The Putative Tumor Suppressor microRNA-101 Modulates the Cancer Epigenome by Repressing the Polycomb Group Protein EZH2 (original) (raw)

Molecular Biology, Pathobiology, and Genetics| March 17 2009

1Department of Urology, Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, and

Search for other works by this author on:

Gangning Liang;

1Department of Urology, Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, and

Search for other works by this author on:

Chun-Chi Liu;

2Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California

Search for other works by this author on:

Erika M. Wolff;

1Department of Urology, Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, and

Search for other works by this author on:

Yvonne C. Tsai;

1Department of Urology, Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, and

Search for other works by this author on:

Wei Ye;

1Department of Urology, Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, and

Search for other works by this author on:

Xianghong Zhou;

2Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California

Search for other works by this author on:

Peter A. Jones

1Department of Urology, Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine, and

Search for other works by this author on:

Requests for reprints: Peter A. Jones, University of Southern California/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Los Angeles, California 90033. Phone: 323-865-0816; Fax: 323-865-0102; E-mail: jones_p@ccnt.usc.edu and Gangning Liang, University of Southern California/Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, NOR7346, Los Angeles, CA 90033. Phone: 323-865-0470; Fax: 323-865-0102; E-mail: gliang@usc.edu.

Received: August 11 2008

Revision Received: December 31 2008

Accepted: January 02 2009

Online ISSN: 1538-7445

Print ISSN: 0008-5472

2009

Cancer Res (2009) 69 (6): 2623–2629.

Article history

Revision Received:

December 31 2008

Accepted:

January 02 2009

Split-Screen
Views Icon Views
Open the PDF for in another window
Tools Icon Tools
Search Site
Article Versions Icon Versions
- Version of Record March 17 2009
- Proof March 3 2009

Abstract

The Polycomb Repressive Complex 2 (PRC2) mediates epigenetic gene silencing by trimethylating histone H3 lysine 27 (H3K27me3) and is known to aberrantly silence tumor suppressor genes in cancer. EZH2, the catalytic subunit of PRC2, enhances tumorigenesis and is commonly overexpressed in several types of cancer. Our microRNA profiling of bladder transitional cell carcinoma (TCC) patient samples revealed that microRNA-101 (miR-101) is down-regulated in TCC, and we showed that miR-101 inhibits cell proliferation and colony formation in TCC cell lines. Furthermore, our results confirm that miR-101 directly represses EZH2 and stable EZH2 knockdowns in TCC cell lines create a similar growth suppressive phenotype. This suggests that abnormal down-regulation of miR-101 could lead to the overexpression of EZH2 frequently seen in cancer. We conclude that miR-101 may be a potent tumor suppressor by altering global chromatin structure through repression of EZH2. [Cancer Res 2009;69(6):2623–9]

Introduction

Polycomb group proteins are chromatin-modifying enzymes that are important in stem cell maintenance, X-inactivation, imprinting, and development, and many Polycomb group proteins are dysregulated in cancer (1). The Polycomb group protein EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), which is a critical part of the cellular machinery involved in epigenetically regulating gene transcription (2). PRC2 represses genes by trimethylating the core histone H3 lysine 27 (H3K27me3) at and around the promoter regions of target genes (1).

EZH2 enhances proliferation and neoplastic transformation (3), is overexpressed in many cancers, and is strongly associated with metastatic breast and prostate cancers (3–7). Recent work has shown that overexpression of EZH2 is directly responsible for the de novo suppression of multiple genes in human cancer (8, 9). However, the cause of EZH2 overexpression in cancer is not clear. Intriguingly, a significant subset of PRC2 target genes in cancer were also targets of PRC2 in embryonic stem cells (10). This illustrates a strong association between the function of PRC2 in cancer and stem cells, which represent dedifferentiated and proliferative cell states. Therefore, EZH2 overexpression might cause a normal cell to dedifferentiate back to a stem cell–like state by epigenetically repressing cell fate–regulating genes and tumor-suppressor genes leading to tumor development (1, 9, 11).

