Yin-Yuan Mo

Long noncoding RNAs (lncRNAs), as a new class of transcripts, have been recently shown to be pervasively transcribed in the genomes of human and mouse. In particular, accumulating evidence link the expression of lncRNAs to diverse human diseases, highlighting their potential as biomarkers and therapeutic targets. However, there are still many missing dots in our current understanding of lncRNA function. Thus, investigation of the expression patterns of lncRNAs is a crucial step to understanding of their roles in many model systems. To this end, we developed a qPCR based disease-related human lncRNA profiler, which allows for the quantification of differential expression of 83 individual lncRNAs among various experimental RNA samples. All 83 lncRNAs chosen for the array are based on publications and they are implicated in diseases ranging from neurodegeneration to cancer. The array plate also includes the house keeping genes and small RNA transcripts for normalization purposes. To determine the utility of this lncRNA profiler, we profiled their expression in breast cancer specimens compared to matched normal tissue and identified several lncRNAs are dysregulated, suggesting that lncRNAs may function as oncogenes or tumor suppressors. Gene expression profiling with this lncRNA profiler in a series of breast cell lines such as MCF10A, MCF7 and MDA-MB-231 cells identified differential expression patterns unique to each cell line, which may provide a basis for future analysis of these differentially expressed lncRNAs. Furthermore, we determined the effect of p53 on lncRNA expression in HCT-116 and MCF-7 cells. Initial testing indicated that at least three lncRNAs are induced by p53 in both cell lines. Experiments are underway to further characterize these p53 inducible lncRNAs. Together, these results suggest that disease-related human lncRNA profiler is a valuable research tool that can be tailored for specific needs in different research laboratories. The features of this profiler are easy, convenient, sensitive and specific. Accordingly it serves the first step toward the understanding of the role of lncRNAs in human diseases. Citation Format: Fangting Wu, Yin-Yuan Mo. Profiling long noncoding RNA expression in cancer cells by real-time PCR arrays [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A19.

MicroRNAs are gene regulators that work through a posttranscriptional repression mechanism. Dysregulation of microRNA expression could lead to a variety of disorders, in particular, human cancer, and has also been implicated in antihormone therapy resistance. However, little is known whether microRNAs have a role in estrogen-independent growth, leading to tamoxifen resistance in estrogen receptor (ER)-positive tumors. In this study, we use an in vivo selection system against a microRNA library using the MCF-7 model and demonstrate that miR-101 promotes estrogen-independent growth and causes the upregulation of phosphorylated Akt (pAkt) without impacting the ER level or activity. Importantly, although miR-101 suppresses cell growth in normal estradiol (E2)-containing medium, it promotes cell growth in E2-free medium. Moreover, estrogen deprivation greatly enhances miR-101-mediated Akt activation. Finally, we show that MAGI-2 (membrane-associated guanylate kinase), a scaffold protein required for PTEN (phosphatase and tensin homolog) activity, is a direct target for miR-101; suppression of MAGI-2 by miR-101 reduces PTEN activity, leading to Akt activation. Taken together, these results not only establish a role for miR-101 in estrogen-independent signaling but also provide a mechanistic link between miR-101 and Akt activation.

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that control gene expression at the post-transcriptional level. To date, over 700 human miRNA precursors with over 1,000 mature miRNAs have been reported. miRNAs are related to, but distinct from, short interfering RNAs (siRNAs). A key difference between siRNAs and miRNAs is that siRNAs require almost identical sequences to targets to exert their silencing function, whereas miRNAs usually bind to the 3-untranslated region (3-UTR) of target genes through partial sequence homology. Because of this unique feature, a single miRNA has multiple targets and thus, miRNAs could regulate a large fraction of proteincoding genes. This may explain why miRNAs play a fundamental role in regulation of diverse cellular processes such as cell growth, proliferation, differentiation and apoptosis and thus, deregulation of miRNA expression can lead to a variety of disorders including cancer. In this regard, miRNAs can function as either oncogenes or tumor suppressors. For example, miR-21 is an oncogenic miRNA which has been shown to promote tumor growth and metastasis in several types of cancers by targeting multiple tumor suppressors. Therefore, targeting miR-21 may provide a promising strategy for cancer intervention. In this review, we will discuss our current understanding of miR-21-mediated gene silencing, tumor growth and metastasis, and the potential of miR-21 as a cancer biomarker. We will also provide a perspective on targeting miR-21 for cancer therapy.

