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1.  Extracellular activation of Wnt signaling through epigenetic dysregulation of Wnt inhibitory factor-1 (Wif-1) is associated with pathogenesis of adrenocortical tumor 
Oncotarget  2014;5(8):2198-2207.
Wnt/β-catenin signaling is considered to be an essential regulator of adrenocortical oncogenesis. Wnt inhibitory factor-1 (Wif-1), an extracellular regulator of Wnt signaling, is frequently down-regulated by hypermethylation of the promoter CpG. We investigated epigenetic regulation of Wif-1 and its association with adrenocortical (AC) tumor pathogenesis in light of Wnt activation. The AC tumors showed a high prevalence of Wif-1 promoter methylation and low prevalence of Wif-1 mRNA transcription as compared to the normal adrenal (NA) samples. Furthermore, a significant correlation was found between Wif-1 promoter methylation and mRNA transcription in the tumors. Either intracellular β-catenin accumulation or β-catenin mRNA transcription was significantly elevated in the AC tumors, which also showed an inverse correlation with Wif-1 mRNA transcription. Cyclin D1, a target gene of Wnt signaling, was also up-regulated in the AC tumors as compared with the NA samples. In addition, down-regulation of Wif-1was correlated with increased cyclin D1 at both mRNA and protein levels. However, despite the proposed activation of Wnt signaling in AC tumors, only 2 of 20 with intracellular β-catenin accumulation showed β-catenin mutations. Thus, genetic alterations of β-catenin and epigenetics-related Wif-1 promoter hypermethylation may be important mechanisms underlying AC tumor formation though aberrant canonical Wnt/β-catenin signaling activation.
PMCID: PMC4039156  PMID: 24755523
adrenocortical tumor; Wif-1; epigenetics; Wnt signaling; cyclin D1
2.  Genistein reverses hypermethylation and induces active histone modifications in tumor suppressor gene B-cell Translocation Gene 3 (BTG3) in prostate cancer 
Cancer  2010;116(1):66-76.
BTG3/ANA/APRO4 is a candidate tumor suppressor gene in some malignancies. We report here that BTG3 is transcriptionally down-regulated in prostate cancer and the mechanism of inactivation is through promoter hypermethylation.
Prostate cancer and normal cell lines were treated with different doses of genistein and 5-aza-2’-deoxycytidine (5Aza-C). BTG3 mRNA expression was determined by quantitative real-time PCR in tissues and cell lines. BS-PCR, cloning and sequencing were used to examine promoter methylation in tumor samples and cell lines. Enzyme activity/inhibition assays were done to check the effect of genistein and 5Aza-C on DNA methyltransferases. ChIP assay was performed to analyze chromatin modifications caused by genistein treatment.
BTG3 mRNA expression was down-regulated in cancer tissues and cells. Genistein and 5Aza-C induced BTG3 mRNA expression in all PC cell lines. Complete methylation of BTG3 promoter in tumor samples and cancer cell lines was observed. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Genistein and 5Aza-C increased levels of acetylated histones 3, 4, 2H3K4, 3H3K4 and Pol II, decreased DNMTase, MBD2 activity and increased HAT activity.
This is the first report to show that BTG3 is silenced in prostate cancer and can be reactivated by genistein induced promoter demethylation and active histone modification. Genistein showed similar effects to that of 5Aza-C, which is currently undergoing phase II clinical trials as a treatment for prostate cancer. Since genistein is a natural, non-toxic, dietary isoflavone, these results indicate that genistein is a novel, advantageous therapeutic agent for treating prostate cancer.
PMCID: PMC3954042  PMID: 19885928
Prostate cancer; genistein; BTG3; DNA methylation; Histone modification
3.  MicroRNA-23b represses proto-oncogene Src kinase and functions as methylation-silenced tumor suppressor with diagnostic and prognostic significance in prostate cancer 
Cancer research  2012;72(24):6435-6446.
MicroRNAs (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 (PCa). We demonstrated that miR-23b expression is controlled by promoter methylation and has great promise as a diagnostic and prognostic biomarker in PCa. High levels of miR-23b expression are positively correlated with higher overall and recurrence-free survival in PCa patients. Further we elucidated the tumor suppressor role of miR-23b using in vitro and in vivo models. We demonstrated 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 PCa. Over-expression 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 to a control miR. These findings suggest that miR-23b is a methylation-silenced tumor suppressor that may be useful biomarker in PCa. Loss of miR-23b may confer proliferative advantage and promote PCa migration and invasion and re-expression of miR-23b may contribute to the epigenetic therapy for PCa.
