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Catechol-estrogens can cause genetic mutations and to counteract their oncogenicity, the catechol-O-methyltransferase (COMT) gene is capable of neutralizing these reactive compounds. In this study, we determined the functional effects and regulation of COMT in prostate cancer. Both the Cancer Genome Atlas (TCGA) and immunohistochemical analysis of clinical specimens demonstrated a reduction of COMT expression in prostate cancer. Also, western analyses of prostate cancer cell lines show COMT levels to be minimal in DuPro and DU145 and thus, these cells were used for further analyses. Re-expression of COMT led to suppressed migration ability (wound healing assay) and enhanced apoptosis (flow cytometric analyses), and when challenged with 4-hydroxyestradiol, a marked reduction of cell proliferation (MTT assay) was observed. Xenograft growth in athymic mice also resulted in inhibition due to COMT. As a mechanism, western analyses show cleaved CASP3 and BID were increased whereas XIAP and cIAP2 were reduced due to COMT. As COMT expression is low in prostate cancer, its regulation was determined. Databases identified several miRNAs capable of binding COMT and of these, miR-195 was observed to be increased in prostate cancer according to TCGA. Real-time PCR validated upregulation of miR-195 in clinical prostate cancer specimens as well as DuPro and DU145 and interestingly, luciferase reporter showed miR-195 capable of binding COMT and overexpressing miR-195 could reduce COMT in cells. These results demonstrate COMT to play a protective role by activating the apoptosis pathway and for miR-195 to regulate its expression. COMT may thus be a potential biomarker and gene of interest for therapeutic development for prostate cancer. [ABSTRACT FROM AUTHOR]

The Abelson (c-Abl) proto-oncogene encodes a highly conserved nonreceptor protein tyrosine kinase that plays a role in cell proliferation, differentiation, apoptosis and cell adhesion. c-Abl represents a specific anti-cancer target in prostate cancer as aberrant activity of this kinase has been implicated in the stimulation of prostate cancer growth and progression. However, the mechanism of regulation of c-Abl is not known. Here we report that Abl kinases are regulated by a novel microRNA, miR-4723, in prostate cancer. Expression profiling of miR-4723 expression in a cohort of prostate cancer clinical specimens showed that miR-4723 expression is widely attenuated in prostate cancer. Low miR-4723 expression was significantly correlated with poor survival outcome and our analyses suggest that miR-4723 has significant potential as a disease biomarker for diagnosis and prognosis in prostate cancer. To evaluate the functional significance of decreased miR-4723 expression in prostate cancer, miR-4723 was overexpressed in prostate cancer cell lines followed by functional assays. miR-4723 overexpression led to significant decreases in cell growth, clonability, invasion and migration. Importantly, miR-4723 expression led to dramatic induction of apoptosis in prostate cancer cell lines suggesting that miR-4723 is a pro-apoptotic miRNA regulating prostate carcinogenesis. Analysis of putative miR-4723 targets showed that miR-4723 targets integrin alpha 3 and Methyl CpG binding protein in addition to Abl1 and Abl2 kinases. Further, we found that the expression of Abl kinase is inversely correlated with miR-4723 expression in prostate cancer clinical specimens. Also, Abl1 knockdown partially phenocopies miR-4723 reexpression in prostate cancer cells suggesting that Abl is a functionally relevant target of miR-4723 in prostate cancer. In conclusion, we have identified a novel microRNA that mediates regulation of Abl kinases in prostate cancer. This study suggests that miR-4723 may be an attractive target for therapeutic intervention in prostate cancer. [ABSTRACT FROM AUTHOR]

