Enhancing RECK Expression Through miR-21 Inhibition: A Promising Strategy for Bladder Carcinoma Control.

Bladder carcinoma (BC) is the tenth most frequent malignancy worldwide, with high morbidity and mortality rates. Despite recent treatment advances, high-grade BC and muscle-invasive BC present with significant progression and recurrence rates, urging the need for alternative treatments. The microRNA-21 (miR-21) has superexpression in many malignancies and is associated with cellular invasion and progression. One of its mechanisms of action is the regulation of RECK, a tumor suppressor gene responsible for inhibiting metalloproteinases, including MMP9. In a high-grade urothelial cancer cell line, we aimed to assess if miR-21 downregulation would promote RECK expression and decrease MMP9 expression. We also evaluated cellular migration and proliferation potential by inhibition of this pathway. In a T24 cell line, we inhibited miR-21 expression by transfection of a specific microRNA inhibitor (anti-miR-21). There were also control and scramble groups, the last with a negative microRNA transfected. After the procedure, we performed a genetic expression analysis of miR-21, RECK, and MMP9 through qPCR. Migration, proliferation, and protein expression were evaluated via wound healing assay, colony formation assay, flow cytometry, and immunofluorescence.After anti-miR-21 transfection, miR-21 expression decreased with RECK upregulation and MMP9 downregulation. The immunofluorescence assay showed a significant increase in RECK protein expression (p < 0.0001) and a decrease in MMP9 protein expression (p = 0.0101). The anti-miR-21 transfection significantly reduced cellular migration in the wound healing assay (p < 0.0001). Furthermore, in the colony formation assay, the anti-miR-21 group demonstrated reduced cellular proliferation (p = 0.0008), also revealed in the cell cycle analysis by flow cytometry (p = 0.0038). Our results corroborate the hypothesis that miR-21 is associated with BC cellular migration and proliferation, revealing its potential as a new effective treatment for this pathology.

Expression of micro-RNAs and genes related to angiogenesis in ccRCC and associations with tumor characteristics.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the third most common urological cancer in adults. Our aim is to evaluate genes and miRNAs expression profiles involved with angiogenesis and tumor characteristics in ccRCC. METHODS: The expression levels of miRNAs miR-99a, 99b, 100; 199a; 106a; 106b; 29a; 29b; 29c; 126; 200a, 200b and their respective target genes: mTOR, HIF1-α, VHL, PDGF, VEGF, VEGFR1 and VEGFR2 were analyzed using qRT-PCR in tumor tissue samples from 56 patients with ccRCC. Five samples of benign renal tissue were utilized as control. The expression levels of miRNAs and genes were related to tumor size, Fuhrman nuclear grade and microvascular invasion. RESULTS: miR99a was overexpressed in most samples and its target gene mTOR was underexpressed, this also occurs for miRNAs 106a, 106b, and their target gene VHL. An increase in miR-200b was correlated with high-risk tumors (p = 0.01) while miR-126 overexpression was associated with Fuhrman's low grade (p = 0.03). CONCLUSIONS: Our results show that in ccRCC there are changes in miRNAs expression affecting gene expression that could be important in determining the aggressiveness of this lethal neoplasia.

Loss of TIMP-1 immune expression and tumor recurrence in localized prostate cancer.

INTRODUCTION AND OBJECTIVE: Overexpression of MMPs has been related to biochemical recurrence after radical prostatectomy. TIMP1 and TIMP2 are controllers of MMPs and the aim of this study is to evaluate the expression levels of MMPs and their regulators using immunohistochemistry in tissue microarray of localized prostate cancer (PC). MATERIALS AND METHODS: Immune-expression of MMP-9, MMP-2, TIMP1, TIMP-2, MMP-14 and IL8, were analyzed by immunohistochemistry in radical prostatectomy specimens of 40 patients with localized PC who underwent surgery between September 1997 and February 2000. Protein expression was considered as categorical variables, negative or positive. The results of the immune-expression were correlated to Gleason score (GS), pathological stage (TNM), pre-operatory PSA serum levels and biochemical recurrence in a mean follow up period of 92.5 months. RESULTS: The loss of TIMP1 immune-expression was related to biochemical recurrence. When TIMP1 was negative, 56.3% patients recurred versus 22.2% of those whose TIMP1 was positive (p=0.042). MMP-9, MMP-2, IL8 and MMP-14 were positive in the majority of PC. TIMP-2 was negative in all cases. CONCLUSION: Negative immune-expression of TIMP1 is correlated with biochemical recurrence in patients with PC possibly by failing to control MMP-9, an important MMP related to cancer progression.

Downregulation of miR-29b is associated with Peyronie's disease.

BACKGROUND: Peyronie's disease (PD) is characterized by the formation of fibrous plaque in tunica albuginea, causing several problems in patients. The etiology of this disease is not fully understood, and there are few effective treatments. To better understand the molecular pathways of PD, we studied miR-29b, a microRNA that could be involved with this illness. MicroRNAs are endogenous molecules that act by inhibiting messenger RNA. MiR-29b regulates 11 of 20 collagen genes and the TGF-ß1 gene, which are related to PD progression. METHODS: We compared miR-29b expression in 11 patients with PD and 14 patients without PD (control group). For the patients with PD, we utilized samples from the fibrous plaque (n = 9), from the tunica albuginea (n = 11), and from the corpus cavernosum (n = 8). For the control group, we utilized samples from the tunica albuginea (n = 14) and from the corpus cavernosum (n = 10). MiR-29b expression was determined by q-PCR. RESULTS: We found a downregulation of miR-29b in the fibrous plaque, tunica albuginea and corpus cavernosum of patients with PD in comparison with the control group (p = 0.0484, p = 0.0025, and p = 0.0016, respectively). CONCLUSION: Although our study has a small sample, we showed for the first time an evidence that the downregulation of miR-29b is associated with PD.

