Int J Stem Cells.  2021 May;14(2):229-239. 10.15283/ijsc20136.

Improved Differentiation Ability and Therapeutic Effect of miR-23a-3p Expressing Bone Marrow-Derived Mesenchymal Stem Cells in Mice Model with Acute Lung Injury

• Zhang P
• Liu L
• Yao L
• Song X

• KMID: 2515999
• DOI: http://doi.org/10.15283/ijsc20136

Abstract

Background and Objectives
Implantation of bone marrow-derived mesenchymal stem cells (BMSCs) has been recognized as an effective therapy for attenuating acute lung injury (ALI). This study aims to discover microRNA (miRNA)-mediated improvement of BMSCs-based therapeutic effects.
Methods and Results
Mice were treated with lipopolysaccharide (LPS) for induction of ALI. BMSCs with lentivirus-mediated expression of miR-23b-3p or fibroblast growth factor 2 (FGF2) were intratracheally injected into the mice with ALI. The expressions of miR-23b-3p, FGF2, Occludin, and surfactant protein C (SPC) in lung tissues were analyzed by immunoblot or quantitative reverse transcription polymerase chain reaction. Histopathological changes in lung tissues were observed via hematoxylin-eosin staining. Lung edema was assessed by the ratio of lung wet weight/body weight (LWW/BW). The levels of interleukin (IL)-1β, IL-6, IL-4, and IL-8 in bronchoalveolar lavage fluid (BALF) were assessed by ELISA. LPS injection downregulated the expressions of miR-23b-3p, SPC and Occludin in the lung tissues, increased the LWW/BW ratio and aggravated histopathological abnormalities, while upregulating IL-1β, IL-6, IL-4, and IL-8 in the BALF. Upregulated miR-23b-3p counteracted LPS-induced effects, whereas downregulated miR-23b-3p intensified LPS-induced effects. FGF2, which was downregulated by miR-23b-3p upregulation, was a target gene of miR-23b-3p. Overexpressing FGF2 downregulated the expressions of miR-23b-3p, SPC and Occludin, increased the LWW/BW ratio and aggravated histopathological abnormalities, while upregulating IL-1β, IL-6, IL-4, and IL-8, and it offset miR-23b-3p upregulation-caused effects on the ALI mice.
Conclusions
Overexpression of miR-23b-3p in BMSCs strengthened BMSC-mediated protection against LPS-induced mouse acute lung injury via targeting FGF2.

Keyword

miR-23b-3p; Acute lung injury; Fibroblast growth factor 2; Lung injury repair; Inflammatory responses

Figure

• 

  Fig. 1 MiR-23b-3p-overexpressing BMSCs promoted the differentiation of BMSCs into TypeII pneumocytes and alleviated injuries in ALI lung tissues. (A, B, D, E, H) The expressions of miR-23b-3p, SPC, Oct4, Nanog and Occludin in the lung tissues of LPS-induced ALI mice were analyzed by qRT-PCR, after injection of BMSCs with no altered gene expression or BMSCs with miR-27a-3p overexpression or underexpression. (C, F, I) The expressions of SPC, Oct4, Nanog and Occludin in the lung tissues of LPS-induced ALI mice were analyzed by immunoblot, with GAPDH serving as a reference gene, after injection of BMSCs with no altered gene expression or BMSCs with miR-27a-3p overexpression or underexpression. (G) The ratio of LWW to BW of LPS-induced ALI mice was calculated, after injection of BMSCs with no altered gene expression or BMSCs with miR-27a-3p overexpression or underexpression. (J) Histopathological changes in the lung tissues of LPS-induced ALI mice were observed via hematoxylin-eosin staining, after injection of BMSCs with no altered gene expression or BMSCs with miR-27a-3p overexpression or underexpression (scale: 100 μm; magnification: ×100). (K∼P) The levels of IL-1β, IL-6, IL-4, IL-8, TNF-α and IL-10 in the bronchoalveolar lavage fluid of LPS-induced ALI mice were assessed by ELISA, after injection of BMSCs with no altered gene expression or BMSCs with miR-27a-3p overexpression or underexpression. #p<0.05; ##p<0.01; ***p or ###p<0.001; * vs. Control ; # vs. LPS＋NC (LPS: lipopolysaccharide, SPC: surfactant protein C, NC: negative control, M: miR-23b-3p mimic, I: miR-23b-3p inhibitor, qRT-PCR: Quantitative reverse transcription polymerase chain reaction, ELISA: Enzyme-linked immunosorbent assay, ALI: acute lung injury, LWW/BW: lung wet weight/body weight, BMSCs: bone marrow-derived mesenchymal stem cells).

