressive character. However, the molecular mechanisms underlying the heterogeneity in this disease are poorly understood. Glioma stem-like cells (GSCs) are considered able to aberrantly differentiate into diverse cell types and may contribute to the establishment of tumor heterogeneity. Using a GSC model, we investigated differentially expressed microRNAs (miRNAs) and associated epigenetic mechanisms that regulate the differentiation of GSCs. miRNA profiling using microarray technology showed that 13 and 34 miRNAs were commonly up-regulated and down-regulated in two independent GSC lines during differentiation, respectively. Among this set of miRNAs, quantitative PCR analysis showed that miRNA-1275 (miR-1275) was consistently down-regulated during GSC differentiation, along with the up-regulation of its target, CLDN11, an important protein during oligodendroglial lineage differentiation. Inhibition of miR-1275 with a specific antisense oligonucleotide (anti-miR-1275) in GSCs increased the expression of CLDN11, together with significant growth suppression. Epigenetic analysis revealed that gain of histone H3 lysine 27 trimethylation (H3K27me3) in the primary microRNA-1275 promoter was closely associated with miR-1275 expression. Treatment with 3-deazaneplanocin A, an inhibitor of H3K27 methyltransferase, attenuated CLDN11 induction by serum stimulation in parallel with sustained miR-1275 expression. Our results have illuminated the epigenetic regulatory pathways of miR-1275 that are closely associated with oligodendroglial differentiation, which may contribute to the tissue heterogeneity seen in the formation of glioblastomas. Given that inhibition of miR-1275 induces expression of oligodendroglial lineage proteins and suppresses tumor cell proliferation, this may be a potential therapeutic target for glioblastomas.

FIGURE 1.

miRNA microarray analysis in GSC lines. A, heat map showing 13 miRNAs and 34 miRNAs that were commonly up-regulated (red) and down-regulated (blue) after serum exposure in two GSC lines (1228- and 316-GSCs). hsa, Homo sapiens. B, the predicted binding sites of miR-1275 are indicated (arrowheads) in the CLDN11 3′-UTR. Sequence alignments of miR-1275 with their corresponding potential binding sites in the CLDN11 3′-UTR are presented in each rectangle. Complementary sequence between CLDN11 and miR-1275 are indicated above or beneath the arrowheads. Nucleotide positions of each target sites are indicated as relative to the position of the stop codon of CLDN11 (The first nucleotide after the stop codon of CLDN11 is defined as 1). C, qPCR analysis of miR-1275 expression in GSC and S-BTC (continuous serum exposure for 21 days). Expression levels were normalized to internal RNU6B control. Levels of miR-1275 expression in the three GSC cell lines were similar, and values are expressed relative to abundance of GSC (upper panel). Lower panel, qPCR analysis of CLDN11 expression in GSC and S-BTC (continuous serum exposure for 21 days). Expression levels were normalized to internal GAPDH control. The assays were performed in three cell lines (1228-, 316-, and 222-GSCs), and the error bars indicate S.D. *, p < 0.05. D, Western blotting analysis of CLDN11 expression in GSC and S-BTC. β-Actin was used as a loading control.

FIGURE 2.

Interaction between miR-1275 and its binding sites in the CLDN11 3′-UTR. A, expression of miR-1275 in five human cancer cell lines (T98, MDA-MB-231, MCF7, SKBR3, and PC3) and human NSCs. Expression levels were normalized by RNU6B expression. B, Dual-Luciferase assay with pmir-CLDN11-3′-UTR reporter vector in cancer cell lines. Schemes of reporter vector with (pmir-CLDN11 3′-UTR) and without CLDN11 3′-UTR sequence (pmir-emp) are shown (upper panel). Black triangles indicate two miR-1275 binding sites within the 3′-UTR of the CLDN11 gene. Luciferase values are indicated as relative to abundance of luciferase activity of pmir-emp (bottom). Assays were performed in triplicate. Error bars indicate S.D. *, p < 0.05. C, Dual-Luciferase assay with the pmir-CLDN11 3′-UTR reporter vector in T98 cells. Cells were co-transfected with 30 nm or 100 nm precursor molecules pre-miR-1275 (Pre) or negative control miRNA precursor (NC). Left, expression levels of miR-1275 are indicated. Right, values are indicated relative to abundance of luciferase activity of pmir-emp control. The assays were performed in triplicate. Error bars indicate S.D. Asterisks mean that repression of luciferase activity was significantly depressed in the indicated treatments as compared with pmir-emp control (p < 0.05).

