In MSC and iPSC lines was first established by RT-PCR (Fig. 3a). TP53INP1 was found

In MSC and iPSC lines was first established by RT-PCR (Fig. 3a). TP53INP1 was found to be expressed in both MSCs andTable 1 Number of ESC-like and AP+Nanog+ colonies obtained on OSKM/mir-524 co-transduction of HFF-1 cellsTransduction OSKM only OSKM + CD511 OSKM + mir-524 Colonies in experiment (n) 1 4 6 8 2 3 4 7 3 6 3 12 12 12 27* 1.53 1.53 2.65 Total colonies (n) SD Reprogramming efficiency ( ) 0.005 0.005 0.012*ESC-like and AP+Nanog+ colonies in triplicate wells in a 12-well plate were counted under a Naramycin A custom synthesis microscope as described in Methods. Reprogramming efficiency was calculated as the total number of AP+Nanog+ colonies generated from the total number of transduced HFF-1 cells in three independent experiments. *p < 0.05 compared with OSKM only. AP alkaline phosphatase, ESC embryonic stem cell, SD standard deviationNguyen et al. Stem Cell Research Therapy (2017) 8:Page 7 ofFig. 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 boxesiPSCs, albeit at higher levels in MSCs than in the derived iPSCs. Interestingly, our previous study has shown that miR-524-5p was abundantly expressed in iPSCs whereas miR-524-5p expression was undetected or detected at very low levels in MSC cell lines [7], suggesting an inverse correlation between the expression of miR524-5p and TP53INP1 and negative regulation of TP53INP1 by miR-524-5p. ESCs and placenta, which comprehensively express all the C19MC miRNAs and, therefore, miR-524-5p [7], also expressed TP53INP1, and in higher levels in ESCs. Surprisingly, the two cancer cell line controls, the colorectal HCT-15 and the breast cancer MCF-7, also expressed TP53INP1 to different levels (Fig. 3a). Notably, all these cell lines, except for the placenta HS799.PI cells, also expressed various levels of miR-524-5p [7], but correlation could not be made between the TP53INP1 and miR-524-5p expression levels. The observation of the lack of inverse correlation between miR-524-5p and TP53INP1 in some cell lines suggests that, besides regulation by miR-524-5p, other factors are involved in regulating TP53INP1 expression, particularly in cancer cells (see Discussion). When HCT-15 cells were transfected with a miR524-5p mimic to achieve a 16-fold upregulation of the miR-524-5p level 48 h post-transfection (Fig. 3b), the TP53INP1 mRNA and protein levels were assayed in qRT-PCR and Western blots (Fig. 3c and d). As controls, a miRNA negative transfection control (NC) and a siRNA to knockdown TP53INP1 expression were also included. Similar to siRNA-mediated TP53INP1 suppression, forced overexpression of miR-524-5p significantlydownregulated PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27465830 TP53INP1 at both the mRNA and protein levels compared with the mock control (Fig. 3c and d), further confirming a reverse relationship between miR524-5p and TP53INP1 expression. The data further suggested that miR-524-5p regulated TP53INP1 expression probably via downregulation of the TP53INP1 transcript. Interestingly, when the TP53INP1 transcript sequences of various species were compared, TP53INP1 sequences of the primate (human and chimpanzee) showed a tight clustering with a high sequence homology of 98.6 and late evolutionary emergence in comparison with other mammalian orthologs (Fig. 4a). Keeping in mind that miR-524-5p bel.