hsa‐miR‐210 is a marker of tumor hypoxia and a prognostic factor in head and neck cancer

BACKGROUND:

Hypoxia is an important mechanism of treatment resistance in head and neck squamous cell carcinoma (HNSCC). MicroRNAs are short noncoding RNAs that regulate multiple mRNAs and are frequently dysregulated in cancer. The authors have investigated the role of 3 microRNAs, including the hypoxia‐induced hsa‐miR‐210, as potential markers of hypoxia or prognosis.

METHODS:

Three hypoxia‐related microRNAs, hsa‐miR‐210, hsa‐miR‐21, and hsa‐miR‐10b, were measured in 46 samples from patients with HNSCC. Expression levels were correlated with clinicopathological variables and other markers of hypoxia: a published 99‐gene hypoxia metagene, individual hypoxia‐related genes such as TWIST1, and immunohistochemical expression of hypoxia‐inducible factor 1 and its target gene carbonic anhydrase 9. We then performed survival analyses to investigate the prognostic significance of these microRNAs.

RESULTS:

Only the level of hsa‐miR‐210 was significantly correlated with other markers of hypoxia, including the 99‐gene hypoxia metagene (rho = 0.67, P < .001). We found no association between hsa‐miR‐210, hsa‐miR‐21, or hsa‐miR‐10b and clinicopathological variables such as tumor size, differentiation, and stage. However, high levels of hsa‐miR‐210 were associated with locoregional disease recurrence (P = .001) and short overall survival (P = .008). hsa‐miR‐21 and hsa‐miR‐10b had no prognostic significance.

CONCLUSIONS:

Expression of hsa‐miR‐210 in head and neck cancer correlates with other approaches for assessing hypoxia and is associated with prognosis. This warrants further study as a classification marker of patients for therapies involving modulation of hypoxia. Cancer 2010. © 2010 American Cancer Society.     

A MDM2‐dependent positive‐feedback loop is involved in inhibition of miR‐375 and miR‐106b induced by Helicobacter pylori lipopolysaccharide

Dysregulation of microRNAs (miRNAs) has been linked to virulence factors of Helicobacter pylori and shown to contribute to the progression of gastric cancer. However, the mechanisms of these processes remain poorly understood. The aim of this study was to investigate the mechanisms by which lipopolysaccharide (LPS), a virulence factor of H. pylori, regulates miR‐375 and miR‐106b expression in gastric epithelial cells. The results show that LPS from H. pylori 26695 downregulated the expression of miR‐375 and miR‐106b in gastric epithelial cells, and low levels of Dicer were also observed. Downregulation of miR‐375 was found to increase expression of MDM2 with SP1 activation. Overexpression of MDM2 inhibited Dicer by repressing p63 to create a positive‐feedback loop involving SP1/MDM2/p63/Dicer that leads to inhibition of miR‐375 and miR‐106b expression. In addition, we demonstrated that JAK1 and STAT3 were downstream target genes of miR‐106b. H. pylori LPS also enhanced the tyrosine phosphorylation of JAK1, JAK2 and STAT3. Together, these results provide insight into the regulatory mechanisms of MDM2 on H. pylori LPS‐induced specific miRNAs, and furthermore, suggest that gastric epithelial cells treated with H. pylori LPS may be susceptible to JAK/STAT3 signal pathway activation via inhibition of miR‐375 and miR‐106b.     

