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The molecular heterogeneity of renal cell carcinoma (RCC) complicates the therapeutic interventions for advanced metastatic disease and thus its management remains a significant challenge. This study investigates the role of the lncRNA CDKN2B-AS1 and miR-141-3p interactions in the progression and metastasis of kidney cancer. Human renal cancer cell lines (ACHN and Caki1), normal RPTEC cells, tissue cohorts, and a series of in vitro assays and in vivo mouse model were used for this study. An overexpression of CDKN2B-AS1 was observed in RCC compared to normal samples in TCGA and our in-house SFVAMC tissue cohorts. Reciprocally, we observed reduced expression of miR-141 in RCC compared to normal in the same cohorts. CDKN2B-AS1 shares regulatory miR-141 binding sites with CCND1 and CCND2 genes. Direct interactions of CDKN2B-AS1/miR-141/Cyclin D1-D2 were confirmed by RNA immunoprecipitation and luciferase reporter assays indicating that CDKN2B-AS1/miR-141/Cyclin D1-D2 acts as a ceRNA network in RCC. Functionally, attenuation of CDKN2B-AS1 and/or overexpression of miR-141 inhibited proliferation, clonogenicity, migration/invasion, induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model. Further, overexpression of CDKN2B-AS1 is positively correlated with poor overall survival of RCC patients. Expression of miR-141 also robustly discriminated malignant from non-malignant tissues and its inhibition in normal RPTEC cells induced pro-cancerous characteristics. CDKN2B-AS1 attenuation or miR-141 overexpression decreased CCND1/CCND2 expression, resulting in reduced RAC1/pPXN that are involved in migration, invasion and epithelial-mesenchymal transition. This study, for the first time, deciphered the role of CDKN2B-AS1/miR-141/Cyclin D axis in RCC and highlights this network as a promising therapeutic target for the regulation of EMT driven metastasis in RCC.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and how lifestyle choices may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population was analysed at polymorphic sites of the 5' untranslated region of CYP1B1 using TaqMan genotyping assays. Overall, drinker status and minor alleles at rs2551188, rs2567206 and rs10175368 were associated with prostate cancer. Linkage was observed between rs2551188, rs2567206, rs2567207 and rs10175368, and the G-C-T-G haplotype (major allele at respective sites) was decreased in cancer. Interestingly when classified by lifestyle factors, no associations of genotypes were found for non-smokers and non-drinkers, whereas on the contrary, minor type at rs2567206 and rs10175368 increased and major G-C-T-G decreased risk for cancer among smokers and drinkers. Interestingly, rs2551188, rs2567206 and rs10175368 minor genotypes correlated with increased tissue CYP1B1 as determined by immunohistochemistry. Further, rs10175368 enhanced luciferase activity and mobility shift show stronger binding of nuclear factor for the minor allele. These results demonstrate smoking and alcohol consumption to modify the risks of CYP1B1 polymorphisms for prostate cancer which may be through rs10175368, and this is of importance in understanding their role in the pathogenesis and as a biomarker for this disease.

Non-coding RNA (ncRNA) has been shown to regulate diverse cellular processes and functions through controlling gene expression. Long non-coding RNAs (lncRNAs) act as a competing endogenous RNAs (ceRNAs) where microRNAs (miRNAs) and lncRNAs regulate each other through their biding sites. Interactions of miRNAs and lncRNAs have been reported to trigger decay of the targeted lncRNAs and have important roles in target gene regulation. These interactions form complicated and intertwined networks. Certain lncRNAs encode miRNAs and small nucleolar RNAs (snoRNAs), and may regulate expression of these small RNAs as precursors. SnoRNAs have also been reported to be precursors for PIWI-interacting RNAs (piRNAs) and thus may regulate the piRNAs as a precursor. These miRNAs and piRNAs target messenger RNAs (mRNAs) and regulate gene expression. In this review, we will present and discuss these interactions, cross-talk, and co-regulation of ncRNAs and gene regulation due to these interactions.

