Secreted frizzled-related protein-5 is epigenetically downregulated and functions as a tumor suppressor in kidney cancer.

Secreted frizzled-related protein-5 (sFRP-5) has been identified as 1 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.

MiR-96 and miR-183 detection in urine serve as potential tumor markers of urothelial carcinoma: correlation with stage and grade, and comparison with urinary cytology.

A new diagnostic marker for urothelial carcinoma (UC) is needed to avoid painful cystoscopy during the initial diagnosis and follow-up period. However, the current urine markers are useless because of the low sensitivities and specificities for UC detection. MiR-96 and miR-183 were differentially upregulated microRNA in our previous microRNA screening for UC. The expression levels of miR-96 and miR-183 in the urine samples were significantly higher in 100 UC than in healthy controls (miR-96, P=0.0059; and miR-183, P=0.0044). The receiver-operating characteristic curve analyses demonstrated that each microRNA had good sensitivity and specificity for distinguishing UC patients from non-UC patients (miR-96, 71.0% and 89.2%; and miR-183, 74.0% and 77.3%). Our cohort included 78 UC patients who had undergone urinary cytology. MiR-96 was positively detected in 27 of 44 patients who had had a "negative" urinary cytology diagnosis. We combined the miR-96 detection data with the urinary cytology data, and diagnosed 61 of 78 cases as UC; sensitivity rose from 43.6% to 78.2%. We found significant stepwise increases in miR-96 and miR-183 expression with advancing tumor grade (miR-96, P=0.0057; and miR-183, P=0.0036) and pathological stage (miR-96, P=0.0332; and miR-183, P=0.0117). The expression levels of the microRNA were significantly lower in urine collected after surgery (miR-96, P=0.0241; and miR-183, P=0.0045). In conclusion, miR-96 and miR-183 in urine are promising tumor markers for UC. In particular, miR-96 may be a good diagnostic marker in combination with urinary cytology.

SWAP70, actin-binding protein, function as an oncogene targeting tumor-suppressive miR-145 in prostate cancer.

BACKGROUND: MiR-145 is down-regulated in various human cancers. We previously demonstrated that some actin-binding proteins were targeted by several microRNAs (miRNAs), including miR-145, in bladder and prostate cancer (CaP). The aim of this study is to determine a novel oncogenic gene targeted by miR-145 by focusing on actin-binding proteins in CaP. METHODS: We focused on the SWAP switching B-cell complex 70 kDa subunit (SWAP70), which is an F-actin binding protein involved in activating B-cell transformation. A luciferase reporter assay was used to identify the actual binding sites between miR-145 and SWAP70 mRNA. Cell viability was evaluated by cell proliferation, wound healing, and matrigel invasion assays in si-SWAP70 transfectants. A total of 75 clinical prostate specimens were subjected to immunohistochemistry of SWAP70. RESULTS: Molecular target searches of this miRNA and the luciferase reporter assay showed that SWAP70 was directly regulated by miR-145. Silencing of SWAP70 studies demonstrated significant inhibitions of cell migration and invasion in CaP cell lines. The SWAP70 positive-staining was significantly higher in percentage in the CaP than in benign prostate hyperplasia tissue. CONCLUSIONS: Down-regulation of miR-145 was a frequent event in CaP, and it may have a tumor suppressive function. SWAP70 may be a target of miR-145, and it might have a potential oncogenic function. The novel molecular networks though which miR-145 acts, may provide new insights into the underlying molecular mechanisms of CaP.

Tumor suppressive microRNA-375 regulates oncogene AEG-1/MTDH in head and neck squamous cell carcinoma (HNSCC).

Our microRNA (miRNA) expression signatures of hypopharyngeal squamous cell carcinoma, maxillary sinus squamous cell carcinoma and esophageal squamous cell carcinoma revealed that miR-375 was significantly reduced in cancer tissues compared with normal epithelium. In this study, we focused on the functional significance of miR-375 in cancer cells and identification of miR-375-regulated novel cancer networks in head and neck squamous cell carcinoma (HNSCC). Restoration of miR-375 showed significant inhibition of cell proliferation and induction of cell apoptosis in SAS and FaDu cell lines, suggesting that miR-375 functions as a tumor suppressor. We adopted genome-wide gene expression analysis to search for miR-375-regulated molecular targets. Gene expression data and luciferase reporter assays revealed that AEG-1/MTDH was directly regulated by miR-375. Cancer cell proliferation was significantly inhibited in HNSCC cells transfected with si-AEG-1/MTDH. In addition, expression levels of AEG-1/MTDH were significantly upregulated in cancer tissues. Therefore, AEG-1/MTDH may function as an oncogene in HNSCC. The identification of novel tumor suppressive miRNA and its regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC oncogenesis.

