MicroRNA-145 Gene Modification Enhances the Retention of Bone Marrow-Derived Mesenchymal Stem Cells within Corpus Cavernosum by Targeting Krüppel-Like Factor 4

Abstract

Purpose: The poor retention and ambiguous differentiation of stem cells (SCs) within corpus cavernosum (CC) limit the cell application in erectile dysfunction (ED). Herein, the effects and mechanism of microRNA-145 (miR-145) gene modification on modulating the traits and fate of bone marrow-derived mesenchymal stem cells (BMSCs) were investigated.

Materials and methods: The effects of miR-145 on cell apoptosis, proliferation, migration, and differentiation were determined by flow cytometry, cell counting kit-8, transwell assays and myogenic induction. Then, the age-related ED rats were recruited to four groups including phosphate buffer saline, BMSC, vector-BMSC, overexpressed-miR-145-BMSC groups. After cell transplantation, the CC were harvested and prepared to demonstrate the retention and differentiation of BMSCs by immunofluorescent staining. Then, the target of miR-145 was verified by quantitative real-time polymerase chain reaction and immunohistochemical. After that, APTO-253, as an inducer of Krüppel-like factor 4 (KLF4), was introduced for rescue experiments in corpus cavernosum smooth muscle cells (CCSMCs) under the co-culture system.

Results: In vitro, miR-145 inhibited the migration and apoptosis of BMSCs and promoted the differentiation of BMSCs into smooth muscle-like cells with stronger contractility. In vivo, the amount of 5-ethynyl-2'-deoxyuridine (EdU)⁺cells within CC was significantly enhanced and maintained in the miR-145 gene modified BMSC group. The EdU/CD31 co-staning was detected, however, no co-staining of EdU/α-actin was observed. Furthermore, miR-145, which secreted from the gene modified BMSCs, dampened the expression of KLF4. However, the effects of miR-145 on CCSMCs could be rescued by APTO-253.

Conclusions: Overall, miR-145 modification prolongs the retention of the transplanted BMSCs within the CC, and this effect might be attributed to the modulation of the miR-145/KLF4 axis. Consequently, our findings offer a promising and innovative strategy to enhance the local stem cell-based treatments.

Keywords: Apoptosis; Cell differentiation; Erectile dysfunction; MicroRNAs; Stem cells.

Fig. 1. The construction of EdU⁺ BMSCs and gene modified BMSCs. (A) Fluorescence images of EdU (green) labeling of BMSCs. (B) The quantification of EdU⁺ cells. (C) The cell viability of the labeled BMSCs with EdU at different concentrations for 24 hours. (D) Fluorescence images of miR-145 gene modified BMSCs. (E) The qRT-PCR results of miR-145. The data is represented as the mean±standard deviation. EdU: 5-ethynyl-2′-deoxyuridine, BMSCs: bone marrow-derived mesenchymal stem cells, miR-145: microRNA-145, qRT-PCR: quantitative real-time polymerase chain reaction. ^*p<0.05 compared with the 20 µm/24 hours group and ^**p<0.01 compared with the control group, scale=100 µm, n=3 per group.

Fig. 2. The apoptosis and migration of BMSCs were inhibited by miR-145. (A) The apoptosis results illustrated by flow cytometry. (B) The apoptotic rates were calculated. (C) The proliferation of BMSCs of each group was assessed by using CCK-8 assays. (D) The migration of BMSCs of each group was evaluated by transwell assays (crystal violet staining, ×200). (E) The cell migration results were quantified. The data is represented as the means±standard deviation. BMSCs: bone marrow-derived mesenchymal stem cells, miR-145: microRNA-145, CCK-8: cell counting kit-8. ^**p<0.01 compared with the control group, scale=100 µm, n=3 per group.

