Effects of hypoxia and its downstream factor mirRNA-145 on differentiation of umbilical cord mesenchymal stem cells into type II alveolar epithelial cells / 吉林大学学报(医学版)

ABSTRACT

Objective: To explore the influence of hypoxia and transforming growth factor β (TGF-β) on the differentiation of umbilical cord mesenchymal stem cells (UCMSC) into the type II alverolar epithelial cells (AT II) and the regulatory effect of miR-145 in this process, and to illuminate the differentiation mechanism of UCMSC into AT II under the double stimulation of both hypoxia and TGF-β in the damaged lung tissue microenvironment. Methods: The UCMSC were isolated in vitro and co-cultured with human lung cancer cell line A549 to induce the differentiation of UCMSC into AT II. Cobaltous chloride (C0CI2) was used to mimic the hypoxia condition, and the induced cells were divided into normoxia group and hypoxia group. In hypoxia group, phase contrast microscope was used to observe the changes in cell morphology and flow cytometry was used to detect the percentage of AT II in the induced cells. qPCR and Western blotting methods were applied to test the expression levels of AT II specific genes, fibrosis-related genes and miR-145 in normoxia group and hypoxia group. In hypoxia group, the cells were divided into inh-145 group, inh-145 scramble group and control group, the expression levels of fibrosis-related genes in the cells in three groups were tested by qPCR and Western blotting methods. The pre-145 and pre-145 scramble were transfected into the 293T cells, and then the dual luciferase reporter gene system was used to check the relative luciferase unit (RLU) of TGF-βRII 37-UTR wild type and mutants. Results: In hypoxia group, the fibroblast-like UCMSC became flat and spindle, and finally showed a morphology of cobblestone-like epithelial cells 8 d later, and the percentage of the induced cells with high expression of AT II surface marker SpC was up to (94. 50 + 3. 37) %. The expression levels of specific genes of AT II (KGF, CK18, SpA, SpB and SpC) and miR-145 were obviously upregulated in hypoxia group compared with normoxia group (P<0.05). After stimulating the UCMSC-AT II differentiation with TGF-fil, the expression levels of fibrosis-related genes Col-I, TGF-βSR II and its downstream signaling factors p-Smad2 and p-Smad3 in hypoxia group were significantly down-regulated compared with normoxia group (P<0. 05). The expression levels of Col-I and TGF-βR II in inh-145 group were significantly raised compared with inh-145 scramble group and control group under hypoxia induction (P<0. 05). The RLU of TGF-βSRII 3'-UTR wild type was decreased after treated with pre-145 compared with TGF-βR II mutants (P< 0. 05). Conclusion: Hypoxia can promote the differentiation of UCMSC into the AT II and inhibit the TGF-β1-induced fibrosis. Its mechanism may be related to the inhibition of TGF-β1/TGFβR II signaling pathway through the down-regulation of TGF-βRII expression by hypoxia-induced miR-145.