Effect of miR-130b-3p on the Reprogramming of Human Dental Origin iPSCs

Xiaobing TAN; Min ZHANG; Yu GUO; Linling DU; Qingyuan DAI

doi:10.12259/j.issn.2095-610X.S20221204

Volume 43 Issue 12

Dec. 2022

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Xiaobing TAN, Min ZHANG, Yu GUO, Linling DU, Qingyuan DAI. Effect of miR-130b-3p on the Reprogramming of Human Dental Origin iPSCs[J]. Journal of Kunming Medical University, 2022, 43(12): 18-22. doi: 10.12259/j.issn.2095-610X.S20221204

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Effect of miR-130b-3p on the Reprogramming of Human Dental Origin iPSCs

doi: 10.12259/j.issn.2095-610X.S20221204

1.
Center of Stomatology，The 1st People’s Hospital of Yunnan Province/The Affiliated Hospital of Kunming University of Science and Technology，Kunming Yunnan 650032
2.
Dept. of Cardiology，The 1st Affiliated Hospital of Kunming Medical University，Kunming Yunnan 650032，China

Received Date: 2022-07-27
Available Online: 2022-12-05
Publish Date: 2022-12-25

Abstract

Abstract

Objective To explore the effect of hsa-miR-130b-3p on the reprogramming of human dental iPSCs. Methods LV3 （H1/GFP&Puro）-hsa-miR-130b-3p-mimics plasmid was designed and synthesized. The plasmid was transduced into human DPSCs by Lipofectamine 3000 kit according to the manufacturer’s instructions. Fluorescence microscopy was used to observe the cells and transfection efficiency was verified by RT-qPCR. Sendai reprogramming kit was used to induce two groups of DPSCs （miR-130b-3p-DPSCs in the experimental group and DPSCs in the control group） into iPSCs. The morphology and reprogramming efficiency were compared and the expression of Oct4/Nanog/KOS/Klf4/c-Myc was detected by RT-PCR in two groups of iPSCs. Results hsa-miR-130b-3p overexpression plasmid was significantly expressed in DPSCs 48h after transfection, indicating high transfection efficiency （P < 0.01）. The two groups of iPSCs obtained by Sev reprogramming showed flat and dense round clones with clear and smooth edges, uniform cell morphology and large nucleolus in the colonies. RT-PCR results showed that cells in both groups could express the specific markers of stem cells Oct4 and Nanog. At the same time, exogenous virus SeV or transcription factors KOS/Klf4/c-Myc were no longer expressed. RT-PCR showed that cells in both groups all expressed specific markers Oct4 and Nanog, while SeV or KOS/Klf4/ C-MYC were not detected. The reprogramming efficiency of the experimental group was higher than that of the control group （0.037% and 0.018% respectively, P < 0.05）. Conclusion hsa-miR-130b-3p could promote the reprogramming efficiency of human DPSCs. Our research provides theoretical foundation for dental iPSCs application in dental pulp regenerative therapy.
- Dental pulp stem cells,
- Reprogramming,
- Induced pluripotent stem cells,
- microRNAs,
- Overexpression

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