miR-524-5p of the primate-specific C19MC miRNA cluster targets TP53IPN1- and EMT-associated genes to regulate cellular reprogramming

Abstract

Background: Introduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is able to 'reprogram' somatic cells to become induced pluripotent stem cells (iPSCs). Several microRNAs (miRNAs) are known to enhance reprogramming efficiency when co-expressed with the OSKM factors. The primate-specific chromosome 19 miRNA cluster (C19MC) is essential in primate reproduction, development, and differentiation. miR-524-5p, a C19MC member, is highly homologous to the reprogramming miR-520d-5p; we also reported that miR-524-5p was expressed in iPSCs but not mesenchymal stem cells (MSCs). This study aimed to elucidate possible contributions of miR-524-5p to the reprogramming process.

Methods: A miR-524-5p precursor was introduced into human fibroblast HFF-1 in the presence of OSKM, and the relative number of embryonic stem cell (ESC)-like colonies that stained positively with alkaline phosphatase (AP) and Nanog were quantified to determine reprogramming efficiency. A miR-524-5p mimic was transfected to MSCs to investigate the effects of miR-524-5p on TP53INP1, ZEB2, and SMAD4 expression by real-time polymerase chain reaction (PCR) and Western blot. Direct gene targeting was confirmed by luciferase activity. A phylogenetic tree of TP53INP1 was constructed by the Clustal method. Contribution of miR-524-5p to cell proliferation and apoptosis was examined by cell counts, BrdU, MTT, and cell death assays, and pluripotency gene expression by real-time PCR.

Results: Co-expressing the miR-524 precursor with OSKM resulted in a two-fold significant increase in the number of AP- and Nanog-positive ESC-like colonies, indicating a role for miR-524-5p in reprogramming. The putative target, TP53INP1, showed an inverse expression relationship with miR-524-5p; direct TP53INP1 targeting was confirmed in luciferase assays. miR-524-5p-induced TP53INP1 downregulation enhanced cell proliferation, suppressed apoptosis, and upregulated the expression of pluripotency genes, all of which are critical early events of the reprogramming process. Interestingly, the TP53INP1 gene may have co-evolved late with the primate-specific miR-524-5p. miR-524-5p also promoted mesenchymal-to-epithelial transition (MET), a required initial event of reprogramming, by directly targeting the epithelial-to-mesenchymal transition (EMT)-related genes, ZEB2 and SMAD4.

Conclusions: Via targeting TP53INP1, ZEB2, and SMAD4, miR-524-5p contributes to the early stage of inducing pluripotency by promoting cell proliferation, inhibiting apoptosis, upregulating expression of pluripotency genes, and enhancing MET. Other C19MC miRNAs may have similar reprogramming functions.

Keywords: Apoptosis; C19MC; Cell proliferation; MET; Pluripotency genes; Reprogramming efficiency; SMAD4; TP53INP1; ZEB2; miR-524-5p.

Fig. 1

Overexpression of mir-524 precursor promotes OKSM-driven iPSC generation at the early stage of induction. a High degree of sequence homology (bold letters) between miR-524-5p and miR-520d-5p. b Upregulated miR-524-5p levels in OSKM/mir-524 co-transduced relative to OSKM-transduced HFF-1 cells. *p < 0.05. c ESC-like morphology of two representative colonies formed at passage 1. d ESC-like alkaline phosphatase (AP)-positive colonies on day 14 that co-stained with Nanog on day 18 post-transduction with OSKM only, or OSKM in combination with either the blank vector CD511 or with a mir-524 precursor construct

Fig. 2

Predicted miR-524-5p-targeted genes regulate the G1 to S transition phase of the cell cycle. The predicted target genes were derived by interrogation of a variety of miRNA target prediction algorithms including the TargetScan, miRanda, and DIANA-microT. Putative miR-524-5p target genes are shown in yellow boxes

Fig. 3

Inverse relationship between expression of miR-524-5p and TP53INP1. a Expression of TP53INP1 in different cell lines determined by 25 cycles of RT-PCR. iPSC (MH#1) and iPSC (ASC-iPSC) were derived from the adipose-MSC cell lines, ASC Lonza and ASC-Inv, respectively. ESC (H9), placenta (HS799.PI), colon cancer (HCT-15), and breast cancer (MCF-7) cell lines were included for comparison and as controls. b Efficient transfection and overexpression of the miR-524-5p mimic in HCT-15 cells. c,d Effects of miR-524-5p overexpression on TP53INP1 expression. A miR-524-5p mimic was transfected into HCT-15 cells for 48 h before the cells were harvested for 40 cycles of real-time RT-PCR (c) or Western blot analysis (d) to determine TP53INP1 expression. As a control, a TP53INP1 siRNA (siTP53INP1) was also used in the transfection. *p < 0.05, relative to mock transfection. ASC adipose-derived stem cell, ESC embryonic stem cell, iPSC induced pluripotent stem cell, MSC mesenchymal stem cell, NC negative control