MicroRNAs (miRNA) are ∼22 nucleotide noncoding RNA molecules that usually function as endogenous repressors of target genes. In animals, miRNAs can bind with imperfect complementarity to the 3′ untranslated region (3′ UTR) of the target mRNA via the RNA-induced silencing complex. The resulting gene repression occurs by multiple mechanisms including enhanced mRNA degradation and translational repression (12). Developmental timing, cell death, proliferation, hematopoiesis, insulin secretion, and the immune response are just a few examples of critical biological events that depend on faithful miRNA expression (13).

In the past few years, miRNA profiling of various human cancers has revealed many miRNAs that function as tumor suppressors, such as let-7, miR-15a/16-1, and the miR-34 family, or oncogenes such as miR-155 and the miR-17-92 cluster (14, 15). In addition, miRNA profiles were better able to predict tumor type than were mRNA profiles (16). However, miRNA profiling of bladder cancer with normal and matched tumor tissues and a functional study of differentially expressed miRNAs in bladder cancer remains to be conducted. Bladder cancers in the United States are almost exclusively transitional cell carcinomas (TCC), and in 2007, TCC was the fifth most common cancer diagnosis according to the National Cancer Institute (NCI).

Therefore, we sought to generate a miRNA expression profile for TCC by comparing primary TCCs to their corresponding normal urothelium. We found many differentially expressed miRNAs, several of which showed putative tumor suppressor functions. The miRNA that most consistently and dramatically suppressed growth was miR-101, which we confirmed can directly target EZH2 and repress H3K27me3. Furthermore, our results indicate that a significant subset of genes is regulated by both miR-101 and EZH2.

Materials and Methods

Detailed protocols and primer sequences are in the Supplementary Data.

Cell lines and primary tumors. T24, UM-UC-3, and TCCSUP cells were obtained from the American Type Culture Collection (ATCC) and cultured according to ATCC protocols. Patient samples were obtained through University of Southern California/Norris Tissue Procurement Core Resource after informed consent and Institutional Review Board approval (IRB #886005 and #926041) at the University of Southern California/Norris Comprehensive Cancer Center.

miRNA microarray. One microgram of total RNA from each of nine TCCs was pooled and the same was done with nine matched normal tissues. miRNA microarray analysis was done as previously described (17).

Reverse transcription and Taqman qPCR. miRNA Taqman assays (Applied Biosystems) were used according to the manufacturer's protocol. EZH2, U6 primers are provided in the supplemental data.

Expression vectors. Expression vectors were made by cloning ∼200 bp surrounding the precursor miRNA into pcDNA3.1(+) (Invitrogen). Expression vectors containing shRNA to EZH2 were commercially available (Sigma).

Cell proliferation and colony formation assays. Cell proliferation and colony formation assays were conducted as described previously (18, 19).

Western blot. Western blots were performed as previously described (17).

Luciferase assay. Reporter vectors were created by cloning the wild-type or mutated 3′UTR of EZH2 into the XbaI site of the pGL3-control vector (Promega). Firefly and renilla luciferase activity was analyzed using the Dual Luciferase Reporter assay system (Promega) as previously described (17).

Messenger RNA microarray. UM-UC-3 cells were transfected with pre–miR-101, control precursors, siRNA to EZH2, and control siRNA in triplicate. Total RNA was prepared 72 h after transient transfection and the Illumina human 6 v 2 array was used for gene expression analysis. The Norris Cancer Center CORE facility performed the amplification and hybridization according to the manufacturer's protocol (Illumina).

Results and Discussion

Differential miRNA expression in TCCs. We used a miRNA microarray to examine differentially expressed miRNAs in a pool of nine TCCs and a pool of the corresponding normal samples. miR-1, miR-101, miR-143, miR-145, and miR-29c were the most down-regulated transcripts in the tumors, and miR-182, miR-183, miR-203, miR-224, and miR-196a were the most up-regulated (Table 1; Fig. 1A). miR-127 was included in the table because our previous work revealed it is down-regulated in human cancers (17).

Table 1.

