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Let-7a microRNA suppresses therapeutics-induced cancer cell death by targeting caspase-3

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Abstract

MicroRNAs (miRNA) are endogenously expressed non-coding RNAs that regulate gene expression post-transcriptionally. Let-7a miRNA is a founding member in the let-7 family and its down-regulation in association with over-expression of RAS and HMGA2 oncogenes has previously been reported. In the present study, caspase-3, the executioner caspase, was confirmed to be the target of let-7a as ectopic expression of let-7a decreased the luciferase activity of a reporter construct containing the 3′ untranslated region of caspase-3 and at the same time repressed the enzyme expression in human squamous carcinoma A431 cells and hepatocellular carcinoma HepG2 cells. Moreover, let-7a was over-expressed while caspase-3 was down-regulated in A10A cells, a doxorubicin-resistant A431 subline. Enforced let-7a expression increased the resistance in A431 cells and HepG2 cells to apoptosis induced by therapeutic drugs such as interferon-gamma, doxorubicin and paclitaxel. On the other hand, down-regulation of let-7a by the anti-let-7a inhibitor increased the doxorubicin-induced apoptosis in A431 parent cells, A10A cells and HepG2 cells while the increase was suppressed by caspase-3 inhibitor. Both anti-let-7a inhibitor and caspase-3 inhibitor however failed to affect the drug-induced apoptosis in human breast cancer MCF7 cells, the cells that do not express caspase-3. Therefore, let-7a by targeting caspase-3 may play a functional role in modulating drug-induced cell death in human cancer cells.

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Acknowledgment

This work was supported by the Earmarked Grants (CUHK4270/04 M, CUHK4428/05 M, CUHK4669/08 M) from Hong Kong Research Grants Council.

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Correspondence to Tim Tak Kwok.

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10495_2008_256_MOESM1_ESM.tif

MOESM1 [The predicted binding of let-7 family members with caspase-3 3′UTR by TargetScan [24]. The matched sequences are shown in bold letter] (TIFF 86 kb)

10495_2008_256_MOESM2_ESM.tif

MOESM2 [Relative expressions of different let-7 members in A431 cells and HepG2 cells. Total RNA was isolated by using the mirVana RNA isolation kit (Ambion, Austin, TX). 50 μg of total RNA was then enriched for small RNA species, 3′amine-modified tailed by using mirVana miRNA labeling kit (Ambion), and fluorescently labeled with amine-reactive Cy3 dyes (GE healthcare, Singapore). Thereafter fluorescent-labeled RNAs were hybridized for 14 h at 42°C on mirVana miRNA Bioarray (Ambion). The microarray was washed as recommended by the manufacturer and fluorescence signals were scanned by using the GenePix scanner] (TIFF 65 kb)

10495_2008_256_MOESM3_ESM.tif

MOESM3 [The firefly luciferase activity in A431 cells and HepG2 cells transiently transfected with pGL3-CASP3 3′UTR or pGL3-Ras 3′UTR luciferase reporter constructs and the let-7a precursor or the anti-let-7a inhibitor. Transfection with control precursor or control inhibitor was done in parallel. The firefly luciferase activity was measured by dual-luciferase reporter assay (Promega). Data represented the mean value of three independent experiments. Mean ± SEM. *P < 0.05. ** P < 0.01] (TIFF 835 kb)

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Tsang, W.P., Kwok, T.T. Let-7a microRNA suppresses therapeutics-induced cancer cell death by targeting caspase-3. Apoptosis 13, 1215–1222 (2008). https://doi.org/10.1007/s10495-008-0256-z

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  • DOI: https://doi.org/10.1007/s10495-008-0256-z

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