Fig. 4

Direct TP53INP1 targeting by and possible co-evolution with miR-524-5p. a Phylogenetic tree alignment (top panel) and sequence comparison (bottom panel) of the 3’-UTR of TP53INP1 ortholog transcript sequences in different species. b Identification of four putative miR-534-5p target sites (red vertical bars) in the 3’-UTR of the human TP53INP1 transcript. c Experimental validation of miR-524-5p targeting TP53INP1 in luciferase assays. In the top panel, alignment of miR-524-5p with the putative target site 2 (boxed and in bold letters) in the 3’-UTR of the TP53INP1 transcript (see text) is shown, so are the mutations (in italics and underlined) in the luciferase construct Mut2. HCT-15 transfected with the wild-type (WT2) or the mutant (Mut2) luciferase constructs alone, or in the presence of the miR-524-5p mimic or a validated negative control (NC), was performed before luciferase assays. The data shown were derived from two independent experiments in triplicate. *p < 0.05. d Alignment of sequences around the active target site 2 of TP53INP1 in different species. The miR-524-5p miRNA sequence is shown in blue above the TP53IPN1 sequences. The targeted core sequences are underlined and shown in bold. Similar nucleotides relative to the human gene are shown in italics and in bold letters. CDS coding sequence.

Fig. 5

miR-524-5p regulates critical features of cellular reprogramming via targeting TP53INP1. a Endogenous transcript levels of miR-524-5p and TP53INP1 in WJ0706 cells. Before (mock) or after transfection of a miR-524-5p mimic, WJ0706 cells were harvested 48 h post-transfection for analysis; miR-524-5p copy number was determined by Taqman qRT-PCR (left panel); TP53INP1 transcript level was determined by direct RT-PCR (right panel). b–f The miR-524-5p mimic-transfected WJ0706 cells were subjected to assays to determine cell proliferation (b,c), cell viability (d), apoptosis after oxidative stress induced with 200 μM H₂O₂ (e), and expression of selected pluripotency genes (f). All the experiments also included mock transfection with a negative control (NC) miRNA mimic, or a TP53INP1 siRNA (siTP53INP1) included as controls. For effects on cell proliferation, cell counts at different days post-transfection (b), or by BrdU ELISA measurements (c) were performed. For apoptosis assay (d,e), cell viability 2 h after incubation with H₂O₂ was determined by the MTT assay (d), or the histone-associated DNA fragments of apoptotic cells were quantified by ELISA assay 6 h after H₂O₂ treatment (e). f Expression of pluripotency genes in the transfected cells was analyzed by real-time RT-PCR 48 h post-transfection. Relative absorbance unit and mRNA level were determined as the relative absorbance units and expression, respectively to the value of the mock and negative control experiments, respectively, which was set as 1.0. *p < 0.05, **p < 0.01

Fig. 6

EMT-related genes ZEB2 and SMAD4 are direct targets of miR-524-5p. a,b Effects of miR-524-5p overexpression on the expression of the EMT-related genes. A miR-524-5p mimic was transfected to WJ0706 cells for 48 h before the cells were harvested for analysis. The analysis of the expression of ZEB2 and SMAD4 by real-time RT-PCR (a) and Western blot analysis (b). In both experiments, TP53INP1 was included as a control. c,d Experimental validation of miR-524-5p targeting of ZEB2 and SMAD4 in luciferase assays. Based on the predicted miR-524-5p binding sites (red vertical lines) in the 3’-UTRs of the ZEB2 and SMAD4 transcripts, a 3’-UTR luciferase construct of each gene was generated (boxed) (c). The blank pmiRGlo and 3’-UTR luciferase constructs were transfected alone, or in the presence of the miR-524-5p mimic, or a validated negative control (NC) in colon cancer cell line HCT-15 prior to luciferase assays (d). *p < 0.05, relative to the pmiRGlo-only control

Fig. 7

A proposed scheme of miR-524-5p regulation of the early stage of the reprogramming process [2, 3]. In the scheme, miR-524-5p promotes reprogramming by downregulating TP53INP1 to mediate processes associated with cell cycle, apoptosis, and expression of pluripotency genes, which are essential for the early stage of reprogramming. Furthermore, miR-524-5p also enhances MET, a required process for initial reprogramming, by targeting the EMT-related genes ZEB2 and SMAD4. See text for further description of the proposed scheme. EMT epithelial-to-mesenchymal transition, iPSC induced pluripotent stem cell, ROS reactive oxygen species