Frequent co-expression of miRNA-5p and -3p species and cross-targeting in induced pluripotent stem cells

Abstract

Background: A miRNA precursor generally gives rise to one major miRNA species derived from the 5' arm, and are called miRNA-5p. However, more recent studies have shown co-expression of miRNA-5p and -3p, albeit in different concentrations, in cancer cells targeting different sets of transcripts. Co-expression and regulation of the -5p and -3p miRNA species in stem cells, particularly in the reprogramming process, have not been studied.

Methods: In this work, we investigated co-expression and regulation of miRNA-5p and -3p species in human induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and embryonic stem cells (ESC) using a nanoliter-scale real-time PCR microarray platform that included 1,036 miRNAs.

Results: In comparing iPSC and ESC, only 32 miRNAs were found to be differentially expressed, in agreement of the ESC-like nature of iPSC. In the analysis of reprogramming process in iPSCs, 261 miRNAs were found to be differentially expressed compared with the parental MSC and pre-adipose tissue, indicating significant miRNA alternations in the reprogramming process. In iPSC reprogrammed from MSC, there were 88 miRNAs (33.7%), or 44 co-expressed 5p/3p pairs, clearly indicating frequent co-expression of both miRNA species on reprogramming. Of these, 40 pairs were either co-up- or co-downregulated indicating concerted 5p/3p regulation. The 5p/3p species of only 4 pairs were regulated in reverse directions. Furthermore, some 5p/3p species of the same miRNAs were found to target the same transcript and the same miRNA may cross-target different transcripts of proteins of the G1/S transition of the cell cycle; 5p/3p co-targeting was confirmed in stem-loop RT-PCR.

Conclusion: The observed cross- and co-regulation by paired miRNA species suggests a fail-proof scheme of miRNA regulation in iPSC, which may be important to iPSC pluripotency.

Keywords: Induced pluripotent stem cells; cell cycle control; miRNA-5p/3p species; reprogramming.

Figure 1

Hierarchical clustering analysis of miRNA profiles of MSC and iPSC. Level of gene expression is shown in the colour code shown at the bottom, ranging from minimal expression levels in green, average or weak miRNA expression in black and maximal levels in red. The analysis was performed using miScript miRNA PCR Array Data Analysis Web Portal.

Figure 2

Cell cycle control by co-expressed miRNA pairs iPSC. (A) Simplified view of the G1 to S phase of the cell cycle passing through the restricted (R) point. Factors that are in colored boxes are those that are further investigated. (B) Targeting of the cell cycle factors, E2F1, 2 and 3 and CDK6 by co-expressed miRNA pairs that were down-regulated in iPSC. In E2F targeting, (1), (2) and (3) indicate E2F1, E2F2 and E2F3, respectively. Solid and dashed lines indicate targeting by the 5p or 3p species, respectively. Asterisks indicated validated miRNA targeting. (C) Cell cycle promotion by co-expressed miRNA pairs that were up-regulated in iPSC. See also legend to (C) above. (D) Predicted miRNA targeting sites in the 3'UTR sequences of CDK6 and TGFβ2 as determined by DIANA-microT v5.0. Only 7-and 8-mericalignments between the miRNA seed sequence and the target transcript are depicted. (E) Co-expression of miRNA pairs as determined by stem-loop RT-PCR. PCR products were analyzed in 4% agarose gels. The U6 snRNA was used as an internal control. HWP, human white pre-adipocyte; HWP-iPSC, HWP-derived induced pluripotent stem cell; ASC, adipose stem cell, ASC-iPSC: ASC-derived iPSC; MSC, mesenchymal stem cell from adipose tissue; MSC-iPSC, MSC-derived iPSC.