BTG3 tumor suppressor gene promoter demethylation, histone modification and cell cycle arrest by genistein in renal cancer.

BTG3/ANA/APRO4 has been reported to be a tumor suppressor gene in some malignancies. It constitutes important negative regulatory mechanism for Src-mediated signaling, a negative regulator of the cell cycle and inhibits transcription factor E2F1. We report that BTG3 is downregulated in renal cancer and that the mechanism of inactivation is through promoter hypermethylation. Quantitative real-time polymerase chain reaction (PCR) showed that BTG3 was downregulated in cancer tissues and cells. Genistein and 5-aza-2'-deoxycytidine (5Aza-C) induced BTG3 messenger RNA (mRNA) expression in A498, ACHN and HEK-293 renal cell carcinoma (RCC) cell lines. Bisulfite-modified PCR and DNA sequencing results showed complete methylation of BTG3 promoter in tumor samples and cancer cell lines. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Chromatin immunoprecipitation assay revealed that genistein and 5Aza-C increased levels of acetylated histones 3, 4, 2H3K4, 3H3K4 and RNA polymerase II at the BTG3 promoter indicative of active histone modifications. Enzymatic assays showed genistein and 5Aza-C decreased DNA Methyltransferase, methyl-CpG-binding domain 2 activity and increased HAT activity. Cell cycle and 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide cell proliferation assays showed that genistein has antiproliferative effect on cancer cell growth through induction of cell cycle arrest. This is the first report to show that BTG3 is epigenetically silenced in RCC and can be reactivated by genistein-induced promoter demethylation and active histone modification. Genistein had similar effects to that of 5Aza-C, which is a potent demethylating agent with high toxicity and instability. Genistein being a natural, non-toxic, dietary isoflavone is effective in retarding the growth of RCC cells, making it a promising candidate for epigenetic therapy in renal carcinoma.

Identification of novel microRNA targets based on microRNA signatures in bladder cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate protein-coding genes. To identify miRNAs that have a tumor suppressive function in bladder cancer (BC), 156 miRNAs were screened in 14 BCs, 5 normal bladder epithelium (NBE) samples and 3 BC cell lines. We identified a subset of 7 miRNAs (miR-145, miR-30a-3p, miR-133a, miR-133b, miR-195, miR-125b and miR-199a*) that were significantly downregulated in BCs. To confirm these results, 104 BCs and 31 NBEs were subjected to real-time RT-PCR-based experiments, and the expression levels of each miRNA were significantly downregulated in BCs (p < 0.0001 in all). Receiver-operating characteristic curve analysis revealed that the expression levels of these miRNAs had good sensitivity (>70%) and specificity (>75%) to distinguish BC from NBE. Our target search algorithm and gene-expression profiling in BCs (Kawakami et al., Oncol Rep 2006;16:521-31) revealed that Keratin7 (KRT7) mRNA was a common target of the downregulated miRNAs, and the mRNA expression levels of KRT7 were significantly higher in BCs than in NBEs (p = 0.0004). Spearman rank correlation analysis revealed significant inverse correlations between KRT7 mRNA expression and each downregulated miRNA (p < 0.0001 in all). Gain-of-function analysis revealed that KRT7 mRNA was significantly reduced by transfection of 3 miRNAs (miR-30-3p, miR-133a and miR-199a*) in the BC cell line (KK47). In addition, significant decreases in cell growth were observed after transfection of 3 miRNAs and si-KRT7 in KK47, suggesting that miR-30-3p, miR-133a and miR-199a* may have a tumor suppressive function through the mechanism underlying transcriptional repression of KRT7.

Polymorphisms of MLH1 in benign prostatic hyperplasia and sporadic prostate cancer.