MicroRNAs (miRNAs) have emerged as key players in cancer progression and metastatic initiation yet their importance in regulating prostate cancer (PCa) metastasis to bone has begun to be appreciated. We employed multimodal strategy based on in-house PCa clinical samples, publicly available TCGA cohorts, a panel of cell lines, in silico analyses, and a series of in vitro and in vivo assays to investigate the role of miR-466 in PCa. Expression analyses revealed that miR-466 is under-expressed in PCa compared to normal tissues. Reconstitution of miR-466 in metastatic PCa cell lines impaired their oncogenic functions such as cell proliferation, migration/invasion and induced cell cycle arrest, and apoptosis compared to control miRNA. Conversely, attenuation of miR-466 in normal prostate cells induced tumorigenic characteristics. miR-466 suppressed PCa growth and metastasis through direct targeting of bone-related transcription factor RUNX2. Overexpression of miR-466 caused a marked downregulation of integrated network of RUNX2 target genes such as osteopontin, osteocalcin, ANGPTs, MMP11 including Fyn, pAkt, FAK and vimentin that are known to be involved in migration, invasion, angiogenesis, EMT and metastasis. Xenograft models indicate that miR-466 inhibits primary orthotopic tumor growth and spontaneous metastasis to bone. Receiver operating curve and Kaplan-Meier analyses show that miR-466 expression can discriminate between malignant and normal prostate tissues; and can predict biochemical relapse. In conclusion, our data strongly suggests miR-466-mediated attenuation of RUNX2 as a novel therapeutic approach to regulate PCa growth, particularly metastasis to bone. This study is the first report documenting the anti-bone metastatic role and clinical significance of miR-466 in prostate cancer.

Silibinin is the major active constituent of silymarin, an extract of milk thistle seeds. Silibinin has been shown to have significant anti-cancer effects in a variety of malignancies. However, the molecular mechanisms of silibinin action in bladder cancer have not been studied extensively. In the present study, we found that silibinin (10 μM) significantly suppressed proliferation, migration, invasion and induced apoptosis of T24 and UM-UC-3 human bladder cancer cells. Silibinin down-regulated the actin cytoskeleton and phosphatidylinositide 3-kinase (PI3K)/Akt signaling pathways in these cancer cell lines. These pathways were found to crosstalk through RAS cascades. We found that silibinin suppressed levels of trimethylated histone H3 lysine 4 and acetylated H3 at the KRAS promoter. Furthermore, silibinin targets long non-coding RNA: HOTAIR and ZFAS1, which are known to play roles as oncogenic factors in various cancers. This study shows that silibinin exerts anti-cancer effects through down-regulation of actin cytoskeleton and PI3K/Akt pathways and thus suppresses bladder cancer growth and progression.

The incidence of prostate cancer (PCa) among African-Americans (AfA) is significantly higher than Caucasian-Americans (CaA) but the genetic basis for this disparity is not known. To address this problem, we analyzed miRNA expression in AfA (n = 81) and CaA (n = 51) PCa patients. Here, we found that miR-24 is differentially expressed in AfA and CaA PCa patients and attempt to clarify its role in AfA patients. Also, the public sequencing data of the miR-24 promoter confirmed that it was highly methylated and down-regulated in PCa patients. Utilizing a VAMCSF and NDRI patient cohorts, we discovered that miR-24 expression was linked to a racial difference between AfA/CaA PCa patients. Interestingly, miR-24 was restored after treatment of PCa cells with 5Aza-CdR in an AfA cell line (MDA-PCa-2b), while restoration of miR-24 was not observed in CaA cells, DU-145. Ectopic expression of miR-24 showed decreased growth and induced apoptosis, though the effect was less in the CaA cell line compared to the AfA cell line. Finally, we found unique changes in biological pathways and processes associated with miR-24 transfected AfA cells by quantitative PCR-based gene expression array. Evaluation of the altered pathways showed that AR, IGF1, IGFBP5 and ETV1 were markedly decreased in the AfA derived cell line compared with CaA cells, and there was a reciprocal regulatory relationship of miR-24/target expression in prostate cancer patients. These results demonstrate that miR-24 may be a central regulator of key events that contribute to race-related tumorigenesis and has potential to be a therapeutic agent for PCa treatment.