Summary of the most differentially expressed miRNAs in TCC compared with normal bladder tissue from Fig. 1A

MiRNA	Fold change (tumor/normal)
hsa-miR-183	38
hsa-miR-182	37
hsa-miR-203	36
hsa-miR-224	23
hsa-miR-196a	23
hsa-miR-10a	10
hsa-miR-127	−3
hsa-miR-29c	−9
hsa-miR-145	−9
hsa-miR-143	−11
hsa-miR-101	−16
hsa-miR-1	−54

MiRNA	Fold change (tumor/normal)
hsa-miR-183	38
hsa-miR-182	37
hsa-miR-203	36
hsa-miR-224	23
hsa-miR-196a	23
hsa-miR-10a	10
hsa-miR-127	−3
hsa-miR-29c	−9
hsa-miR-145	−9
hsa-miR-143	−11
hsa-miR-101	−16
hsa-miR-1	−54

NOTE: Data are presented as the fold change of miRNA expression from the tumor/normal for the most differentially expressed transcripts (including miR-127) that were validated with RT-qPCR in additional patient samples.

Figure 1.

miRNAs are dysregulated in TCC. A, plot for miRNA microarray of the Tumor signal versus Normal signal shows all transcripts with those showing fold change of >8 in black. B, miRNA RT-qPCR of 28 clinical TCC samples and matched normal urothelium. All reactions were done in duplicate and U6 was the internal control. The graph shows the ratio of miRNA expression of Tumor/Normal on a logarithmic scale. *, statistical significance; error bars, 95% confidence interval. C, cell proliferation assays were conducted by transferring equal cell numbers to 10-cm dishes 48 h posttransfection with miRNA expression vectors or empty vector (e.v.) control. After 13 d under G418 selection, total cells were counted and normalized to the empty vector. D, colony formation assays were conducted by seeding equal cell numbers 48 h posttransfection into 6-well plates. Colonies were stained and counted after 13 d under G418 selection and normalized to the empty vector control. *, P < 0.02 according to Dunnet's method (except UM-UC-3 miR-145, P value = 0.044; C and D); columns, mean; bars, SE.

Figure 1.

Close modal

We conducted RT-qPCR for 12 differentially expressed miRNAs on 28 additional TCC patients. miR-1, miR-101, miR-143, miR-145, miR-29c, and miR-127 were significantly down-regulated in the tumors, whereas miR-224, miR-182, and miR-183 were significantly up-regulated in the tumors (Fig. 1B). miR-196a, miR-10a, and miR-203 showed no significant differences. Our miRNA microarray analysis and RT-qPCR results showed that there is severe and consistent miRNA misexpression in TCC, and our miRNA panel likely constitutes a TCC miRNA signature. The miRNA that was most down-regulated in TCC, miR-1, and miR-29c were down-regulated in hepatocellular carcinoma and lung cancer, respectively (20, 21). Furthermore, our study supports the link between down-regulation of miR-143 and miR-145 and cancer, which was previously shown in various malignancies (22, 23).

Restored expression of miRNAs in TCC cell lines reveals putative tumor suppressors. We used RT-qPCR to analyze miRNA expression in 10 TCC cell lines. miR-1, miR-101, miR-127, miR-143, and miR-145 were expressed at low levels in all cell lines (data not shown), indicating that these miRNAs are promising tumor suppressor candidates. After creating miRNA expression vectors, T24, TCCSUP, and UM-UC-3 cells were transfected and the enhanced miRNA expression from each vector was confirmed by RT-qPCR (Supplementary Fig. S1). The expression levels for miR-101 were found to be of similar levels to those found in adjacent normal tissues (Supplementary Fig. S2).

Proliferation assays were done by stably transfecting cells with the miRNA expression vectors to determine the functional effects of miRNA misexpression. The results showed a strong inhibition of cell proliferation by miR-101. In T24 cells, miR-101 suppressed proliferation by 57% (Fig. 1C). Proliferation was significantly inhibited by miR-101, miR-1, and miR-145 in UM-UC-3 and TCCSUP cells (Fig. 1C). These results indicate that miR-101 is the most potent growth suppressor, although miR-1 and miR-145 also significantly inhibited cell proliferation.

We determined the effect of restored miRNA expression on colony formation, and again, the results varied depending on the cell line and miR-101 was the most potent suppressor of colony formation. In T24 cells, miR-101 suppressed colony formation by 45% (Fig. 1D). miR-101, miR-127, miR-143, and miR-145 significantly reduced colonies in UM-UC-3 cells, whereas in TCCSUP cells, miR-101, miR-1, and miR-143 showed significant suppression (Fig. 1D). Clearly, restored expression of miR-101 potently suppresses colony formation in these cell lines, whereas other miRNAs also suppress colony formation, although less substantially and consistently. The cell proliferation and colony formation assays indicate that miR-101 is a putative tumor suppressor and may be a therapeutic target for cancer.