Mismatch repair is one of several DNA repair pathways of which defects may lead to cancer. We hypothesize that polymorphisms of the MLH1 gene can be a risk factor for benign prostatic hyperplasia (BPH) and prostate cancer. The genetic distribution of MLH1 polymorphisms that lead to amino acid changes at codons 132, 219, 384, and 723 were analyzed in BPH and sporadic prostate cancer patients, and compared to healthy controls from an Asian population. These experiments demonstrate a protective role for the codon 384 variant allele against prostate cancer (P=0.031) but not BPH when compared to normal controls and furthermore, an inverse association was observed with stage (P=0.074) and grade (P=0.056) of cancer. This is the first report that demonstrates a protective effect for the race-related MLH1 polymorphism at codon 384 against prostate cancer and these results are important in understanding their role in this disease.

Restoring erectile function by antioxidant therapy in diabetic rats.

PURPOSE: Diabetes mediates an increase in reactive oxygen species that can lead to impaired endothelial function, decreased smooth muscle in the diabetic corpus cavernosum and increased apoptosis. We hypothesized that antioxidant therapy may restore erectile function by inhibiting apoptosis in diabetic rat crura. MATERIALS AND METHODS: A total of 40 male Sprague-Dawley rats were randomized to 5 groups of 8 each, including healthy controls, rats with diabetes, and rats with diabetes with the antioxidant tempol (4-hydroxytetramethyl-piperidine-1-oxyl) (Sigma-Aldrich), with insulin, and with tempol and insulin. Intracavernous pressure was measured for functional analysis. Smooth muscle and collagen fiber levels in the rat penile corpus cavernosum were assessed by hematoxylin and eosin, and Masson's trichrome staining. Endothelial cells were assessed by CD31 staining. Reactive oxygen species related genes were analyzed by cDNA microarray. We confirmed mRNA and protein expression profiles for these genes in diabetic and treated rats using real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. TUNEL assay was done to analyze apoptosis status. RESULTS: Intracavernous pressure in diabetic rats was significantly decreased vs controls. After treatment with tempol or insulin alone intracavernous pressure was significantly increased compared to that in untreated diabetic rats. In the diabetic group mean smooth muscle area significantly decreased but was restored after combined tempol and insulin. Endothelial cell area in diabetic rats significantly decreased and was not restored by any treatments. However, apoptosis was restored to normal by combined insulin and tempol. Of 84 reactive oxidative stress and antioxidant genes 32 were identified specific to diabetic rats compared to healthy controls. UCP3 expression was significantly increased in diabetic rats and normal levels were restored by all treatments. CONCLUSIONS: To our knowledge this is the first report that tempol and insulin can restore erectile function in diabetic rats by inhibiting apoptosis.

CpG hypermethylation of collagen type I alpha 2 contributes to proliferation and migration activity of human bladder cancer.

In our microarray screening of methylated genes in bladder cancer (BC), the collagen type 1 alpha 2 (COL1A2) gene was the most up-regulated among the 30,144 genes screened. We hypothesize that inactivation of the COL1A2 gene through CpG methylation contributes to proliferation and migration activity of human BC. We subjected a bladder cancer cell line (BOY) and 67 BC specimens and 10 normal bladder epitheliums (NBEs) to conventional or real-time methylation quantitative polymerase chain reaction (PCR) and to real-time reverse transcriptase (RT)-PCR. We also established a stable COL1A2 transfectant for evaluating cell proliferation and migration activity. After 5-aza-dC treatment, the expression levels of COL1A2 mRNA transcript markedly increased in BOY. Our cell proliferation assays consistently demonstrated growth inhibition in the COL1A2 transfectant compared with control and wild-type BOY cells (p<0.0001). Wound healing assays also showed significant wound healing inhibition in the COL1A2 transfectant compared to the counterparts (p=0.0016). We demonstrated by bisulfite DNA sequencing that the promoter hypermethylation of COL1A2 was a frequent event in clinical BCs. The methylation index of COL1A2 was significantly higher in the 67 BCs than in the 10 NBEs (p=0.0011). Conversely, COL1A2 mRNA transcript was significantly lower in the BCs than in the NBEs (p=0.0052). The mechanism of COL1A2 down-regulation in BC is through CpG hypermethylation of the promoter region. COL1A2 gene inactivation through CpG hypermethylation may contribute to proliferation and migration activity of BC.

Nerve growth factor combined with vascular endothelial growth factor enhances regeneration of bladder acellular matrix graft in spinal cord injury-induced neurogenic rat bladder.

