Dataset Information

Bone marrow mesenchymal stem cells derived miRNA-130b enhances epithelial sodium channel by targeting PTEN.

ABSTRACT:

Aims

Acute lung injury (ALI) is a clinical syndrome with high morbidity and mortality, and severe pulmonary edema is one of the characteristics. Epithelial sodium channel (ENaC) located on the apical side of alveolar type 2 epithelial (AT2) cells is the primary rate limiting segment in alveolar fluid clearance. Many preclinical studies have revealed that mesenchymal stem cells (MSCs) based therapy has great therapeutic potential for ALI, while the role of ENaC in this process is rarely known.

Methods

We studied the effects of bone marrow-derived MSCs (BMSCs) on the protein/mRNA expression and activity of ENaC in primary mouse AT2 and human H441 cells by co-culture with them, respectively. Moreover, the changes of miRNA-130b in AT2 cells were detected by qRT-PCR, and we studied the involvement of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and the downstream PI3K/AKT pathway in the miRNA-130b regulation of ENaC.

Results

Our results demonstrated that BMSCs could increase ENaC protein expression and function, as well as the expression level of miRNA-130b. The dual luciferase target gene assay verified that PTEN was one of the target genes of miR-130b, which showed adverse effects on the protein expression of ?/?-ENaC and PTEN in AT2 cells. Upregulating miR-130b and/or knocking down PTEN resulted in the increase of ?/?-ENaC protein level, and the protein expression of p-AKT/AKT was enhanced by miR-130b. Both ? and ?-ENaC protein expressions were increased after AT2 cells were transfected with siPTEN, which could be reversed by the co-administration of PI3K/AKT inhibitor LY294002.

Conclusion

In summary, miRNA-130b in BMSCs can enhance ENaC at least partially by targeting PTEN and activating PI3K/AKT pathway, which may provide a promising new direction for therapeutic strategy in ALI.

SUBMITTER: Zhang H

PROVIDER: S-EPMC7731743 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Publications

Bone marrow mesenchymal stem cells derived miRNA-130b enhances epithelial sodium channel by targeting PTEN.

Zhang Honglei H Ding Yan Y Hou Yapeng Y Liu Yanhong Y Zhou Zhiyu Z Nie Hongguang H

Respiratory research 20201211 1

<h4>Aims</h4>Acute lung injury (ALI) is a clinical syndrome with high morbidity and mortality, and severe pulmonary edema is one of the characteristics. Epithelial sodium channel (ENaC) located on the apical side of alveolar type 2 epithelial (AT2) cells is the primary rate limiting segment in alveolar fluid clearance. Many preclinical studies have revealed that mesenchymal stem cells (MSCs) based therapy has great therapeutic potential for ALI, while the role of ENaC in this process is rarely k ...[more]

PMID: 33308227

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Project description:BackgroundOne of the characteristics of acute lung injury (ALI) is severe pulmonary edema, which is closely related to alveolar fluid clearance (AFC). Mesenchymal stem cells (MSCs) secrete a wide range of cytokines, growth factors, and microRNA (miRNAs) through paracrine action to participate in the mechanism of pulmonary inflammatory response, which increase the clearance of edema fluid and promote the repair process of ALI. The epithelial sodium channel (ENaC) is the rate-limiting step in the sodium-water transport and edema clearance in the alveolar cavity; the role of bone marrow-derived MSC-conditioned medium (BMSC-CM) in edema clearance and how miRNAs affect ENaC are still seldom known.MethodsCCK-8 cell proliferation assay was used to detect the effect of BMSC-CM on the survival of alveolar type 2 epithelial (AT2) cells. Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect the expression of ENaC in AT2 cells. The effects of miR-34c on lung fluid absorption were observed in LPS-treated mice in vivo, and the transepithelial short-circuit currents in the monolayer of H441 cells were examined by the Ussing chamber setup. Dual luciferase reporter gene assay was used to detect the target gene of miR-34c.ResultsBMSC-CM could increase the viability of mouse AT2 cells. RT-PCR and western blot results showed that BMSC-CM significantly increased the expression of the γ-ENaC subunit in mouse AT2 cells. MiR-34c could restore the AFC and lung wet/dry weight ratio in the ALI animal model, and Ussing chamber assay revealed that miR-34c enhanced the amiloride-sensitive currents associated with ENaC activity in intact H441 cell monolayers. In addition, we observed a higher expression of miR-34c in mouse AT2 cells administrated with BMSC-CM, and the overexpression or inhibition of miR-34c could regulate the expression of ENaC protein and alter the function of ENaC. Finally, we detected that myristoylated alanine-rich C kinase substrate (MARCKS) may be one of the target genes of miR-34c.ConclusionOur results indicate that BMSC-CM may alleviate LPS-induced ALI through miR-34c targeting MARCKS and regulate ENaC indirectly, which further explores the benefit of paracrine effects of bone marrow-derived MSCs on edematous ALI.