Branched rolling circle amplification method for measuring serum circulating microRNA levels for early breast cancer detection

Serum circulating microRNAs (c‐miRNAs) are serving as useful biomarkers for cancer diagnosis. Here, we describe the development of a one‐step branched rolling circle amplification (BRCA) method to measure serum c‐miRNAs levels for early diagnosis of breast cancer. Four c‐miRNAs, c‐miRNA16 (c‐miR‐16), c‐miRNA21 (c‐miR‐21), c‐miRNA155 (c‐miR‐155), and c‐miRNA195 (c‐miR‐195) were isolated from the serum of 49 breast cancer patients (stages I‐IV) and 19 healthy controls, and analyzed using one‐step BRCA. The serum levels of c‐miR16, c‐miR21, c‐miR155, and c‐miR195 were higher (P < 0.0001) in stage I breast cancer patients than healthy controls. These levels were also higher in several breast cancer molecular subtypes (HER‐2 over‐expression, Luminal A, Luminal B, and triple negative breast cancer) than in healthy control subjects. The diagnostic accuracy of c‐miR16, c‐miR21, c‐miR155, and c‐miR195 for early diagnosis of breast cancer was confirmed by receiver operating characteristic (ROC) curve assay. These results show that the BRCA method can be used to measure serum c‐miRNAs levels, and that this method has high accuracy, sensitivity, and specificity. Moreover, both BRCA approach and quantitative real‐time PCR (qRT‐PCR) method show that the serum levels of c‐miR16, c‐miR21, c‐miR155, and c‐miR195 could be used as biomarkers to improve the early diagnosis of breast cancer, and distinguish different breast cancer molecular subtypes.     

Circulating microRNAs, miR-21, miR-122, and miR-223, in patients with hepatocellular carcinoma or chronic hepatitis

Numerous studies have shown that aberrant microRNA (miRNA) expression is associated with the development and progression of various types of human cancer and serum miRNAs are potential biomarkers. This study examined whether some commonly deregulated miRNAs in hepatocellular carcinoma (HCC) are presented in serum of patients with HCC and can serve as diagnostic markers. Serum miRNAs (miR‐21, miR‐122, and miR‐223) were quantified by real‐time quantitative RT‐PCR in 101 patients with HCC and 89 healthy controls. In addition, 48 patients with chronic type B hepatitis were also analyzed for comparison. We found that the median levels of miR‐21, miR‐122, and miR‐223 were significantly higher in patients with HCC than those in healthy controls (P = 7.48 × 10^?13, P = 6.93 × 10^?9, and P = 3.90 × 10^?12, respectively). However, these elevated serum miRNAs were also detected in patients with chronic hepatitis (P = 2.05 × 10^?12, P = 4.52 × 10^?16, and P = 1.65 × 10^?11, respectively). Moreover, serum miR‐21 and miR‐122 in patients with chronic hepatitis were higher than in patients with HCC (P = 3.99 × 10^?4 and P = 4.97 × 10^?8), although no such significant difference was found for miR‐223. Receiver‐operator characteristic (ROC) curve analyses suggest that these serum miRNAs may be useful markers for discriminating patients with HCC or chronic hepatitis from healthy controls, but not patients with HCC from patients with chronic hepatitis. Our results indicate that serum miR‐21, miR‐122 and miR‐223 are elevated in patients with HCC or chronic hepatitis and these miRNAs have strong potential to serve as novel biomarkers for liver injury but not specifically for HCC. © 2010 Wiley‐Liss, Inc.     

The tumor suppressor microRNA‐29c is downregulated and restored by celecoxib in human gastric cancer cells

MicroRNAs (miRNAs) are small noncoding RNAs that function as endogenous silencers of target genes and play critical roles during carcinogenesis. The selective cyclooxygenase‐2 (COX‐2) inhibitor celecoxib has been highlighted as a potential drug for treatment of gastrointestinal tumors. The aim of this study was to investigate the role of miRNAs in gastric carcinogenesis and the feasibility of a new therapeutic approach for gastric cancer. miRNA expression profiles were examined in 53 gastric tumors including gastric adenomas (atypical epithelia), early gastric cancers and advanced gastric cancers and in gastric cancer cells treated with celecoxib. miRNA microarray analysis revealed that miR‐29c was significantly downregulated in gastric cancer tissues relative to nontumor gastric mucosae. miR‐29c was significantly activated by celecoxib in gastric cancer cells. Downregulation of miR‐29c was associated with progression of gastric cancer and was more prominent in advanced gastric cancers than in gastric adenomas and early gastric cancer. In addition, expression of the oncogene Mcl‐1, a target of miR‐29c, was significantly increased in gastric cancer tissues relative to nontumor gastric mucosae. Activation of miR‐29c by celecoxib induced suppression of Mcl‐1 and apoptosis in gastric cancer cells. These results suggest that downregulation of the tumor suppressor miR‐29c plays critical roles in the progression of gastric cancer. Selective COX‐2 inhibitors may have clinical promise for the treatment of gastric cancer via restoration of miR‐29c.     