OBJECTIVE: To determine the combined effects of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) on regeneration of the bladder acellular matrix graft (BAMG) in spinal cord injury (SCI)-mediated neurogenic bladder in rats. MATERIALS AND METHODS: In all, 40 female Sprague-Dawley rats were used. At 8 weeks after spinalization surgery (neurogenic bladder), they were divided into five groups consisting of untreated controls and those whose bladders were injected with either no growth factor, NGF (2 microg/rat), VEGF (2 microg/rat) or both at partial BAMG replacement surgery. After 8 weeks, bladder function was assessed by urodynamic studies and the bladders were harvested for histological examination. Smooth muscle induction, collagen and nerve fibre regeneration were assessed immunohistochemically using antibodies to smooth muscle actin (alpha-actin), Masson's trichrome and protein gene product 9.5, respectively. RESULTS: Bladder capacity and compliance were significantly increased in all BAMG groups 8 weeks after surgery compared with that before bladder replacement surgery. Bladder capacity and compliance were much higher in the VEGF and NGF combined group than in the control, or NGF and VEGF alone groups. There was no significant difference in the residual volume ratio among all groups. CONCLUSIONS: This is the first report showing that NGF has a significant synergistic effect on the development, differentiation and functional restoration of the BAMG when administered with VEGF in neurogenic bladder. Our results indicate that NGF may be a useful cytokine for enhancing the regeneration of a functional bladder following acellular matrix grafting in a neurogenic rat model.

CpG hypermethylation of the UCHL1 gene promoter is associated with pathogenesis and poor prognosis in renal cell carcinoma.

PURPOSE: Aberrant DNA hypermethylation has been reported in renal cell carcinoma. We performed microarray analysis in the renal cancer cell line ACHN treated with the demethylating agent 5-aza-2'-deoxycytidine and investigated the UCHL1 gene involved in the regulation of cellular ubiquitin levels. MATERIALS AND METHODS: We subjected 131 renal cell carcinoma and 61 corresponding normal kidney tissue samples to real-time reverse transcriptase-polymerase chain reaction, quantitative methylation specific polymerase chain reaction and immunohistochemistry. We also established a stable UCHL1 transfectant to evaluate cell growth. RESULTS: We identified 10 genes that were up-regulated more than 2.5-fold in 5-aza-2'-deoxycytidine treated vs untreated ACHN cells. UCHL1 expression was increased 3.41-fold by 5-aza-2'-deoxycytidine treatment. In clinical samples the UCHL1 methylation index was significantly higher in renal cell carcinoma than in normal kidney tissue (p = 0.011). Conversely UCHL1 mRNA expression was significantly lower in renal cell carcinoma than in normal kidney tissue (p <0.0001). There was a negative correlation between mRNA expression and the UCHL1 methylation index (p = 0.017). The immunostaining score for UCHL1 was significantly higher in normal kidney tissue than in renal cell carcinoma (p <0.0001). Kaplan-Meier analysis showed that a positive UCHL1 methylation index had a significant adverse effect on prognosis (p = 0.048). Significant growth inhibition in UCHL1 transfectant compared to that in WT ACHN (p <0.0001) suggests that UCHL1 functions as a potential tumor suppressor gene in human renal cell carcinoma. CONCLUSIONS: To our knowledge we report the first study demonstrating that the mechanism of UCHL1 down-regulation in renal cell carcinoma is through CpG hypermethylation of the promoter region and methylation of the UCHL1 gene is associated with a poor prognosis in patients with renal cell carcinoma.

Identification of differentially expressed genes in human bladder cancer through genome-wide gene expression profiling.

