Abstract
ObjectiveTo investigate the involvement of miR-520e in the modulation of cancer-promoting cyclinD1 in breast cancer.MethodsA reverse transcription-polymerase chain reaction (RT-PCR) was applied to test the regulation of miR-520e on cyclinD1. The binding of miR-520e to 3’-untranslated region (3’UTR) of cyclinD1 mRNA was predicted by an online bioinformatics website. The effect of miR-520e on the luciferase reporters with binding sites of miR-520e and 3’UTR of cyclinD1 mRNA was revealed using a luciferase reporter gene assay. The correlation between miR-520e and cyclinD1 in clinical breast cancer samples was detected through quantitative real-time PCR.ResultsThe expression of cyclinD1 was gradually reduced as the dose of miR-520e increased. Anti-miR-520e obviously induced cyclinD1 in breast cancer cells. After anti-miR-520e was introduced into the cells, the inhibition of cyclinD1 expression mediated by miR-520e was reversed. The binding of miR-520e with cyclinD1 was revealed via bioinformatics. Under the treatment of dose-increasing miR-520e or anti-miR-520e, the luciferase activities of cyclinD1 3’UTR vector were lower or higher by degrees. However, the activity of the mutant vector was not affected at all. Finally, in clinical breast cancer tissues the negative correlation of miR-520e with cyclinD1 was revealed.ConclusionIn conclusion, cyclinD1 is a new target of miR-520e in breast cancer.
Highlights
After antimiR-520e was introduced into the cells, the inhibition of cyclinD1 expression mediated by miR-520e was reversed
In clinical breast cancer tissues the negative correlation of miR-520e with cyclinD1 was revealed
A report has revealed that miR-520e makes breast cancer cells sensitive to complement attacks, resulting in the suppression of breast cancer [1]
Summary
A reverse transcription-polymerase chain reaction (RT-PCR) was applied to test the regulation of miR520e on cyclinD1. The correlation between miR520e and cyclinD1 in clinical breast cancer samples was detected through quantitative real-time PCR. 2.4 RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR. For the detection of the cyclinD1 mRNA level, reverse transcription was performed by GoScriptTM Reverse Transcriptase (Promega, USA) and GAPDH was used as a loading control. The relative cyclinD1 mRNA levels were analyzed by normalizing the threshold cycle (Ct) value to that of the internal loading control, GAPDH. For miR-520e, its level was measured using a TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher, Carlsbad, CA, USA) and TaqMan gene expression master mix (Thermo Fisher, Carlsbad, CA, USA) according to the manufacturer’s protocol. The level of miR520e was analyzed by normalizing the threshold cycle (Ct) value to that of the internal loading control, U6 snRNA.
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