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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.

Recently miR-182 has been reported to be over-expressed in prostate cancer (PC) tissues, however detailed functional analysis of miR-182-5p has not been carried out. The purpose of this study was to: 1. analyze the function of miR-182-5p in prostate cancer, 2. assess its usefulness as a tumor marker, 3. identify miR-182-5p target genes in PC, 4. investigate the potential for miR-182-5p inhibitor to be used in PC treatment. Initially we found that miR-182-5p expression was significantly higher in prostate cancer tissues and cell lines compared to normal prostate tissues and cells. Moreover high miR-182-5p expression was associated with shorter overall survival in PC patients. To study the functional significance of miR-182-5p, we knocked down miR-182-5p with miR-182-5p inhibitor. After miR-182-5p knock-down, prostate cancer cell proliferation, migration and invasion were decreased. We identified FOXF2, RECK and MTSS1 as potential target genes of miR-182-5p using several algorithms which was confirmed by 3'UTR luciferase assay and Western analysis. Knock-down of miR-182-5p also significantly decreased in vivo prostate tumor growth. In conclusion this is the first report documenting that over-expression of miR-182-5p is associated with prostate cancer progression and potentially useful as a prognostic biomarker. Also knock down of miR-182-5p in order to increase expression of tumor suppressor genes FOXF2, RECK and MTSS1 may be of therapeutic benefit in prostate cancer treatment.

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.

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.

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.

miR-23b is located on chromosome number 9 and plays different roles in different organs especially with regards to cancer development. However, the functional significance of miR-23b-3p in renal cell carcinoma (RCC) has not been reported.We measured miR-23b-3p levels in 29 pairs of renal cell carcinoma and their normal matched tissues using real-time PCR. The expression level of miR-23b-3p was correlated with the 5 year survival rate of renal cancer patients. In 15 cases (52%), miR-23b-3p expression was found to be high. All patients with moderate to low miR-23b-3p expression survived 5 years, while those with high miR-23b-3p expression, only 50% survived. After knocking down miRNA-23b-3p expression in RCC cell lines, there was an induction of apoptosis and reduced invasive capabilities. MiR-23b-3p was shown to directly target PTEN gene through 3'UTR reporter assays. Inhibition of miR-23b-3p induces PTEN gene expression with a concomitant reduction in PI3-kinase, total Akt and IL-32. Immunohistochemistry showed the lack of PTEN protein expression in cancerous regions of tissue samples where the expression of miR-23b-3p was high. We studied the in vitro effects of the dietary chemo preventive agent genistein on miR-23b-3p expression and found that it inhibited expression of miR-23b-3p in RCC cell lines.The current study shows that miR-23b-3p is an oncogenic miRNA and inhibits PTEN tumor suppressor gene in RCC. Therefore, inhibition of miR-23b-3p may be a useful therapeutic target for the treatment of renal cell carcinoma.

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.

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.We measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time 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.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.

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 3'UTR 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.

Background: Cytochrome P450 1B1 (CYP1B1) has been shown to be up-regulated in many types of cancer including renal cell carcinoma (RCC). Several reports have shown that CYP1B1 can influence the regulation of tumor development; however, its role in RCC has not been well investigated. The aim of the present study was to determine the functional effects of CYP1B1 gene on tumorigenesis in RCC.Methods: Expression of CYP1B1 was determined in RCC cell lines, and tissue microarrays of 96 RCC and 25 normal tissues. To determine the biological significance of CYP1B1 in RCC progression, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses.Results: First, we confirmed that CYP1B1 protein expression was significantly higher in RCC cell lines compared to normal kidney tissue. This trend was also observed in RCC samples (p < 0.01). Interestingly, CYP1B1 expression was associated with tumor grade and stage. Next, we silenced the gene in Caki-1 and 769-P cells by RNA interference and performed various functional analyses to determine the biological significance of CYP1B1 in RCC progression. Inhibition of CYP1B1 expression resulted in decreased cell proliferation, migration and invasion of RCC cells. In addition, reduction of CYP1B1 induced cellular apoptosis in Caki-1. We also found that these anti-tumor effects on RCC cells caused by CYP1B1 depletion may be due to alteration of CDC20 and DAPK1 expression based on gene microarray and confirmed by real-time PCR. Interestingly, CYP1B1 expression was associated with CDC20 and DAPK1 expression in clinical samples.Conclusions: CYP1B1 may promote RCC development by inducing CDC20 expression and inhibiting apoptosis through the down-regulation of DAPK1. Our results demonstrate that CYP1B1 can be a potential tumor biomarker and a target for anticancer therapy in RCC. [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]