There are a plethora of mechanisms that could lead to decreased miRNA expression in cancer including copy number alterations (16), epigenetic silencing (17), and _trans_-acting factors (15). There are two copies of miR-101, with miR-101-1 located at chromosome 1p31 and miR-101-2 located at chromosome 9p24. The two precursors have different sequences, but the mature forms are identical. We found that miR-101 silencing in TCC cell lines was probably not due to epigenetic phenomena because treatment with the DNA demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor 4-phenylbutyric acid did not induce expression of miR-101 (data not shown). Intriguingly, loss of heterozygosity (LOH) at chromosome 1p occurs in many different solid tumors and is negatively associated with survival (24), whereas LOH at chromosome 9p also commonly occurs in cancer, particularly TCC (25). While this article was being revised, genomic loss was found to be a mechanism for miR-101 down-regulation in prostate cancer (26). This suggests that DNA copy number may regulate miR-101 expression, although a _trans_-acting mechanism should be investigated in the future.

miR-101 represses the Polycomb group protein EZH2. We used the prediction algorithm TargetScan3

to identify targets of miR-101 and found the histone methyltransferase EZH2 had a very high score, a highly conserved sequence, and two predicted sequence matches to the miR-101 seed. To make a clear comparison, we plotted miR-101 RT-qPCR data from 20 individual patient samples from Fig. 1B above the mRNA levels of EZH2 that were obtained by RT-qPCR (Fig. 2A). Our results showed that EZH2 was up-regulated in the tumor samples relative to the adjacent normal specimens, whereas miR-101 is down-regulated in these same samples (Fig. 2A).

Figure 2.

miR-101 directly targets EZH2. A, RT-qPCR analysis of 20 TCC samples shows that EZH2 is up-regulated in tumors compared with adjacent normal tissue. The data from Fig. 1B showing miR-101 levels was modified to show individual patient samples and this was compared with EZH2 levels in these same samples. Each experiment was duplicated and the value for miR-101 or EZH2 was normalized to U6. Data are presented as individual samples with the line indicating the mean (for EZH2 paired t test, P = 0.004). B, Western blot analysis of TCC cell lines after transient transfection with pre–miR-101 or control precursors at a final concentration of 50 or 5 nmol/L where indicated. Lysates were prepared 48 h after transfection and membranes were probed with antibodies to EZH2, H3K27me3, and β-actin as a loading control. Bands were quantitated by Quantity One software (Bio-Rad). C, the highly conserved sequence of the 3′UTR of EZH2 for human, mouse, rat, dog, and chicken are shown. *, nucleotides that were mutated for the luciferase insert. D, luciferase assay conducted in UM-UC-3 cells 24 h after cotransfection with pre–miR-101, renilla luciferase vector pRL-SV40, and either the firefly luciferase reporter pGL3-control containing wild-type EZH2 3′UTR insert (GUACUGU) or mutated EZH2 3′UTR insert (_C_UA_G_U_C_U). Relative luciferase activity was normalized to the no insert control (*, P < 0.01). Columns, mean; bars, SE.

Figure 2.

Close modal

We transiently transfected miR-101 precursors (pre–miR-101) into T24, TCCSUP, and UM-UC-3 cells and visualized EZH2 levels by Western blot. In TCCSUP and UM-UC-3 cells, EZH2 levels decreased by 86% and 91%, respectively, whereas H3K27me3 decreased by 58% in both cell lines (Fig. 2B). In T24 cells, there was a 52% decrease in EZH2 levels and the H3K27me3 levels remained the same, which is likely due to lower transfection efficiencies that yielded 50% less mature miR-101 levels when compared with UM-UC-3 cells (data not shown). We determined the effect of physiologic levels of miR-101 on EZH2 by transfecting 5 nmol/L pre–miR-101 in UM-UC-3 cells. The results showed a 39% down-regulation of EZH2 (Fig. 2B) and mature miR-101 levels comparable with the average normal patient sample (Supplementary Fig. S2). These results indicate that miR-101 can repress EZH2 in TCC cell lines even at levels that are similar to those found in normal urothelium (Fig. 2B).

