MicroRNA-124-3p affects myogenic differentiation of adipose-derived stem cells by targeting Caveolin-1 during pelvic floor dysfunction in Sprague Dawley rats.

ABSTRACT

BACKGROUND: The aim of this study was to investigate using myogenic differentiation of adipose stem cells for the treatment of female pelvic floor dysfunction (PFD) and aimed to further study the influences of microRNA-124-3p (miR-124-3p) in the process of myogenic differentiation of adipose-derived stem cells (ADSCs) through targeting Caveolin-1 (Cav1) during PFD in Sprague Dawley (SD) rats. METHODS: The ADSCs were separated from 6-8-week-old female SD rats (n=25) and were cultivated. Then, we observed the cell status and conducted fat and osteogenic experiments. We then constructed an ADSC-green fluorescent protein (GFP) stable transfer strain. Flow cytometry was used to identify the positive rates of CD44, CD90, and CD45 in ADSCs and ADSC-GFP. Real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting were used to mRNA and protein expression levels. Myogenic differentiation of ADSCs was measured with immunofluorescence methods. A dual-luciferase reporter assay was executed to affirm whether Cav1 was a target of miR-124-3p. RESULTS: The isolated ADSCs cells were in good condition under the microscope. The results of flow cytometry showed that the positive rate of CD44 and CD90 was high, and the positive rate of CD45 was low in ADSCs and ADSC-GFP. Under normal culture conditions, ADSCs-GFP cells can be massively adipated and osteogenic. After 5-Aza induced ADSC-GFP myogenic differentiation, the level of miR-124-3p was significantly increased. We found that MiR-124-3p mimics promoted the myogenic differentiation of ADSCs. Moreover, we discovered that Cav1 was a target gene of miR-124-3p and was negatively regulated by miR-124-3p. The results of leak point pressure (LPP), hematoxylin and eosin (HE), and Masson showed that the collagen fiber content of the PFD group was lower than that of the control group; the collagen fiber content of ADSC-GFP, 5-Aza, or miR-124-3p mimics were increased after intervention. Furthermore, the outcomes qRT-PCR, western blotting, and immunofluorescence suggested that miR-124-3p facilitated the survival ADSC-GFP fat transplantation by regulating many key factors in vivo. CONCLUSIONS: These results proofed that miR-124-3p could accelerate myogenic differentiation of ADSCs by down-regulating Cav1 to improve PFD in SD rats, which will pave the way for therapeutic delivery of miRNA targeting PFD disease.