PMCID: PMC3940348  PMID: 23074286
MicroRNA-23b; Src kinase; prostate cancer
4.  Mismatch repair gene MSH3 polymorphism is associated with the risk of sporadic prostate cancer 
The Journal of urology  2008;179(5):2020-2024.
The mismatch repair (MMR) system is a DNA repair mechanism that corrects mispaired bases during DNA replication errors. Cancer cells deficient in the MMR proteins have a 102 –103-fold increase in the mutation rate. Single nucleotide polymorphisms (SNPs) of MMR genes have been shown to cause a reduction in DNA repair activity. We hypothesized that mismatch repair gene polymorphism could be a risk factor for prostate cancer (PC) and that p53 Pro/Pro genotype carriers could influence MSH3 and MSH6 polymorphisms.
Material and Methods
DNA samples from 110 cases of prostate cancer and healthy controls (n=110) were analyzed by SSCP and PCR-RFLP to determine the genotypic frequency of five different polymorphic loci on two MMR genes (MSH3 and MSH6) and p53 codon72. The chi-square test was applied to compare the genotype frequency between patients and controls.
A significant increase in the G/A+A/A genotype of MSH3 Pro222Pro was observed in patients compared to controls (OR, 1.87; 95% CI, 1.0–3.5). The frequency of A/G + G/G genotypes of MSH3 exon23 Thr1036Ala also tended to increase in patients (OR, 1.57; 95% CI, 0.92–2.72). Among p53 codon72 Arg/Pro + Pro/Pro carriers, the frequency of the AG + GG genotype of MSH3 exon23 was significantly increased in patients compared to controls (OR = 2.1, 95% CI; 1.05–4.34).
This is the first report on the association of MSH3 gene polymorphisms in prostate cancer. These results suggest that the MSH3 polymorphism may be a risk factor for prostate cancer.
PMCID: PMC3940351  PMID: 18355840
Polymorphism; mismatch repair gene; prostate cancer; p53
5.  MicroRNA-205 inhibits Src-mediated oncogenic pathways in renal cancer 
Cancer research  2011;71(7):2611-2621.
The Src family of protein kinases (SFKs) plays key roles in regulating fundamental cellular processes, including cell growth, differentiation, cell shape, migration and survival, and specialized cell signals in various malignancies. The pleotropic functions of SFKs in cancer make them promising targets for intervention. Here we sought to investigate the role of miR-205 in inhibition of Src-mediated oncogenic pathways in renal cancer. We report that expression of miR-205 was significantly suppressed in renal cancer cell lines and tumors when compared with normal tissues and a non-malignant cell line, and is correlated inversely with the expression of SFKs. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3’UTR sequences complementary to either Src, Lyn or Yes, which was abolished by mutations in these 3’UTR regions. Over-expression of miR-205 in A498 cells reduced Src, Lyn and Yes expression both at mRNA and protein levels. Proliferation of renal cancer cells was suppressed by miR-205, mediated by the phosphoSrc-regulated ERK1/2 pathway. Cell motility factor- FAK and STAT3 activation was also inhibited by miR-205. Transient as well as stable over-expression of miR-205 in A498 cells resulted in induction of G0/G1 cell cycle arrest and apoptosis as indicated by decreased levels of cyclin D1 and cMyc, suppressed cell proliferation, colony formation, migration, and invasion in renal cancer cells. miR-205 also inhibited tumor cell growth in vivo. This is the first study demonstrating that miRNA-205 inhibits protooncogenic Src family of kinases indicating a therapeutic potential of miR-205 in the treatment of renal cancer.
PMCID: PMC3940352  PMID: 21330408
MicroRNA-205; Src; Lyn; Yes; renal cancer
6.  Secreted frizzled-related protein-5 (sFRP-5) is epigenetically downregulated and functions as a tumor suppressor in kidney cancer 
Secreted frizzled-related protein-5 (sFRP-5) has been identified as one of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.
