A large shRNA library approach identifies lncRNA Ntep as an essential regulator of cell proliferation

Abstract

The mammalian cell cycle is a complex and tightly controlled event. Myriads of different control mechanisms are involved in its regulation. Long non-coding RNAs (lncRNA) have emerged as important regulators of many cellular processes including cellular proliferation. However, a more global and unbiased approach to identify lncRNAs with importance for cell proliferation is missing. Here, we present a lentiviral shRNA library-based approach for functional lncRNA profiling. We validated our library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria we identified lncRNA NR_015491.1 out of 3842 different RNA targets represented in our library. We termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, we discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, our study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach.

Figure 1

Experimental design and selection strategy for the identification of lncRNAs essential for proliferation. (a) Schematic workflow of proliferation-based lncRNA shRNA library screen in 3T3 cells. (b) Library infection quality control: Scatter plot of the log 2-transformed DNA sequencing read values from baseline sample (5 days post library infection) normalized to 20 million reads versus the sequencing reads of the input pooled plasmid library normalized to 20 million reads. (c) Sequencing reads of shRNA barcodes against lncRNA NR_015491.1 (Ntep) in CFSE-low and -high sub-populations. Data are fold-change (FC) relative to barcode reads of shRNAs in each sub-population against luciferase control. (d) Scheme of the selection strategy to narrow down initial lncRNA candidates that influence proliferation. UCE, ultraconserved elements

Figure 2

Ntep is a mainly nuclear lncRNA. (a) Graphical representation of Ntep showing multiple exons with different lengths according to GRCm38/mm10. Primers used for qPCR analysis are shown in purple, individual shRNAs from shRNA library are shown in blue and GapmeRs used to target Ntep are shown in orange. (b) Subcellular distribution of Ntep in 3T3 subfractions, 18s, GAPDH, β-Actin and Xist expression levels were measured as positive controls. Data are % distribution calculated to complete amount of transcript in qPCR analysis±S.E.M. (n=3 independent experiments). Piechart showing the subcellular distribution of Ntep in 3T3 cells. Data are % distribution calculated to complete amount of transcript in qPCR analysis (n=3 independent experiments). (c) Expression of Ntep in different organs from C57BL6J mice (n=3). Normalized expression level was measured with qPCR±S.E.M. *P<0.05; **P<0.01; ***P<0.001

Figure 3

Ntep inhibition impairs fibroblast functions. (a) Cell number counted after treatment of 3T3 cells with GapmeR Ntep and GapmeR control. Six images per well taken, three wells per condition. (b) Example pictures of 4′,6-diamidino-2-phenylindole (DAPI)-stained 3T3 cells 48h after treatment with GapmeR Ntep and GapmeR control. (c) Expression level of P21 mRNA, protein and Ntep, and level of proliferation of 3T3 cells after 48h of GapmeR Ntep treatment. Expression level was measured by qPCR. Ct values are given for Ntep expression above the respective bars. Protein level was assessed using western blotting. Proliferation rate was measured in BrdU enzyme-linked immunosorbent assays. (d) Expression level of Col1a1, Col3a1, A-sma, Ctgf, Mmp2, Tgfβ1 and Tgfβ3 mRNA in 3T3 cells treated with GapmeR Ntep and GapmeR control for 48 h. Expression level was measured with qPCR. (e) Example pictures of 3T3 cells treated with GapmeR Ntep or GapmeR control for 48 h and scratched to assay migration capacity. Pictures show time point 0 and 6 h after scratch wound was carried out. Migration index of 3T3 cells from three independent experiments. (f) Example picture of petri dishes seeded with single cells for colony-forming assay and stained with crystal violet. Cells were treated with GapmeR Ntep or GapmeR control 11 days before staining and 200 cells were seeded per Petri dish. Colony numbers of 3T3 cells from three independent experiments. All data are mean fold-change (FC) relative to control±S.E.M. (n=3 independent experiments), except stated otherwise. *P<0.05; **P<0.01; ***P<0.001. Student’s t-test