EZH2 is a direct target of miR-101. To confirm that EZH2 is a direct target of miR-101, we created luciferase reporter vectors by cloning either the wild-type or a mutated portion of the 3′UTR of EZH2 into the 3′UTR of the pGL3-control vector. The mutated 3′UTR had three bases changed, from GUACUGU to _C_UA_G_U_C_U, at each of the two putative miR-101 binding sites (Fig. 2C). We transfected these vectors with pre–miR-101 into UM-UC-3 cells and the lysates were analyzed 24 hours later. The results showed a 42% decrease in luciferase activity for the wild-type 3′UTR of EZH2, whereas the mutated 3′UTR showed no repression when compared with the empty reporter as was also found previously (Fig. 2D; ref. 27). Therefore, miR-101 represses EZH2 by binding to the 3′UTR of EZH2 in a direct and sequence specific manner.

Knockdown of EZH2 shows phenotypic overlap with the restoration of miR-101 expression in TCC cells. After confirming that miR-101 leads to a decrease in H3K27me3 levels by targeting EZH2, we examined if there was a phenotypic overlap between stable miR-101 transfection and knockdown of EZH2 by shRNA in TCC cells. We conducted cell proliferation assays and colony formation assays in three TCC cell lines using four expression vectors (clones 74, 75, 76, and 77) containing distinct shRNAs targeting EZH2. The results showed that two of four, one of four, and four of four shRNAs suppressed cell proliferation in T24, TCCSUP, and UM-UC-3 cells, respectively (Fig. 3A). The colony formation results were more striking as all four shRNAs suppressed colony formation in T24 and TCCSUP cells, whereas three of four shRNAs suppressed colony formation in UM-UC-3 cells (Fig. 3B and C).

Figure 3.

Knockdown of EZH2 decreases cell proliferation and colony formation in TCC cell lines. A, cell proliferation assays were conducted by transferring equal cell numbers to 10-cm dishes 48 h posttransfection with 4 different expression vectors (clones 74, 75, 76, and 77) containing distinct shRNAs to EZH2 or control shRNA vector. After 13 d under puromycin selection, total cells were counted and normalized to the empty vector. B, colony formation assays were conducted by seeding equal cell numbers 48 h posttransfection into 10-cm dishes. Colonies were stained and counted after 13 d under puromycin selection and normalized to the control shRNA vector. *, P < 0.05 according to t test; columns, mean; bars, SE. C, photographs of representative colony formation assays.

Figure 3.

Close modal

To examine additional phenotypic overlap, expression microarrays were conducted on UM-UC-3 cells transfected with pre–miR-101 or siRNA to EZH2. We identified a significant overlap of 43 up-regulated genes (P < 10−11 by hypergeometric distribution), which suggests that restoring miR-101 expression in cancer cells re-expresses a subset of genes that are repressed by EZH2 (Fig. 4B). The extra decrease in EZH2 and H3K27me3 from the siRNA to EZH2 may explain why there were 62 up-regulated genes that did not overlap with the genes up-regulated by pre–miR-101 transfection (Fig. 4A and B). In contrast, we expect the pre–miR-101 transfection to have much wider effects on gene expression than the siRNA to EZH2 because miRNAs are more promiscuous and likely repress several targets that influence the transcriptome. We conducted chromatin immunoprecipitation analysis on the promoters for the Polycomb target genes FAM84 and DDIT4 after treating UM-UC-3 cells with siRNA to EZH2 and pre–miR-101 for 72 hours. The results showed that both treatments depleted the promoters of H3K27me3 (Supplementary Fig. S3), indicating that miR-101 regulates histone methylation through EZH2.

Figure 4.

Pre–miR-101 transfection and siRNA to EZH2 lead to the up-regulation of overlapping genes. A, Western blot analysis of UM-UC-3 cells transfected with siRNA to EZH2, control siRNA, pre–miR-101, or control precursors to a final concentration of 50 nmol/L. Total protein was extracted 72 h after transfection and membranes were probed with antibodies to EZH2, H3K27me3, and β-actin as a loading control. B, Illumina Human 6 v 2 chips were used to interrogate the mRNA levels from UM-UC-3 cells 72 h after transfection with pre–miR-101, control precursors, siRNA to EZH2, and control siRNA. Based on the criteria fold change of >1.5 and t test P value of <0.05, 1,092 genes were up-regulated after pre–miR-101 transfection, whereas 105 genes were up-regulated after treatment with siRNA to EZH2. There was an overlap of 43 genes (P < 10−11 based on hypergeometric distribution).