MicroRNA expression profiling is a potential diagnostic tool for thyroid cancer

BACKGROUND:

Approximately 30% of fine‐needle aspiration (FNA) biopsies of thyroid nodules are indeterminate or nondiagnostic. Recent studies suggest microRNA (miRNA, miR) is differentially expressed in malignant tumors and may have a role in carcinogenesis, including thyroid cancer. The authors therefore tested the hypothesis that miRNA expression analysis would identify putative markers that could distinguish benign from malignant thyroid neoplasms that are often indeterminate on FNA biopsy.

METHODS:

A miRNA array was used to identify differentially expressed genes (5‐fold higher or lower) in pooled normal, malignant, and benign thyroid tissue samples. Real‐time quantitative polymerase chain reaction was used to confirm miRNA array expression data in 104 tissue samples (7 normal thyroid, 14 hyperplastic nodule, 12 follicular variant of papillary thyroid cancer, 8 papillary thyroid cancer, 15 follicular adenoma, 12 follicular carcinoma, 12 Hurthle cell adenoma, 20 Hurthle cell carcinoma, and 4 anaplastic carcinoma cases), and 125 indeterminate clinical FNA samples. The diagnostic accuracy of differentially expressed genes was determined by analyzing receiver operating characteristics.

RESULTS:

Ten miRNAs showed >5‐fold expression difference between benign and malignant thyroid neoplasms on miRNA array analysis. Four of the 10 miRNAs were validated to be significantly differentially expressed between benign and malignant thyroid neoplasms by quantitative polymerase chain reaction (P < .002): miR‐100, miR‐125b, miR‐138, and miR‐768‐3p were overexpressed in malignant samples of follicular origin (P < .001), and in Hurthle cell carcinoma samples alone (P < .01). Only miR‐125b was significantly overexpressed in follicular carcinoma samples (P < .05). The accuracy for distinguishing benign from malignant thyroid neoplasms was 79% overall, 98% for Hurthle cell neoplasms, and 71% for follicular neoplasms. The miR‐138 was overexpressed in the FNA samples (P = .04) that were malignant on final pathology with an accuracy of 75%.

CONCLUSIONS:

MicroRNA expression differs for normal, benign, and malignant thyroid tissue. Expression analysis of differentially expressed miRNA could help distinguish benign from malignant thyroid neoplasms that are indeterminate on thyroid FNA biopsy. Cancer 2011. © 2011 American Cancer Society.     

Identification of serum miRNAs as novel non-invasive biomarkers for detection of high risk for early gastric cancer

Background:

Many micro-RNAs (miRNAs) are differentially expressed in Helicobacter pylori-infected gastric mucosa and in gastric cancer tissue and previous reports have suggested the possibility of serum miRNAs as complementary tumour markers. The aim of the study was to investigate serum miRNAs and pepsinogen levels in individuals at high risk for gastric cancer both before and after H. pylori eradication.

Methods:

Patients with recent history of endoscopic resection for early gastric cancer and the sex- and age-matched controls were enrolled. Serum was collected from subjects before or after eradication and total RNA was extracted to analyse serum levels of 24 miRNAs. Serum pepsinogen (PG) I and II levels were measured using enzyme-linked immunosorbent assay kits.