Large-scale gene expression profiling is an effective strategy for understanding the progression of bladder cancer (BC). The aim of this study was to identify genes that are expressed differently in the course of BC progression and to establish new biomarkers for BC. Specimens from 21 patients with pathologically confirmed superficial (n = 10) or invasive (n = 11) BC and 4 normal bladder samples were studied; samples from 14 of the 21 BC samples were subjected to microarray analysis. The validity of the microarray results was verified by real-time RT-PCR. Of the 136 up-regulated genes we detected, 21 were present in all 14 BCs examined (100%), 44 in 13 (92.9%), and the other 71 in 12 BCs (85.7%). Of 69 down-regulated genes, 25 were found in all 14 BCs (100%), 22 in 13 (92.9%), and the other 22 in 12 BCs (85.7%). Functional annotation revealed that of the up-regulated genes, 36% were involved in metabolism and 14% in transcription and processing; 25% of the down-regulated genes were linked to cell adhesion/surface and 21% to cytoskeleton/cell membrane. Real-time RT-PCR confirmed the microarray results obtained for the 6 most highly up- and the 2 most highly down-regulated genes. Among the 6 most highly up-regulated genes, CKS2 was the only gene with a significantly greater level of up-regulation in invasive than in superficial BC (p = 0.04). To confirm this result, we subjected all 21 BC samples to real-time PCR assay for CKS2. We found a considerable difference between superficial and invasive BC (p = 0.001). Interestingly, there was a considerable difference between the normal bladder and invasive BC (p = 0.001) and less difference between the normal bladder and superficial BC (p = 0.005). We identified several genes as promising candidates for diagnostic biomarkers of human BC and the CKS2 gene not only as a potential biomarker for diagnosing, but also for staging human BC. This is the first report demonstrating that CKS2 expression is strongly correlated with the progression of human BC.

MiR-133a induces apoptosis through direct regulation of GSTP1 in bladder cancer cell lines.

OBJECTIVE: We previously demonstrated that miR-133a is a tumor-suppressive microRNA (miRNA) and is commonly down-regulated in human bladder cancer (BC). The aim of this study is to determine a novel oncogenic gene targeted by miR-133a in BC. METHODS: To identify genes targeted by miR-133a, an oligo-microarray analysis was performed using the miR-133a-transfected BC cell lines. For gain/loss-of-function studies, miR-133a/si-glutathione S-transferase π1 (GSTP1)-transfectants were subjected to XTT assay and flow cytometry to evaluate their cell viability and apoptosis status. The luciferase reporter assay was used to confirm the actual binding sites between miR-133a and GSTP1 mRNA. The mRNA and protein expression of GSTP1 in BC cell lines and clinical samples were evaluated by real-time RT-PCR and Western blot, respectively. RESULTS: MiR-133a transfection induced cell viability inhibition and apoptosis in BC cell lines. We focused on the GSTP1 gene that was the top 7 down-regulated one in the gene profile from the miR-133a-transfectants. MiR-133a transfection repressed expression levels of mRNA and protein levels of GSTP1. A luciferase reporter assay suggested that the actual binding may occur between miR-133a and GSTP1 mRNA. Cell viability inhibition and apoptosis were induced in the si-GSTP1 transfectants compared with the controls (P < 0.005). GSTP1 mRNA expression levels in 43 clinical BCs were significantly higher than those in eight normal bladder epitheliums (P = 0.0277). CONCLUSION: Our data suggest that tumor suppressive miR-133a directly regulated oncogenic GSTP1 gene in BC, and that an anti-apoptotic effect mediated by GSTP1 is maintained by miR-133a down-regulation in human BC.

Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer.

OBJECTIVE: Our previous study demonstrated that fascin homolog 1 (FSCN1) might have an oncogenic function in bladder cancer (BC) and that its expression was regulated by specific microRNAs (miRNAs). Recently, LIM and SH3 protein 1 (LASP1) as well as FSCN1 have been reported as actin filament bundling proteins in the same complexes attached to the inner surfaces of cell membranes. We hypothesize that LASP1 as well as FSCN1 have an oncogenic function and that is regulated by miRNAs targeting LASP1 mRNA. METHODS: The expression levels of LASP1 mRNA in 86 clinical samples were evaluated by real-time RT-PCR. LASP1-knockdown BC cell lines were transfected by siRNA in order to examine cellular viability by XTT assay, wound healing assay, and matrigel invasion assay. We employed web-based software in order to search for candidate miRNAs targeting LASP1 mRNA, and we focused on miR-1, miR-133a, miR-145, and miR-218. The luciferase reporter assay was used to confirm the actual binding sites between the miRNAs and LASP1 mRNA. RESULTS: Real-time RT-PCR showed that LASP1 mRNA expression was higher in 76 clinical BC specimens than in 10 normal bladder epitheliums (P < 0.05). Loss-of-function studies using si-LASP1-transfected BC cell lines demonstrated significant cell viability inhibition (P < 0.0005), cell migration inhibition (P < 0.0001), and a decrease in the number of invading cells (P < 0.005) in the transfectants compared with the controls. Transient transfection of three miRNAs (miR-1, miR-133a, and miR-218), which were predicted as the miRNAs targeting LASP1 mRNA, repressed the expression levels of mRNA and protein levels of LASP1. The luciferase reporter assay demonstrated that the luminescence intensity was significantly decreased in miR-1, miR-133a, and miR-218 transfectants (P < 0.05), suggesting that these miRNAs have actual target sites in the 3' untranslated region of LASP1 mRNA. Furthermore, significant cell viability inhibitions occurred in miR-218, miR-1, and miR-133a transfectants (P < 0.001). CONCLUSION: Our data indicate that LASP1 may have an oncogenic function and that it might be regulated by miR-1, miR-133a, and miR-218, which may function as tumor suppressive miRNAs in BC.

