We aimed for the construction of a miRNA expression library that fulfills four criteria: 1) the library contains all human miRNAs; 2) each miRNA is represented in a separate stock to allow for arrayed screening; 3) the library can be used over a wide range of different cell types; 4) the miRNAs are swiftly and stably overexpressed. The first two criteria will be discussed below. The third and fourth criteria were met by choosing a lentiviral expression system, employing the pCDH vector. The lentiviral particles express VSV-G, a glycoprotein that grants broad tropism 41 . The glycoprotein's receptor is a lipid component of the plasma membrane that occurs on most cell types over a wide range of species. Lentiviruses integrate into the host genome 42 , but unlike other retroviruses, lentiviruses do not require cell division for genomic integration 43 44 . For instance, the lentivirus is able to transduce quiescent stem cells and terminally differentiated neurons 43 . By integrating into the host genome, the construct is retained through cell divisions and can be stably expressed for an indefinite period of time. We chose a lentiviral backbone with a puromycin-resistance cassette to be able to select for successfully transduced cells 45 .

To create a comprehensive library containing all human miRNAs, we included all miRNAs known at the time we started composing the library. We obtained genomic loci from the widely adopted miRBase miRNA repository (version 14) 46 . We also included a set of candidate miRNAs from previous experiments 10 40 . Although we did not update the library after viral particles for the entire library were produced, some of the candidate miRNAs have since entered the registry. See additional file 1, table S1 for a list of the current annotation of all miRNAs in the library.

All miRNA loci, containing the full-length hairpin and ~100 nt flanking sequence on each side, were cloned into pCDH behind the CMV promoter. Care was taken to clone each miRNA separately, even when this miRNA resides in close proximity to another miRNA. After construction of the plasmid library, all constructs were packaged into lentiviral particles and portioned into individual tubes organized in ready-to-use 96-well format; the fact that tubes can be accessed separately adds versatility to the library. All virus supernatants were concentrated to titers of ~5*10⁸ IFU/mL, in order to achieve efficient transduction with only small volumes of virus supernatant. This eliminates the need for infection under centrifugation or replacement of medium with virus-conditioned medium. While lentiviral particles were produced for all constructs individually, it should be mentioned the original plasmid library can also be applied in its own right. It can also be used for plasmid-based screens, recloning purposes, or to create a pool of constructs containing all or a subset of miRNA constructs for generating mixed lentiviral stocks. Figure 1 is a schematic of the library construction and its most important features.

The first step in the characterization of the library is the determination of transduction efficiency and optimal titration of the lentiviral particles. To this end, we used a variant of pCDH containing a GFP cassette in place of the puromycin-resistance cassette. We infected A375 melanoma cells with different virus concentrations, in combination with several concentrations of polybrene. While transduction efficiency reached over 90% under several conditions, increased amounts of virus led to brighter GFP expression within the population of cells (Figure 2a). This indicates multiple copies are integrated per cell when using higher titers. GFP expression was higher at 72 hours after infection than after 48 hours. This implies 72 hours post infection is a more sensitive time point for determination of transduction efficiency, rather than earlier timepoints. We further optimized conditions for virus infection in a panel of seven cell lines (Figure 2b). Notably, this panel includes the diploid fetal lung fibroblast line IMR-90; a non-transformed cell line 47 . Optimal transduction efficiencies were reached between 10⁶ and 10⁷ IFU/mL.

As mentioned, lentivirus is a favorable vector for its ability to transduce cells that are hard to transfect, such as stem cells. We have successfully transduced primary human intestinal cells using a GFP-encoding vector. These cells were subsequently used to create intestinal organoids in vitro 48 . After prolonged culture, entire organoid bodies turned fluorescent, indicating the stem cells from which the organoids developed were also successfully transduced (Figure 2c).