Results:

Using miR-16 as an endogenous control, the relative levels of miR-106 and let-7d before and after H. pylori eradication and miR-21 after eradication were significantly higher in the high-risk group than in the controls. H. pylori eradication significantly decreased miR-106b levels and increased let-7d only in the control group. After eradication, the combination MiR-106b with miR-21 was superior to serum pepsinogen and the most valuable biomarker for the differentiating high-risk group from controls.

Conclusion:

Serum miR-106b and miR-21 may provide a novel and stable marker of increased risk for early gastric cancer after H. pylori eradication.     

Plasma miRNAs as early biomarkers for detecting hepatocellular carcinoma

The early detection of hepatocellular carcinoma (HCC) presents a challenge because of the lack of specific biomarkers. Serum/plasma microRNAs (miRNAs) can discriminate HCC patients from controls. We aimed to identify and evaluate HCC‐associated plasma miRNAs originating from the liver as early biomarkers for detecting HCC. In this multicenter three‐phase study, we first performed screening using both plasma (HCC before and after liver transplantation or liver hepatectomy) and tissue samples (HCC, para‐carcinoma and cirrhotic tissues). Then, we evaluated the diagnostic potential of the miRNAs in two case–control studies (training and validation sets). Finally, we used two prospective cohorts to test the potential of the identified miRNAs for the early detection of HCC. During the screening phase, we identified ten miRNAs, eight of which (miR‐20a‐5p, miR‐25‐3p, miR‐30a‐5p, miR‐92a‐3p, miR‐132‐3p, miR‐185‐5p, miR‐320a and miR‐324‐3p) were significantly overexpressed in the HBV‐positive HCC patients compared with the HBV‐positive cancer‐free controls in both the training and validation sets, with a sensitivity of 0.866 and specificity of 0.646. Furthermore, we assessed the potential for early HCC detection of these eight newly identified miRNAs and three previously reported miRNAs (miR‐192‐5p, miR‐21‐5p and miR‐375) in two prospective cohorts. Our meta‐analysis revealed that four miRNAs (miR‐20a‐5p, miR‐320a, miR‐324‐3p and miR‐375) could be used as preclinical biomarkers (p_meta < 0.05) for HCC. The expression profile of the eight‐miRNA panel can be used to discriminate HCC patients from cancer‐free controls, and the four‐miRNA panel (alone or combined with AFP) could be a blood‐based early detection biomarker for HCC screening.     

Regulation of antitumor miR‐144‐5p targets oncogenes: Direct regulation of syndecan‐3 and its clinical significance

In the human genome, miR‐451a, miR‐144‐5p (passenger strand), and miR‐144‐3p (guide strand) reside in clustered microRNA (miRNA) sequences located within the 17q11.2 region. Low expression of these miRNAs is significantly associated with poor prognosis of patients with renal cell carcinoma (RCC) (miR‐451a: P = .00305; miR‐144‐5p: P = .00128; miR‐144‐3p: P = 9.45 × 10^?5). We previously reported that miR‐451a acted as an antitumor miRNA in RCC cells. Involvement of the passenger strand of the miR‐144 duplex in the pathogenesis of RCC is not well understood. Functional assays showed that miR‐144‐5p and miR‐144‐3p significantly reduced cancer cell migration and invasive abilities, suggesting these miRNAs acted as antitumor miRNAs in RCC cells. Analyses of miR‐144‐5p targets identified a total of 65 putative oncogenic targets in RCC cells. Among them, high expression levels of 9 genes (FAM64A, F2, TRIP13, ANKRD36, CENPF, NCAPG, CLEC2D, SDC3, and SEMA4B) were significantly associated with poor prognosis (P < .001). Among these targets, expression of SDC3 was directly controlled by miR‐144‐5p, and its expression enhanced cancer cell aggressiveness. We identified genes downstream by SDC3 regulation. Data showed that expression of 10 of the downstream genes (IL18RAP, SDC3, SH2D1A, GZMH, KIF21B, TMC8, GAB3, HLA‐DPB2, PLEK, and C1QB) significantly predicted poor prognosis of the patients (P = .0064). These data indicated that the antitumor miR‐144‐5p/oncogenic SDC3 axis was deeply involved in RCC pathogenesis. Clustered miRNAs (miR‐451a, miR‐144‐5p, and miR‐144‐3p) acted as antitumor miRNAs, and their targets were intimately involved in RCC pathogenesis.     

