MicroRNA-126 inhibits invasion in bladder cancer via regulation of ADAM9

Background:

The miRNA deregulation is commonly observed in human malignancies, where they act as tumour suppressors or oncogenes. Despite the association of several miRNAs with bladder cancer, little is known about the miRNAs that contribute to bladder cancer progression from non-muscle invasive (NMI) to muscle-invasive (MI) disease.

Methods:

We first profiled the expression of miRNAs and mRNAs in a cohort of urothelial carcinomas and further characterised the role of miR-126 in invasion, as it emerged as the most downregulated miRNA between MI and NMI tumours.

Results:

We found that restoration of miR-126 levels attenuated the invasive potential of bladder cancer cells. Mechanistically, we identified the role of miR-126 in invasion through its ability to target ADAM9. Notably, a significant inverse correlation between miR-126 and ADAM9 expression was observed, where ADAM9 was upregulated in MI bladder cancer cells. While knockdown of ADAM9 attenuated the invasiveness of cells with low miR-126 levels, experimental upregulation of ADAM9 recapitulated the invasive phenotype. Furthermore, ADAM9 expression assessed by immunohistochemistry significantly correlated with poor prognosis in patients with urothelial carcinoma.

Conclusions:

In this study we describe the role of miR-126 in bladder cancer progression, identifying miR-126 and ADAM9 as potential clinical biomarkers of disease aggressiveness.     

MicroRNA-145 inhibits tumour growth and metastasis in colorectal cancer by targeting fascin-1

Background:

Recent studies have reported miR-145 dysregulated in colorectal cancer (CRC). In this study, miR-145 profiles were compared between CRC and corresponding non-tumour tissues.

Methods:

The expression levels of miR-145 were analysed in CRC cell lines and tumour tissues by real-time PCR. A luciferase reporter assay confirmed direct targets. The functional effects of miR-145 were examined in transfected CRC cells in vitro and in vivo using established assays.

Results:

Downregulation of miR-145 was detected in most primary CRC tumours, and was significantly correlated with a more aggressive phenotype of CRC in patients. In CRC cell lines, ectopic overexpression of miR-145 inhibited cell proliferation, motility and invasion in vitro. Stable overexpression of miR-145 suppressed tumour growth and pulmonary metastasis in vivo. Further studies indicated that miR-145 may directly interact with the 3′-untranslated region (3′-UTR) of Fascin-1 messenger RNA (mRNA), downregulating its mRNA and protein expression levels. In clinical specimens, Fascin-1 expression was negatively correlated with miR-145 expression.

Conclusions:

MiR-145 has a critical role in the inhibition of invasive and metastatic capacities of CRC, probably through directly targeting Fascin-1. This miRNA may be involved in the development and progression of CRC.     

MicroRNA-15b is induced with E2F-controlled genes in HPV-related cancer

Background:

MicroRNAs (miRNAs) are important regulators of cellular processes and are found to be deregulated in many cancers. We here analysed the miRNA expression in anal carcinomas. In a previous study, we found that our anal carcinoma tumours were divided into two groups based on the expression of E2F-regulated genes. Therefore, we searched for miRNAs that could reproduce this grouping.

Methods:

A global screen of the miRNA population was performed using real-time quantitative PCR (RT–qPCR) array methods and differentially expressed miRNAs were identified. Real-time–qPCR was used to verify the expression levels of selected miRNAs and genes in a larger collection of biopsies. A siRNA-mediated knockdown of human papilloma virus (HPV)16 E7 in a cervical cell line was performed to assess the effect of E7 on miR-15b.

Results:

The grouping of tumours into two groups based on the expression of E2F-controlled genes was confirmed in a larger collection of anal carcinoma tumours. The expression of miR-15b was shown to be highly correlated with that of five selected E2F-induced genes (CCNA2, CCNB1, CCNB2, MSH6 and MCM7). A knockdown of HPV16 E7 resulted in decreased levels of miR-15b in Ca Ski cells.

Conclusion:

MiR-15b expression correlates with E2F-regulated genes in anal carcinoma and appears to be part of the E2F-regulatory network.     

miRNA profiling in metastatic renal cell carcinoma reveals a tumour-suppressor effect for miR-215

Background:

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney. Metastatic RCC is difficult to treat. The 5-year survival rate for metastatic RCC is ⩽10%. Recently, microRNAs (miRNAs) have been shown to have a role in cancer metastasis and potential as prognostic biomarkers in cancer.

