Volume 137, Issue 4, Pages 647-658 (May 2009) MicroRNA-145 Regulates OCT4, SOX2, and KLF4 and Represses Pluripotency in Human Embryonic Stem Cells  Na Xu, Thales Papagiannakopoulos, Guangjin Pan, James A. Thomson, Kenneth S. Kosik  Cell  Volume 137, Issue 4, Pages 647-658 (May 2009) DOI: 10.1016/j.cell.2009.02.038 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 The Expression of miR-145 in hESCs and Target Validation of the OCT4, SOX2, and KLF4 3′ UTRs (A) The relative level of miR-145 increased after the hESC differentiation. In Taqman real-time RT-PCR, the snoRNA h-NR003022 was used as an internal normalization control. EB, embryoid bodies. (B) The in situ expression of miR-145 RNA in the day 8 differentiated hESCs by EB formation. Red fluorescent signal was from CY3-miR-145 RNA probe, while blue fluorescent signal was from nuclear DNA counterstained with DAPI. The scale bar represents 50 μm. (C) Summary of miR-145 target sites in the 3′ UTRs of KLF4, POU5F1 (encoding for OCT4), and SOX2. H, human; R, rat; M, mouse; D, dog. The underlined nucleotides (target sites) were deleted in the mutant 3′ UTR constructs. (D) The 3′ UTR reporters in target validation. Luc, firefly luciferase; pA, polyadenylation signal; WT, wild-type. Mutant UTR has a 6 bp deletion of the miR-145 target site. (E) miR-145 specifically represses its targets in the luciferase assay in HeLa. Relative luciferase level = (Sluc/Srenilla)/(Cluc/Crenilla). Luc, raw firefly luciferase activity; Renilla, internal transfection control renilla activity; S, sample; C, control pre-scramble. Student's t test compared two data sets marked by brackets in the panel. ∗∗ p < 0.01. The error bar represents the standard deviation (SD) from three independent experiments. +, wild-type; MT, mutant UTRs. Cell 2009 137, 647-658DOI: (10.1016/j.cell.2009.02.038) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Endogenous miR-145 Represses the 3′ UTRs of OCT4, SOX2, and KLF4 in hESCs (A–C) The relative luciferase level of 3′ UTR luciferase reporters of OCT4 (A), SOX2 (B), and KLF4 (C) in hESCs under self-renewal conditions at 24 hr or 48 hr after transfection. Mut, mutant UTR with a 6 bp deletion of the miR-145 target sites; Control, the basal luciferase reporter without UTR. ∗∗ p < 0.01; ∗ p < 0.05. The error bar represents the standard deviation (SD) from three independent experiments. (D–F) The difference between 3′ UTR mutant and wild-type luciferase reporters of OCT4 (D), SOX2 (E), and KLF4 (F) depends on miR-145 in hESCs. On the y axis, the mutant reporter level is normalized against the average of the wild-type reporter level to reflect the magnitude of repression. (G–I) The repression of OCT4 (G) and KLF4 (I) but not SOX2 (H) UTR reporter by miR-145 increased during hESC differentiation from day 2–8. Cell 2009 137, 647-658DOI: (10.1016/j.cell.2009.02.038) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Effect of miR-145 on Endogenous OCT4, KLF4, and SOX2 in hESCs (A) Isolation of the GFP+ hESCs under self-renewal conditions expressing the control Lenti-scr or Lenti-miR-145 by FACS. (B and C) Relative mRNA (B) and protein (C) levels of OCT4, SOX2, and KLF4 in control Lenti-scr or Lenti-miR-145 GFP+ cells. (D) Isolation of the Alexa488-positive hESCs under self-renewal conditions transfected with Alexa488-conjugated LNA-scr control or LNA-miR-145 by FACS. (E and F) Relative mRNA (E) and protein (F) levels of OCT4, SOX2, and KLF4 in LNA-scr or LNA-miR-145 transfected cells. In real-time RT-PCR, GAPDH mRNA is the normalization control (B and E). In western blot (C and F), protein level quantification was normalized to GAPDH. Cell 2009 137, 647-658DOI: (10.1016/j.cell.2009.02.038) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Overexpression of miR-145 Inhibits hESC Self-Renewal and Colony Formation (A) Inhibition of self-renewal during hESC maintenance by ectopic miR-145 in the Lenti-miR-145-expressing/GFP+ cells. After regular mechanical splitting of the hESC colonies, the cells were transduced with lentiviruses and maintained under self-renewal conditions for 6 days. Self-renewal is assessed by flow cytometry analysis of the SSEA4 staining in lentiviral GFP expressing cells (GFP+) cells. Flow cytometry data collection was in two channels: GFP in green channel and SSEA4 in yellow channel. The self-renewal percentage was N(GFP+SSEA4+) / (N(GFP+ SSEA+) + N(GFP+ SSEA−)). N, number of events. t test compared shOCT4 or Lenti-miR-145 to control Lenti-scr. ∗∗ p < 0.01. n = 6. (B) Increased apoptosis in Lenti-miR-145 cells after 6 days, assessed by flow cytometry analysis of the annexin V staining in lentiviral GFP-expressing (GFP+) cells. The percentage was N(GFP+Annexin+) / (N(GFP+ Annexin+) + N(GFP+ Annexin−)). ∗∗ p < 0.01. n = 6. (C) Decreased S phase population in Lenti-miR-145 GFP+ cells sorted after 6 days. The cell cycle is analyzed by flow cytometry after propidium iodide (PI) staining, and the data are processed with the ModFit LT program. (D) The morphologies of the hESC colonies 11 days after single cell transduction. Left column, bright field images; right column, GFP