Independent and tissue-specific prognostic impact of miR-126 in nonsmall cell lung cancer: coexpression with vascular endothelial growth factor-A predicts poor survival

BACKGROUND:

Angiogenesis is pivotal in tumor development. Vascular endothelial growth factor‐A (VEGF‐A) is considered one of the most important angiogenic factors, but lately several microRNAs (miRs) have been associated with vascular development. miR‐126 has been related to tumor angiogenesis and in the regulation of VEGF‐A. The authors aimed to investigate the prognostic impact of miR‐126 and its co‐expression with VEGF‐A in nonsmall cell lung cancer (NSCLC) patients.

METHODS:

Tumor tissue samples from 335 resected stage I to IIIA NSCLC patients were obtained and tissue microarrays (TMAs) were constructed with 4 cores from each tumor specimen. VEGF‐A expression was evaluated by immunohistochemistry, and in situ hybridization was used to evaluate the expression of miR‐126.

RESULTS:

In the total material, miR‐126 was a significant negative prognostic factor in both univariate (P = .005) and multivariate analyses (hazard ratio [HR] 1.8, 95% confidence interval [CI] 1.2‐2.8, P = .01). Stratified by histology, miR‐126 was only significant in squamous cell carcinomas (univariate: P < .001; multivariate: HR 3.1, CI 95% 1.7‐5.6, P<.001). Stratified by lymph node status, miR‐126 was significant only in the lymph node‐positive subgroup (univariate: P<.001; multivariate: HR 4.1, CI 95% 2.0‐8.4, P < .001). High miR‐126 expression correlated significantly with high VEGF‐A expression (P = .037). The co‐expression of miR‐126 and VEGF‐A had a significant prognostic impact (P = .002), with 5‐year survival rates of 68%, 51%, and 42% for low/low (n = 150), mixed combinations (n = 129), and high/high (n = 35) expression, respectively.

CONCLUSIONS:

miR‐126 is a strong and independent negative prognostic factor in NSCLC, and its prognostic impact appears related primarily to histology and nodal status. Cancer 2011. © 2011 American Cancer Society.     

CDH1 methylation in preoperative peritoneal washes is an independent prognostic factor for gastric cancer

Background and Objectives

To investigate the clinical value of CDH1 methylation in preoperative peritoneal washes (PPW) from gastric cancer patients.

Methods

CDH1 methylation was detected by real‐time methylation specific‐PCR in tumor tissues and corresponding PPW from 92 gastric cancer patients, gastric mucosa from 40 chronic gastritis patients and 48 normal persons.

Results

CDH1 methylation was found in 75 of 92 (81.5%) gastric cancer tissues, which significantly correlated with size, growth pattern, differentiation, lymphatic invasion, venous invasion, invasion depth, lymph node metastasis, distant metastasis, and TNM stage of tumor (all P < 0.05), but its relationship to age, gender, tumor site, and H. pylori infection was not found (all P > 0.05). The percentage of CDH1 methylation in PPW was 48.9%, of which the Aζ value of ROC curve was 0.8 compared to that in gastric cancer tissues. Kaplan–Meier analysis showed that there was a significant difference in disease‐free survival (DFS) between the patients with or without methylated CDH1 in their PPW (χ² = 109.64, P < 0.000). Cox regression analysis revealed CDH1 methylation in PPW was an independent risk factor for gastric cancer patients, with a remarkable decrease in DFS after postoperative 30 months.

Conclusions

Methylated CDH1 in PPW predicts poor prognosis for gastric cancer patients. J. Surg. Oncol. 2012; 106:765–771. © 2012 Wiley Periodicals, Inc.     