Method:

We performed a miRNA microarray to identify a miRNA signature characteristic of metastatic compared with primary RCCs. We validated our results by quantitative real-time PCR. We performed experimental and bioinformatic analyses to explore the involvement of miR-215 in RCC progression and metastasis.

Results:

We identified 65 miRNAs that were significantly altered in metastatic compared with primary RCCs. We validated our results by examining the expression of miR-10b, miR-126, miR-196a, miR-204 and miR-215, in two independent cohorts of patients. We showed that overexpression of miR-215 decreased cellular migration and invasion in an RCC cell line model. In addition, through gene expression profiling, we identified direct and indirect targets of miR-215 that can contribute to tumour metastasis.

Conclusion:

Our analysis showed that miRNAs are altered in metastatic RCCs and can contribute to kidney cancer metastasis through different biological processes. Dysregulated miRNAs represent potential prognostic biomarkers and may have therapeutic applications in kidney cancer.     

Different miRNA signatures of oral and pharyngeal squamous cell carcinomas: a prospective translational study

Background:

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV).

Methods:

Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature.

Results:

One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%.

Conclusion:

MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours.     

miR-375 is upregulated in acquired paclitaxel resistance in cervical cancer

Background:

Chemo-resistance is one of the key causal factors in cancer death and emerging evidences suggest that microRNAs (miRNAs) have critical roles in the regulation of chemo-sensitivity in cancers. Cervical cancer is one of the most common malignancies in women and insensitive to chemotherapy clinically.

Methods:

The differentially expressed miRNAs in cervical squamous cell carcinoma tissues were screened by using a microarray platform (μParaflo Sanger miRBase release 13.0). The expression of miR-375 was determined by stem-loop RT–PCR using 23 clinical cervical cancer samples and 2 cervical cancer cell lines. We exogenously upregulated miR-375 expression in SiHa and Caski cells using a pre-miRNA lentiviral vector transfection and observed its impact on paclitaxel sensitivity using MTS. The cells that stably overexpressed miR-375 were subcutaneously injected into mice to determine tumour growth and chemo-sensitivity in vivo.

Results:

Twenty-one differentially expressed miRNAs were found by miRNA microarray between pro- and post-paclitaxel cervical cancer tissues. Of those, miR-375 showed consistent high expression levels across paclitaxel-treated cervical cells and tissues. Paclitaxel induced upregulated miR-375 expression in a clear dose-dependent manner. Forced overexpression of miR-375 in cervical cancer cells decreased paclitaxel sensitivity in vitro and in vivo.

Conclusion:

Collectively, our results suggest that miR-375 might be a therapeutic target in paclitaxel-resistant cervical cancer.     

The functional significance of microRNA-145 in prostate cancer

Background:

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in numerous cellular processes. Recent studies have shown aberrant expression of miRNAs in prostate cancer tissues and cell lines. On the basis of miRNA microarray data, we found that miR-145 is significantly downregulated in prostate cancer.

Methods and results:

We investigated the expression and functional significance of miR-145 in prostate cancer. The expression of miR-145 was low in all the prostate cell lines tested (PC3, LNCaP and DU145) compared with the normal cell line, PWR-1E, and in cancerous regions of human prostate tissue when compared with the matched adjacent normal. Overexpression of miR-145 in PC3-transfected cells resulted in increased apoptosis and an increase in cells in the G2/M phase, as detected by flow cytometry. Investigation of the mechanisms of inactivation of miR-145 through epigenetic pathways revealed significant DNA methylation of the miR-145 promoter region in prostate cancer cell lines. Microarray analyses of miR-145-overexpressing PC3 cells showed upregulation of the pro-apoptotic gene TNFSF10, which was confirmed by real-time PCR and western analysis.

Conclusion:

One of the genes significantly upregulated by miR-145 overexpression is the proapoptotic gene TNFSF10. Therefore, modulation of miR-145 may be an important therapeutic approach for the management of prostate cancer.     