Abstract   Objective To determine the value of histology in identifying Lynch syndrome among those patients with early onset of colorectal cancer (CRC). Methods Demographic, clinical and cancer history data from patients diagnosed with CRC before 60 years of age, and treated at our institution between 1997 and 2005, were collected from medical records and direct interview. Their tumors were assessed to identify histological features suggestive of high frequency microsatellite instability (MSI-H): tumor infiltrating lymphocytes, Crohn’s like inflammatory reaction, mucinous, signet ring cells, medullary growth pattern and then, tested for microsatellite instability (MSI) and MLH1/ MSH2 protein expression. Results Sixty-five patients were included in the study. The mean age at diagnosis was 48 ± 9.9 years. Overall, 28 (43%) patients, including 13 of 35 diagnosed between ages 50 and 60, had tumor demonstrating one or more histological features suggestive of MSI-H. These patients were younger (45 vs. 50 years, P = 0.02) and more commonly had family history of Lynch syndrome-related cancers (36 vs. 19%), though the latter feature did not reach statistical significance (P = 0.07). Eleven of 25 tumors with MSI-H histology, but only 1 of 29 tumors without special histological features were found to be MSI-H (P Conclusions Limiting MSI analysis only to those tumors with suggestive histology would have reduced the need for testing by nearly 60% of all tumors from patients that met the revised Bethesda guidelines. [ABSTRACT FROM AUTHOR]

Recently, long noncoding RNAs (lncRNA) have emerged as new gene regulators and prognostic markers in several cancers, including renal cell carcinoma (RCC). In this study, we investigated the contributions of the lncRNA MALAT1 in RCC with a specific focus on its transcriptional regulation and its interactions with Ezh2 and miR-205.Wefound thatMALAT1 expression was higher in human RCC tissues, where it was associated with reduced patient survival. MALAT1 silencing decreased RCC cell proliferation and invasion and increased apoptosis. Mechanistic investigations showed that MALAT1 was transcriptionally activated by c-Fos and that it interacted with Ezh2. After MALAT1 silencing, β-catenin expression was increased, whereas b-catenin expression was decreased through Ezh2. Reciprocal interaction between MALAT1 and miR-205 was also observed. Lastly, MALAT1 bound Ezh2 and oncogenesis facilitated by MALAT1 was inhibited by Ezh2 depletion, thereby blocking epithelial-mesenchymal transition via β-catenin recovery and b-catenin downregulation. Overall, our findings illuminate how overexpression of MALAT1 confers an oncogenic function in RCC that may offer a novel theranostic marker in this disease. [ABSTRACT FROM AUTHOR]

HOTAIR is a long non-coding RNA that interacts with the polycomb repressive complex and suppresses its target genes. HOTAIR has also been demonstrated to promote malignancy. MicroRNA-141 (miR-141) has been reported to play a role in the epithelial to mesenchymal transition process, and the expression of miR-141 is inversely correlated with tumorigenicity and invasiveness in several human cancers. We found that HOTAIR expression is inversely correlated to miR-141 expression in renal carcinoma cells. HOTAIR promotes malignancy, including proliferation and invasion, whereas miR-141 suppresses malignancy in human cancer cells. miR-141 binds to HOTAIR in a sequence-specific manner and suppresses HOTAIR expression and functions, including proliferation and invasion. Both HOTAIR and miR-141 were associated with the immunoprecipitated Ago2 (Argonaute2) complex, and the Ago2 complex cleaved HOTAIR in the presence of miR-141. These results demonstrate that HOTAIR is suppressed by miR-141 in an Ago2-dependent manner. [ABSTRACT FROM AUTHOR]

The miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control multiple genes and potential to influence cellular behavior. Here, we identified that miR-23b is a methylation-silenced tumor suppressor in prostate cancer. We showed that miR-23b expression is controlled by promoter methylation and has great promise as a diagnostic and prognostic biomarker in prostate cancer. High levels of miR-23b expression are positively correlated with higher overall and recurrence-free survival in patients with prostate cancer. Furthermore, we elucidated the tumor suppressor role of miR-23b using in vitro and in vivo models. We showed that proto-oncogene Src kinase and Akt are direct targets of miR-23b. Increased expression of miR-23b inhibited proliferation, colony formation, migration/invasion, and triggered G0-G1 cell-cycle arrest and apoptosis in prostate cancer. Overexpression of miR-23b inhibited epithelial-to-mesenchymal transition (EMT) causing a decline in mesenchymal markers Vimentin and Snail and increasing the epithelial marker, E-cadherin. Depletion of Src by RNA interference conferred similar functional effects as that of miR-23b reconstitution. miR- 23b expression caused a dramatic decrease in tumor growth in nude mice and attenuated Src expression in excised tumors compared with a control miR. These findings suggest that miR-23b is a methylation-silenced tumor suppressor that may be a useful biomarker in prostate cancer. Loss of miR-23b may confer proliferative advantage and promote prostate cancer migration and invasion, and re-expression of miR-23b may contribute to the epigenetic therapy for prostate cancer. [ABSTRACT FROM AUTHOR]