MicroRNA-mediated regulation of Ubc9 expression in cancer cells.
OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
These results provide new insight into regulation of Ubc9 in cancer cells.
Wu
Fangting
F
Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois 62794, USA.
Zhu
Shuomin
S
Ding
Yanna
Y
Beck
William T
WT
Mo
Yin-Yuan
YY
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D049109:Cell Proliferation; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007124:Immunoenzyme Techniques; D008156:Luciferases / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D035683:MicroRNAs / Q000494:pharmacology*; D016612:Paraffin Embedding; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D020413:3' Untranslated Regions; D035683:MicroRNAs; D008156:Luciferases; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9