Olonies of the OSKM-transduced cells (Fig. 1b). After transduction, the cells were cultured under standard conditions for human iPSC induction but in the presence of Dox, and the transduced cells were monitored daily for morphological changes. ESC-like colonies began to form on day 7 which, when passaged on day 30 and cultured under Dox-free conditions, showed clear and round borders (Fig. 1c). Furthermore, these colonies showed AP staining on day 14 and Nanog at day 18 culturing under Dox-dependent medium (Fig. 1d). Colonies that displayed ESC-like morphology and that were both APand Nanog-positive were considered bona fide iPSC colonies (Fig. 1c and d). In each of the three independent experiments, the total number of ESC-like and AP + Nanog+ colonies observed varied between three to six in the triplicate wells of the 12-well plate transduced with OSKM alone, or OSKM with the blank vector CD511, and from seven to twelve colonies on OSKM/ mir-524 transduction (Table 1). Taken together, OSKM/ mir?24 co-transduction generated a total of 27 ESClike/AP+Nanog+ colonies in the three independent transduction experiments, with a calculated reprogramming efficiency of 0.012 , and was 2.25-fold that of OSKM or OSKM/CD511 transduction, which was within the range of reprogramming efficiencies RR6 site reported by others [25, 26]. The data thus support the notion that miR-524 enhanced OKSM-induced reprogramming of HFF-1 fibroblast cells.Bioinformatics analysis of miRNA-524-5p and predicted target mRNA interactionsIn our previous work, we have described the bioinformatics analysis of C19MC miRNAs, including the most significantly enriched gene ontology terms associated with biological process and molecular functions and the KEGG pathways [7]. In the same study, our data showed that C19MC could play an important role in regulating stemness. Since cell cycle, more critically the G1-to-S transition phase, is an important feature of the regulation of stem cell self-renewal [13, 27] we focused in this work on determining possible functions of miR-5245p in relation to the G1-S phase of the cell cycle. Based on the earlier bioinformatics analysis [7], eight predicted G1-to-S transition-related genes, namely TGFR1, Smad2/3/4, Rb1, PTEN, HIPK2, and TP53INP1, were identified to be targeted by miR-524-5p (Fig. 2). Repression of the PI3K/PKB/Akt/mTOR and TGF pathways, such as PTEN, p21/CDK1NA, TGFR1, and SMAD2/3/4, has been reported to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26104484 promote self-renewal and proliferation by blocking the G1 to S transition boundary of the cell cycle [13, 28?0]. Furthermore, p53, a pro-apoptotic, antiproliferative, and antioxidant regulator, is indirectly regulated by miR-524-5p through targeting TP53INP1 and HIPK2 (Fig. 2). Suppression of HIPK2 could inhibit p53 expression [31] whereas TP53INP1 is a major mediator of p53-driven responses to oxidative stress [32]. Besides p53, miR-524-5p was also predicted to target a member of the Rb family, RB1, an event that regulates the cell cycle by enhancing G1 to S transition and proliferation. TP53INP1 plays important roles not only in reprogramming by regulating p53 [33, 34] but also in cancer stem cells in which TP53INP1 deficiency results in increased self-renewal and acquisition of cancer stem cell phenotype [35, 36]. Thus, the possible correlation between miR-524-5p and TP53INP1 was further investigated.TP53INP1 is a direct target of miR-524-5pTo investigate the relationship between miR-524-5p and TP53INP1, endogenous TP53INP1 expression.
