Liu Zhe, Yang Yuan, Ju Jihui, Zhang Guangliang, Zhang Ping, Ji Pengxiang, Jin Qianheng, Cao Gaobiao, Zuo Rui, Wang Hongyu, Yu Chenghao, Zhang Zhiqiang, Le Yingying, Fu Yi, Hou Ruixing
Suzhou Medical College of Soochow University, Suzhou, China.
Department of Hand Surgery, Ruihua Affiliated Hospital of Soochow University, Suzhou, China.
Stem Cells Int. 2022 Aug 21;2022:1474273. doi: 10.1155/2022/1474273. eCollection 2022.
Skin epidermal stem cells (EpSCs) play a critical role in wound healing and are ideal seed cells for skin tissue engineering. Exosomes from human adipose-derived stem cells (ADSC-Exos) promote human EpSC proliferation, but the underlying mechanism remains unclear. Here, we investigated the effect of miR-100-5p, one of the most abundant miRNAs in ADSC-Exos, on the proliferation of human EpSCs and explored the mechanisms involved. MTT and BrdU incorporation assays showed that miR-100-5p mimic transfection promoted EpSC proliferation in a time-dependent manner. Cell cycle analysis showed that miR-100-5p mimic transfection significantly decreased the percentage of cells in the G1 phase and increased the percentage of cells in the G2/M phase. Myotubularin-related protein 3 (MTMR3), a lipid phosphatase, was identified as a direct target of miR-100-5p. Knockdown of MTMR3 in EpSCs by RNA interference significantly enhanced cell proliferation, decreased the percentage of cells in the G1 phase and increased the percentage of cells in the S phase. Overexpression of MTMR3 reversed the proproliferative effect of miR-100-5p on EpSCs, indicating that miR-100-5p promoted EpSC proliferation by downregulating MTMR3. Mechanistic studies showed that transfection of EpSCs with miR-100-5p mimics elevated the intracellular PIP3 level, induced AKT and ERK phosphorylation, and upregulated cyclin D1, E1, and A2 expression, which could be attenuated by MTMR3 overexpression. Consistently, intradermal injection of ADSC-Exos or miR-100-5p-enriched ADSC-Exos into cultured human skin tissues significantly reduced MTMR3 expression and increased the thickness of the epidermis and the number of EpSCs in the basal layer of the epidermis. The aforementioned effect of miR-100-5p-enriched ADSC-Exos was stronger than that of ADSC-Exos and was reversed by MTMR3 overexpression. Collectively, our findings indicate that miR-100-5p promotes EpSC proliferation through MTMR3-mediated elevation of PIP3 and activation of AKT and ERK. miR-100-5p-enriched ADSC-Exos can be used to treat skin wound and expand EpSCs for generating epidermal autografts and engineered skin equivalents.
皮肤表皮干细胞（EpSCs）在伤口愈合中起关键作用，是皮肤组织工程的理想种子细胞。人脂肪来源干细胞外泌体（ADSC - Exos）可促进人EpSC增殖，但其潜在机制尚不清楚。在此，我们研究了ADSC - Exos中最丰富的miRNA之一miR - 100 - 5p对人EpSCs增殖的影响，并探讨了其相关机制。MTT和BrdU掺入试验表明，miR - 100 - 5p模拟物转染以时间依赖性方式促进EpSC增殖。细胞周期分析表明，miR - 100 - 5p模拟物转染显著降低了G1期细胞百分比，增加了G2/M期细胞百分比。肌管素相关蛋白3（MTMR3），一种脂质磷酸酶，被鉴定为miR - 100 - 5p的直接靶点。通过RNA干扰敲低EpSCs中的MTMR3可显著增强细胞增殖，降低G1期细胞百分比，增加S期细胞百分比。MTMR3的过表达逆转了miR - 100 - 5p对EpSCs的促增殖作用，表明miR - 100 - 5p通过下调MTMR3促进EpSC增殖。机制研究表明，用miR - 100 - 5p模拟物转染EpSCs可提高细胞内PIP3水平，诱导AKT和ERK磷酸化，并上调细胞周期蛋白D1、E1和A2的表达，而MTMR3的过表达可减弱这种作用。同样，将ADSC - Exos或富含miR - 100 - 5p的ADSC - Exos皮内注射到培养的人皮肤组织中，可显著降低MTMR3表达，并增加表皮厚度和表皮基底层中EpSCs的数量。富含miR - 100 - 5p的ADSC - Exos的上述作用比ADSC - Exos更强，且可被MTMR3的过表达逆转。总体而言，我们的研究结果表明，miR - 100 - 5p通过MTMR3介导的PIP3升高和AKT及ERK激活促进EpSC增殖。富含miR - 100 - 5p的ADSC - Exos可用于治疗皮肤伤口，并扩增EpSCs以生成表皮自体移植物和工程皮肤替代物。
