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J. Yu, D. G. Ryan, M. Oliveira-Fernandes, A. Fatima, R. M. Lavker; Corneal Epithelial-Enriched MicroRNAs Combine to Regulate the Lipid Phosphatase SHIP2: A Novel Paradigm for MicroRNA Function. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5043.
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© ARVO (1962-2015); The Authors (2016-present)
MicroRNAs (miRNAs) play important regulatory roles in development, differentiation, cellular stress responses and cancer. We reported that miRNA-184 (mir-184) was the most abundant corneal epithelial miRNA; mir-205 was the second most abundant. We have identified a common target of mir-184 and -205 activity and show a unique regulatory role for mir-184.
We used luciferase reporter assays in HeLa cells to validate potential targets of mir-184 and -205 identified by bioinformatics. To confirm these findings we conducted gain- and loss-of-function experiments with mir-184 and -205 in HeLa cells and human keratinocytes, in conjunction with Northern, Western, and immunohistochemical analyses.
We screened for targets to mir-184 and -205. Marked reduction (>60%) in luciferase activity was seen in cells co-transfected with mir-205 and a reporter construct carrying the 3’UTR for SHIP2. To validate SHIP2 as a target of mir-205, we used a mir-205 mimic to knock-down SHIP2 in HeLa cells, which have negligible endogenous levels of mir-205. Treatment with the mir-205 mimic resulted in a marked reduction in endogenous SHIP2 expression on Western blots, while a non-targeting mimic had no effect. Similarly, SHIP2 staining was markedly diminished after transfection with the mir-205 mimic. To test whether mir-205 regulates SHIP2 in keratinocytes, we used an antagomir to mir-205 (anti-mir-205). We determined by Northern analysis that anti-mir-205 could down-regulate mir-205 in cultured keratinocytes, and that such a treatment resulted in a marked increase in SHIP2 staining when compared with cells treated with anti-mir124 (an irrelevant antagomir). Bioinformatics indicates that only mir-184 and -205 are potential regulators of SHIP2. Interestingly, no reduction in luciferase activity for the SHIP2 construct was observed in HeLa cells co-transfected with mir-184; however, co-transfection of both mir-184 and -205 inhibited mir-205-mediated downregulation of the luciferase activity for the SHIP2 construct. Mutation of the bases required for mir-184 and -205 binding on the SHIP2 molecule revealed that mir-184 negatively regulates the activity of mir-205 on SHIP2.
These findings establish that: (i) SHIP2, which is a negative regulator of the PI3K-PKB/AKT pathway, is directly regulated by mir-205; and (ii) mir-205 undergoes a unique form of regulation in the corneal epithelium through an interaction with the corneal-specific mir-184. This is the first demonstration that one miRNA plays a major role as a negative regulator of another miRNA.
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