MicroRNA-mediated Regulation of Ubc9 Expression in Cancer Cells

Abstract

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.

Mir-30 reduction maintains self-renewal and inhibits apoptosis in breast tumor-initiating cells

“…This is in line with a previous report suggesting that Ubc9 is a direct target of mir-30, and the latter regulates Ubc9 expression mainly through translation repression (Wu et al, 2009). We confirm these findings by assays with a luciferase reporter carrying the Ubc9 3 0 -UTR, which indicates that mir-30 directly suppresses the luciferase activity of the vector with wild-type Ubc9 3 0 -UTR but not the one with mutated sequence at the mir-30e target site.…”

“…Introduction of mir-30 into BT-ICs not only reduces their ability of self-renewal in vitro and in vivo, but also inhibits their capacity of anoikis resistance and increased apoptosis. Ubc9 (Wu et al, 2009) and ITGB3 are two direct target genes of mir-30, and they are both upregulated in BT-ICs because of mir-30 reduction. Furthermore, Ubc9 elevation maintains the self-renewing capacity of BT-IC, whereas overexpression of both Ubc9 and ITGB3 contributes to the anoikis resistance and apoptosis inhibition in the T-ICs.…”

Attenuation of the beta-catenin/TCF4 complex in colorectal cancer cells induces several growth-suppressive microRNAs that target cancer promoting genes

“…miR-30e-3p shares its seed region with miR-30d-3p and miR-30a-3p, raising the possibility of some overlap in target repertoire. Our results, indicating that these miRNAs exhibit tumor suppressor activities, aligns with previous reports showing that overexpression of the miR-30e hairpin (encoding both -3p and -5p miRNA variants) reduced proliferation of breast cancer cells (Wu et al, 2009), and likewise miR-30a-3p inhibited growth of bladder cancer cells (Ichimi et al, 2009). Because of the pleiotropic effects of miRNAs on target genes, also evident from our proteomic and microarray results, it is possible that their biological effects are the net result of the complex modulation of numerous targets belonging to multiple pathways.…”

Role of microRNAs in breast cancer

“…A similar correlation is also seen in breast tumor specimens where Ubc9 expression is about 6-fold higher than in the matched normal breast tissue. 58 Moreover, enforced expression of miR-30 can inhibit self-renewal capacity of BT-ICs through Ubc9 and induces apoptosis by targeting ITGB3 expression. Particularly, BT-IC xenograft overexpressing miR-30 can reduce tumorigenesis and lung metastasis in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model.…”