Serum macrophage migration‐inhibitory factor as a diagnostic and prognostic biomarker for gastric cancer

BACKGROUND:

This study aimed to determine the potential diagnostic value of migration‐inhibitory factor (MIF) for gastric cancer in patients presenting with dyspepsia and its prognostic value for gastric cancer.

METHODS:

A cohort of 97 patients with histologically confirmed gastric adenocarcinoma and 222 patients with dyspepsia were recruited. Enzyme‐linked immunosorbent assay was used to measure serum MIF and carcinoembryonic antigen (CEA).

RESULTS:

The serum MIF concentrations were 6554.0 ± 204.1 pg/mL and 1453.7 ± 79.9 pg/mL, respectively, in gastric cancer patients and dyspeptic patients (P < .001). Serum MIF levels increased with the advancing gastric pathologies (P < .001). With the cutoff value of 3230 pg/mL, serum MIF had sensitivity, specificity, and accuracy of 83.5%, 92.3%, and 89.7%, respectively, in diagnosing gastric cancer, whereas the rates were 60.8%, 83.3%, and 76.5%, respectively, for serum CEA. Gastric cancer patients with serum MIF levels above 6600 pg/mL had a lower 5‐year survival rate than those with serum MIF level below that level (P = .012). Higher serum CEA levels were also associated with poor survival. The prediction for 5‐year survival was even better (P = .0001), using a combination of serum MIF and CEA.

CONCLUSIONS:

Serum MIF level, which correlates with gastric MIF expression, is a better molecular marker than CEA in diagnosing gastric cancer in patients presenting with dyspepsia. A combination of serum MIF and CEA predicts 5‐year survival better than the individual test. Cancer 2009. © 2009 American Cancer Society.     

Circulating microRNAs from the miR-106a–363 cluster on chromosome X as novel diagnostic biomarkers for breast cancer

Purpose

Novel noninvasive biomarkers with high sensitivity and specificity for the diagnosis of breast cancer (BC) are urgently needed in clinics. The aim of this study was to explore whether miRNAs from the miR-106a–363 cluster can be detected in the circulation of BC patients and whether these miRNAs can serve as potential diagnostic biomarkers.

Methods

The expression of 12 miRNAs from the miR-106a–363 cluster was evaluated using qRT-PCR in 400 plasma samples (from 200 BC patients and 200 healthy controls (HCs)) and 406 serum samples (from 204 BC patients and 202 HCs) via a three-phase study. The identified miRNAs were further examined in tissues (32 paired breast tissues), plasma exosomes (from 32 BC patients and 32 HCs), and serum exosomes (from 32 BC patients and 32 HCs).

Results

Upregulated levels of four plasma miRNAs (miR-106a-3p, miR-106a-5p, miR-20b-5p, and miR-92a-2-5p) and four serum miRNAs (miR-106a-5p, miR-19b-3p, miR-20b-5p, and miR-92a-3p) were identified and validated in BC. A plasma 4-miRNA panel and a serum 4-miRNA panel were constructed to discriminate BC patients from HCs. The areas under the receiver-operating characteristic curves of the plasma panel were 0.880, 0.902, and 0.858, and those of the serum panel were 0.910, 0.974, and 0.949 for the training, testing, and external validation phases, respectively. Two overlapping miRNAs (miR-106a-5p and miR-20b-5p) were consistently upregulated in BC tissues. Except for the expression of the plasma-derived exosomal miR-20b-5p, the expression patterns of exosomal miRNAs were concordant between plasma and serum, indicating the potential use of exosomal miRNAs as biomarkers.

Conclusion

We identified four plasma miRNAs and four serum miRNAs from the miR-106a–363 cluster as promising novel biomarkers for the diagnosis of BC.