Figure 4.

Close modal

The molecular similarities between cancer and stem cells are becoming ever more apparent. For example, EZH2 is critical for the maintenance of proliferation and pluripotency in stem cells (1). Global levels of Ezh2 and H3K27me3 decrease when mouse embryonic stem cells differentiate and PRC2 target genes specifically lose Ezh2 binding and H3K27me3 enrichment (28). In addition, two groups found that miR-101 was up-regulated after differentiation of human embryonic stem cell lines (29, 30). These reports present a strong association between miR-101 activation and EZH2 repression upon differentiation of stem cells, suggesting that miR-101 might be part of the complex network, which includes Polycomb group proteins, that determines developmental outcome. Therefore, in addition to its potential role in cancer, miR-101 might be involved in normal differentiation by directly repressing EZH2 and re-expressing cell fate regulating genes.

The role of miRNAs in controlling epigenetics is just emerging. Embryonic stem cell–specific miRNAs control de novo DNA methylation during differentiation by targeting Rbl2 (31, 32). A recent report suggests that the down-regulation of the miR-29 family in lung cancer could lead to the overexpression of DNA methyltransferases 3A and 3B and subsequent DNA hypermethylation and gene silencing (21). This study shows another instance in which miRNA-mediated epigenetic mechanisms can be dysregulated in cancer with global consequences.

Our results and previously published data examining miR-101 in lung cancer, breast cancer, colorectal cancer, and prostate cancer (22, 23, 33, 34) indicate that miR-101 is down-regulated in the five most frequently diagnosed cancers in the United States (NCI), demonstrating that miR-101 might be part of a solid tumor signature. This report suggests that aberrant silencing of miR-101 may be a cause of the overexpression of EZH2 seen in cancer and that restoring miR-101 expression could hinder EZH2-mediated neoplastic progression.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Grant support: NIH grant RO1 CA 83867 and the NIH/National Institute of General Medical Sciences–supported training program in Cellular, Biochemical and Molecular Sciences at the University of Southern California.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

All tissue procurement was performed by the University of Southern California/Norris Comprehensive Cancer Center Translational Pathology Core Facility, supported by the Cancer Center Support grant P30 CA 014089.

References

Sparmann A, van Lohuizen M. Polycomb silencers control cell fate, development and cancer.

Nat Rev Cancer

2006

;

846

–56.

Pasini D, Bracken AP, Helin K. Polycomb group proteins in cell cycle progression and cancer.

Cell Cycle

2004

;

396

–400.

Kleer CG, Cao Q, Varambally S, et al. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells.

Proc Natl Acad Sci U S A

2003

;

100

11606

–11.

Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer.

EMBO J

2003

;

5323

–35.

Raman JD, Mongan NP, Tickoo SK, Boorjian SA, Scherr DS, Gudas LJ. Increased expression of the polycomb group gene, EZH2, in transitional cell carcinoma of the bladder.

Clin Cancer Res

2005

;

8570

–6.

Varambally S, Dhanasekaran SM, Zhou M, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer.

Nature

2002

;

419

624

–9.

Rhodes DR, Yu J, Shanker K, et al. Large-scale meta-analysis of cancer microarray data identifies common transcriptional profiles of neoplastic transformation and progression.

Proc Natl Acad Sci U S A

2004

;

101

9309

–14.

Gal-Yam EN, Egger G, Iniguez L, et al. Frequent switching of Polycomb repressive marks and DNA hypermethylation in the PC3 prostate cancer cell line.

Proc Natl Acad Sci U S A

2008

;

105

12979

–84.

Kondo Y, Shen L, Cheng AS, et al. Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation.

Nat Genet

2008

;

741

–50.

Yu J, Yu J, Rhodes DR, et al. A polycomb repression signature in metastatic prostate cancer predicts cancer outcome.

Cancer Res

2007

;

10657

–63.