PMCID: PMC3940353  PMID: 20340127
RCC; sFRP-5; methylation; histone modification; tumor suppressor
7.  Superoxide dismutase analog (TEMPOL: 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine 1-oxyl) treatment restores erectile function in diabetes-induced impotence 
We hypothesized that administration of the superoxide dismutase (SOD) mimetic Tempol (4-hydroxy-2, 2, 6, 6-tetramethylpiperidine 1-oxyl) may reverse diabetes induced ED(erectile dysfunction). To test this hypothesis, ROS related genes (SOD1, SOD2, GPx1, CAT, NOS2, NOS3), erectile functional studies, and immunohistochemical analysis were performed in diabetic rats treated with or without Tempol. Thirty Sprague-Dawley (3–4 months old) rats were divided into 3 groups (n=10 each), 20 with diabetes (diabetic control and Tempol treatment) and 10 healthy controls. Twelve weeks after induction of diabetes by streptozotocin and Tempol treatment, all groups underwent in vivo cavernous nerve stimulation. Rat crura were harvested and expression of antioxidative defense enzymes examined by semi-quantitative RT-PCR. To confirm the RT-PCR results, we performed immunohistochemistry (IHC) for catalase (CAT) and iNOS (NOS2). Nitration of tyrosine groups in proteins was also examined by IHC. Mean intracavernous pressure in the diabetic group was significantly lower than in healthy controls (p<0.001) and was reversed by Tempol treatment (p<0.0108). NOS2 protein expression was significantly increased in diabetic animals compared to healthy controls and Tempol restored NOS2 protein level. Nitrotyrosine was also higher in diabetic animals and though Tempol treatment decreased its formation, it remained higher than that found in healthy controls. This study suggests that Tempol treatment increased erectile function through modulating oxidative stress related genes in diabetic rats. This is the first report about the relationship between diabetes induced erectile dysfunction and oxidative stress, and anti-oxidative therapy using the superoxide dismutase mimetic, Tempol to restore erectile function.
PMCID: PMC3940356  PMID: 19554009
oxidative stress; nitric oxide; superoxide; diabetes; erectile dysfunction
8.  MicroRNA-708 induces apoptosis and suppresses tumorigenicity in renal cancer cells 
Cancer research  2011;71(19):6208-6219.
Cancer pathogenesis is restricted by stresses that compromise cell division and survival. In this study we identify miR-708, a little studied member of a set of microRNAs that have been implicated in stress control, as an important tumor suppressor in renal cell carcinoma (RCC). miR-708 expression was attenuated widely in human RCC specimens. Restoration of miR-708 expression in RCC cell lines decreased cell growth, clonability, invasion, migration and elicited a dramatic increase in apoptosis. Moreover, intratumoral delivery of miR-708 was sufficient to trigger in vivo regression of established tumors in a murine xenograft model of human RCC. Investigation of the targets of miR-708 identified the small inhibitor of apoptosis protein survivin as important. siRNA-mediated knockdown of survivin partially phenocopied miR-708 overexpression suggesting that the pro-apoptotic role of miR-708 may be mediated primarily through survivin regulation. Additionally, we identified the E-cadherin regulators ZEB2 and BMI1 as likely miR-708 targets. Taken together, our findings define a major tumor suppressive role for miR-708, which may offer an attractive new target for prognostic and therapeutic intervention in RCC.
PMCID: PMC3940359  PMID: 21852381
miR-708; renal cancer; apoptosis; survivin
9.  MicroRNA-205 directed transcriptional activation of tumor suppressor genes in prostate cancer 
Cancer  2010;116(24):5637-5649.
MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of approximately 30% of all human genes. They play important roles in numerous cellular processes including development, proliferation, and apoptosis. It is currently believed that miRNAs elicit their effect by silencing the expression of target genes. Here we show that microRNA-205 (miR-205) induces the expression of IL24 and IL32 tumor suppressor genes by targeting specific sites in their promoters.
Methods used in this study include transfection of small RNAs, quantitative-real-time-PCR, in-situ hybridization, fluorescence labeled in-situ hybridization, cell cycle, apoptosis, cell viability, migratory, clonability and invasion assays, immunoblotting, luciferase reporter, nuclear run-on and chromatin immunoprecipitation assays.
Our results revealed that miR-205 is silenced in prostate cancer. Its re-expression induced apoptosis and cell cycle arrest. It also impaired cell growth, migration, clonability and invasiveness of prostate cancer cells. MicroRNA-205 induced tumor suppressor genes IL24 and IL32 at both mRNA and protein levels. Induction of in-vitro transcription and enrichment of markers for transcriptionally active promoters in IL24 and IL32 genes was observed in response to miR-205.
