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

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.

Caveolin-1 scaffolding domain peptide prevents corpus cavernosum fibrosis and erectile dysfunction in bilateral cavernous nerve injury-induced rats.

BACKGROUND: Corpus cavernosum (CC) fibrosis significantly contributes to post-radical prostatectomy erectile dysfunction (pRP-ED). Caveolin-1 scaffolding domain (CSD)-derived peptide has gained significant concern as a potent antagonist of tissue fibrosis. However, applying CSD peptide on bilateral cavernous nerve injury (BCNI)-induced rats remains uninvestigated. AIM: The aim was to explore the therapeutic outcome and underlying mechanism of CSD peptide for preventing ED in BCNI rats according to the hypothesis that CSD peptide may exert beneficial effects on erectile tissue and function following BCNI through limiting collagen synthesis in CC smooth muscle cells (CCSMCs) and CC fibrosis. METHODS: After completing a random assignment of male Sprague Dawley rats (10 weeks of age), BCNI rats received either saline or CSD peptide treatment, as opposed to sham-operated rats. The evaluations of erectile function (EF) and succedent collection and histological and molecular biological examinations of penile tissue were accomplished 3 weeks postoperatively. In addition, the fibrotic model of CCSMCs was used to further explore the mechanism of CSD peptide action in vitro. OUTCOMES: The assessments of EF, SMC/collagen ratio, α-smooth muscle actin, caveolin-1 (CAV1), and profibrotic indicators expressions were conducted. RESULTS: BCNI rats exhibited significant decreases in EF, SMC/collagen ratio, α-SMA, and CAV1 levels, and increases in collagen content together with transforming growth factor (TGF)-ß1/Smad2 activity. However, impaired EF, activated CC fibrosis, and Smad2 signaling were attenuated after 3 weeks of CSD peptide treatment in BCNI rats. In vitro, TGF-ß1-induced CCSMCs underwent fibrogenetic transformation characterized by lower expression of CAV1, higher collagen composition, and phosphorylation of Smad2; then, the delivery of CSD peptide could significantly block CCSMC fibrosis by inactivating Smad2 signaling. CLINICAL IMPLICATIONS: Based on available evidence of CSD peptide in the prevention of ED in BCNI rats, this study can aid in the development and clinical application of CSD peptide targeting pRP-ED. STRENGTHS AND LIMITATIONS: This study provides data to suggest that CSD peptide protects against BCNI-induced deleterious alterations in EF and CC tissues. However, the available evidence still does not fully clarify the detailed mechanism of action of CSD peptide. CONCLUSION: Administration of CSD peptide significantly retarded collagen synthesis in CCSMCs, limited CC fibrosis, and prevented ED via confrontation of TGF-ß1/Smad signaling in BCNI rats.

Heterogeneity of fibroblasts is a hallmark of age-associated erectile dysfunction.

BACKGROUND: The prevalence of age-associated erectile dysfunction (ED) increases pronouncedly with age. However, the cellular composition and transcriptomic changes of aging penile corpus cavernosum remain largely unclear. METHODS: Herein, we performed single cell sequencing penile corpus cavernosum from five young with normal erectile response and five old rats with ED. RESULTS: Clustering analysis identified 19 cell types, such as fibroblasts, myofibroblasts and immune cells. We next revealed their transcriptomic alterations and investigated novel subpopulations of major cell types. Among them, fibroblasts possessed the largest cell number and showed apparent heterogeneity. By performing single-cell entropy analysis on fibroblasts, we observed the age-associated decrease of entropy, and aged fibroblasts were found to adopt senescent secretory phenotype, as evidenced by the high expression of genes associated with the senescence-associated secretory phenotype (SASP). Finally, we constructed a comprehensive intercellular communication network and highlighted key mediators of crosstalk between fibroblasts and other cell types. CONCLUSIONS: We plotted a cellular atlas of aging cells within penile corpus cavernosum, especially fibroblasts. Our work will deepen the understanding of the heterogeneity among certain cell types within aged penile corpus cavernosum, which will generate positive effects on the future treatment of age-associated ED.

Upregulated IGFBP3 with Aging Is Involved in Modulating Apoptosis, Oxidative Stress, and Fibrosis: A Target of Age-Related Erectile Dysfunction.