MicroRNAs are important gene regulators that could play a profound role in tumorigenesis. Our previous studies indicate that miR-145 is a tumor suppressor capable of inhibiting tumor cell growth both in vitro and in vivo. In this study, we show that miR-145 exerts its function in a cell-specific manner. Although miR-145 inhibits cell growth in MCF-7 and HCT-116 cells, it has no significant effect on cell growth in metastatic breast cancer cell lines. However, miR-145 significantly suppresses cell invasion in these cells; in contrast, the antisense oligo against miR-145 increases cell invasion. miR-145 is also able to suppress lung metastasis in an experimental metastasis animal model. This miR-145–mediated suppression of cell invasion is in part due to the silencing of the metastasis gene mucin 1 (MUC1). Using luciferase reporters carrying the 3′-untranslated region of MUC1 combined with Western blot and immunofluorescence staining, we identify MUC1 as a direct target of miR-145. Moreover, ectopic expression of MUC1 enhances cell invasion, which can be blocked by miR-145. Of interest, suppression of MUC1 by miR-145 causes a reduction of β-catenin as well as the oncogenic cadherin 11. Finally, suppression of MUC1 by RNAi mimics the miR-145 action in suppression of invasion, which is associated with downregulation of β-catenin and cadherin 11. Taken together, these results suggest that as a tumor suppressor, miR-145 inhibits not only tumor growth but also cell invasion and metastasis. Cancer Res; 70(1); 378–87

Ubc9 is an E2-conjugating enzyme that transfers the activated small ubiquitin-like modifier (SUMO) to protein substrates, and thus it has an important function in sumoylation-mediated cellular pathways. We have earlier reported that Ubc9 promotes tumor growth in the xenograft mouse model using breast cancer cell line MCF-7 in part through regulation of Bcl-2 expression. In this study, we show that ectopic expression of wild-type Ubc9 (Ubc9-WT) promotes cell invasion and metastasis. Surprisingly, the dominant negative mutant Ubc9 (Ubc9-DN) also causes the same phenotype, indicating that the ability of Ubc9 to promote invasion and metastasis is distinct from its ability to conjugate SUMO to protein substrates. Of considerable interest, several microRNAs such as miR-224 are regulated by Ubc9. Although ectopic expression of Ubc9 causes downregulation of miR-224, suppression of Ubc9 by Ubc9-siRNAs leads to its upregulation. We further show that miR-224 can inhibit cell invasion and directly targets CDC42 and CXCR4, and that suppression of CDC42 and CXCR4 by RNAi causes inhibition of Ubc9-mediated invasion. Together, these results show a molecular link between Ubc9 and the metastasis genes such as CDC42 and CXCR4, and thus provide new insight into the mechanism by which Ubc9 promotes tumor invasion and metastasis.

Background: MicroRNAs are the endogenous small non-coding RNA molecules capable of silencing protein coding genes at the posttranscriptional level. Based on computer-aided predictions, a single microRNA could have over a hundred of targets. On the other hand, a single protein-coding gene could be targeted by many potential microRNAs. However, only a relatively small number of these predicted microRNA/mRNA interactions are experimentally validated, and no systematic validation has been carried out using a reporter system. Methods: In this study, we used luciferease reporter assays to validate microRNAs that can silence cyclin D1 (CCND1) because CCND1 is a well known proto-oncogene implicated in a variety of types of cancers. We chose miRanda http://www.microRNA.org as a primary prediction method. We then cloned 51 of 58 predicted microRNA precursors into pCDH-CMV-MCS-EF1-copGFP and tested for their effect on the luciferase reporter carrying the 3'-untranslated region (UTR) of CCND1 gene. Results: Real-time PCR revealed the 45 of 51 cloned microRNA precursors expressed a relatively high level of the exogenous microRNAs which were used in our validation experiments. By an arbitrary cutoff of 35% reduction, we identified 7 microRNAs that were able to suppress Luc-CCND1-UTR activity. Among them, 4 of them were previously validated targets and the rest 3 microRNAs were validated to be positive in this study. Of interest, we found that miR-503 not only suppressed the luciferase activity, but also suppressed the endogenous CCND1 both at protein and mRNA levels. Furthermore, we showed that miR-503 was able to reduce S phase cell populations and caused cell growth inhibition, suggesting that miR-503 may be a putative tumor suppressor. Conclusion: This study provides a more comprehensive picture of microRNA/CCND1 interactions and it further demonstrates the importance of experimental target validation.