In this study we identify a new function for miR-205 to specifically activate tumor suppressor genes by targeting specific sites in their promoters. These results corroborate a new function that miRNAs have in regulating gene expression at the transcriptional level. The specific activation of tumor suppressor genes (e.g., IL24, IL32) or other dysregulated genes by microRNAs may contribute to the novel therapeutic approach in the treatment of prostate cancer.
PMCID: PMC3940365  PMID: 20737563
MicroRNA-205; IL24; IL32; Transcriptional activation; Prostate cancer
10.  MicroRNA-34b inhibits prostate cancer through demethylation, active chromatin modifications and AKT pathways 
MicroRNAs (miRNAs) can act as oncomirs or tumor-suppressor miRs in cancer. This study was undertaken to investigate the status and role of miR-34b in prostate cancer.
Experimental Design
Profiling of miR-34b was performed in human prostate cancer (PCa) cell lines and clinical samples by quantitative-real-time-PCR and in situ hybridization. Statistical analyses were done to assess diagnostic/prognostic potential. Biological significance was elucidated by performing a series of experiments in vitro and in vivo.
We report that miR-34b is silenced in human PCa and the mechanism is through CpG hypermethylation. miR-34b directly targeted methyltransferases (DNMT) and deacetylases (HDACs) resulting in a positive feedback loop inducing partial demethylation and active chromatin modifications. miR-34b expression could predict overall and recurrence free survival such that patients with high miR-34b levels had longer survival. Functionally miR-34b inhibited cell proliferation, colony formation, migration/invasion and triggered G0/G1 cell cycle arrest and apoptosis by directly targeting the AKT and its downstream proliferative genes. miR-34b caused a decline in the mesenchymal markers Vimentin, ZO1, N-cadherin and Snail with an increase in E-cadherin expression, thus inhibiting epithelial to mesenchymal transition. Finally we demonstrated the antitumor effect of miR-34b in vivo. MiR-34b caused a dramatic decrease in tumor growth in nude mice compared to cont-miR.
These findings offer new insight into the role of miR-34b in the inhibition of PCa through demethylation, active chromatin modification and AKT pathways and may provide a rationale for the development of new strategies targeting epigenetic regulation of miRNAs for the treatment of PCa.
PMCID: PMC3910324  PMID: 23147995
MicroRNA-34b; Methylation; DNMT; HDAC; prostate cancer
11.  MicroRNAs 221/222 and Genistein mediated regulation of ARHI tumor suppressor gene in prostate cancer 
ARHI, an imprinted tumor suppressor gene, is expressed in normal immortalized prostate epithelial cells, but is dramatically down-regulated in prostate cancer cell lines. Here we investigated the mechanisms of ARHI silencing in prostate cancer through miRNA and genistein mediated pathways.
We evaluated ARHI mRNA and protein levels by real time PCR and immunostaining of prostate tissue array. Then, ARHI was over-expressed in prostate cancer PC-3 cells followed by functional studies. Finally, miRNA inhibitor studies and dual luciferase pMIR-REPORT assay were performed to prove the direct target of miR-221&222 to ARHI.
Both ARHI mRNA and protein levels were down regulated in prostate cancer tissues compared to adjacent normal tissues. Over-expression of ARHI can inhibit cell proliferation, colony formation, invasion and induced apoptosis. Further studies on a new mechanism of ARHI down regulation showed a significant inverse relationship between ARHI and miR-221 & 222 which were up-regulated in cancer cell lines. Transfection of miR-221 & 222 inhibitors into PC-3 cells caused a significant induction of ARHI expression. A direct interaction of miR-221 or 222 with a target site on the 3’UTR of ARHI was confirmed by a dual luciferase pMIR-REPORT assay.
ARHI is a tumor suppressor gene down regulated in prostate cancer and over-expression of ARHI can inhibit cell proliferation, colony formation and invasion. This study demonstrates for the first time that prostate cancer cells have decreased level of ARHI which could be caused by direct targeting of 3’UTR of ARHI by miR221/222.
PMCID: PMC3894108  PMID: 21071579
ARHI; prostate cancer; miRNA; tumor suppressor gene
12.  Combining Molecular Targeted Drugs to Inhibit Both Cancer Cells and Activated Stromal Cells in Gastric Cancer1 
Neoplasia (New York, N.Y.)  2013;15(12):1391-1399.
