Micro-vesicles from Mesenchymal Stem Cells Over-expressing miR-34a Inhibit Transforming Growth Factor-b1-Induced Epithelial-Mesenchymal Transition in Renal Tubular Epithelial Cells In Vitro
Kidney disease remains one of the most significant challenges in healthcare systems worldwide. Despite advancements in medical care, mortality rates for patients with acute kidney injury or chronic kidney disease (CKD) have not seen a significant reduction in recent decades. Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic strategy due to their ability to self-renew, proliferate, and differentiate into multiple cell lineages. The therapeutic effects of MSCs are largely attributed to the release of beneficial factors that promote organ repair. Among these factors, micro-vesicles (MVs) secreted by MSCs have shown potential in promoting functional improvement in injured kidneys. This study focuses on the effects of miR-34a-modified MSC-MVs on transforming growth factor (TGF)-b1-induced fibrosis and apoptosis in renal tubular epithelial cells in vitro.
MSCs were isolated from the bone marrow of male C57BL/6J mice and cultured according to established protocols. The cells were characterized using flow cytometry to confirm the presence of MSC markers (CD105) and the absence of hematopoietic markers (CD34, CD45, and CD11b). The multi-potential differentiation capacity of MSCs was confirmed through osteogenic and adipogenic induction assays, with successful differentiation indicated by Alizarin Red S and Oil Red O staining, respectively.
To overexpress miR-34a in MSCs, lentiviral particles encoding miR-34a or a negative control (miR-nc) were used to infect the cells. The expression of miR-34a in the infected MSCs was significantly increased, as confirmed by real-time PCR. MVs were then collected from the culture media of these MSCs using ultracentrifugation. The MVs were characterized by transmission electron microscopy, which confirmed their size to be less than 200 nm. The miR-34a content in the MVs was also quantified, showing a three-fold increase in miR-34a-enriched MVs compared to control MVs.
Human Kidney-2 (HK-2) renal tubular epithelial cells were used as the in vitro model to study the effects of miR-34a-enriched MSC-MVs on TGF-b1-induced epithelial-mesenchymal transition (EMT). HK-2 cells were treated with 6 ng/mL recombinant TGF-b1 to induce EMT, and the effects of MSC-MVs were assessed through various assays. The expression levels of epithelial markers (tight junction protein 1 [TJP1] and E-cadherin) and mesenchymal markers (smooth muscle actin alpha [a-SMA] and fibronectin) were measured using Western blot analysis and immunofluorescence assays. Additionally, changes in the Notch-1/Jagged-1 signaling pathway were analyzed using Western blotting.
The results showed that TGF-b1 treatment significantly reduced the expression of epithelial markers (TJP1 and E-cadherin) and increased the expression of mesenchymal markers (a-SMA and fibronectin) in HK-2 cells. However, treatment with miR-34a-enriched MSC-MVs partially restored the expression of epithelial markers and reduced the expression of mesenchymal markers. The effects of miR-34a-enriched MSC-MVs on EMT markers were more pronounced than those of control MSC-MVs. Furthermore, TGF-b1-induced upregulation of Notch-1 and Jagged-1 was significantly inhibited by miR-34a-enriched MSC-MVs, suggesting that miR-34a targets the Notch-1/Jagged-1 signaling pathway.
In addition to EMT, TGF-b1 also induced apoptosis in HK-2 cells. The effects of miR-34a-enriched MSC-MVs on cell viability and apoptosis were assessed using MTT and Annexin V/PI assays. While both miR-34a-enriched and control MSC-MVs were able to enhance cell survival and reduce apoptosis, the protective effects of miR-34a-enriched MSC-MVs were less significant than those of control MSC-MVs. This phenomenon may be attributed to the pro-apoptotic effects of miR-34a, which targets pro-survival molecules such as Bcl-2.
The study also explored the role of Jagged-1 in the anti-fibrotic effects of miR-34a-enriched MSC-MVs. Overexpression of Jagged-1 in HK-2 cells using a Jagged-1 encoding vector weakened the anti-fibrotic effects of miR-34a-enriched MSC-MVs, further supporting the notion that miR-34a exerts its effects through the inhibition of the Jagged-1/Notch-1 signaling pathway.
In conclusion, this study demonstrates that miR-34a-enriched MSC-MVs inhibit TGF-b1-induced EMT in renal tubular epithelial cells, likely through the suppression of the Jagged-1/Notch-1 signaling pathway. The findings suggest that genetic modification of MSC-MVs with anti-fibrotic molecules such as miR-34a represents a novel strategy for the treatment of renal fibrosis. However, the pro-apoptotic effects of miR-34a may limit its therapeutic potential, highlighting the need for further research to optimize the use of miR-34a-enriched MSC-MVs in kidney disease.
doi.org/10.1097/CM9.0000000000000720
Was this helpful?
0 / 0