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MicroRNAs are small noncoding RNA molecules that control expression of target genes. Our previous studies show that mir-21 is overexpressed in tumor tissues compared with the matched normal tissues. Moreover, suppression of mir-21 by antisense oligonucleotides inhibits tumor cell growth both in vitro and in vivo. However, it remains largely unclear as to how mir-21 affects tumor growth, because our understanding of mir-21 targets is limited. In this study, we performed two-dimensional differentiation in-gel electrophoresis of tumors treated with anti-mir-21 and identified the tumor suppressor tropomyosin 1 (TPM1) as a potential mir-21 target. In agreement with this, there is a putative mir-21 binding site at the 3-untranslated region (3-UTR) of TPM1 variants V1 and V5. Thus, we cloned the 3-UTR of TPM1 into a luciferase reporter and found that although mir-21 down-regulated the luciferase activity, anti-mir-21 up-regulated it. Moreover, deletion of the mir-21 binding site abolished the effect of mir-21 on the luciferase activity, suggesting that this mir-21 binding site is critical. Western blot with the cloned TPM1-V1 plus the 3-UTR indicated that TPM1 protein level was also regulated by mir-21, whereas real-time quantitative reverse transcription-PCR revealed no difference at the mRNA level, suggesting translational regulation. Finally, overexpression of TPM1 in breast cancer MCF-7 cells suppressed anchorage-independent growth. Thus, down-regulation of TPM1 by mir-21 may explain, at least in part, why suppression of mir-21 can inhibit tumor growth, further supporting the notion that mir-21 functions as an oncogene. MicroRNAs (miRNAs)2 are a class of naturally occurring small noncoding RNAs that regulate gene expression by targeting mRNAs for translational repression or cleavage (1, 2). Like protein-coding mRNAs, miRNAs are transcribed as long primary transcripts in the nucleus. However, unlike protein-coding mRNAs, miRNAs are subsequently cleaved to produce stem-loop-structured precursor molecules of ϳ70 nucleotides in length (pre-miRNAs) by the nuclear RNase III enzyme Drosha (3). The pre-miRNAs are then exported to the cytoplasm, where the RNase III enzyme Dicer further processes them into mature miRNAs (ϳ22 nucleotides). Thus, miRNAs are related to, but distinct from, short inferring RNAs (siRNAs) (4, 5). A key difference between siRNAs and miRNAs is that siRNAs require almost identical sequences to targets to exert their silencing function, whereas miRNAs bind through partial sequence homology to the 3Ј-untranslated region (3Ј-UTR) of target genes. Because of this unique feature, a single miRNA has multiple targets. Thus, miRNAs could regulate a large fraction of protein-coding genes, and as high as 30% of all genes could be miRNA targets (6).As a new layer of gene regulation mechanism, miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation, and apoptosis (7,8). Hence, deregulation of miRNA expression may lead to a variety of disorders. Aberrant expression of miR...

Ubc9 is an E2 conjugating enzyme that transfers the activated SUMO (small ubiquitin-related modifier) to protein substrates, and thus it plays a critical role in sumoylation-mediated cellular pathways. We have previously 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. While ectopic expression of Ubc9 causes downregulation of miR-224, and 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 demonstrate 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.

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.

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