Prognostic value of TERF1 expression in prostate cancer.

BACKGROUND: Telomere dysfunction is one of the hallmarks of cancer and is crucial to prostate carcinogenesis. TERF1 is a gene essential to telomere maintenance, and its dysfunction has already been associates with several cancers. TERF1 is a target of miR-155, and this microRNA can inhibit its expression and promotes carcinogenesis in breast cancer. We aim to analyze TERF1, in gene and mRNA level, involvement in prostate cancer progression. RESULTS: Alterations in TERF1 DNA were evaluated using datasets of primary tumor and castration-resistant tumors (CRPC) deposited in cBioportal. The expression of TERF1 mRNA levels was assessed utilizing TCGA datasets, clinical specimens, and metastatic prostate cancer cell lines (LNCaP, DU145, and PC3). Six percent of localized prostate cancer presents alterations in TERF1 (the majority of that was amplifications). In the CRPC cohort, 26% of samples had TERF1 amplification. Patients with TERF1 alterations had the worst overall survival only on localized cancer cohort (p = 0.0027). In the TCGA cohort, mRNA levels of TERF1 were downregulated in comparison with normal tissue (p = 0.0013) and upregulated in tumors that invade lymph nodes (p = 0.0059). The upregulation of TERF1 is also associated with worst overall survival (p = 0.0028) and disease-free survival (p = 0.0023). There is a positive correlation between TERF1 and androgen receptor expression in cancer tissue (r = 0.53, p < 0.00001) but not on normal tissue (r = - 0.16, p = 0.12). In the clinical specimens, there is no detectable expression of TERF1 and upregulation of miR-155 (p = 0.0348). In cell lines, TERF1 expression was higher in LNCaP and was progressively lower in DU145 and PC3 (p = 0.0327) with no differences in miR-155 expression. CONCLUSION: Amplification/upregulation of TERF1 was associated with the worst prognostic in localized prostate cancer. Our results corroborate that miR-155 regulates TERF1 expression in prostate cancer. TERF1 has the potential to become a biomarker in prostate cancer.

Tissue expression of MMP-9, TIMP-1, RECK, and miR338-3p in prostate gland: can it predict cancer?

Prostate cancer is the most frequent malignancy affecting men worldwide. Due to the low sensitivity and specificity of the prostate-specific antigen test and the digital rectal exam as screening modalities, several alternatives are being studied. This study aimed to evaluate the application of MMP-9 and its regulators (TIMP-1, RECK, and miR-338-3p) as diagnostic and prognostic indicators of prostate cancer. A total of 134 randomly selected patients under investigation for prostate cancer submitted to a transrectal ultrasound-guided prostate biopsy were enrolled in the study; of these, 61 were positive for the disease (cases), and 73 were negative (control group). The tissue samples were further analyzed by gene and miR-338-3p expression analysis using qRT-PCR (one randomly selected fragment of each patient). Approximately 58% of the patients with prostate cancer presented MMP9 upregulation, while 73%, 65%, and 69% downregulated IMP-1, RECK, and miR-338-3p, respectively. MiR-338-3p was expressed at lower levels in patients with PSA concentrations exceeding 20 ng/mL (p=0.045) and abnormal DRE (p=0.006), while the RECK was more expressed in patients with abnormal DRE (p=0.01). We found that most patients with prostate cancer overexpressed MMP-9; on the other hand, most of them underexpressed TIMP-1, RECK, and miR-338-3p. MiR-338-3p presented as a possible predictor of poor prognosis. Further studies are warranted to evaluate these biomarkers as prognosis factors better.

Loss of TIMP-1 immune expression and tumor recurrence in localized prostate cancer

Introduction and objective: Overexpression of MMPs has been related to biochemical recurrence after radical prostatectomy. TIMP1 and TIMP2 are controllers of MMPs and the aim of this study is to evaluate the expression levels of MMPs and their regulators using immunohistochemistry in tissue microarray of localized prostate cancer (PC). Materials and Methods: Immune-expression of MMP-9, MMP-2, TIMP1, TIMP-2, MMP-14 and IL8, were analyzed by immunohistochemistry in radical prostatectomy specimens of 40 patients with localized PC who underwent surgery between September 1997 and February 2000. Protein expression was considered as categorical variables, negative or positive. The results of the immune-expression were correlated to Gleason score (GS), pathological stage (TNM), pre-operatory PSA serum levels and biochemical recurrence in a mean follow up period of 92.5 months. Results: The loss of TIMP1 immune-expression was related to biochemical recurrence. When TIMP1 was negative, 56.3% patients recurred versus 22.2% of those whose TIMP1 was positive (p=0.042). MMP-9, MMP-2, IL8 and MMP-14 were positive in the majority of PC. TIMP-2 was negative in all cases. Conclusion: Negative immune-expression of TIMP1 is correlated with biochemical recurrence in patients with PC possibly by failing to control MMP-9, an important MMP related to cancer progression.