Aging has been deemed the primary factor in erectile dysfunction (ED). Herein, age-related changes in the erectile response and histomorphology were detected, and the relationship between aging and ED was investigated based on gene expression levels. Thirty male Sprague-Dawley (SD) rats were randomly divided into 6 groups, and intracavernous pressure (ICP) and mean arterial pressure (MAP) were measured. Subsequently, the corpus cavernosum (CC) was harvested and prepared for histological examinations of apoptosis, oxidative stress (OS), and fibrosis. Then, the microarray dataset (GSE10804) was analyzed to identify differentially expressed genes (DEGs) in ED progression, and hub genes were selected. In addition, aged CC smooth muscle cells (CCSMCs) were isolated to evaluate the function of the hub gene by siRNA interference, qRT-PCR, immunofluorescence staining, enzyme-linked immunosorbent assay, western blot analysis, CCK-8 assay, EdU staining, and flow cytometry approaches. The ICP/MAP and smooth muscle cell (SMC)/collagen ratios declined with aging, while apoptosis and OS levels increased with aging. The enriched functions and pathways of the DEGs were investigated, and 15 hub genes were identified, among which IGFBP3 was significantly upregulated. The IGFBP3 upregulation was verified in the CC of aging rats. Furthermore, aged CCSMCs were transfected with siRNA to knock down IGFBP3 expression. The viability and proliferation of the CCSMCs increased, while apoptosis, OS, and fibrosis decreased. Our findings demonstrate that the erectile response of SD rats declines in parallel with enhanced CC apoptosis, OS, and fibrosis with aging. Upregulation of IGFBP3 plays an important role; furthermore, downregulation of IGFBP3 improves the viability and proliferation of CCSMCs and alleviates apoptosis, OS, and fibrosis. Thus, IGFBP3 is a potential therapeutic target for age-related ED.

MicroRNA-145 engineered bone marrow-derived mesenchymal stem cells alleviated erectile dysfunction in aged rats.

BACKGROUND: Aging is one of the dominant factors contributing to erectile dysfunction (ED), and effective treatments for age-associated ED are urgently demanded. In this study, the therapeutic efficiency of bone marrow-derived mesenchymal stem cells (BMSCs) overexpressing microRNA-145 (miR-145) was evaluated in ED. METHODS: Sixty male Sprague-Dawley rats (24 months old) were randomly divided into 4 treatment groups (n = 15/group): PBS (control), BMSCs, BMSCs transfected with a blank vector (vector-BMSCs), and BMSCs transfected with a lentivirus overexpressing miR-145 (OE-miR-145-BMSCs). Fourteen days after transplantation of BMSCs, erectile function was evaluated by measuring intra-cavernous pressure (ICP) and mean arterial pressure (MAP). Subsequently, penile erectile tissues were harvested and subjected to Masson staining, qRT-PCR, immunofluorescence staining, dual luciferase assay, and Western blot analysis. RESULTS: Fourteen days after transplantation, the ICP/MAP was 0.79 ± 0.05 in the OE-miR-145-BMSC group, 0.61 ± 0.06 in the BMSC group, 0.57 ± 0.06 in the vector-BMSC group, and 0.3 ± 0.01 in the PBS group. Treatment with OE-miR-145-BMSCs significantly improved ED (P < 0.05), and the treatment increased the smooth muscle content in the penis tissues of ED rats (P < 0.05). In the OE-miR-145-BMSC group, the expression levels of α-SMA, desmin, and SM-MHC were higher than they were in the other ED groups (P < 0.05). In addition, the levels of collagen 1, MMP2, and p-Smad2 in the BMSC-treated group, especially in the OE-miR-145-BMSC group, were lower than those in the control group (P < 0.05). CONCLUSIONS: MicroRNA-145 engineered BMSCs effectively attenuate age-related ED. Transplantation of miR-145-overexpressing BMSCs may provide a promising novel avenue for age-associated ED therapy.

Thyroid Receptor-Interacting Protein 13 is Correlated with Progression and Poor Prognosis in Bladder Cancer.