African-Americans are diagnosed with more aggressive prostate cancers and have worse survival than Caucasians, however a comprehensive understanding of this health disparity remains unclear. To clarify the mechanisms leading to this disparity, we analyzed the potential involvement of miR-34b expression in African-Americans and Caucasians. miR-34b functions as a tumor suppressor and has a multi-functional role, through regulation of cell proliferation, cell cycle and apoptosis. We found that miR-34b expression is lower in human prostate cancer tissues from African-Americans compared to Caucasians. DNA hypermethylation of the miR-34b-3p promoter region showed significantly higher methylation in prostate cancer compared to normal samples. We then sequenced the promoter region of miR-34b-3p and found a chromosomal deletion in miR-34b in African-American prostate cancer cell line (MDA-PCA-2b) and not in Caucasian cell line (DU-145). We found that AR and ETV1 genes are differentially expressed in MDA-PCa-2b and DU-145 cells after overexpression of miR-34b. Direct interaction of miR-34b with the 3' untranslated region of AR and ETV1 was validated by luciferase reporter assay. We found that miR-34b downregulation in African-Americans is inversely correlated with high AR levels that lead to increased cell proliferation. Overexpression of miR-34b in cell lines showed higher inhibition of cell proliferation, apoptosis and G1 arrest in the African-American cells (MDA-PCa-2b) compared to Caucasian cell line (DU-145). Taken together, our results show that differential expression of miR-34b and AR are associated with prostate cancer aggressiveness in African-Americans.

MicroRNAs (miRNAs) have emerged as key players in cancer progression and metastatic initiation yet their importance in regulating prostate cancer (PCa) metastasis to bone has begun to be appreciated. We employed multimodal strategy based on in-house PCa clinical samples, publicly available TCGA cohorts, a panel of cell lines, in silico analyses, and a series of in vitro and in vivo assays to investigate the role of miR-466 in PCa. Expression analyses revealed that miR-466 is under-expressed in PCa compared to normal tissues. Reconstitution of miR-466 in metastatic PCa cell lines impaired their oncogenic functions such as cell proliferation, migration/invasion and induced cell cycle arrest, and apoptosis compared to control miRNA. Conversely, attenuation of miR-466 in normal prostate cells induced tumorigenic characteristics. miR-466 suppressed PCa growth and metastasis through direct targeting of bone-related transcription factor RUNX2. Overexpression of miR-466 caused a marked downregulation of integrated network of RUNX2 target genes such as osteopontin, osteocalcin, ANGPTs, MMP11 including Fyn, pAkt, FAK and vimentin that are known to be involved in migration, invasion, angiogenesis, EMT and metastasis. Xenograft models indicate that miR-466 inhibits primary orthotopic tumor growth and spontaneous metastasis to bone. Receiver operating curve and Kaplan-Meier analyses show that miR-466 expression can discriminate between malignant and normal prostate tissues; and can predict biochemical relapse. In conclusion, our data strongly suggests miR-466-mediated attenuation of RUNX2 as a novel therapeutic approach to regulate PCa growth, particularly metastasis to bone. This study is the first report documenting the anti-bone metastatic role and clinical significance of miR-466 in prostate cancer.

Genomic loss of chromosome (chr) 8p21 region, containing prostate-specific NKX3.1 gene, is a frequent alteration of the prostate cancer (PCa) oncogenome. We propose a novel, paradigm shifting hypothesis that this frequently deleted locus is also associated with a cluster of microRNA genes- miR-3622a/b- that are lost in PCa and play an important mechanistic role in progression and metastasis. In this study, we demonstrate the role of miR-3622b in prostate cancer. Expression analyses in a cohort of PCa clinical specimens and cell lines show that miR-3622b expression is frequently lost in prostate cancer. Low miR-3622b expression was found to be associated with tumor progression and poor biochemical recurrence-free survival. Further, our analyses suggest that miR-3622b expression is a promising prostate cancer diagnostic biomarker that exhibits 100% specificity and 66% sensitivity. Restoration of miR-3622b expression in PCa cell lines led to reduced cellular viability, proliferation, invasiveness, migration and increased apoptosis. miR-3622b overexpression in vivo induced regression of established prostate tumor xenografts pointing to its therapeutic potential. Further, we found that miR-3622b directly represses Epidermal Growth Factor Receptor (EGFR). In conclusion, our study suggests that miR-3622b plays a tumor suppressive role and is frequently downregulated in prostate cancer, leading to EGFR upregulation. Importantly, miR-3622b has associated diagnostic, prognostic and therapeutic potential. Considering the association of chr8p21 loss with poor prognosis, our findings are highly significant and support a novel concept that associates a long standing observation of frequent loss of a chromosomal region with a novel miRNA in prostate cancer.