images. The scale bar represents 100 μm. (E) Reduction of hESC colony formation by miR-145. Total, sum of total colony number in three experiments; Differentiation, the differentiated colony number. In each experiment, 105 single cells were transduced (MOI equaled 20) for 24 hr, and further cultured in fresh conditioned medium with bFGF for 11 days. (F) Reduction of hESC self-renewal in Lenti-miR-145-expressing/GFP+ cells after 11 days. ∗∗ p < 0.01. n = 6. Cell 2009 137, 647-658DOI: (10.1016/j.cell.2009.02.038) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 miR-145 Promotes Differentiation of hESCs (A–C) Increased mesoderm and ectoderm differentiation in Lenti-miR-145 infected hESCs. First column, GFP images; second column, mesoderm marker SMA (A), ectoderm marker TUJ1 (B), endoderm marker AFP (C). (D) Relative mRNA level of differentiation marker genes MIXL1, NODAL, GATA4, FABP1, VIMENTIN, OTX2, and CG-α. GAPDH was used as internal normalization in SYBR qRT-PCR. ∗∗ p < 0.01. n = 6. In (A)–(D), hESC colonies were infected with lentivirus and cultured under self-renewal conditions for 11 days. (E) Upregulation of miR-145 elevates differentiation rate. Percentage of differentiation = 100% − percentage of self-renewal (assayed by SSEA4 expression flow cytometry). In (E)–(I), hESC colonies were cultured under differentiation conditions without conditioned media and FGF. (F) Effect of miR-145 on differentiation is mediated by OCT4 and SOX2. Expression of the OCT4/SOX2 rescue constructs completely inhibits the effect of ectopic miR-145 in promoting differentiation. (G and H) Lenti-miR-145 has no obvious effect on hESC growth (G) and death (I) during differentiation. (I) Lenti-miR-145 induces more differentiated colonies on differentiation day 12. Cell 2009 137, 647-658DOI: (10.1016/j.cell.2009.02.038) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Necessity of miR-145 during hESC Differentiation (A) LNA-miR-145 inhibition decreased the endogenous miR-145 upregulation during differentiation at 48 hr, 96 hr, and 144 hr after transfection compared to control LNA-scr. The snoRNA h-NR003022 was used as an internal normalization control in qRT-PCR. In Figure 6, hESC colonies were cultured under differentiation conditions without conditioned media and FGF. (B) miR-145 depletion by LNA-miR-145 delayed hESC differentiation at 48 hr and 96 hr after transfection. Percentage of differentiation = 100% − percentage of self-renewal (assayed by SSEA4 expression flow cytometry). (C) miR-145 depletion resulted in increased S phase population during the cell cycle. The cell cycle is analyzed by flow cytometry after PI staining, and the data are processed with the ModFit LT program. (D) FACS isolation of the Alexa488-positive hESCs under differentiation conditions transfected with Alexa488-LNA-scr control or LNA-miR-145. (E) Western blot of Alexa488-LNA sorted hESCs. Protein level quantification was normalized to GAPDH and is provided under each band. (F) The differences in differentiation between LNA-scr and LNA-miR-145 was rescued by shOCT4 and shSOX2, two vectors that downregulate OCT4 and SOX2. (G) The alkaline phosphatase staining of hESC differentiation at 96 hr after LNA transfection. (H) LNA-miR-145 elevated the target gene expression of OCT4, KLF4 and disrupted the expression of differentiation markers. Cell 2009 137, 647-658DOI: (10.1016/j.cell.2009.02.038) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 miR-145 Promoter Transcription Is Regulated by OCT4 (A) Ectopic OCT4 represses miR-145 promoter transcription in HeLa cells. The promoter construct P-miR-145 contains a putative OCT4 site (“Site 1”), which was deleted in P-del. For derivation of the relative luciferase level, the raw luciferase activity was first normalized to the internal transfection control Renilla and then divided by the average value of the basal construct P-control. Open triangle indicates increasing amounts of OCT4 proteins expressed from a pSin-EF2-Oct4-Pur vector. n = 3. (B) EMSA shows the interaction between the miR-145 site 1 and OCT4 in hESCs. NE, nuclear extracts; Ab, antibodies; Ng, negative control NANOG antibody; Comp, cold competitor probes; Hs27, negative control human fibroblast cells which lack OCT4. Top arrows, shifted probes; bottom arrows, free probes. Lanes 1, 5, 9 and 13 are nuclear extract-free negative controls without any shift. (C) Chromatin IP shows high OCT4 enrichment at the miR-145 promoter in hESCs, but not in BMP4-differentiated cells (hESC-BMP4). Relative enrichment is normalized to control IgG. The positions of each PCR amplicon relative to the transcription start site of each promoter are labeled. n = 3. (D) Proposed model of the double-negative feedback loop by miR-145 and three factors. miR-145 inhibits self-renewal and pluripotency factors OCT4, SOX2, and KLF4 and controls differentiation. Grey color, low level of either miR-145 or pluripotency factors; bold font, high level of either miR-145 or pluripotency factors. Cell 2009 137, 647-658DOI: (10.1016/j.cell.2009.02.038) Copyright © 2009 Elsevier Inc. Terms and Conditions