Speakers - 2026

Title: Therapeutic mechanism of hucMSCs via circANKRD42-miR-136-5p-YAP1 in pulmonary fibrosis

Abstract

Objectives:
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic interstitial pneumonia of unknown etiology that is confined to the lungs and occurs in the elderly population. The median survival time is only 3-5 years. Pirfenidone and nintedanib which are the currently used anti-pulmonary fibrosis drugs are only effective to patients in the early and middle stage of the disease. Although pirfenidone and nintedanib can improve clinical symptoms, it can not cure or reverse fibrosis and prolong survival. Therefore, the unclear pathogenesis and poor drug efficacy are the major challenge of IPF. Our study will investigate the anti-pulmonary fibrosis potential of human umbilical cord mesenchymal stem cells (hucMSCs), and explore the non-coding RNA-mediated disease mechanisms and provide detailed theoretical basis and experimental data to prove that mesenchymal stem cells (MSCs) can be used as a potential new clinical treatment for IPF.

Methods:
1.Seven discarded donor umbilical cords were collected and MSCs were isolated by tissue block apposition method, and hucMSCs were identified by flow cytometry, adipogenic and osteogenic differentiation experiments.
2.HucMSCs were labeled with fluorescent probes and injected into C57BL/6 mice via tail vein. Frozen sections confirmed that hucMSCs were targeted to the lungs of mice.
3.Forty C57BL/6 mices were randomly divided into normal group, model group, control group and treatment group. The in vivo model group was established by nebulization of 5 mg/kg BLM through mouse lung drug delivery device, and 1.0×105 hucMSCs were injected into the mice through tail vein the third day for treatment. MRC-5 cells were stimulated with 5 nM serum-free TGF-β1 for 72 h to establish an in vitro model, and 1 ml hucMSCs were added into the cell-embedded dishes for co-culture. MicroCT imaging system, H&E and Masson staining, lung function analysis, cell proliferation assay, scratch assay and Western blot were applied to observe the anti-pulmonary fibrosis effect of hucMSCs in vivo and the inhibition of fibrosis-related functions in vitro. Phalloidin staining, atomic force microscopy (AFM) and Western blot were applied to clarify the effect of hucMSCs on cytoskeleton and mechanical stiffness.
4.QRT-PCR was used to assess the expression changes of circANKRD42 by hucMSCs treatment. Immunofluorescence and Western blot were used to determine the intranuclear expression changes of hnRNP L before and after hucMSCs treatment. Rescue assays and RNA binding protein immunoprecipitation (RIP) were used to clarify the binding ability of hnRNP L to pre-mRNA (ANKRD42) before and after hucMSCs treatment.
5.The circANKRD42 overexpression plasmid was constructed to transfect MRC-5 cells and randomly divided into model group, treatment group, control group and circANKRD42 overexpression group. circANKRD42 overexpression or mutation adenovirus was constructed to transfect mice and fifty C57BL/6 mice were randomly divided into model group, treatment group, blank plasmid group, overexpression group and mutation group. MicroCT imaging system, H&E and Masson staining, lung function analysis, cell proliferation assay, scratch assay, phalloidin staining, AFM, Western blot and RNA Antisense Purification (RAP) assay were performed to explore the circANKRD42-mediated molecular mechanism of anti-pulmonary fibrosis in hucMSCs.