Prostate carcinogenesis involves alterations in several signaling pathways, the most prominent being the PI3K/AKT pathway. This pathway is constitutively active and drives prostate cancer (PCa) progression to advanced metastatic disease. PTEN, a critical tumor and metastasis suppressor gene negatively regulates cell survival, proliferation, migration and angiogenesis via the PI3K/Akt pathway. PTEN is mutated, downregulated/dysfunctional in many cancers and its dysregulation correlates with poor prognosis in PCa. Here, we demonstrate that microRNA-4534 (miR-4534) is overexpressed in PCa and show that miR-4534 is hypermethylated in normal tissues and cell lines compared to PCa tissues/cells. miR-4534 exerts its oncogenic effects partly by downregulating the tumor suppressor PTEN gene. Knockdown of miR-4534 impaired cell proliferation, migration/invasion and induced G0/G1 cell cycle arrest and apoptosis in PCa. Suppression of miR-4534 and its effects on tumor growth was confirmed in a xenograft mouse model. We performed parallel experiments in non-cancer RWPE1 cells by overexpessing miR-4534 followed by functional assays. Overexpression of miR-4534 induced pro-cancerous characteristics in this non-cancer cell line. Statistical analyses revealed that miR-4534 has potential to independently distinguish malignant from normal tissues and positively correlated with poor overall and PSA recurrence free survival. Taken together, our results show that depletion of miR-4534 in PCa induces a tumor suppressor phenotype partly through induction of PTEN. These results have important implications for identifying and defining the role of new PTEN regulators such as microRNAs in prostate tumorigenesis. Understanding aberrantly overexpressed miR-4534 and its downregulation of PTEN will provide mechanistic insight and therapeutic targets for PCa therapy.

Cytochrome P450 (CYP) 1A1 is a phase I enzyme that can activate various compounds into reactive forms and thus, may contribute to carcinogenesis. In this study, we investigated the expression, methylation status, and functional role of CYP1A1 on prostate cancer cells. Increased expression of CYP1A1 was observed in all cancer lines (PC-3, LNCaP, and DU145) compared to BPH-1 (P < 0.05); and was enhanced further by 5-aza-2'-deoxycytidine treatment (P < 0.01). Methylation-specific PCR (MSP) and sequencing of bisulfite-modified DNA of the xenobiotic response element (XRE) enhancer site XRE-1383 indicated promoter methylation as a regulator of CYP1A1 expression. In tissue, microarrays showed higher immunostaining of CYP1A1 in prostate cancer than normal and benign prostatic hyperplasia (BPH; P < 0.001), and methylation analyses in clinical specimens revealed significantly lower methylation levels in cancer compared to BPH at all enhancer sites analyzed (XRE-1383, XRE-983, XRE-895; P < 0.01). Interestingly, smoking affected the XRE-1383 site where the methylation level was much lower in cancer tissues from smokers than non-smokers (P < 0.05). CYP1A1 levels are thus increased in prostate cancer and to determine the functional effect of CYP1A1 on cells, we depleted the gene in LNCaP and DU145 by siRNA. We observe that CYP1A1 knockdown decreased cell proliferation (P < 0.05) and increased apoptosis (P < 0.01) in both cell lines. We analyzed genes affected by CYP1A1 silencing and found that apoptosis-related BCL2 was significantly down-regulated. This study supports an oncogenic role for CYP1A1 in prostate cancer via promoter hypomethylation that is influenced by tobacco smoking, indicating CYP1A1 to be a promising target for prostate cancer treatment.

