Highly-expressed micoRNA-21 in adipose derived stem cell exosomes can enhance the migration and proliferation of the HaCaT cells by increasing the MMP-9 expression through the PI3K/AKT pathway

Objective: Wound healing remains a challenge in burns and trauma fields. Adipose derived stem cells exosomes (AD-exos) had been confirmed to have a positive effect on the wound healing and the migration and proliferation of keratinocyte. However, the mechanism of the AD-exos is still unclear. The objective of this article is to observe the function of the miR-21 expressed in the adipose AD-exos and the effect on migration and proliferation of the HaCaT cells.

Materials and methods: The full layer dermal wound of BALb/c mouse was used to observe the vitro effect of the AD-exos and detect the expression of miR-21.The co-culture systems were established by transwell plates for observing the migration, proliferation, apoptosis rate, detecting the RNA, and protein expression in different treated groups. MiR-21 plasmid was used to over-express miR-21 by transfection of HaCaT cells. GW4869 was used to inhibit the secreting of exosomes from ADSCs.

Results: The results showed that both ADSCs and the AD-exos could improve the wound healing process of BALb/c mouse full layer skin wound at a similar level, especially at the 7th day post surgery when compared to the control group (p < 0.01) and the highly expressed miR-21 was detected (p < 0.01 compared with control group and p < 0.001 compared to other microRNAs) in the treated groups at the same time point. AD-exos could obviously enhance the migration and proliferation of the HaCaT cells (p < 0.01), and fell back to the same level when the exosomes inhibitor--GW4869 was added compared with control group (p > 0.05). Over-expressed miR-21 could also significantly improve the migration and proliferation of HaCaT cells. But both AD-exos and miR-21 had no significantly effect on the apoptosis rate of HaCaT cells (p > 0.05 compared with each other). Over-expression of miR-21 plasmid could decrease the TGF-βI protein level (p < 0.001 vs. control group) in HaCaT cells while TGF-βI protein level increased again when antagomiR-21 was added in (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 expression of HaCaT cells could be increased by the transfect ion of miR-21 plasmid (p < 0.001 vs. empty plasmid group) and decreased by antagomiR-21 (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 appeared to have influence on MMP-9 and TIMP-2 (p < 0.001 compared to control group and p < 0.001 compared to TGF-βI group) but not MMP-2 and TIMP-1 (p > 0.05 compared to control group and TGF-βI group). These processes might act through PI3K/AKT pathway.

Conclusion: This research provide the experimental evidence that the miR-21 is highly expressed in AD-exos and can significantly accelerate the wound healing process and enhance the migration and proliferation of the HaCaT cells. Over-expressed miR-21 can inhibit the TGF-βI expression and excess TGF-βI can also have negative feedback influence on miR-21. We have found a reliable evidence that these two factors can act on HaCaT cells by influencing MMP-2 and TIMP-1 protein expression through the PI3K/AKT signal pathway. These results may provide a potential perspectives on improving the wound healing.