Figure 4

Ntep inhibition leads to decreased proliferation in murine fibroblasts, but not in cardiomyocytes. (a) Expression level of Ntep and proliferation rate of MEFs treated with GapmeR Ntep and GapmeR control for 48 and 72 h. Expression level was measured by qPCR. Proliferation rate was measured in BrdU enzyme-linked immunosorbent assays. (b) Expression level of Ntep, Col1a1, Col3a1 and Tgfβ3 mRNA after 48 h transfection of MEF cells with GapmeR Ntep and GapmeR control. Expression level was measured by qPCR. (c) Expression level of Ntep and (d) proliferation rate in L929 fibroblasts treated with GapmeR Ntep and GapmeR control for 48 h. Expression level was measured by qPCR. Proliferation rate was measured in BrdU enzyme-linked immunosorbent assays. (e) Expression level of Ntep and (f) proliferation rate in HL-1 cardiomyocytes treated with GapmeR Ntep and GapmeR control for 48 h. Expression level was measured by qPCR. Proliferation rate was measured in BrdU enzyme-linked immunosorbent assays. All data are mean fold-change (FC) relative to control±S.E.M. (n=3 independent experiments). *P<0.05; **P<0.01; ***P<0.001. Student’s t-test

Figure 5

Microarray analysis reveals GapmeR-mediated P53 pathway activation. (a) Heatmaps of indicated gene sets with false discover rate (FDR) of <25% after GSEA showing the 20 most differentially expressed, core-enriched genes of 3T3 cells treated with GapmeR Ntep or GapmeR control. Matching GSEA plots for each pathway are shown beneath. Genes are ranked in GSEA plot according to expression level in the indicated sample. Genes on the left side are relatively highly expressed in GapmeR Ntep cells compared with controls, whereas genes on the opposite site are underrepresented. The red to blue horizontal bar represents the ranked list. According to the amount of genes enriched for each gene set, an enrichment score is calculated, which is shown by the green line in the GSEA plots. (b) Validation of P53 protein level in 3T3 cells treated with GapmeR Ntep and GapmeR control for 48 h. Expression level was measured using western blotting. Representative picture of a western blot showing the band of P21 and the housekeepeing gene GAPDH. (c) Caspase-3/7 activity after treatment of 3T3 cells with GapmeR Ntep and GapmeR control. Activity was measured using luminescent assays. All data are mean fold-change (FC) relative to control±S.E.M. (n=3 independent experiments). *P<0.05; **P<0.01; ***P<0.001. Student’s t-test

Figure 6

Effect of Ntep inhibition is not detectable in p53-deficient fibroblasts. (a) Expression level of Ntep, (b) proliferation rate of and (c) caspase-3/7 activity of p53-deficient primary MEF treated with GapmeR Ntep and GapmeR control for 48 h. Expression level was measured by qPCR. Proliferation rate was measured in BrdU enzyme-linked immunosorbent assays. Caspase activity was measured using luminescent assays. All data are mean fold-change (FC) relative to control±S.E.M. (n=3 independent experiments). *P<0.05; **P<0.01; ***P<0.001. Student’s t-test

Figure 7

Ntep is essential for successful progression through the cell cycle. (a) Expression level of P53, Apaf-1, Bax, Bak mRNA and Ntep in 3T3 cells treated with GapmeR control and dimethyl sulphoxide (DMSO) and GapmeR Ntep and DMSO/pifithrin-α. Expression level was measured with qPCR. (b) Caspase-3/7 activity after treatment of 3T3 cells with GapmeR control and DMSO and GapmeR Ntep and DMSO/pifithrin-α. Activity was measured using luminescent assays. (c) TUNEL-positive cells after treatment of 3T3 cells with GapmeR control and DMSO and GapmeR Ntep and DMSO/pifithrin-α. TUNEL-positive cells were counted and normalized to 4′,6-diamidino-2-phenylindole (DAPI) signal. (d) Representative pictures from DAPI/TUNEL staining of 3T3 cells treated with GapmeR control and DMSO and GapmeR Ntep and DMSO/pifithrin-α. (e) Propidium iodide staining of 3T3 cells treated for 48 h with GapmeR Ntep and GapmeR control to analyse the cell cycle using fluorescence-activated cell sorter (FACS). Plots showing % of cells in the G0/G1 phase, cells in the S phase and cells in the G2/M phase. (f) Cyclin B1 protein level in 3T3 cells treated with GapmeR Ntep and GapmeR control for 48 h. Expression level was measured using western blotting. Representative picture of a western blot showing the band of cyclin B1 and the housekeeping gene GAPDH. (g) Expression level of Ntep in 3T3 cells stably expressing cyclin B1 coupled with GFP. Cells were sorted according to GFP signal in GFP-positive and GFP-negative cells. Expression level was measured with qPCR. *P<0.05; **P<0.01; ***P<0.001. Student’s t-test or one-way anaylsis of variance (ANOVA) for three groups. All data are mean fold-change (FC) relative to control±S.E.M. (n=3 independent experiments)