Objective: Genistein is a soy isoflavone that has antitumor activity both in vitro and in vivo. It has been shown that genistein inhibits many type of cancers including prostate cancer (PCa) by regulating several cell signaling pathways and microRNAs (miRNAs). Recent studies suggest that the long non-coding RNAs (lncRNAs) are also involved in many cellular processes. At present there are no reports about the relationship between gensitein, miRNAs and lncRNAs. In this study, we focused on miRNAs, lncRNA that are regulated by genistein and investigated their functional role in PCa. Method: Microarray (SurePrint G3 Human GE 8×60K) was used for expression profiling of genistein treated and control PCa cells (PC3 and DU145). Functional assay (cell proliferation, migration, invasion, apoptosis and cell cycle assays) were performed with the PCa cell lines, PC3 and DU145. Both in vitro and in vivo (nude mouse) models were used for growth assays. Luciferase reporter assays were used for binding of miR-34a to HOTAIR. Results: LncRNA profiling showed that HOTAIR was highly regulated by genistein and its expression was higher in castration-resistant PCa cell lines than in normal prostate cells. Knockdown (siRNA) of HOTAIR decreased PCa cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. miR-34a was also up-regulated by genistein and may directly target HOTAIR in both PC3 and DU145 PCa cells. Conclusions: Our results indicated that genistein inhibited PCa cell growth through down-regulation of oncogenic HOTAIR that is also targeted by tumor suppressor miR-34a. These findings enhance understanding of how genistein regulates lncRNA HOTAIR and miR-34a in PCa. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and translation. In this study we show that miRNA-23b (miR-23b) acts as a tumor suppressor in bladder cancer. Quantitative real-time PCR analysis showed that miR-23b is significantly down-regulated in bladder cancer cell lines and tumor tissues compared to non-malignant cells and normal tissue samples. We also demonstrate that miR-23b expression has a potential to be diagnostic and prognostic biomarker in bladder cancer. High miR-23b expression is positively correlated with higher overall survival of bladder cancer patients as revealed by Kaplan-Meier analysis. ROC analysis showed that miR-23b expression can distinguish between normal and bladder cancer tissues. Further we elucidated the biological significance of miR-23b in bladder cancer. Over-expression of miR-23b in bladder cancer cells inhibited cell proliferation and impaired colony formation. Fluorescence activated cell sorting (FACS) analysis revealed that re-expression of miR-23b in bladder cancer cells induced G0/G1 cell cycle arrest and apoptosis while inhibiting cell migration and invasion. Luciferase reporter assays demonstrated that Zeb1, a crucial regulator of epithelial-to-mesenchymal transition (EMT), is a direct target of miR-23b in bladder cancer. These results show that loss of miR-23b confers a proliferative advantage and promotes bladder cancer cell migration and invasion. Furthermore, re-expression of miR-23b may be a beneficial therapeutic strategy for the treatment of human bladder cancer. [ABSTRACT FROM AUTHOR]

Genistein has been shown to inhibit cancers both in vitro and in vivo, by altering the expression of several microRNAs (miRNAs). In this study, we focused on tumor suppressor miRNAs regulated by genistein and investigated their function in prostate cancer (PCa) and target pathways. Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion in vitro and in vivo. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as ‘Pathways in cancer’, ‘Jak-STAT signaling pathway’, and ‘Wnt signaling pathway’. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3′ UTR of several target genes (such as RAC1, EGFR and EP300) that are components of ‘Pathways in cancer’. Quantitative real-time PCR and Western analysis showed that the mRNA and protein expression levels of the three target genes in PCa cells were markedly down-regulated with miR-574-3p. Loss-of-function studies demonstrated that the three target genes significantly affect cell proliferation, migration and invasion in PCa cell lines. Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are non-protein-coding sequences that can function as oncogenes or tumor suppressor genes. This study documents the tumor suppressor role of miR-1280 in bladder cancer. Quantitative real-time PCR and in situ hybridization analyses showed that miR-1280 is significantly down-regulated in bladder cancer cell lines and tumors compared to a non-malignant cell line or normal tissue samples. To decipher the functional significance of miR-1280 in bladder cancer, we ectopically over-expressed miR-1280 in bladder cancer cell lines. Over-expression of miR-1280 had antiproliferative effects and impaired colony formation of bladder cancer cell lines. FACS (fluorescence activated cell sorting) analysis revealed that re-expression of miR-1280 in bladder cancer cells induced G2-M cell cycle arrest and apoptosis. Our results demonstrate that miR-1280 inhibited migration and invasion of bladder cancer cell lines. miR-1280 also attenuated ROCK1 and RhoC protein expression. Luciferase reporter assays demonstrated that oncogene ROCK1 is a direct target of miR-1280 in bladder cancer. This study also indicates that miR-1280 may be of diagnostic and prognostic importance in bladder cancer. For instance, ROC analysis showed that miR-1280 expression can distinguish between malignant and normal bladder cancer cases and Kaplan-Meier analysis revealed that patients with miR-1280 high expression had higher overall survival compared to those with low miR-1280 expression. In conclusion, this is the first study to document that miR-1280 functions as a tumor suppressor by targeting oncogene ROCK1 to invasion/migration and metastasis. Various compounds are currently being used as ROCK1 inhibitors; therefore restoration of tumor suppressor miR-1280 might be therapeutically useful either alone or in combination with these compounds in the treatment of bladder cancer. [ABSTRACT FROM AUTHOR]

