RPL27A is a target of miR-595 and may contribute to the myelodysplastic phenotype through ribosomal dysgenesis

We investigated the functional consequences following deletion of a microRNA (miR) termed miR-595 which resides on chromosome 7q and is localised within one of the commonly deleted regions identified for Myelodysplasia (MDS) with monosomy 7 (−7)/isolated loss of 7q (7q-). We identified several targets for miR-595, including a large ribosomal subunit protein RPL27A. RPL27A downregulation induced p53 activation, apoptosis and inhibited proliferation. Moreover, p53-independent effects were additionally identified secondary to a reduction in the ribosome subunit 60s. We confirmed that RPL27A plays a pivotal role in the maintenance of nucleolar integrity and ribosomal synthesis/maturation. Of note, RPL27A overexpression, despite showing no significant effects on p53 mRNA levels, did in fact enhance cellular proliferation. In normal CD34+ cells, RPL27A knockdown preferentially blocked erythroid proliferation and differentiation. Lastly, we show that miR-595 expression appears significantly downregulated in the majority of primary samples derived from MDS patients with (−7)/(7q-), in association with RPL27A upregulation. This significant downregulation of miR-595 is also apparent when higher risk MDS cases are compared to lower risk cases. The potential clinical importance of these findings requires further validation.


Culture of primary cells and flow cytometry analysis
Peripheral Blood Mononuclear Cells (PBMCs) were isolated using Ficoll density-gradient centrifugation. CD34+ isolation was performed using a CD34+ MicroBead Kit following the manufacturers protocol (Milteny Biotech). The isolated CD34+ cells were cultured in two phases in liquid culture. For the first 6 days, cells were cultured in Serum-Free expansion medium (StemCell Technologies), supplemented with 100 U/mL penicillin/ streptomycin, 2 mM glutamine, 100 ng/mL stem cell factor (SCF), 10 ng/mL interleukin-3 (IL-3), 10 ng/mL interleukin-6 (IL-6) and 0.5U/mL erythropoietin (EPO). On day 6, cells were collected for both RNA and protein extraction; the remaining cells were split for two different liquid culture differentiation systems. To induce erythroid and myeloid differentiation, 3 U/mL EPO, 15 ng/mL granulocyte colony-stimulating factor (G-CSF) and 40 ng/mL FLT3 was added. Cells were harvested on day 10 or 11 (day 4 of the differentiation cycle) and stained for subsequent flow cytometry analysis. For flow cytometry the cells were washed with Phosphate Buffered Saline (PBS).
Cell pellets were resuspended in 1mL PBS and mixed with 1 μL of live/ dead stain (eFluor ® 780) and incubated in the dark for 30 minutes. Next, cells were washed twice with FACS buffer and stained with the corresponding marker antibodies and incubated for 30 minutes in the dark followed by a further two washes. The resultant supernatant was removed and the cell pellet was resuspended in FACS buffer and analyzed by FACS scan.

Quantitative RT-PCR
RNA from cell lines was extracted using the standard TRIzol (Invitrogen) methodology. The RNA from CD34+ cells was purified using a RNeasy Micro kit (Qiagen) according to protocol. The first strand cDNA was generated from 500 ng of total RNA using SuperScript ® VILO™ cDNA Synthesis Kit (Invitrogen) following the manufacturer's protocol. Quantitative RT-PCR was performed using a Universal Probe Library (UPL) from Roche. The PCR primers and compatible probes were designed using the LightCycler ® Probe Design Software 2.0, available on the Roche website. Relative mRNA expression was determined using the ΔΔCT method. A list of primers and probes utilised is provided in Supplementary Table S3.
The miRNA reverse transcription and quantitative PCR was performed as previously described [2]. miR-595 and RNUB6 (endogenous control) primers were obtained from Applied Biosystems. cDNA was generated from 5ng of RNA using TaqMan miRNA Reverse Transcription Kit (Applied Biosystems). The qPCR was performed using Taqman Universal Mastermix (Applied Biosystem).

Flow cytometry for apoptosis and cell cycle analysis
Apoptosis was assessed by staining with Annexin V and 7-anti actinomycin D (7-AAD) (Biolegend) according to the manufacturer's instructions. For cell cycle distribution analysis, cells were stained with propidium iodide/RNAse staining solution. Flow cytometric analysis was performed on a FACS Canto2™ platform and data was analysed using FlowJo™ software.

Immunoflorescence analysis of nucleoli structure
A total of 5 × 10 4 of the cells under study were re-suspended in 200 μL PBS and subsequently spun onto slides using a Cytospin. Following an Abcam immunofluorescence protocol, Anti-Fibrillarin antibody (Abcam, ab5821) was used at 1:200 dilutions in PBS supplemented with 1% Tween-20 (TPBS). Slides were then analysed via confocal microscopy to visualize nucleoli signals.

Methylcellulose colony assays
At day 6 post infection, cells were plated in methylcellulose media containing SCF, IL-3, IL-6 and EPO (MethoCult GF M3434; StemCell Technologies). Medium was layered on a 6-well tissue culture plate. The plate was incubated for 14-16 days in a humidified incubator with 5% CO 2 at 37°C to allow colony formation and subsequent assessment.

Cell viability and proliferation assays
Cell viability was performed using standard MTT assay techniques.
Cellular proliferation was determined at 48-hour intervals. Cells were mixed with Trypan Blue dye and loaded into a Haemocytometer (Neubauer cell counting chamber, depth 0.1 μl) to facilitate cell counting. Figure S1: Representative PCR amplification of extracted genomic DNA. The breast carcinoma MCF7 cell line, lacking miR-595, was transfected with cDNA library (Target ID Library, Sigma) and underwent zeocin selection. Expanded zeocin resistant cells underwent infection with either a pBabePuro vector expressing miR-595 or empty vector. After 48 hours, cells underwent puromycin selection and subsequent ganciclovir (GCV) counter-selection. Genomic DNAs were isolated from GCV resistant cells from 21 flasks and PCR amplified using vector specific primers. PCR products were visualized on a 2% (w/v) agarose gel and the gel image is representative of PCR products from individual flasks. Each lane represents DNA from one flask. Some of the bands are present in multiple samples and potentially represent identical targets in differing transfections. Bands of different sizes were hence purified and sequenced. A BLAST search of the sequences obtained identified two transcripts; RPL27a (≈ 500 bp) and HSPA14 (≈ 800 bp) as indicated. Figure S2: RPL27A depletion using lentivirus short-hairpin RNA. RPL27A mRNA and protein expression was examined in HCT-116 and HCT-116 p53 −/− cells infected with four different shRNAs to determine efficiency (A + B). Attenuated RPL27A mRNA and protein expression (using tubulin as a control) followed infection with RPL27A-sh2 and RPL27A-sh4 compared to controls. (C) RPL27A mRNA expression in HEL and K562 cell lines following infection with either RPL27A-sh2 and RPL27A-sh4 also displayed differential reductions in expression. (D) RPSA14 and RPL5 mRNA expression in HEL and K562 cell lines following infection with either RPS14 shRNA and RPL5 shRNA also displayed differential reductions in expression mRNA expression was normalized to GAPDH mRNA, calibrated to the non infected cells and compared with empty vector. Bars