Int J Stem Cells.  2021 Nov;14(4):423-433. 10.15283/ijsc21080.

miR-200c-141 Enhances Sheep Kidney Cell Reprogramming into Pluripotent Cells by Targeting ZEB1

• Zhang Y
• He Y
• Wu P
• Hu S
• Zhang Y
• Chen C

• KMID: 2522561
• DOI: http://doi.org/10.15283/ijsc21080

Abstract

Background and Objectives
Sheep-induced pluripotent stem cells (siPSCs) have low reprogramming efficiency, thereby hampering their use in biotechnology and agriculture. Several studies have shown that some microRNAs play an important role in promoting somatic reprogramming in mouse and human. In this study, we investigated the effect of miR-200c-141 on somatic reprogramming in sheep and explored the mechanism of promoting the reprogramming.
Methods and Results
The lentivirus system driven by tetracycline (TET)-on carrying Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, hTERT, and SV40LT (OSKMNLST) could reprogram sheep kidney cells into pluripotent cells. Overexpression of miR-200c-141 in combination with OSKMNLST could significantly improve the efficiency of sheep iPSC generation (p＜0.01). Sheep iPSCs derived from miR-200c-141 showed embryonic stem cell (ESC)-like pluripotent properties, were positive for alkaline phosphatase and some pluripotent markers by quantitative real-time PCR (qRT-PCR) and immunofluorescence, and were able to differentiate into three germ layers in vitro. Oar-miR-200c was transfected into HEK293FT cells and was able to target the zinc finger E-box-binding homeobox 1 (ZEB1) 3’UTR using dual luciferase reporting analysis. Overexpression of oar-miR-200c in SKCs significantly reduced the expression of ZEB1, but increased the expression of E-cadherin by qRT-PCR and western blotting analysis.
Conclusions
These results suggest that miR-200c-141 can promote the reprogramming of sheep somatic cells to iPSCs, and oar-miR-200c targeted ZEB1 3’UTR, significantly decreased expression of ZEB1, and increased expression of E-cadherin. Oar-miR-200c may improve the MET process by affecting the TGF-β signaling pathway, thus improving the efficiency of somatic cell reprogramming in sheep.

Keyword

Sheep; iPSC; miR-200c; ZEB1; E-cadherin

Figure

• 

  Fig. 1 Process of sheep induced pluripotent stem cell (siPSC) generation and reprogramming efficiency calculation of siPSC. (A) Schematic diagram of sheep kidney cells (SKCs) reprogramming protocol used. (B) AP staining of siPSC generation with 8 factors (Oct4/Sox2/Klf4/c-Myc/Nanog/Lin28/hTERT/SV40LT). (C) AP staining of siPSC generation with 8 factors plus miR200c-141. (D) Magnification image of representative AP-positive siPSC colonies (100×). (E) The number of AP-positive colonies was counted to calculate the reprogramming efficiency. Data presented as the mean±standard deviation (SD), n=3, **p<0.01.

• 

  Fig. 2 Characterization of siPSCs by viral transduction of miR-200c-141 plus 8 factors. (A) SKCs expressed high levels of green fluorescent protein at 48 h after lentivirus transduction. (B) Typical morphology of siPSC colonies. (C) Magnification of the siPSC colony (200×). (D) siPSCs expressed pluripotency markers using immunofluorescence (200×).

• 

  Fig. 3 Relative mRNA expression levels of pluripotency markers in siPSCs by quantitative real-time PCR (qRT-PCR). Data presented as the mean±SD, n=3, **p<0.01.

• 

  Fig. 4 Characterization and differentiation ability of siPSCs. (A) Repre-sentative images of embryoid body (EB) (100×). (B) Detection of differentiation markers for the three germ layers in the EB using reverse transcriptase-PCR. (C) Detection of differentiation markers for the three germ layers in the EB using immuno-fluorescence. (D) Karyotype analysis of siPSC. (E) Bisulfite genomic sequencing of the promoter regions of Nanog (open circles: non-methylation; closed circles: methylation).

• 

  Fig. 5 Oar-miR-200c hinders ZEB1 expression and upregulates E-cadherin expression during SKCs repro-gramming. (A) Schematic representation of ZEB1 3’UTR cloned into luciferase reporter vector-pmiRGLO. The 6 binding sites sequence in the 3’UTR and the corresponding mutations for miR-200c are underlined. (B) miR-200c directly targeted the ZEB1 3’UTR by dual-luciferase reporter assay. (C) Overexpressing miR-200c in SKCs down-regulated of ZEB1 and up-re-gulated of E-cadherin transcription levels by qRT-PCR. (D) Overex-pressing miR-200c in SKCs down-regulated of ZEB1 and up-regulated of E-cadherin protein expression by western blotting. β-actin is the loading control. Data presented as the mean±SD, n=3, *p<0.05, **p< 0.01. ZEB1: zinc finger E-box-binding homeobox 1.

Reference

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