• 

  Fig. 2 MiR-23b-3p directly targeted FGF2. (A) The putative binding sites of miR-23b-3p on FGF2 were predicted by TargetScan V7.2. (B) The interaction between miR-23b-3p and FGF2 was validated by dual-luciferase reporter assay. ***p<0.001; * vs. NC (NC: negative control, M: miR-23b-3p mimic, WT: wild type, MUT: mutant type, FGF2: fibroblast growth factor 2).

• 

  Fig. 3 FGF2 expression was inhibited by miR-23b-3p overexpressing BMSCs and it inhibited the differentiation of BMSCs into TypeII pneumocytes. (A, C) The expressions of FGF2 and SPC in the lung tissues of LPS-induced ALI mice were analyzed by qRT-PCR, after injection of BMSCs with no altered gene expression, BMSCs with overexpression of miR-27a-3p or FGF2 alone, or BMSCs with co-overexpression of miR-27a-3p and FGF2. (B, D) The expressions of FGF2 and SPC in the lung tissues of LPS-induced ALI mice were analyzed by immunoblot, with GAPDH serving as a reference gene, after injection of BMSCs with no altered gene expression, BMSCs with overexpression of miR-27a-3p or FGF2 alone, or BMSCs with co-overexpression of miR-27a-3p and FGF2. #p<0.05; **p or ##p<0.01; ***p or ###p or ^^^p<0.001; * vs. NC＋FGF2-NC ; # vs. M＋FGF2-NC; ^ vs. NC＋FGF2 (LPS: lipopolysaccharide, SPC: surfactant protein C, FGF2: fibroblast growth factor 2, NC: negative control, M: miR-23b-3p mimic, qRT-PCR: Quantitative reverse transcription polymerase chain reaction, BMSCs: bone marrow-derived mesenchymal stem cells).

• 

  Fig. 4 FGF2 reversed the effect of the implantation of BMSCs overexpressing miR-23b-3p on ALI lung tissues. (A) The ratio of LWW to BW of LPS-induced ALI mice was calculated, after injection of BMSCs with no altered gene expression, BMSCs with overexpression of miR-27a-3p or FGF2 alone, or BMSCs with co-overexpression of miR-27a-3p and FGF2. (B) The expression of Occludin in the lung tissues was analyzed by qRT-PCR, after injection of BMSCs with no altered gene expression, BMSCs with overexpression of miR-27a-3p or FGF2 alone, or BMSCs with co-overexpression of miR-27a-3p and FGF2. (C) The expression of Occludin in the lung tissues of LPS-induced ALI mice was analyzed by immunoblot, with GAPDH serving as a reference gene, after injection of BMSCs with no altered gene expression, BMSCs with overexpression of miR-27a-3p or FGF2 alone, or BMSCs with co-overexpression of miR-27a-3p and FGF2. (D) Histopathological changes in the lung tissues of LPS-induced ALI mice were observed via hematoxylin-eosin staining, after injection of BMSCs with no altered gene expression, BMSCs with overexpression of miR-27a-3p or FGF2 alone, or BMSCs with co-overexpression of miR-27a-3p and FGF2 (scale: 100 μm; magnification: ×100). (E∼J) The levels of IL-1β, IL-6, IL-4, IL-8, TNF-α and IL-10 in the bronchoalveolar lavage fluid of LPS-induced ALI mice were assessed by ELISA, after injection of BMSCs with no altered gene expression, BMSCs with overexpression of miR-27a-3p or FGF2 alone, or BMSCs with co-overexpression of miR-27a-3p and FGF2. *p or #p<0.05; **p or ##p or ^^p<0.01; ***p or ###p or ^^^p<0.001; * vs. NC＋FGF2-NC ; # vs. mimic＋FGF2-NC; ^ vs. NC＋FGF2 (LPS: lipopolysaccharide, FGF2: fibroblast growth factor 2, NC: negative control, M: miR-23b-3p mimic, qRT-PCR: Quantitative reverse transcription polymerase chain reaction, ELISA: Enzyme-linked immunosorbent assay, ALI: acute lung injury, LWW/BW: lung wet weight/body weight, BMSCs: bone marrow-derived mesenchymal stem cells).

Reference

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