miR-33a hinders the differentiation of adipose mesenchymal stem cells towards urothelial cells in an inductive condition by targeting β‑catenin and TGFR

Abstract

Tissue engineering technology offers an appealing approach for tissue reconstruction of the urothelium. Adipose‑derived mesenchymal stem cells (ADSCs) represent an abundant source for tissue engineering applications. However, ASCs primarily possess mesoderm lineage differentiation potential. It is difficult to induce differentiation of ASCs towards urothelial cells that are derived from the endoderm, although a recent findings have reported that a conditioned medium may drive ADSCs towards differentiation into the urothelium phenotype. In the present study, human ADSCs were isolated from abdominal adipose tissues and incubated in this conditioned medium for indicated time periods. Western blotting showed that protein expression levels of urothelial specific marks, including CK7, CK20 and UPIII, were increased after seven days' incubation, but immunofluorescence microscopy determined that cells with CK7 and UPIII staining were scarce, which suggested a low‑efficiency for the differentiation. Prolonging the incubation time did not further increase CK20 and UPIII expression. Furthermore, miR‑33a expression was increased with ADSC differentiation. Using synthetic miRNAs to mimic or inhibit the action of miR‑33a revealed that miR‑33a hinders the differentiation of ADSCs towards urothelial cells. Furthermore, luciferase reporter assay confirmed that β‑catenin and transforming growth factor‑β receptor (TGFR) are targets of miR‑33a. Inhibition of miR‑33a expression increased β‑catenin and TGFR expression and improved the efficiency of ADSCs towards differentiation into the urothelium phenotype. The present novel finding suggests that miR‑33 may be an important target in tissue engineering and regenerative medicine for urothelium repair.

Figure 1.

Adipose-derived mesenchymal stem cells (ADSCs) identification. Human ADSCs were isolated from abdominal adipose tissues. Flow cytometry analysis detected the expression of surface markers, including CD29, CD90, and CD34 for ADSCs identification. Quad, quadrant; Tota, total.

Figure 2.

Urothelial differentiation of adipose-derived mesenchymal stem cells (ADSCs). For urothelial differentiation, ADSCs were incubated in the conditioned medium for 14 days. The un-induced ADSCs were used as the negative control (NC). (A) Cell viability was evaluated during the differentiation. (B) PCR assay was conducted to evaluate miR-33a and miR-33b expression. (C) Western blot assay was performed to detect levels of β-catenin, TGFR, CK7, CK20 and UPIII expression. *P<0.05 and **P<0.01 vs. control.

Figure 3.

IF assay. For urothelial differentiation, adipose-derived mesenchymal stem cells (ADSCs) were incubated in the conditioned medium for 14 days. The un-induced ADSCs were used as the negative control (NC). IF assay was performed to detect CK20 and UPIII expression. Green fluorescence stands for positive cells with CK20 and UPIII expression and blue fluorescence (DAPI) shows cell nuclei.

Figure 4.

β-catenin and TGFR are targets of miR-33a. Luciferase reporter assay was performed to determine that β-catenin and TGFR are targets of miR-33a. The predicted miR-33a-binding site sequence (miRNA response element, MRE) on the 3′ untranslated regions (3′UTR) of β-catenin and TGFR1 mRNA were subcloned into pmirGLO respectively to construct miR-33a/β-catenin and miR-33a/TGFR1 MRE luciferase reporters. These reporters were transfected into adipose-derived mesenchymal stem cells (ADSCs) alone or in combination with miR-33a mimics. The activity of firefly luciferase was normalized to that of renilla luciferase. *P<0.05 and **P<0.01 vs. control.

Figure 5.

miR-33a blocks urothelial differentiation of adipose-derived mesenchymal stem cells (ADSCs). For urothelial differentiation, ADSCs were incubated in the conditioned medium for 14 days. To determine the role of miR-33a in the differentiation, this study used synthetic miRNAs to mimic or inhibit the action of miR-33a. (A) Cell viability was evaluated during the differentiation. (B) PCR assay was conducted to evaluate miR-33a and miR-33b expression. (C) Western blot assay was performed to detect levels of β-catenin, TGFR, CK7, CK20 and UPIII expression. *P<0.05 and **P<0.01 vs. control. *P<0.05 and **P<0.01 vs. control, ^#P<0.05 and ^##P<0.01 vs. the ID group. Con, Control; ID, induction of urothelial differentiation; MI, miR-33a mimics; IN, miR-33a inhibitors.

Figure 6.

IF measurement. For urothelial differentiation, adipose-derived mesenchymal stem cells (ADSCs) were incubated in the conditioned medium for 14 days. To determine the role of miR-33a in the differentiation, this study used synthetic miRNAs to mimic or inhibit the action of miR-33a. IF assay was performed to detect CK20 and UPIII expression. Con, control; ID, induction of urothelial differentiation; MI, miR-33a mimics; IN, miR-33a inhibitors.