     

miR‐212 is downregulated and suppresses methyl‐CpG‐binding protein MeCP2 in human gastric cancer

To clarify the role of micro (mi) RNAs in gastric carcinogenesis, we studied the expression and function of miRNAs in gastric carcinoma (GC) cells. Initially, we performed microarray analysis using total RNA from 3 human GC cell lines and noncancerous gastric tissue. Among the downregulated miRNAs in GC cells, miR‐212 expression was decreased in all 8 GC cell lines examined and a significant decrease of miR‐212 expression in human primary GC tissues was also observed in 6 of 11 cases. Transfection of the precursor miR‐212 molecule induced decreased growth of 3 GC cell lines. Using 3 different databases, methyl‐CpG‐binding protein MeCP2 was postulated to be a target of miR‐212. As seen on reporter assaying, miR‐212 repressed the construct with the MECP2 3′‐UTR. Ectopic expression of miR‐212 repressed expression of the MeCP2 protein but not the MECP2 mRNA level. These data suggest that downregulation of miR‐212 may be related to gastric carcinogenesis through its target genes, such as MECP2.     

Gastric Carcinoma: Recent Trends in Diagnostic Biomarkers and Molecular Targeted Therapies

Gastric cancer is generally associated with poor survival rates and accounts for a remarkable proportion of global cancer mortality. The prevalence of gastric carcinoma varies in different regions of world and across teh various ethnic groups. On the basis of pathological assessment, gastric cancer can be categorized as intestinal and diffuse carcinomas. The etiology is diverse, including chemical carcinogen exposure, and high salt intake Helicobacter pylori also plays a vital role in the pathogenesis of certain gastric carcinomas. The development of gastric cancer involves various alterations in mRNAs, genes (GOLPH3, MTA2) and proteins (Coronins). miRNAs, Hsamir135b, MiR21, miR106b, miR17, miR18a, MiR21, miR106b, miR17, miR18a and MiRNA375, miRNA1955p are the latest diagnostic biomarkers which can facilitate the early diagnosis of gastric carcinomas. Recent development in the treatment strategies for gastric carcinoma include the introduction of monoclonal antibodies, TKI inhibitors, inhibitors of PDGFR , VEGFR1, VEGFR2, AntiEGFR and antiHER2 agents which can be applied along with conventional therapies.     

microRNA expression profile in a large series of bladder tumors: Identification of a 3‐miRNA signature associated with aggressiveness of muscle‐invasive bladder cancer

The aim of this study was to evaluate the expression levels of microRNAs (miRNAs) in bladder tumors in order to identify miRNAs involved in bladder carcinogenesis with potential prognostic implications. Expression levels of miRNAs were assessed by quantitative real‐time RT‐PCR in 11 human normal bladder and 166 bladder tumor samples (86 non‐muscle‐invasive bladder cancer (NMIBC) and 80 muscle‐invasive bladder cancer (MIBC)). The expression level of 804 miRNAs was initially measured in a well‐defined series of seven NMIBC, MIBC and normal bladder samples (screening set). The most strongly deregulated miRNAs in tumor samples compared to normal bladder tissue were then selected for RT‐PCR validation in a well‐characterized independent series of 152 bladder tumors (validation set), and in six bladder cancer cell lines. Expression levels of these miRNAs were tested for their association with clinical outcome. A robust group of 15 miRNAs was found to be significantly deregulated in bladder cancer. Except for two miRNAs, miR‐146b and miR‐9, which were specifically upregulated in MIBC, the majority of miRNAs (n = 13) were deregulated in the same way in the two types of bladder tumors, irrespective of pathological stage : three miRNAs were upregulated (miR‐200b, miR‐182 and miR‐138) and the other 10 miRNAs were downregulated (miR‐1, miR‐133a, miR‐133b, miR‐145, miR‐143, miR‐204, miR‐921, miR‐1281, miR‐199a and miR‐199b). A 3‐miRNA signature (miR‐9, miR‐182 and miR‐200b) was found to be related to MIBC tumor aggressiveness and was associated with both recurrence‐free and overall survival in univariate analysis with a trend to significance in the multivariate analysis (p = 0.05). Our results suggested a promising individual prognostic value of these new markers.