Abstract
It is well known that microRNAs play a very important role in regulating reprogramming, pluripotency and cell fate decisions. Porcine induced pluripotent stem cells (piPSCs) are now available for studying the pluripotent regulation network in pigs. Two types of piPSCs have been derived from human and mouse embryonic stem cell (ESC) culture conditions: hpiPSCs and mpiPSCs, respectively. The hpiPSCs were morphologically similar to human ESCs, and the mpiPSCs resembled mouse ESCs. However, our current understanding of the role of microRNAs in the development of piPSCs is still very limited. Here, we performed small RNA sequencing to profile the miRNA expression in porcine fibroblasts (pEFs), hpiPSCs and mpiPSCs. There were 22 differential expressed (DE) miRNAs down-regulated in both types of piPSCs compared with pEFs, such as ssc-miR-145-5p and ssc-miR-98. There were 27 DE miRNAs up-regulated in both types of piPSCs compared with pEFs. Among these up-regulated DE miRNAs in piPSCs, ssc-miR-217, ssc-miR-216, ssc-miR-142-5p, ssc-miR-182, ssc-miR-183 and ssc-miR-96-5p have much higher expression levels in mpiPSCs, while ssc-miR-106a, ssc-miR-363, ssc-miR-146b, ssc-miR-195, ssc-miR-497, ssc-miR-935 and ssc-miR-20b highly expressed in hpiPSCs. Quantitative stem-loop RT-PCR was performed to confirm selected DE miRNAs expression levels. The results were consistent with small RNA sequencing. Different expression patterns were observed for key miRNA clusters, such as the miR-17-92 cluster, the let-7 family, the miR-106a-363 cluster and the miR-182-183 cluster, in the mpiPSCs and hpiPSCs. Novel miRNAs were also predicted in this study, including a putative porcine miR-302 cluster: ssc_38503, ssc_38503 and ssc_38501 (which resemble human miR-302a and miR-302b) found in both types of piPSCs. The miR-106a-363 cluster and putative miR-302 cluster increased the reprogramming efficiency of pEFs. The study revealed significant differences in the miRNA signatures of hpiPSCs and mpiPSCs under different pluripotent states that were derived from different culture conditions. These differentially expressed miRNAs may play important roles in pluripotent regulation in pigs, and this information will facilitate the understanding of the mechanism of pluripotency in pigs.
Highlights
The pig is an excellent model for human disease because of its similarity to humans in terms of its organ morphology and function
Venn diagram showed the numbers of differential expressed (DE) miRNAs in pluripotent stem cells (piPSCs) compared with porcine fibroblasts (pEFs) (Fig 2C)
Compared with pEFs, there were 50 DE miRNAs in both types of piPSCs. Among these DE miRNAs, there 22 were down-regulated in both types of piPSCs compared with pEFs (Table 1), while 27 DE miRNAs up-regulated in both types of piPSCs versus pEFs (Table 2)
Summary
The pig is an excellent model for human disease because of its similarity to humans in terms of its organ morphology and function. MicroRNAs, which have 20 to 22 nucleotides RNA sequences, play a major role in posttranscriptional gene regulation in higher eukaryotes. It was recently reported that microRNAs can bind the CDS region to regulate gene expression [7]. Families of microRNAs with highly conserved sequences are found in many organisms, and clusters of miRNAs located in discrete genomic loci can be coordinately expressed. Studies have shown that miRNAs and miRNA clusters play critical roles in the reprogramming process and maintenance of pluripotent stem cells in humans and mice. Naïve and primed murine pluripotent stem cells have distinct miRNA expression profiles [12]. Human embryonic stem cells (hESCs) have a special miRNA expression pattern that differs from that of mESCs [13,14]. The primed hESCs can be converted to a naïve state following a change in miRNA expression [15]
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