Genistein has been shown to suppress the growth of several cancers through modulation of various pathways. However, the effects of genistein on the regulation of oncogenic microRNA-151 (miR-151) have not been reported. In this study, we investigated whether genistein could alter the expression of oncogenic miR-151 and its target genes that are involved in the progression and metastasis of prostate cancer (PCa). Real-time RT-PCR showed that the expression of miR-151 was higher in PC3 and DU145 cells compared with RWPE-1 cells. Treatment of PC3 and DU145 cells with 25 µM genistein down-regulated the expression of miR- 151 compared with vehicle control. Inhibition of miR-151 in PCa cells by genistein significantly inhibited cell migration and invasion. In-silico analysis showed that several genes (CASZ1, IL1RAPL1, SOX17, N4BP1 and ARHGDIA) suggested to have tumor suppressive functions were target genes of miR-151. Luciferase reporter assays indicated that miR-151 directly binds to specific sites on the 39UTR of target genes. Quantitative real-time PCR analysis showed that the mRNA expression levels of the five target genes in PC3 and DU145 were markedly changed with miR-151 mimics and inhibitor. Kaplan-Meier curves and log- rank tests revealed that high expression levels of miR-151 had an adverse effect on survival rate. This study suggests that genistein mediated suppression of oncogenic miRNAs can be an important dietary therapeutic strategy for the treatment of PCa. [ABSTRACT FROM AUTHOR]

Background: MicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically. Methods and Results: We measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by realtime PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation. Conclusions: The current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21. [ABSTRACT FROM AUTHOR]

MicroRNA-34a (miR-34a), a potent mediator of tumor suppressor p53, has been reported to function as a tumor suppressor and miR-34a was found to be downregulated in prostate cancer tissues. We studied the functional effects of miR-34a on c- Myc transcriptional complexes in PC-3 prostate cancer cells. Transfection of miR-34a into PC-3 cells strongly inhibited in vitro cell proliferation, cell invasion and promoted apoptosis. Transfection of miR-34a into PC-3 cells also significantly inhibited in vivo xenograft tumor growth in nude mice. miR-34a downregulated expression of c-Myc oncogene by targeting its 3' UTR as shown by luciferase reporter assays. miR-34a was found to repress RhoA, a regulator of cell migration and invasion, by suppressing c-Myc-Skp2-Miz1 transcriptional complex that activates RhoA. Overexpression of c-Myc reversed miR-34a suppression of RhoA expression, suggesting that miR-34a inhibits invasion by suppressing RhoA through c-Myc. miR-34a was also found to repress c-Myc-pTEFB transcription elongation complex, indicating one of the mechanisms by which miR- 34a has profound effects on cellular function. This is the first report to document that miR-34a suppresses assembly and function of the c-Myc-Skp2-Miz1 complex that activates RhoA and the c-Myc-pTEFB complex that elongates transcription of various genes, suggesting a novel role of miR-34a in the regulation of transcription by c-Myc complex. [ABSTRACT FROM AUTHOR]