Jones PA, Baylin SB. The epigenomics of cancer.

Cell

2007

;

128

683

–92.

Valencia-Sanchez MA, Liu J, Hannon GJ, Parker R. Control of translation and mRNA degradation by miRNAs and siRNAs.

Genes Dev

2006

;

515

–24.

Ambros V. The functions of animal microRNAs.

Nature

2004

;

431

350

–5.

Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a role in cancer.

Nat Rev Cancer

2006

;

259

–69.

Hermeking H. p53 enters the microRNA world.

Cancer Cell

2007

;

414

–8.

Calin GA, Croce CM. MicroRNA signatures in human cancers.

Nat Rev Cancer

2006

;

857

–66.

Saito Y, Liang G, Egger G, et al. Specific activation of microRNA-127 with downregulation of the proto-oncogene BCL6 by chromatin-modifying drugs in human cancer cells.

Cancer Cell

2006

;

435

–43.

Robertson KD, Jones PA. Tissue-specific alternative splicing in the human INK4a/ARF cell cycle regulatory locus.

Oncogene

1999

;

3810

–20.

Kim TY, Zhong S, Fields CR, Kim JH, Robertson KD. Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma.

Cancer Res

2006

;

7490

–501.

Datta J, Kutay H, Nasser MW, et al. Methylation mediated silencing of MicroRNA-1 gene and its role in hepatocellular carcinogenesis.

Cancer Res

2008

;

5049

–58.

Fabbri M, Garzon R, Cimmino A, et al. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B.

Proc Natl Acad Sci U S A

2007

;

104

15805

–10.

Iorio MV, Ferracin M, Liu CG, et al. MicroRNA gene expression deregulation in human breast cancer.

Cancer Res

2005

;

7065

–70.

Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis.

Cancer Cell

2006

;

189

–98.

Ragnarsson G, Eiriksdottir G, Johannsdottir JT, Jonasson JG, Egilsson V, Ingvarsson S. Loss of heterozygosity at chromosome 1p in different solid human tumours: association with survival.

Br J Cancer

1999

;

1468

–74.

Wolff EM, Liang G, Jones PA. Mechanisms of disease: genetic and epigenetic alterations that drive bladder cancer.

Nat Clin Pract Urol

2005

;

502

–10.

Varambally S, Cao Q, Mani RS, et al. Genomic Loss of microRNA-101 Leads to Overexpression of Histone Methyltransferase EZH2 in Cancer.

Science

2008

;

322

1695

–9.

Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of mammalian microRNA targets.

Cell

2003

;

115

787

–98.

Lee ER, Murdoch FE, Fritsch MK. High histone acetylation and decreased polycomb repressive complex 2 member levels regulate gene specific transcriptional changes during early embryonic stem cell differentiation induced by retinoic acid.

Stem Cells

2007

;

2191

–9.

Bar M, Wyman SK, Fritz BR, et al. MicroRNA Discovery and profiling in human embryonic stem cells by deep sequencing of small RNA libraries.

Stem Cells

2008

;

2496

–505.

Lakshmipathy U, Love B, Goff LA, et al. MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells.

Stem Cells Dev

2007

;

1003

–16.

Sinkkonen L, Hugenschmidt T, Berninger P, et al. MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells.

Nat Struct Mol Biol

2008

;

259

–67.

Benetti R, Gonzalo S, Jaco I, et al. A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases.

Nat Struct Mol Biol

2008

;

268

–79.

Porkka KP, Pfeiffer MJ, Waltering KK, Vessella RL, Tammela TL, Visakorpi T. MicroRNA expression profiling in prostate cancer.

Cancer Res

2007

;

6130

–5.

Schepeler T, Reinert JT, Ostenfeld MS, et al. Diagnostic and prognostic microRNAs in stage II colon cancer.

Cancer Res

2008

;

6416

–24.

2009

Supplementary data

747 Views

324 Web of Science

The Putative Tumor Suppressor microRNA-101 Modulates the Cancer Epigenome by Repressing the Polycomb Group Protein EZH2 (original) (raw)

Abstract

Introduction

Materials and Methods

Results and Discussion

Disclosure of Potential Conflicts of Interest

Acknowledgments

References

Supplementary data

Citing articles via

Email alerts