Results:
1.Flow cytometry detected positive expression of surface antigens CD44, CD73 and CD90 in the extracted MSCs, with expression rates of 99.7%, 100% and 99.9%, respectively, while negative expression of CD34, CD45 and HLA-DR, with expression rates of 0.6%, 1.9% and 0.8%, respectively. In vitro induction experiments showed that MSCs could differentiate towards osteoblasts and adipocytes. They were identified as hucMSCs according to the standards of the International Society of Cell Therapy.
2.Results of animal experiments in vivo. The results of animal experiments showed that the tail vein injection of hucMSCs treatment group significantly reduced the degree of both lung fibrosis on chest CT imaging compared to the BLM group. Histopathologically, the alveolar structure of mice in the BLM model group was destroyed, filled with fibrous tissue and had collagen deposition, while the alveolar structure was significantly improved and collagen deposition was significantly reduced after BLM+hucMSCs treatment. In terms of lung function, the hucMSCs treatment group significantly improved FVC of fibrotic mice. Body weight monitoring results showed that hucMSCs treatment mices significantly gained weight. HucMSCs also suppressed the expression of fibrosis-related protein α-SMA, vimentin, collagen I, collagen III and transdifferentiation-related proteins S100A4 and FAP1. In addition, hucMSCs treatment also attenuated Young's modulus of lung tissue.
3.Results of cell experiments in vitro. The results of IncuCyte S3 real-time dynamic cell imaging analysis system showed that cell proliferation was significantly enhanced in the TGF-β1 group compared with the normal group, while cell proliferation was inhibited in the TGF-β1 and hucMSCs co-culture group. The results of cell scratch assay showed that the migration ability of MRC-5 cells treated with TGF-β1 was significantly increased, while the migration ability of cells in the TGF-β1 and hucMSCs co-culture group was diminished. Western blot results showed that the protein expression of collagen I, collagen III, vimentin, α-SMA, FAP1 and S100A4 were significantly increased in the TGF-β1 group compared with the normal group, while the expression of all the above proteins was decreased in the TGF-β1+hucMSCs co-culture group. In addition, AFM results showed that Young's modulus of cells in the TGF-β1 group increased, and hucMSCs treatment decreased the Young's modulus of cells. Phalloidin staining results showed that cytoskeletal protein expression was significantly increased in the TGF-β1 group compared with the normal group, while cytoskeletal protein expression was significantly decreased after treatment with hucMSCs.
4.Mechanism research. Firstly, hucMSCs affects the splicing generation of circANKRD42 by reducing the intranuclear expression of hnRNP L. RNA pull-down results indicated that hnRNP L was a binding protein of circANKRD42. Dual luciferase reporter analysis assay showed that si-hnRNP L reduced the fluorescence intensity of circANKRD42. Immunofluorescence experiments showed that hnRNP L was mainly located in the nucleus after TGF-β1 treatment, and hucMSCs treatment inhibited the expression of hnRNP L in the nucleus. The results of RIP experiment showed that hnRNP L was significantly combined with pre-mRNA (ANKRD42) in TGF-β1 group, and the binding amount of hnRNP L to pre-mRNA (ANKRD42) was reduced after hucMSCs treatment.
5.Secondly, the qRT-PCR results in vivo assay showed that the expression of circANKRD42 was increased in the lung tissue of BLM group mice compared with the sham group, and decreased after hucMSCs treatment. The microCT results of small animals showed that the degree of pulmonary fibrosis and consolidation of mice was aggravated in the overexpressed circANKRD42 group compared with the hucMSCs treatment group. H&E and Masson staining showed that the degree of alveolar structural destruction, fibroblast filling and collagen deposition of mice were aggravated in the circANKRD42 overexpression group compared with the treatment group. Meanwhile, overexpression of circANKRD42 reversed the effect of hucMSCs on lung function and body weight in mice with fibrosis. Western blot experiments showed that overexpression of circANKRD42 increased the protein expression of Collagen I, Collagen III, Vimentin, α-SMA, FAP1 and S100A4. In addition, the Young's modulus in the
circANKRD42 overexpression group was increased compared with the hucMSCs treatment group.
6.Thirdly, the proliferation assay in vitro showed that cell proliferation was enhanced after overexpression of circANKRD42. The results of cell scratching assay showed that overexpression of circANKRD42 promoted cell migration. Western blot assay showed that overexpression of circANKRD42 significantly increased Collagen I, Collagen III, vimentin, α-SMA, FAP1 and S100A4 protein expression. In addition, overexpression of circANKRD42 also increased the Young's modulus of cells.
7.Finally, we explored the downstream mechanisms of circANKRD42 with the presence of hucMSCs. Dual luciferase reporter gene assay confirmed the binding of miR-136-5p to circANKRD42. QRT-PCR results showed that the expression of miR-136-5p was significantly increased in the hucMSCs treatment group compared with the TGF-β1/BLM model group. RAP analysis showed that circANKRD42 could adsorb miR-136-5p in the normal group and TGF-β1 treatment further enhanced its adsorption, while this effect was diminished after hucMSCs treatment. TargetScan, miRbase, miRDB database cross-tabulation and dual luciferase reporter gene assay results confirmed that YAP1 was the target gene of miR-136-5p. Western blot results showed that hucMSCs decreased the expression of YAP1 and the expression of Myo1c and F-actin, while the expression of YAP1, Myo1c and F-actin increased after overexpression of circANKRD42.

Conclusion:
1.The results of animal experiments in vivo showed that hucMSCs could significantly improve BLM-induced pulmonary fibrosis of mice through chest imaging, histopathology, pulmonary function, mechanical stiffness and fibrosis-related characteristic proteins.
2.The results of cell experiments in vitro showed that the co-culture of hucMSCs with TGF-β1-stimulated MRC-5 cells attenuated the effects of TGF-β1 on cell proliferation migration, transdifferentiation, mechanical stiffness, cytoskeleton protein, and exerted the effect of inhibition of fibrosis-related functions.
3.Overexpression of circANKRD42 can reverse the therapeutic effect of hucMSCs on pulmonary fibrosis in vivo and in vitro experiments.
4.Mechanistic studies showed that hucMSCs achieve interaction between biochemical signal and mechanical stiffness signal through the circANKRD42-miR-136-5p-YAP1 signaling axis, which inhibit the expression of YAP1 and downstream cytoskeletal proteins and mechanics-related genes, so as to attenuate the occurrence and development of pulmonary fibrosis.