FIGURE 3.

miR-1275 targets CLDN11 in GSCs. A, expression levels of miR-1275 (left) and CLDN11 (right) in three GSC lines are indicated. Cells were transfected with 100 nm anti-miR inhibitor against miR-1275 (Anti-miR-1275), a negative control miRNA inhibitor (Anti-N), or without any antisense molecules (Mock). Expression levels were normalized to internal GAPDH and RNU6B controls, respectively. Values are indicated relative to the levels of mock control. Assays were performed in triplicate. Error bars indicate S.D. *, p < 0.05. B, Western blot analysis of CLDN11 expression in GSC transfected with either 100 nm anti-miR-1275 or 100 nm anti-N. β-Actin was used as a loading control (left panel). Immunofluorescence analysis of CLDN11 in GSCs and S-BTCs was performed. GSCs were transfected with either 100 nm anti-miR-1275 or 100 nm anti-N (middle). Bar, 20 μm. The ratio of immunopositive cells in each sphere is shown (right). The error bars indicate S.D. *, p < 0.01. C, cell proliferation assay of GSC lines (316, 222) transfected with either 100 nm anti-miR-1275 (triangle) or 100 nm anti-N (circle). Values are expressed relative to abundance in 0 days. The error bars indicate S.D. *, p < 0.05. D, cell proliferation assay of GSC lines (316, 222) transfected with either 100 nm siRNA against CLDN11 (triangle, siRNA-CLDN11, siRNA 1; square, siRNA 2) or 100 nm control siRNA (NC, circle) during GSC differentiation (left). Values are expressed relative to abundance at day 0. The error bars indicate S.D. *, p < 0.05. CLDN11 mRNA expression levels (mean ± S.D.) in GSCs transfected with either siRNA-CLDN11 or control siRNA are shown at the right.

FIGURE 4.

miR-1275 is regulated by PRC2-mediated H3K27me3. A, diagrammatic representation of the pri-miR-1275 promoter is shown (upper panel). The transcription start site (arrow) and location of pri-miR-1275 (black box) are indicated. Thick bars represent the region analyzed by ChIP-PCR. H3K27me3, H3K9Ac, and YY1 status in the pri-miR-1275 promoter region before (GSC, white box) and after serum exposure (S-BTC, black box) are shown (lower panel). Values are indicated relative to enrichment of each modification in GSCs. Error bars represent S.D. *, p < 0.01, **, p < 0.05. B, 222-GSCs were treated with DZNep (5 μm) followed by serum exposure. Expression levels of miR-1275 and CLDN11 after continuous serum exposure for 2–7 days (Dif.2 and Dif.7) with DZNep treatment are shown. The assays were performed in triplicate. Error bars indicate S.D. *, p < 0.01.

FIGURE 5.

Immunohistochemistry of CLDN11 and neural lineage-specific marker in clinical samples. A and B, hematoxylin/eosin (HE) staining (A) and corresponding immunohistochemistry of CLDN11 in normal brain (white matter) (B). Bar, 100 μm. C and D, hematoxylin/eosin staining of glioblastoma section (C) and corresponding immunohistochemical analysis of CLDN11 in glioblastoma section (D). Bar, 500 μm. E, the area in panel D is magnified. Bar, 100 μm. F–H, immunofluorescence analysis of CLDN11 (green) and GFAP (red) (F), CLDN11 (green), and MAP2 (red) (G), and CLDN11 (green) and OLIG2 (red) (H) in glioblastoma. Cells are also stained with DAPI to highlight nuclear areas. Bar, 100 μm. I, ratio of immunopositive cells to each neural-lineage marker among CLDN11-positive cells is counted. Error bars indicate S.D.