BACKGROUND Bladder cancer is the fourth most common cancer worldwide. Thyroid receptor-interacting protein 13 (TRIP13) is a member of the AAA+ ATPase family. The upregulation of TRIP13 has been shown to be involved in a few diseases, especially in cancers, but the expression and function of TRIP13 in bladder cancer is still elusive. MATERIAL AND METHODS In our study, the expression of TRIP13 was investigated with immunohistochemistry (IHC). The mRNAs of TRIP13 in bladder cancer and adjacent normal tissues were compared using quantitative real-time polymerase chain reaction (qRT-PCR) and IHC scores. The clinical value of TRIP13 was estimated by evaluating its correlation with other clinicopathological factors using the chi-square test. The prognostic significance of TRIP13 was evaluated using univariate and multivariate analyses. The effect of TRIP13 on proliferation and invasion was evaluated using function assays in vitro. RESULTS In the 139 samples of bladder cancer tissues, the patients with low and high expression of TRIP13 accounted for 64.03% and 35.97%, respectively. Moreover, the mRNA expression of TRIP13 in bladder cancer was significantly higher than in normal tissues. High expression of TRIP13 was remarkably correlated with T stage, metastasis, and poor prognosis. In addition, TRIP13 was demonstrated to promote the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of bladder cancer. CONCLUSIONS TRIP13 is correlated with poor prognosis of bladder cancer by promoting proliferation, invasion, and EMT, indicating that TRIP13 may be a promising drug target in bladder cancer.

GLUT1 regulates cell glycolysis and proliferation in prostate cancer.

BACKGROUND: Glucose transporter 1 (GLUT1) plays a critical role in tumorigenesis and tumor progression in multiple cancer types. However, the specific function and clinical significance of GLUT1 in prostate cancer (PCa) are still unclear. Therefore, in this study, we investigated the role of GLUT1 in PCa. METHODS: GLUT1 protein levels in prostate cancer tissue and tumor-adjacent normal tissues were measured and compared. Furthermore, real-time PCR and Western blot analysis were both used to detect GLUT1 expression levels in different PCa cell lines. Flow cytometry and cell-based assays, such as a glucose uptake and lactate secretion assay, CCK-8 assay, and transwell migration and wound healing assay, were used to monitor cancer cell cycle distribution, glycolysis, proliferation, and motility, respectively. Moreover, a mouse tumor xenograft model was used to investigate the role of GLUT1 in tumor progression in vivo. RESULTS: GLUT1 expression levels are higher in PCa tissues than in tumor-adjacent normal tissues. The results from real-time PCR and Western blot analysis revealed a similar increase in the GLUT1 expression levels in PCa cell lines. Moreover, knockdown of GLUT1 inhibits cell glycolysis and proliferation and leads to cell cycle arrest at G2/M phase in the 22RV1 cell line but not in the PC3 cell line. In vivo experiments further confirmed that GLUT1 knockdown inhibits the growth of tumors derived from the 22RV1 cell line. In addition, we also showed that GLUT1 knockdown has no effect on cell migration in vitro. CONCLUSIONS: GLUT1 may play an important role in PCa progression via mediating glycolysis and proliferation. Our study also indicated a potential crosstalk between GLUT1-mediated glycolysis and androgen sensitivity in PCa.

Increased expression of glycolytic enzymes in prostate cancer tissues and association with Gleason scores.

OBJECTIVE: Recent studies have shown that understanding the differences between Gleason 3+4 and Gleason 4+3 in PCa patients may improve their treatment. This study aimed to evaluate the different expression levels of glycolytic proteins for Gleason score of 4+3 and 3+4. METHODS: A total of 90 PCa patients, including 38 cases with a Gleason score of 7, were included in this study. The expression of glycolytic proteins in both prostate cancer and normal prostate tissues, in GGG2 and GGG3 as well were assessed by immunohistochemical staining. RESULTS: Compared with GGG3, the GGG2 cases displayed significantly lower expression of all proteins (P < 0.05). The correlation among all enzymes showed that the key glycolytic enzyme, HK2, was significantly positively related to another key enzyme, PKM2 (r = 0.550, P < 0.01), and the expression of PFKFB4 was correlated with the expression of HK2 (r = 0.236, P < 0.05) and PKM2 (r = 0.392, P < 0.01). Additionally, neither GLUT1 nor PFKFB3 was correlated with PFKFB4, HK2 or PKM2. Further analysis showed that HK2 (r = 0.297, P < 0.01) and PKM2 (r = 0.431, P < 0.01) were significantly positively related to the Gleason score in PCa tissues. CONCLUSIONS: Glycolytic proteins expression levels were upregulated in PCa tissues. Furthermore, GGG3 exhibits a higher level of glycolysis compared with GGG2 in PCa tissues. Additionally, the key glycolytic enzymes, HK2 and PKM2, are overexpressed simultaneously in PCa and significantly correlate with PCa progression as represented by the GS.