The miR-28-5p targetome discovery identified SREBF2 as one of the mediators of the miR-28-5p tumor suppressor activity in prostate cancer cells

miR-28-5p is downregulated in some tumor tissues in which it has been demonstrated to have a tumor suppressor (TS) activity. Here, we demonstrate that miR-28-5p acts as a TS in prostate cancer (PCa) cells affecting cell proliferation/survival as well as migration and invasion. Using the miRNA pull out assay and next generation sequencing we collected the complete repertoire of miR-28-5p targets obtaining a data set (miR-28-5p targetome) of 191 mRNAs. Filtering the targetome with TargetScan 7, PITA and RNA22 we found that the 61% of the transcripts had miR-28-5p binding sites. To assign a functional value to the captured transcripts, we grouped the miR-28-5p targets into gene families with annotated function and showed that six transcripts belong to the transcription factor category. Among them we selected SREBF2, a gene with increasing importance in PCa. We validated miR-28-5p/SREBF2 interaction demonstrating that SREBF2 inhibition affects almost all the tumor processes affected by the miR-28-5p re-expression, suggesting that SREBF2 is an important mediator of miR-28-5p TS activity. Our findings support the identification of the targetome of cancer-related miRNAs as a tool to discover genes and pathways fundamental for tumor development and potential new targets for anti-tumor therapy. The miRNA pull out assay was performed as described in Rizzo et al. [ref: doi:10.7150/jca.18396]. DU-145 were transfected using Lipofectamine 2000 (Thermo Fisher) with 60 nM of either miR-28-5p duplex (ds-miR-28CT) or a mix of 3’ biotin-tagged miR-28-5p 8tU (nucleotide 8 was a thiouridine) and miR-28-5p 18tU duplexes (ds-miR-28BIO). All oligos were synthetized by Bio-Synthesis Inc. 24 hours after transfection cells were irradiated with UV (365nm, 2J/cm2) using the Bio-Link crosslinking (BLX) (Ambrose Lourmat) and total RNA extracted with TRIzol (Thermo Fisher) directly on adherent cells following the manufacturer’s protocol. 15 μg of RNA was incubated 4 hours at 4°C with 100 μl of streptavidin-conjugated beads (Streptavidin Sepharose high performance, GE Healthcare) and the RNA complexed with the beads was recovered using TRIzol. We performed two biological replicates obtaining three miR-28CT (control) and two miR-28BIO (miR-28-5p) pull out samples consisting of background RNA and ds-miR-28BIO/interacting mRNA complexes respectively. The RNA isolated after the miRNA pullout procedure was used for the construction of the cDNA libraries using the TruSeq Stranded Total RNA Sample Preparation kit (Illumina) according to the manufacturer’s suggestions. cDNA libraries were sequenced by HiSeq2000 (Illumina) in single-reads mode (50bp) by IGA Technology Service, Udine, Italy, obtaining about 20 million of reads for each samples.

miR-28-5p在部分肿瘤组织中呈低表达状态，且已有研究证实其具备肿瘤抑制（tumor suppressor, TS）活性。本研究证实，miR-28-5p在前列腺癌（prostate cancer, PCa）细胞中发挥肿瘤抑制作用，可调控细胞增殖/存活、迁移与侵袭能力。本研究借助miRNA pull-out实验与下一代测序技术，获取了miR-28-5p的全部靶基因集合，最终得到包含191条mRNA的数据集（miR-28-5p靶基因组，miR-28-5p targetome）。通过TargetScan 7、PITA及RNA22三款工具对该靶基因组进行筛选后，我们发现61%的转录本带有miR-28-5p结合位点。为明确所捕获转录本的功能属性，我们将miR-28-5p靶基因按已注释功能划分为不同基因家族，结果显示其中6条转录本属于转录因子类。我们从中筛选出SREBF2，该基因在前列腺癌中的研究价值日益凸显。我们验证了miR-28-5p与SREBF2的靶向结合关系，结果证实抑制SREBF2的表达可模拟miR-28-5p重新表达所介导的几乎全部肿瘤相关生物学过程，这表明SREBF2是miR-28-5p发挥肿瘤抑制活性的关键介导因子。本研究结果表明，对癌症相关miRNA的靶基因组进行鉴定，可作为挖掘肿瘤发生发展核心基因与信号通路、开发新型抗肿瘤治疗靶点的有效手段。miRNA pull-out实验操作参照Rizzo等人发表的方法[参考文献：doi:10.7150/jca.18396]。本研究使用Lipofectamine 2000（赛默飞世尔科技，Thermo Fisher）将60 nM的miR-28-5p双链（ds-miR-28CT），或是由3'端生物素标记的miR-28-5p 8tU（第8位核苷酸为硫代尿苷）与miR-28-5p 18tU双链（ds-miR-28BIO）混合体系转染至DU-145细胞中。所有寡核苷酸序列均由Bio-Synthesis Inc.公司合成。转染24小时后，使用Bio-Link交联仪（BLX，Ambrose Lourmat）以365nm紫外线、2J/cm²的剂量对细胞进行照射，随后直接在贴壁细胞中按照TRIzol（赛默飞世尔科技，Thermo Fisher）的试剂盒说明书提取总RNA。取15 μg总RNA与100 μl链霉亲和素偶联磁珠（Streptavidin Sepharose 高性能型，GE Healthcare）在4℃下孵育4小时，随后使用TRIzol回收与磁珠结合的RNA复合物。本研究设置2次生物学重复，共得到3份miR-28CT（对照组）样本与2份miR-28BIO（miR-28-5p组）pull-out样本：前者仅包含背景RNA，后者则为ds-miR-28BIO与结合的mRNA复合物。经miRNA pull-out实验分离得到的RNA，将按照TruSeq链特异性总RNA样本制备试剂盒（Illumina）的操作指南构建cDNA文库。cDNA文库由意大利乌迪内的IGA Technology Service公司使用HiSeq2000（Illumina）以单端测序模式（50bp）完成测序，每个样本约得到2000万条reads（测序读段）。