Novel oncogenic function of mesoderm development candidate 1 and its regulation by MiR-574-3p in bladder cancer cell lines.

Our previous studies suggested that microRNA (miR)-574-3p is a candidate tumor suppressor microRNA (miRNA) in human bladder cancer (BC). Among 17 down-regulated miRNAs, miR-574-3p is located on chromosome 4p14 where we had identified a chromosomal loss region by array-CGH in BC cell lines. MiR-574-3p expression was down-regulated in BC cell lines. Gain-of-function analysis revealed that cell proliferation, migration and invasion were significantly inhibited in miR­574­3p-transfected BC cell lines. Flow cytometry analysis showed that cell apoptosis was induced in miR-574-3p transfectants. Oligo microarray analysis suggested that the mesoderm development candidate 1 (MESDC1) gene was a target gene in miR-574-3p transfectants. Luciferase assays revealed that miR­574­3p was directly bound to MESDC1 mRNA. MESDC1 is predicted to be a novel actin-binding protein located on chromosome 15q13. Although the gene is conserved among many species, its functional role is still unknown in both human malignancies and normal tissues. Loss-of-function studies demonstrated that cell proliferation, migration and invasion were significantly inhibited in si-MESDC1-transfected BC cell lines. Flow cytometry analysis showed that apoptosis was induced in si-MESDC1 transfectants. We are the first to demonstrate that miR-574-3p is a miRNA with tumor suppressor function and that MESDC1 (which has a potential oncogenic function in BC) may be targeted by miR-574-3p.

The functional significance of miR-1 and miR-133a in renal cell carcinoma.

PURPOSE: The aim of this study was to find a novel molecular network involved in renal cell carcinoma (RCC) development through investigating the functions of miR-1 and miR-133a and their target genes. METHODS: We checked the expression levels of miR-1 and miR-133a in RCC cell lines and specimens (N=40) using real time RT-PCR. MiR-1 and miR-133a transfectants were subjected to a gain-of-function study to identify the functions of the miRNAs. To find the target genes of the miRNAs, we analysed the gene expression profile of their transfectants and performed a luciferase reporter assay. mRNA expression levels of the candidate target gene in the clinical specimens were examined, and loss-of-function studies were performed. RESULTS: The expression levels of miR-1 and miR-133a were significantly suppressed in RCC cell lines and specimens. Ectopic restoration of miR-1 and miR-133a showed significant inhibition of cell proliferation and invasion, and moreover, revealed induction of apoptosis and cell cycle arrest. The luciferase assay revealed transgelin-2 (TAGLN2), selected as a target gene for miR-1 and miR-133a on the basis of the gene expression profile, to be directly regulated by both miR-1 and miR-133a. The loss-of-function studies showed significant inhibitions of cell proliferation and invasion in the si-TAGLN2 transfectant. The expression level of TAGLN2 mRNA was significantly up-regulated in the RCC specimens; in addition, there was a statistically significant inverse correlation between TAGLN2 and miR-1 and miR-133a expression. CONCLUSIONS: Our data indicate that up-regulation of the oncogenic TAGLN2 was due to down-regulation of tumour-suppressive miR-1 and miR-133a in human RCC.

Tumor suppressive microRNA-375 regulates lactate dehydrogenase B in maxillary sinus squamous cell carcinoma.