While ectopic expression of GFP is useful to determine transduction efficiency, it does not address whether miRNAs are also successfully overexpressed. We used a panel of ten test miRNAs to assess miRNA overexpression in five human cell lines: PC-3 (prostate cancer), MDA-MB-231 (breast cancer), A549 (lung cancer), IMR-90 (fetal lung fibroblast), and MCF-7 (breast cancer). Cells were infected with 1.0*10⁷ IFU/mL and subjected to puromycin selection from 24 hours after infection. RNA was collected 48 hours (MCF-7), 72 hours (PC-3, MDA-MB-231 and A549) or 96 hours after infection (IMR-90). Relative mature miRNA expression was measured by qPCR. Values were standardized against the corresponding cell line infected with an empty vector control. We were able to overexpress all miRNAs in all cell lines, with the exception of miR-221 in IMR-90 cells (table 1). The fact that miR-221 is very effectively overexpressed in MCF7 indicates the expression construct is functional. Besides, we were able to overexpress all other miRNAs in IMR-90. The results with miR-221 can at least partially be ascribed to very high endogenous miR-221 levels in this cell line (additional file 2, table S2) and also suggest that overexpression of miRNAs using our standard conditions remains within physiological levels.

Next, we examined miRNA expression kinetics after infection. To determine the optimal conditions for a screen, such as assay length and virus titer, it is important to know when and to what level the miRNA will be expressed. We have tested this extensively for let-7a and miR-372 in A375 cells. While let-7a is abundant in these cells, miR-372 is barely detectable (Figure 3). Both expression profiles show a quick surge of miRNA expression within 4 hours after infection. The early source of these miRNAs is unclear, but they may have been present in the virus supernatant, encapsidated within the virus, or processed intracellularly from the viral RNA genome. miRNA levels exhibit a slight drop, but are quickly overtaken by expression from integrated constructs 16 to 20 hours after infection. Our data demonstrate that already after 24 hours, ectopic expression of miRNAs reaches comparable levels of endogenously predominant miRNAs, as evident from the expression relative to endogenous let-7a and U6 RNA (Figure 3b).

For long-term experiments, it is necessary for miRNA expression to be sustained. 72 hours after infection with let-7a or miR-372, we passaged A375 cells and cultured them either with or without puromycin selection. We examined miRNA expression by miRNA qPCR at 5, 9 and 20 days after infection (Figure 3c). A decline in miRNA expression was observed with both miRNAs between day 3 and day 5 after infection. This may be caused either by passaging or changed culture conditions, but it is also possible that it is a secondary effect of highly elevated miRNA processing. This effect was markedly stronger in the let-7a-infected cells, which may indicate that there is a selection disadvantage for cells with high let-7a expression. Despite the small drop after three days, overexpression of both miRNAs stabilized and persisted for weeks. Even after prolonged culture without puromycin selection, miRNA overexpression was sustained. We conclude there is little or no genetic loss or epigenetic silencing of the integrated construct.

As the goal of the library is to identify biologically relevant functions of miRNAs, we performed a proof-of-principle experiment by testing the well-documented effects of let-7a 49 50 51 as an inhibitor of cell proliferation and miR-372 38 as a stimulator in five cell lines. The experiment was set up in the fashion of a high-content screen. Cells were seeded in 384-well plates and infected after 8 hours. 72 hours after infection, cells were fixed and nuclei were stained with Hoechst. The automated image analysis software not only counts the number of nuclei, but also detects aberrations in nuclear shape and condensed chromatin (Figure 4). Although we did not find any novel effects of the miRNAs on nuclear shape, we were able to confirm the expected effects of let-7a and miR-372 on proliferation in a high-content setup.

As a first use of the complete library we screened miRNAs for their effect on A375 melanoma cell proliferation. We tested whether we could detect proliferation effects in A375 cells in a 96-well setup, again using let-7a and miR-372 as test miRNAs. The effects were highly similar to those seen in Figure 4 (Figure 5a). Every single well infected with either let-7a or miR-372 proved a statistical outlier compared to empty-vector transduced wells (Figure 5a, right panel).

For the entire library, we performed an arrayed screen by MTS assay, which measures the viability of cells in a well 52 . This measure is reported to correlate strongly with cell number. All samples were tested in duplicate, and for each replicate a B-score was calculated 53 . This method calculates how a miRNA scores relative to the other miRNAs on the same plate. It is a robust method as it is not sensitive to outliers. B-scores of replicates are shown in Figure 5b. Also shown are the measures for all let-7a constructs and miR-372. The strong correlation between replicates indicates a high reproducibility of the assay. The effects of let-7a and miR-372 are similar as in the cell count assay. Importantly, all let-7a constructs cluster together. Since these constructs have different backbones but produce the same mature miRNA, we surmise the observed effects are likely to be caused by the mature miRNA. A confounding factor in short-term arrayed screens can be the range of virus titers of the different constructs. However, we saw no correlation between virus titer and cell viability (additional file 3, figure S1). Thus, with these experimental conditions, virus titers were not a significant factor contributing to toxicity.