Project description:BackgroundLong non-coding RNA (lncRNA) plays crucial role in osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), involving in regulation of competing endogenous RNA (ceRNA) mechanisms and conduction of signaling pathways. However, its mechanisms are poorly understood. This study aimed to investigate lncRNAs, miRNAs and mRNAs expression profiles in rat BMMSCs (rBMMSCs) osteogenic differentiation, screen the potential key lncRNA-miRNA-mRNA networks, explore the putative functions and identify the key molecules, as the basis of studying potential mechanism of rBMMSCs osteogenic differentiation driven by lncRNA, providing molecular targets for the management of bone defect.MethodsHigh-throughput RNA sequencing (RNA-seq) was used to determine lncRNAs, miRNAs, and mRNAs expression profiles at 14-day rBMMSCs osteogenesis. The pivotal lncRNA-miRNA and miRNA-mRNA networks were predicted from sequencing data and bioinformatic analysis, and the results were exported by Cytoscape 3.9.0 software. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used for functional exploration. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to validate lncRNAs, miRNAs and mRNAs.ResultsrBMMSCs were identified, and the osteogenic and adipogenic differentiation ability were detected. A total of 8634 lncRNAs were detected by RNA-seq, and 1524 differential expressed lncRNAs, of which 812 up-regulated and 712 down-regulated in osteo-inductive groups compared with control groups. 30 up-regulated and 61 down-regulated miRNAs, 91 miRNAs were differentially expressed in total. 2453 differentially expressed mRNAs including 1272 up-expressed and 1181 down-expressed were detected. 10 up-regulated lncRNAs were chosen to predict 21 down-regulated miRNAs and 650 up-regulated mRNAs. 49 lncRNA-miRNA and 1515 miRNA-mRNA interactive networks were constructed. GO analysis showed the most important enrichment in cell component and molecular function were "cytoplasm" and "protein binding", respectively. Biological process related to osteogenic differentiation such as "cell proliferation", "wound healing", "cell migration", "osteoblast differentiation", "extracellular matrix organization" and "response to hypoxia" were enriched. KEGG analysis showed differentially expressed genes were mainly enriched in "PI3K-Akt signaling pathway", "Signaling pathway regulating pluripotency of stem cells", "cGMP-PKG signaling pathway", "Axon guidance" and "Calcium signaling pathway". qRT-PCR verified that lncRNA Tug1, lncRNA AABR07011996.1, rno-miR-93-5p, rno-miR-322-5p, Sgk1 and Fzd4 were consistent with the sequencing results, and 4 lncRNA-miRNA-mRNA networks based on validations were constructed, and enrichment pathways were closely related to "PI3K-Akt signaling pathway", "Signaling pathway regulating pluripotency of stem cells" and "Wnt signaling pathway".ConclusionslncRNAs, miRNAs and mRNAs expression profiles provide clues for future studies on their roles for BMMSCs osteogenic differentiation. Furthermore, lncRNA-miRNA-mRNA networks give more information on potential new mechanisms and targets for management on bone defect.

Dataset Information

Bone marrow mesenchymal stem cells derived miRNA-130b enhances epithelial sodium channel by targeting PTEN.

Aims

Methods

Results

Conclusion

Publications

Bone marrow mesenchymal stem cells derived miRNA-130b enhances epithelial sodium channel by targeting PTEN.

Similar Datasets

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