Recent studies have revealed that PDGF plays a role in promoting progressive tumor growth in several cancers, including gastric cancer. Cancer-associated fibroblasts, pericytes, and lymphatic endothelial cells in stroma express high levels of PDGF receptor (PDGF-R); cancer cells and vascular endothelial cells do not. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that increases the production of proteins that stimulate key cellular processes such as cell growth and proliferation, cell metabolism, and angiogenesis. In the present study, we examined the effects of PDGF-R tyrosine kinase inhibitor (nilotinib) and mTOR inhibitor (everolimus) on tumor stroma in an orthotopic nude mice model of human gastric cancer. Expression of PDGF-B and PDGF-Rβ mRNAs was associated with stromal volume. Treatment with nilotinib did not suppress tumor growth but significantly decreased stromal reactivity, lymphatic invasion, lymphatic vessel area, and pericyte coverage of tumor microvessels. In contrast, treatment with everolimus decreased tumor growth and microvessel density but not stromal reactivity. Nilotinib and everolimus in combination reduced both the growth rate and stromal reaction. Target molecule-based inhibition of cancer-stromal cell interaction appears promising as an effective antitumor therapy.
PMCID: PMC3884530  PMID: 24403861
13.  MicroRNA-4723 Inhibits Prostate Cancer Growth through Inactivation of the Abelson Family of Nonreceptor Protein Tyrosine Kinases 
PLoS ONE  2013;8(11):e78023.
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.
PMCID: PMC3815229  PMID: 24223753
14.  Genistein Inhibits Prostate Cancer Cell Growth by Targeting miR-34a and Oncogenic HOTAIR 
PLoS ONE  2013;8(8):e70372.
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.
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.
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.
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.
PMCID: PMC3731248  PMID: 23936419
15.  MicroRNA-23b Functions as a Tumor Suppressor by Regulating Zeb1 in Bladder Cancer 
PLoS ONE  2013;8(7):e67686.
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.
PMCID: PMC3699593  PMID: 23844063
16.  Genistein Up-Regulates Tumor Suppressor MicroRNA-574-3p in Prostate Cancer 
PLoS ONE  2013;8(3):e58929.
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.
PMCID: PMC3595226  PMID: 23554959
17.  MicroRNA-34a suppresses malignant transformation by targeting c-Myc transcriptional complexes in human renal cell carcinoma 
Carcinogenesis  2011;33(2):294-300.
We investigated the functional effects of microRNA-34a (miR-34a) on c-Myc transcriptional complexes in renal cell carcinoma. miR-34a down-regulated expression of multiple oncogenes including c-Myc by targeting its 3′ untranslated region, which was revealed by luciferase reporter assays. miR-34a was also found to repress RhoA expression by suppressing the c-Myc–Skp2–Miz1 transcriptional complex that activates RhoA. Overexpression of c-Myc reversed miR-34a suppression of RhoA expression and inhibition of cell invasion, suggesting that miR-34a inhibits invasion by suppressing RhoA through c-Myc. miR-34a was also found to repress the c-Myc–P-TEFb transcription elongation complex, indicating one of the mechanisms by which miR-34a has profound effects on cellular functions. Our results demonstrate that miR-34a suppresses assembly and function of the c-Myc complex that activates or elongates transcription, indicating a novel role of miR-34a in the regulation of transcription by c-Myc.
PMCID: PMC3271269  PMID: 22159222
18.  MicroRNA-182-5p Promotes Cell Invasion and Proliferation by Down Regulating FOXF2, RECK and MTSS1 Genes in Human Prostate Cancer 
PLoS ONE  2013;8(1):e55502.
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.
PMCID: PMC3559583  PMID: 23383207
19.  Oncogenic miRNA-182-5p Targets Smad4 and RECK in Human Bladder Cancer 
PLoS ONE  2012;7(11):e51056.