Some assays do not lend themselves to screening in 96-well format. In such cases the lentiviral library can be used for a pooled screen. There are two approaches: pool virus supernatant of several or all constructs together, or infect cells with the individual constructs and subsequently pool cells. The latter is generally favorable, because it precludes the possibility that two or more virus particles containing different miRNAs infect a single cell. A puromycin selection step can be added to make certain each construct is represented in transduced cells and to reduce background caused by non-transduced cells. We applied this approach to the poorly invasive HCT15 colorectal cell line. HCT15 cells were individually infected, puromycin-selected, and pooled in groups of 40 constructs. A fraction of the pool was used for genomic DNA isolation and the rest was subjected to two rounds of an invasion assay. Cells that successfully transmigrated through extracellular matrix were subcultured and genomic DNA was isolated. To identify the miRNA constructs that were enriched in the transmigrated fraction, we took advantage of the fact that the constructs are integrated into the host genome. We PCR-amplified the constructs from the genomic DNA using universal primers that anneal to the vector backbone (Figure 6a). The PCR products were subjected to high-throughput sequencing. Relative abundance of the various constructs was derived from the sequence reads mapping to them. Hits were selected on the basis of a weighted score calculated from the enrichment and the number of sequence reads in the invasive fraction. We found evidence for 45 miRNAs that may enhance the invasive capacity of HCT15 cells (Figure 6b).

In a pooled invasion screen, as performed above, the possibility exists that a portion of the hits can be "passenger hits" that do not impart invasive capacity, but are present in cells that trail the truly invasive cells as they make their way through the extracellular matrix. To distinguish these two types, we subjected each hit individually to an invasion assay. We were able to confirm 23 miRNAs that increase invasiveness of HCT15 cells (Figure 6b).

Backbone for all constructs in the library is the lentiviral expression construct pCDH (cat. no. CD510B-1, System Biosciences). Individual loci containing a single miRNA hairpin were PCR amplified from human genomic DNA and cloned into the multiple cloning site of the plasmid. Loci were either obtained from miRBase http://mirbase.org/ or from mapping of candidate miRNAs found in previous experiments 10 40 . Primers were designed using Primer3. PCR was performed using Pfu polymerase (Agilent). The cloned fragments contain the full-length miRNA hairpin and approximately 100 flanking base pairs on both sides. An expression construct with EGFP was cloned by excision and ligation of the EGFP sequence from pEGFP-N1 (Clontech) into the multiple cloning site of pCDH. All constructs were packaged into lentiviral particles commercially by System Biosciences using the pPACKH1 HIV-based lentiviral packaging kit (cat. no. LV500A-1, System Biosciences). Viral particles were recieved in concentrated form with a median titer of 5.9*10⁸ IFU/mL in a 96-well format. Sequence of all inserts was confirmed from both the plasmid and the virus supernatant (we were able to obtain high-quality specific sequences from the virus supernatant using universal primers, most likely due to trace amounts of plasmid). A list of all included miRNAs in their current annotation (miRBase17) is available in additional file 1, table S1. Lentiviral particles for pCDH with copGFP instead of puroR (cat. no. CD511B-1) were ordered separately from System Biosciences.

293T, A375, MDA-MB-231, A549, MCF-7, IMR-90, PC-3, and HCT15 cells were maintained on 10% FCS complete medium: DMEM Glutamax (GIBCO) with 10% FCS (Sigma) supplemented with non-essential amino acids (GIBCO) and penicillin/streptomycin (GIBCO). All viral infections followed by RNA isolation were done 8 hours after seeding 10, 000 cells in a 6-well plate in 2 mL 10% FCS complete medium. All transduction efficiency experiments were performed by infection with pCDH-MCS-EF1-copGFP, except for the organoid culture, for which we used pCDH-EGFP-EF1-PuroR. Infection mix contained 2 μL virus supernatant, 12 μL 1 mg/mL polybrene (Sigma), and 86 μL PBS0, unless stated otherwise. Intestinal organoid bodies were cultured as described before 48 . Because the organoids are grown in Matrigel, cells were infected before seeding. Cells were infected in 250 μL Wnt-3a-conditioned medium containing 2 μL virus supernatant. Infection took place during a centrifugation step at 150 rcf for 1 hour at room temperature. Unless indicated otherwise, infections were performed in 96-well plates using the following set-up: 1000 cells were seeded in 100 μL 5% FCS complete medium per well of a 96-well plate and infected after 8 hours with 10 μL infection mix. 10 μL infection mix contained 0.6 μL 1 mg/mL polybrene, 0.5 μL virus supernatant, and 8.9 μL PBS0. In the arrayed screen, medium was replaced with 150 μL fresh medium 24 hours after infection.