The tumor suppressor p53 negatively regulates a number of genes, including the proto-oncogene c-Myc, in addition to activating many other genes. One mechanism of the p53-mediated c-Myc repression may involve transcriptional regulation. However, it is not clear whether microRNAs (miRNAs) play a role in the p53-mediated posttranscriptional regulation of c-Myc. In this study, we show that a putative tumor suppressor, miR-145 , is expressed through the phosphoinositide-3 kinase (PI-3K)/Akt and p53 pathways. Importantly, p53 transcriptionally induces the expression of miR-145 by interacting with a potential p53 response element (p53RE) in the miR-145 promoter. We further show that c-Myc is a direct target for miR-145 . Although miR-145 silences the expression of c-Myc, anti- miR-145 enhances its expression. This specific silencing of c-Myc by miR-145 accounts at least in part for the miR-145 -mediated inhibition of tumor cell growth both in vitro and in vivo. Finally, the blockade of miR-145 by anti- miR-145 is able to reverse the p53-mediated c-Myc repression. Together, these results define the role of miR-145 in the posttranscriptional regulation of c-Myc by p53 and suggest that, as a new member of the p53 regulatory network, miR-145 provides a direct link between p53 and c-Myc in this gene regulatory network.

Purpose: As an E2-conjugating enzyme for sumoylation, Ubc9 plays a critical role in sumoylation-mediated cellular pathways, ultimately impacting cell growth and cancer development. The aim of this study was to investigate the regulation of Ubc9 in cancer cells. Experimental Design: Immunohistochemistry and Western blot were used to determine Ubc9 expression in paraffin-embedded tumor tissue and frozen specimens of the matched tumors from the same patient, respectively. To establish the causal relationship between miR-30e and Ubc9 expression, we overexpressed miR-30e and then determined the resultant effects on Ubc9 expression. To determine whether miR-30e directly targets Ubc9, we did luciferase assays using luciferase reporters carrying the 3′-untranslated region (3′-UTR) of the Ubc9 gene. Results: We found that Ubc9 is up-regulated in breast, head and neck, and lung cancer specimens. In addition, an examination of eight pairs of matched breast tumor specimens by Western blot analysis revealed that, on average, the level of Ubc9 is 5.7-fold higher in tumor than in the matched normal breast tissue. Of interest, we present evidence that Ubc9 is subjected to posttranscriptional regulation by microRNA, and the miR-30 family, such as miR-30e, negatively regulates Ubc9 expression. In contrast to Ubc9, miR-30e is underexpressed in tumors. Moreover, ectopic expression of miR-30e suppresses cell growth, which can be partially reversed by Ubc9. Finally, using luciferase-Ubc9-3′-UTR reporters, we show that Ubc9 is a direct target for miR-30e by interactions with the putative miR-30e binding sites. Conclusion: These results provide new insight into regulation of Ubc9 in cancer cells.

MicroRNAs (miRNAs) are endogenous, small, non-coding RNAs, which are capable of silencing gene expression at the post-transcriptional level. In this study, we report that miR-205 is significantly underexpressed in breast tumor compared to the matched normal breast tissue. Similarly, breast cancer cell lines, including MCF-7 and MDA-MB-231, express a lower level miR-205 than the non-malignant MCF-10A cells. Of interest, ectopic expression of miR-205 significantly inhibits cell proliferation and anchorage independent growth, as well as cell invasion. Furthermore, miR-205 was shown to suppress lung metastasis in an animal model. Finally, western blot combined with the luciferase reporter assays demonstrate that ErbB3 and vascular endothelial growth factor A (VEGF-A) are direct targets for miR-205, and this miR-205-mediated suppression is likely through the direct interaction with the putative miR-205 binding site in the 3′-untranslated region (3′-UTR) of ErbB3 and VEGF-A. Together, these results suggest that miR-205 is a tumor suppressor in breast cancer.