BackgroundCytochrome 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.MethodsExpression 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.ResultsFirst, 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

Genomic studies suggest that deletions at chromosome (chr) 5q region (particularly chr5q14-q23) are frequent in prostate cancer, implicating this region in prostate carcinogenesis. However, the genes within this region are largely unknown. Here, we report for the first time the widespread attenuation of miR-3607, an miRNA gene located at chr5q14 region, in prostate cancer. Expression analyses of miR-3607 in a clinical cohort of prostate cancer specimens showed that miR-3607 is significantly attenuated and low miR-3607 expression is correlated with tumor progression and poor survival outcome in prostate cancer. Our analyses suggest that miR-3607 expression may be a clinically significant parameter with an associated diagnostic potential. We examined the functional significance of miR-3607 in prostate cancer cell lines and found that miR-3607 overexpression led to significantly decreased proliferation, apoptosis induction, and decreased invasiveness. Furthermore, our results suggest that miR-3607 directly represses oncogenic SRC family kinases LYN and SRC in prostate cancer. In view of our results, we propose that miR-3607 plays a tumor-suppressive role in prostate cancer by regulating SRC kinases that in turn regulates prostate carcinogenesis. To our knowledge, this is the first report that: (i) identifies a novel role for miR-3607 located in a frequently deleted region of prostate cancer and (ii) defines novel miRNA-mediated regulation of SRC kinases in prostate cancer. Because SRC kinases play a central role in prostate cancer progression and metastasis and are attractive targets, this study has potential implications in the design of better therapeutic modalities for prostate cancer management.

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.

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.

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.

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 3UTR 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.

BackgroundMicroRNA-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 resultsWe 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.ConclusionsThe 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.

Background/Aims: Genistein, a soy isoflavone, has been shown to have anti-cancer effects in various cancers including renal cancer. Long non-coding RNA, HOX transcript antisense RNA (HOTAIR), is involved in cancer progression and metastasis, such as renal cancer. Our aim was to investigate the effects of genistein on HOTAIR chromatin remodeling functions. Methods: We used MTS assays and Transwell migration assays to study the effects of genistein on cell proliferation and migration respectively in human renal cell carcinoma (RCC) cell lines. We used Western blots to analyze SNAIL and ZO-1 expression. We performed chromatin immunoprecipitation (ChIP) assays to study recruitment of the polycomb repressive complex 2 (PRC2) to the ZO-1 promoter. We performed RNA immunoprecipitation (RIP) assays to study interaction between HOTAIR and PRC2, SMARCB1 or ARID1A. We also performed transfection experiments to overexpress EED, HOTAIR and knockdown SMARCB1. Results: Genistein reduced cell proliferation and migration of human renal cell carcinoma cell lines. ChIP assays indicated that genistein reduces recruitment of the PRC2 to the ZO-1 promoter and increased its expression. RIP assays showed that genistein inhibits HOTAIR interaction with PRC2, leading to tumor suppression. Immunoprecipitation also revealed that genistein reduced EED levels in PRC2, suggesting that decreased EED levels suppress HOTAIR interaction with PRC2. EED overexpression in the presence of genistein restored PRC2 interaction with HOTAIR and reduced ZO-1 transcription, suggesting genistein activates ZO-1 by inhibiting HOTAIR/PRC2 functions. RIP assays also showed that HOTAIR interacts with SMARCB1 and ARID1A, subunits of the human SWI/SNF chromatin remodeling complex and genistein reduces this interaction. Combination of HOTAIR overexpression and SMARCB1 knockdown in the presence of genistein revealed that genistein inhibits SNAIL transcription via the HOTAIR/SMARCB1 pathway. Conclusion: Genistein suppresses EED levels in PRC2 and inhibits HOTAIR/ PRC2 interaction. Genistein suppresses HOTAIR/PRC2 recruitment to the ZO-1 promoter and enhances ZO-1 transcription. Genistein also inhibits SNAIL transcription via reducing HOTAIR/SMARCB1 interaction. We demonstrate that the reduction of HOTAIR interaction with chromatin remodeling factors by genistein represses HOTAIR/chromatin remodeling pathways to suppress RCC malignancy. [ABSTRACT FROM AUTHOR]