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Genomic Loss of microRNA-101 Leads to Overexpression of Histone Methyltransferase EZH2 in Cancer

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Genomic Loss of microRNA-101 Leads to Overexpression of Histone Methyltransferase EZH2 in Cancer

Sooryanarayana Varambally et al. Science.

Abstract

Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes and regulates the survival and metastasis of cancer cells. EZH2 is overexpressed in aggressive solid tumors by mechanisms that remain unclear. Here we show that the expression and function of EZH2 in cancer cell lines are inhibited by microRNA-101 (miR-101). Analysis of human prostate tumors revealed that miR-101 expression decreases during cancer progression, paralleling an increase in EZH2 expression. One or both of the two genomic loci encoding miR-101 were somatically lost in 37.5% of clinically localized prostate cancer cells (6 of 16) and 66.7% of metastatic disease cells (22 of 33). We propose that the genomic loss of miR-101 in cancer leads to overexpression of EZH2 and concomitant dysregulation of epigenetic pathways, resulting in cancer progression.

Figures

Fig. 1
Fig. 1
miR-101 regulates EZH2 transcript and protein expression. (A) Venn diagram displaying miRNAs computationally predicted to target EZH2 from PicTar (blue), miRanda (red), TargetScan (green), and MicroInspector (orange). (B) Schematic of two predicted miR-101 binding sites in the EZH2 3’UTR. (C) miR-101 regulates EZH2 transcript expression. qRT-PCR of EZH2 in SKBr3 cells transfected with precursor miR-101. Control miR and other precursor miRNAs (miR-26a, miR-128a, and miR-217) were also used for transfection. (D), miR-101 regulates Polycomb Group Complex 2 protein expression. miR-101 downregulates EZH2 protein as well as Polycomb members SUZ12 and EED in SKBr3 cells. Control miRs and EZH2 specific siRNA were also used for transfection. The experiment was performed three independent times and a representative result displayed.
Fig. 2
Fig. 2
The role of miR-101 in regulating cell proliferation, invasion and tumor growth. (A) miR-101 overexpression reduces cell proliferation. Cell growth assay of SKBr3 cells treated with either precursor miR-101 or siRNA targeting EZH2. Cell growth relative to the control miRNA and control siRNA duplex was measured. Rescue experiments were performed by overexpressing EZH2 (minus its endogenous 3’UTR) in miR-101-treated cells. (B) miR-101 expression decreases cell invasion of DU145 prostate carcinoma cells. Cells were transfected with miR-101, EZH2 specific siRNA, control miR and non-targeting siRNA. miR-101 was also overexpressed in those cells overexpressing EZH2 by andenoviral infection. All cells were subjected to a matrigel invasion assay. (C) AntagomiRs to miR-101 induce the invasiveness of benign immortalized H16N2 breast epithelial cells. Representative fields of invaded and stained cells are shown in the inset. P-values were calculated between control antagomiR and antagomiR-101i and ii. (D) Overexpression of miR-101 attenuates prostate tumor growth. Overexpression of miR-101 reduces DU145 tumor growth in a mouse xenograft model. Plot of mean tumor volume trajectories over time for the mice inoculated with miR-101 (red) and vector (green) expressing DU145 cells. Error bars represent the standard error of the mean at each time point. Inset displays decrease of EZH2 protein levels in miR-101 expressing cell lines.
Fig. 3
Fig. 3
miR-101 regulation of the cancer epigenome through EZH2 and H3K27 tri-methylation. (A) Chromatin immunoprecipitation (ChIP) assay of the trimethyl H3K27 histone mark when miR-101 is overexpressed. Known PRC2 repression targets were examined in SKBr3 cells. ChIP was performed to test H3K27 trimethylation at the promoters of ADRB2, DAB2IP, CIITA, RUNX3, CDH1 and WNT1. GAPDH, KIAA0066 and NUP214 gene promoters served as controls. (B) qRT-PCR of EZH2 target genes was performed using SKBr3 cells transfected with miR-101. The EZH2 transcript and its known targets including ADRB2, DAB2IP, CIITA, RUNX3 and E-cadherin (CDH1) were measured.
Fig. 4
Fig. 4
Genomic loss of the miR-101 locus may explain overexpression of EZH2 in solid tumors. (A) miR-101 transcript levels are inversely correlated with EZH2 expression in prostate cancer progression. qPCR was performed for miR-101 and miR-217 using total RNA from benign adjacent prostate, prostate cancer (PCA), and metastatic (MET) prostate cancer tissue. EZH2 expression was analyzed from the same RNA samples. (B) Genomic PCR of miR-101-1 and miR-101-2 in prostate cancer progression. Vertical axes represent log (base 2) relative quantification values; dashed lines are shown at the deletion threshold of log2(0.7)≈−0.51. For clarity, points have been horizontally displaced within each sample class. (C) Heatmap representation of matched normal, tumor, and metastatic samples (from right to left) in which miR-101 transcript, EZH2 transcript, and both miR-101-1 and miR-101-2 relative copy number were assessed. miR-101 and EZH2 expression is represented by a color scale highlighting down-regulation (green), no alteration (black), and up-regulation (red) of transcripts. miR-101-1 and miR-101-2 relative quantitation (RQ) of copy number are represented as homozygous loss (< 0.3; bright green), single copy loss (< 0.7; light green), no copy number change (>= 0.7 and <= 1.3; black), single copy gain (> 1.3; light red), and double copy gain (> 1.7; bright red). (D) Evidence that the miR-101-1 locus is somatically lost in tumors samples relative to matched normal samples. Nine metastatic prostate cancers were chosen that have copy number loss in the miR-101-1 locus, and matched normal tissue were analyzed for comparison. Bar heights represent differences in log2(RQ) values between metastatic and matched normal tissues.

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