miR-1915 and miR-1225-5p regulate the expression of CD133, PAX2 and TLR2 in adult renal progenitor cells

Abstract

Adult renal progenitor cells (ARPCs) were recently identified in the cortex of the renal parenchyma and it was demonstrated that they were positive for PAX2, CD133, CD24 and exhibited multipotent differentiation ability. Recent studies on stem cells indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Distinct sets of miRNAs are specifically expressed in pluripotent stem cells but not in adult tissues, suggesting a role for miRNAs in stem cell self-renewal. We compared miRNA expression profiles of ARPCs with that of mesenchymal stem cells (MSCs) and renal proximal tubular cells (RPTECs) finding distinct sets of miRNAs that were specifically expressed in ARPCs. In particular, miR-1915 and miR-1225-5p regulated the expression of important markers of renal progenitors, such as CD133 and PAX2, and important genes involved in the repair mechanisms of ARPCs, such as TLR2. We demonstrated that the expression of both the renal stem cell markers CD133 and PAX2 depends on lower miR-1915 levels and that the increase of miR-1915 levels improved capacity of ARPCs to differentiate into adipocyte-like and epithelial-like cells. Finally, we found that the low levels of miR-1225-5p were responsible for high TLR2 expression in ARPCs. Therefore, together, miR-1915 and miR-1225-5p seem to regulate important traits of renal progenitors: the stemness and the repair capacity.

Figure 1. Characterization of isolated glomerular and tubular ARPCs.

Cytofluorimetric and immunofluorescence analysis of tARPCs showed the expression of: CD133 (A), CD24 (B), CD44 (C), Oct-4 (D), PAX2 (E), BMI-1 (F), Cytofluorimetric and immunofluorescence analysis of gARPCs showed the expression of: CD133 (G), CD24 (H), CD44 (I), Oct-4 (J), PAX2 (K), BMI-1 (L). CD106 expression in glomerular (M) and tubular (N) ARPCs. Original view: X63.

Figure 2. Unsupervised hierarchical clustering and principal component analysis (PCA) of miRNA expression profile.

miRNA expression patterns of 5 tARPC, 5 gARPC and 3 MSC different clones and 3 different RPTEC lines were examined using Agilent array composed of 1205 human miRNAs. A total of 327 miRNA resulted expressed between different cell types (false discovery rate <0.01). The 2-D hierarchical clustering (A) and the PCA (B) showed that miRNA expression profile was different among MSCs, RPTECs and ARPCs, whereas it was very similar between gARPCs and tARPCs.

Figure 3. Principal genes predicted to be targeted by miR-1225-5p and miR-1915.

Figure 4. Validation of miRNA expression levels by qRT-PCR.

The amount of miR-1225-5p, miR-1915, miR-371-5p and miR-196a in an independent set of 5 tARPC, 5 gARPC clones and 3 RPTEC lines was evaluated by real-time PCR (q-RT-PCR). The miRNA relative expressions were normalized to the expression of U6. Expression levels of miR-1225-5p, miR-1915, miR-371-5p and miR-196a were found significantly lower in tARPCs and gARPCs compared to RPTECs. The histograms represent the mean ± SD. *p<0.03; **p<0.01.

Figure 5. miR-1915 regulates CD133 and PAX2 in ARPCs.

(A–B) CD133 and PAX2 expression levels were analyzed by real-time PCR following transfection with miR-1915 mimic. Increasing the amount of miR-1915 within ARPCs resulted in a 1.5 fold reduction of both CD133 and PAX2 mRNA levels. Expression data were normalized on the housekeeping gene β-actin. Data are representative of four independent experiments (means ± SEM), *p<0.01; **p<0.0003. (C) Surface marker expression of CD133, as measured by flow cytometry, resulted in a large reduction (8% vs 96% of mock transfection control) following transfection with 50 nM miR-1915 mimic. Red area represents the transfected condition. Data are representative of three independent experiments (D) Transfection of ARPCs with 100 nM miR-1915 mimic resulted in a 2 fold reduction of PAX2 protein expression, as shown by Western blot. β-actin was used as endogenous control. Data are representative of three independent experiments (means ± SEM). *p<0.02. Mock indicates mock-transfected cells going through the transfection processes without addition of mimic miRNA.

Figure 6. miR-1915 upregulation favors ARPC differentiation.

(A–D) Effect of miR-1915 on adipocyte differentiation of ARPCs. Cells were cultured for 20 days in adipogenic medium with or without miR-1915 mimic (50 nM). Oil red O staining was used to detect mature adipocytes. The increase of miR-1915 levels resulted in an higher number of adipocyte-like cells (B, D, E). (A, C) Representative micrographs showing ARPCs cultured in maintenance medium transfected and not-transfected with miR-1915 mimic. (E) The number of adipocyte was counted and reported as a percentage of total ARPCs. Values are expressed as means ± SEM. *p<0.01. (F–J) ARPC differentiation in epithelial-like cells with and without miR-1915 (50 nM) and stained positively for CK-19. The increase of miR-1915 levels resulted in an higher CK-19 expression (G, I, J). (F, H) Representative micrographs showing ARPCs cultured in maintenance medium transfected and not-transfected with miR-1915 mimic. (K–O) ARPC differentiation in epithelial-like cells with and without miR-1915 (50 nM) and stained positively for ZO-1. (K, M) Representative micrographs showing ARPCs cultured in maintenance medium transfected and not-transfected with miR-1915 mimic. To-pro-3 counterstains nuclei (blue). Original view X63. *p<0.01 vs. not differentiated cells. Mock indicates mock-transfected cells going through the transfection processes without addition of mimic miRNA.

Figure 7. miR-1225-5p regulates TLR2 in ARPCs.

(A) TLR2 expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. (B–C) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.