Fig. 3. The differentiation of BMSCs into SMLCs enhanced by miR-145. (A) Fluorescence images of phalloidin staining (green represents F-actin). (B) Quantitative analysis of phalloidin staining. (C) The qRT-PCR results of α-actin and desmin. (D, E) The western blot results of α-actin and desmin. (F, G) The immunofluorescence results of α-actin. (H, I) The immunofluorescence results of desmin. (J) Cell contraction in each group under the stimulation of carbachol (×200). (K) Quantitative analysis of cell surface areas in each group. The data is represented as the means±standard deviation. BMSCs: bone marrow-derived mesenchymal stem cells, SMLCs: smooth muscle-like cells, DAPI: 4′,6-diamidino-2-phenylindole, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, qRT-PCR: quantitative real-time polymerase chain reaction. ^*p<0.05 and ^**p<0.01 compared with the control group, scale=100 µm, n=3.

Fig. 4. The retention and differentiation of BMSCs within CC after different treatments. (A) Representative images of EdU⁺ cells within CC after 7 days of different treatments. (B) Representative images of EdU⁺ cells within CC after 14 days of different treatments. (C) Quantitative analysis of the amount of EdU positive cells within CC after 7 and 14 days of different treatments. (D) Representative images of EdU/α-actin co-staining cells within CC after 7 days and 14 days of the different treatments. (E) Representative images of EdU/CD31 co-staining cells within CC after 7 days and 14 days of different treatments. (F) Quantitative analysis of EdU/CD31 co-staining cell amount within CC. The EdU (red)/DAPI (blue) fluorescence co-staining represents the EdU⁺ cells. The EdU (red)/CD31 (green)/DAPI (blue) fluorescence co-staining represents the positive cells. The data is represented as the means±standard deviation, n=3. EdU: 5-ethynyl-2′-deoxyuridine, BMSCs: bone marrow-derived mesenchymal stem cells, miR-145: microRNA-145, PBS: phosphate buffer saline, DAPI: 4′,6-diamidino-2-phenylindole, CD31: platelet endothelial cell adhesion molecule-1. ^*p<0.05 and ^**p<0.01 compared with the BMSCs group at the same time point. ^##p<0.01 compared with the same treatment group at the different time point, scale=100 µm.

Fig. 5. The target and function of OE-miR-145-BMSC. (A) The qRT-PCR results of KLF4 after the different treatments. (B, C) The IHC results of KLF4 (immunohistochemical staining, ×200). (D) The migration and apoptosis of OE-miR-145-BMSC of each group were evaluated by transwell and TUNEL assays (crystal violet staining, ×200). (E, F) The quantitative analysis of the results of cell migration and apoptosis. (G, I) The TUNEL staining results of CCSMCs in each group. (H, J) The IF staining of α-actin of CCSMCs in each group. The data is represented as the means±standard deviation. KLF4: Krüppel-like factor 4, miR-145: microRNA-145, BMSCs: bone marrow-derived mesenchymal stem cells, PBS: phosphate buffer saline, CCSMCs: corpus cavernosum smooth muscle cells, IF: immunofluorescence, DAPI: 4′,6-diamidino-2-phenylindole. ^*p<0.05 and ^**p<0.01 compared with the control group, scale=100 µm, n=3.

Fig. 6. Schematic diagram illustrates the mechanism by which miR-145 gene modification modulates the traits and fate of BMSCs. In vitro, miR-145 inhibited the apoptosis and migration of BMSCs and promoted the differentiation of BMSCs into SMLCs. In vivo, after the ICI of OE-miR-145-BMSC, the prolonged retention of BMSCs and endothelioid cells were detected. However, no evidence supported that miR-145 modulated the differentiation of BMSCs into SMLCs in vivo. The apoptosis of the CCSMCs and the expression of cell α-actin were modulated by miR-145/KLF4 axis though paracrine. KLF4: Krüppel-like factor 4, miR-145: microRNA-145, BMSCs: bone marrow-derived mesenchymal stem cells, SRF: serum response factor, EdU: 5-ethynyl-2′-deoxyuridine, SMLCs: smooth muscle-like cells, CCSMCs: corpus cavernosum smooth muscle cells, ICI: intracavernous injection, CD31: platelet endothelial cell adhesion molecule-1.