Five days after infection, 30 μL MTS One Solution (Promega) was added to all samples. After each hour, plates were gently tapped to disperse the coloration of the MTS, and absorbance was measured at 492 nm. Last measurements were taken 4 hours after start of the assay. The time point with measurements showing the highest dynamic range without saturation of signal was used for data analysis.

Five days after infection, 100 μL 8% PFA (Sigma) was added to all samples. Cells were fixed for 15 minutes. Samples were washed once with PBS0 followed by 10 minutes staining in 100 μL PBS0 containing 0.5 μg/mL Hoechst 33342 (Sigma). Cells were washed twice with PBS0 and kept on PBS0 at 4°C. Cells were quantified on a Cellomics ArrayScan VTI using the accompanying software by counting nuclei in 4 fields per well under 10× magnification. Nuclei were identified as shapes with a contiguous Hoechst stain. Nuclear demarcations were used to quantify GFP intensity. All data acquisition was done using adaptions of the TargetActivation program of the ArrayScan software. Data and images displayed in table 1 and Figure 4 were generated by Cenix BioScience, GmbH.

RNA was isolated using TRIzol (Invitrogen) isolation following the manufacturer's protocol. Small RNAs qPCR reactions were performed using the TaqMan MicroRNA reverse transcription kit (ABI), Taqman MicroRNA qPCR assays (ABI) and TaqMan Universal PCR Master Mix, No AmpErase UNG (ABI) using 10 ng total RNA input. qPCR reactions were set up using the suggested reaction conditions on a Bio-Rad MyiQ thermal cycler. U6 was used as a housekeeping control RNA in the experiments concerning Figure 3, RNU6B was used in the experiments concerning table 1. Relative expression was calculated using the 2^-ΔΔCt method. miRNA qPCR data in table 1 were generated by Cenix BioScience GmbH.

HCT15 cells were seeded at 2500 cells per well in a 96-well plate in 10% complete medium. Cells were infected with individual virus supernatants (0.5 μL per well) after 24 hours. 72 hours after infection cells were selected with puromycin-containing medium and subsequently grown to 100% confluency. Pools were made of 40 samples per pool and grown for another 3 days on puromycin-containing medium. Half of the pooled culture was used for genomic DNA isolation, the other half was subjected to two rounds of invasion assay. For each pool, 2*10⁵ cells were applied to the upper compartment of a Boyden chamber (BD Fluoroblok 24-Multiwell, 8 μm pores) coated with extracellular matrix (ECM, Sigma) and containing serum-free medium, and allowed to invade the lower compartment containing 10% FCS medium. Cells were collected from the bottom compartment and expanded for 2-3 weeks on complete medium. 2*10⁵ cells of this subculture were applied to a second round of invasion as described above. Cells in the resulting bottom compartment were subcultured to be used for genomic DNA isolation.

Genomic DNA was isolated from cells using a Qiagen Genomic Tip kit following the manufacterer's instructions. For both the invasive fraction and the control fraction, 10 ng DNA of each pool was pooled together and subjected to PCR-amplification (20 cycles) of integrated constructs using Platinum PCR Supermix (Invitrogen) and primers flanking the inserts. In a subsequent, secondary (3 cycles) and tertiary (3 cycles) PCR the adapters and barcodes used for massively parallel sequencing were added to the flanks of the products. Samples were sequenced on the ABI SOLiD platform. The invasive and control fraction yielded 1.0*10⁶ and 0.9*10⁶ reads that mapped to integrated constructs respectively.

Both the plasmid and virus library are publicly available when requested through InteRNA Technologies. Contact information is available at http://www.interna-technologies.com.