Research On Prolactin Receptor Associated MicroRNA Expression Profiling And Its Regulationg Networks In Breast Cancer

Objective By identifying differentially expressed micro RNA(mi RNA) and novel mi RNA in association with bioinformatics analysis to explore prolactin(PRL) and prolactin receptor(PRLR) signaling pathway associated mi RNA profiling in breast cancer, and further research detail working mechanism of mi R-449c-5p in breast cancer initiation and progression.Method Human breast cancer T-47 D cells were treated by human recombinant PRL, Solexa technology was applied to detect PRL-treated and none-treated cells to acquire s RNA library. Differentially expressed mi RNAs between the twogroups were analyzed and 4 of them(mi R-449c-5p, mi R-16-5p, mi R-15b-5p and mi R-193a-3p) were chosen for verification by Real-time PCR. Bioinformatic sofewares also were applied to predict potential target genes and run GO(Gene Ontology) and KEGG(Kyoto Encyclopedia of Genes and Genomes) analysis. Meanwhile, 3 newly-detected novel mi RNAs(noted as novel-mi R-01, 02, 03) were identified by RT-PCR, and subsequently run target genes prediction, GO and KEGG analysis. Differentially expressed mi R-449c-5p was invited to target prediction and its target genes was identified by dual-luciferase repoter system. After transfection with mi R-449c-5p mimics in T-47 D cells, target gene related molecular were detected by Real-time PCR and Western blotting, and its cellular function on breast cancer also was evalutated by transwell migration assay and wound healing assay.Results Successfully constructed two mi RNA expression profiling of PRL-treated and none-treated T-47 D cells, and 821 and 798 mi RNAs mature sequence were harvested, respectively. In which, 428 mi RNAs were coexpressed, 42 of them weredifferentially expressed, Real-time PCR verified 4 differentially expressed mi RNAs, and their expression levels were identical with Solexa sequencing results. 86 and 115 novel mi RNAs mature sequence were detected from the two libraries,46 novel mi RNAs were corexpressed, and 3 of them were chosen for RT-PCR identification,results showed that they were expressed at different levels. Target genes of the 4 known mi RNAs and 3 novel mi RNAs were subjected to GO and KEGG analysis, all results indicated that they mainly distributed in nucleus, cytoplasma and plasma membrane, functioning in transcriptional regulation, development and signal transduction, involved in JAK-STAT and MAPK signaling pathways, which were highly related with PRLR signaling pathway. PRLR was predicted as a target gene of mi R-449c-5p, and dual-luciferease reporter assay confirmed the prediction, experiment also demonstrated that over expression of mi R-449c-5p inhibit migration of T-47 D cells.Conclusion By Solexa sequencing technology, we successfully constructed mi RNA expression library of human breast cancer T-47 D cells, harvested series of mi RNAs related with PRLR signaling pathway, confirmed PRLR was a target of mi R-449c-5p and it also act as a tumor-suppressor in breast cancer. This research lay a foundation for mi R-449c-5p in breast cancer tumorigenisis and progression. Besides, this research also proved that Solexa sequencing is a promising ideal technology to detect mi RNAs in tissues and cells.