Research Paper Volume 13, Issue 2 pp 3045—3059

Transplantation of bone marrow-derived mesenchymal stem cells with silencing of microRNA-138 relieves pelvic organ prolapse through the FBLN5/IL-1β/elastin pathway

The target relationship between FBLN5 and miR-138 is verified. (A) sequence alignment of miR-138 with potential targeting site in the 3’-UTR of FBLN5 mRNA; (B) Wild-type (Luc-FBLN5-WT) or mutated (Luc-FBLN5-mut) targeting sequences from FBLN5 mRNA 3’-UTR were fused at the 3’ of the luciferase reporter open reading frame (Luc ORF); (C) Luciferase activities of Luc-FBLN5-WT and Luc-FBLN5-mut constructs were measured in BMSCs transfected with mimic NC or miR-138 mimic; (D) miR-138 expression and the mRNA expression of FBLN5 examined in BMSCs transfected with mimic NC or miR-138 mimic by RT-qPCR; (E) the protein expression of FBLN5 examined in BMSCs transfected with mimic NC or miR-138 mimic by western blot analysis; (F) miR-138 expression and the mRNA expression of FBLN5 examined in BMSCs transfected with inhibitor NC or miR-138 inhibitor by RT-qPCR; (G) the protein expression of FBLN5 examined in BMSCs transfected with inhibitor NC or miR-138 inhibitor by western blot analysis; (H) the OD value of BMSCs at the 0-7 d assessed by CCK-8. *p vs. BMSCs treated with mimic NC; #p vs. BMSCs treated with inhibitor NC; statistical data were measurement data, and described as mean ± standard deviation. The unpaired t test was conducted for comparison between two groups. The repeated measures ANOVA was applied for the comparison of data at different time points, followed by Bonferroni’s correction. The experiment was repeated 3 times independently.

Figure 2. The target relationship between FBLN5 and miR-138 is verified. (A) sequence alignment of miR-138 with potential targeting site in the 3’-UTR of FBLN5 mRNA; (B) Wild-type (Luc-FBLN5-WT) or mutated (Luc-FBLN5-mut) targeting sequences from FBLN5 mRNA 3’-UTR were fused at the 3’ of the luciferase reporter open reading frame (Luc ORF); (C) Luciferase activities of Luc-FBLN5-WT and Luc-FBLN5-mut constructs were measured in BMSCs transfected with mimic NC or miR-138 mimic; (D) miR-138 expression and the mRNA expression of FBLN5 examined in BMSCs transfected with mimic NC or miR-138 mimic by RT-qPCR; (E) the protein expression of FBLN5 examined in BMSCs transfected with mimic NC or miR-138 mimic by western blot analysis; (F) miR-138 expression and the mRNA expression of FBLN5 examined in BMSCs transfected with inhibitor NC or miR-138 inhibitor by RT-qPCR; (G) the protein expression of FBLN5 examined in BMSCs transfected with inhibitor NC or miR-138 inhibitor by western blot analysis; (H) the OD value of BMSCs at the 0-7 d assessed by CCK-8. *p < 0.05 vs. BMSCs treated with mimic NC; #p < 0.05 vs. BMSCs treated with inhibitor NC; statistical data were measurement data, and described as mean ± standard deviation. The unpaired t test was conducted for comparison between two groups. The repeated measures ANOVA was applied for the comparison of data at different time points, followed by Bonferroni’s correction. The experiment was repeated 3 times independently.