Onco-miR-182-5p has been reported to be over-expressed in bladder cancer (BC) tissues however a detailed functional analysis of miR-182-5p has not been carried out in BC. Therefore the purpose of this study was to: 1. conduct a functional analysis of miR-182-5p in bladder cancer, 2. assess its usefulness as a tumor marker, 3. identify miR-182-5p target genes in BC. Initially we found that miR-182-5p expression was significantly higher in bladder cancer compared to normal tissues and high miR-182-5p expression was associated with shorter overall survival in BC patients. To study the functional significance of miR-182-5p, we over-expressed miR-182-5p with miR-182-5p precursor and observed that cell proliferation, migration and invasion abilities were increased in BC cells. However cell apoptosis was inhibited by miR-182-5p. We also identified Smad4 and RECK as potential target genes of miR-182-5p using several algorithms. 3′UTR luciferase activity of these target genes was significantly decreased and protein expression of these target genes was significantly up-regulated in miR-182-5p inhibitor transfected bladder cancer cells. MiR-182-5p also increased nuclear beta-catenin expression and while Smad4 repressed nuclear beta-catenin expression. In conclusion, our data suggests that miR-182-5p plays an important role as an oncogene by knocking down RECK and Smad4, resulting in activation of the Wnt-beta-catenin signaling pathway in bladder cancer.
PMCID: PMC3511415  PMID: 23226455
20.  Inhibition of PTEN Gene Expression by Oncogenic miR-23b-3p in Renal Cancer 
PLoS ONE  2012;7(11):e50203.
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.
Methods and Results
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.
PMCID: PMC3506541  PMID: 23189187
21.  MicroRNA-1280 Inhibits Invasion and Metastasis by Targeting ROCK1 in Bladder Cancer 
PLoS ONE  2012;7(10):e46743.
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.
PMCID: PMC3464246  PMID: 23056431
22.  Inhibition of transcellular tumor cell migration and metastasis by novel carba-derivatives of cyclic phosphatidic acid 
Biochimica et biophysica acta  2006;1771(1):103-112.
Cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3-cyclic phosphate; cPA) is a naturally occurring analog of lysophosphatidic acid (LPA) with a variety of distinctly different biological activities from those of LPA. In contrast to LPA, a potent inducer of tumor cell invasion, palmitoyl-cPA inhibits FBS- and LPA-induced transcellular migration and metastasis. To prevent the conversion of cPA to LPA we synthesized cPA derivatives by stabilizing the cyclic phosphate ring; to prevent the cleavage of the fatty acid we generated alkyl ether analogs of cPA. Both sets of compounds were tested for inhibitory activity on transcellular tumor cell migration. Carba derivatives, in which the phosphate oxygen was replaced with a methylene group at either the sn-2 or the sn-3 position, showed much more potent inhibitory effects on MM1 tumor cell transcellular migration and the pulmonary metastasis of B16-F0 melanoma than the natural pal-cPA. The antimetastatic effect of carba-cPA was accompanied by the inhibition of RhoA activation and was not due to inhibition of the activation of LPA receptors.
PMCID: PMC3446789  PMID: 17123862
Cyclic phosphatidic acid; Lysophosphatidic acid; LPA; Invasion; Metastasis
23.  Neuroimaging characteristics and growth pattern on magnetic resonance imaging in a 52-year-old man presenting with pituicytoma: a case report 
Pituicytoma is a rare neoplasm of the neurohypophysis. To the best of our knowledge there have been no reports of pituicytoma in which long-term magnetic resonance imaging observation was performed. We calculated the doubling time of the tumor volume and described the growth pattern of a pituicytoma.
Case presentation
A 52-year-old Japanese man with a history of decreased libido was found to have a sellar and suprasellar mass. He underwent transsphenoidal surgery, but only a small specimen was obtained because of intraoperative bleeding. The tentative histological diagnosis was schwannoma. He noticed bitemporal hemianopsia 7 years later. A follow-up magnetic resonance imaging disclosed a tumor volume doubling time of 3830 days. Transcranial gross-total tumor resection was performed. The lesion consisted of elongated and plump tumor cells that were arranged in a fascicular or storiform pattern and were positive for S-100 protein and focally positive for glial fibrillary acidic protein. The final histological diagnosis was pituicytoma.
Pituicytoma is a slow-growing tumor, but the growth rate may change during follow-up.
PMCID: PMC3537696  PMID: 22989192
Neurohypophysis; Pituicyte; Pituicytoma; Tumor volume doubling time
24.  Genistein Suppresses Prostate Cancer Growth through Inhibition of Oncogenic MicroRNA-151 
PLoS ONE  2012;7(8):e43812.
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.
PMCID: PMC3426544  PMID: 22928040
25.  MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer 
Carcinogenesis  2011;32(5):772-778.
MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2′-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer.
PMCID: PMC3086703  PMID: 21349819

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