The expression of microRNA-375 (miR-375) is significantly reduced in cancer tissues of maxillary sinus squamous cell carcinoma (MSSCC). The aim of this study was to investigate the functional significance of miR-375 and a possible regulatory role in the MSSCC networks. Restoration of miR-375 significantly inhibited cancer cell proliferation and invasion in IMC-3 cells, suggesting that miR-375 functions as a tumor suppressor in MSSCC. Genome-wide gene expression data and luciferase reporter assays indicated that lactate dehydro-genase B (LDHB) was directly regulated by miR-375. Cancer cell proliferation and invasion were significantly inhibited by transfection of si-LDHB into IMC-3 cells, suggesting that LDHB may play a role in MSSCC oncogenic function. In clinical MSSCC specimens, LDHB mRNA levels were up-regulated in cancer tissues, which were inversely correlated with the expression of miR-375. In addition, Kaplan-Meier curves and log-rank tests revealed that the high mRNA expression levels of LDHB had a significant adverse effect on survival rate. The identification of a cancer network regulated by the miR-375 tumor suppressor could provide new insights into the molecular mechanisms of MSSCC oncogenesis.

Identification of novel molecular targets regulated by tumor suppressive miR-1/miR-133a in maxillary sinus squamous cell carcinoma.

Based on our microRNA (miRNA) expression signature analysis of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-1 and miR-133a were significantly reduced in tumor tissues. Quantitative real-time RT-PCR revealed that the expression levels of miR-1 and miR-133a were significantly downregulated in clinical MSSCC tumor tissues compared with normal tissues. We focused on the functional significance of miR-1 and miR-133a in cancer cells and identification of the novel cancer networks regulated by these miRNAs in MSSCC. Restoration of downregulated miRNAs (miR-1 or miR-133a) in cancer cells revealed that both miRNAs significantly inhibited cancer cell proliferation and induced cell apoptosis. Molecular target identification of these miRNAs showed that transgelin 2 (TAGLN2) and purine nucleoside phosphorylase (PNP) were regulated by miR-1 and miR-133a. Both TAGLN2 and PNP mRNA expression levels were significantly upregulated in clinical MSSCC tumor tissues. Silencing studies of target genes demonstrated that both genes inhibited cancer cell proliferation. The identification of novel miR-1/miR-133a-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.

Restoration of miR-145 expression suppresses cell proliferation, migration and invasion in prostate cancer by targeting FSCN1.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression, primarily at the post-transcriptional level. Growing evidence suggests that miRNAs function as oncogenes or tumor suppressors in human cancers. The down-regulation of miR-145 has been reported in many types of human cancer, including prostate cancer (PC), suggesting that miR-145 functions as a tumor suppressor. Using the PC cell lines, PC3 and DU145, gain-of-function assays revealed that miR-145 transfection inhibited cell proliferation, migration and invasion. Fascin homolog 1 (FSCN1), an actin-bundling protein, is a candidate target gene of miR-145 based on genome-wide gene expression analysis. A luciferase reporter assay showed a significantly decreased signal at two miR-145 target sites at the 3'UTR of FSCN1, suggesting that miR-145 directly regulates FSCN1. In FSCN1 loss-of-function assays, cell growth, migration and invasion were all inhibited, implying that FSCN1 is associated with the progression of PC. The identification of tumor suppressive miRNAs and their target genes could provide new insights into the potential mechanisms of prostate carcinogenesis.

miR-1 as a tumor suppressive microRNA targeting TAGLN2 in head and neck squamous cell carcinoma.

Based on the microRNA (miRNA) expression signatures of hypopharyngeal and esophageal squamous cell carcinoma, we found that miR-1 was significantly down-regulated in cancer cells. In this study, we investigated the functional significance of miR-1 in head and neck squamous cell carcinoma (HNSCC) cells and identified miR-1-regulated novel cancer pathways. Gain-of-function studies using miR-1 revealed significant decreases in HNSCC cell proliferation, invasion, and migration. In addition, the promotion of cell apoptosis and cell cycle arrest was demonstrated following miR-1e transfection of cancer cells. A search for the targets of miR-1 revealed that transgelin 2 (TAGLN2) was directly regulated by miR-1. Silencing of TAGLN2 significantly inhibited cell proliferation and invasion in HNSCC cells. Down-regulation of miR-1 and up-regulation of TAGLN2 were confirmed in HNSCC clinical specimens. Our data indicate that TAGLN2 may have an oncogenic function and may be regulated by miR-1, a tumor suppressive miRNA in HNSCC. The identification of novel miR-1-regulated cancer pathways could provide new insights into potential molecular mechanisms of HNSCC carcinogenesis.