MicroRNA profiles classify papillary renal cell carcinoma subtypes

Background:

Besides the conventional clear-cell renal cell carcinoma (ccRCC), papillary RCC (pRCC) is the second most common renal malignancy. Papillary RCCs can further be subdivided into two distinct subtypes. Although a clinical relevance of pRCC subtyping has been shown, little is known about the molecular characteristics of both pRCC subtypes.

Methods:

We performed microarray-based microRNA (miRNA) expression profiling of primary ccRCC and pRCC cases. A subset of miRNAs was identified and used to establish a classification model for ccRCC, pRCC types 1 and 2 and normal tissue. Furthermore, we performed gene set enrichment analysis with the predicted miRNA target genes.

Results:

Only five miRNAs (miR-145, -200c, -210, -502-3p and let-7c) were sufficient to identify the samples with high accuracy. In a collection of 111 tissue samples, 73.9% were classified correctly. An enrichment of miRNA target genes in the family of multidrug-resistance proteins was noted in all tumours. Several components of the Jak-STAT signalling pathway might be targets for miRNAs that define pRCC tumour subtypes.

Conclusion:

MicroRNAs are able to accurately classify RCC samples. Deregulated miRNAs might contribute to the high chemotherapy resistance of RCC. Furthermore, our results indicate that pRCC type 2 tumours could be dependent on oncogenic MYC signalling.     

miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC)

Background:

Hypopharyngeal squamous cell carcinoma (HSCC) is an aggressive malignancy with one of the worst prognoses among all head and neck cancers. Greater understanding of the pertinent molecular oncogenic pathways could help improve diagnosis, therapy, and prevention of this disease. The aim of this study was to identify tumour-suppressive microRNAs (miRNAs), based on miRNA expression signatures from clinical HSCC specimens, and to predict their biological target genes.

Methods:

Expression levels of 365 human mature miRNAs from 10 HSCC clinical samples were screened using stem-loop real-time quantitative PCR. Downregulated miRNAs were used in cell proliferation assays to identify a tumour-suppressive miRNA. Genome-wide gene expression analyses were then performed to identify the target genes of the tumour-suppressive miRNA.

Results:

Expression analysis identified 11 upregulated and 31 downregulated miRNAs. Gain-of-function analysis of the downregulated miRNAs revealed that miR-489 inhibited cell growth in all head and neck cancer cell lines examined. The gene PTPN11 coding for a cytoplasmic protein tyrosine phosphatase containing two Src Homology 2 domains was identified as a miR-489-targeted gene. Knockdown of PTPN11 resulted in the inhibition of cell proliferation in head and neck SCC cells.

Conclusion:

Identification of the tumour-suppressive miRNA miR-489 and its target, PTPN11, might provide new insights into the underlying molecular mechanisms of HSCC.     

The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer

Background:

On the base of the microRNA (miRNA) expression signature of bladder cancer (BC), we found that miR-1 and miR-133a were significantly downregulated in BC. In this study, we focussed on the functional significance of miR-1 and miR-133a in BC cell lines and identified a molecular network of these miRNAs.

Methods and results:

We investigated the miRNA expression signature of BC clinical specimens and identified several downregulated miRNAs (miR-133a, miR-204, miR-1, miR-139-5p, and miR-370). MiR-1 and miR-133a showed potential role of tumour suppressors by functional analyses of BC cells such as cell proliferation, apoptosis, migration, and invasion assays. Molecular target searches of these miRNAs showed that transgelin 2 (TAGLN2) was directly regulated by both miR-1 and miR-133a. Silencing of TAGLN2 study demonstrated significant inhibitions of cell proliferation and increase of apoptosis in BC cell lines. The immunohistochemistry showed a positive correlation between TAGLN2 expression and tumour grade in clinical BC specimens.

Conclusions:

The downregulation of miR-1 and miR-133a was a frequent event in BC, and these miRNAs were recognised as tumour suppressive. TAGLN2 may be a target of both miRNAs and had a potential oncogenic function. Therefore, novel molecular networks provided by miRNAs may provide new insights into the underlying molecular mechanisms of BC.     

Predictive value of miR-9 as a potential biomarker for nasopharyngeal carcinoma metastasis

Background:

Nasopharyngeal carcinoma (NPC) has a distinctive geographic distribution and is characterised by its strong tendency of metastasis. We aimed to examine the microRNA (miRNA) expression profiles in plasma samples of NPC patients to explore their clinical significance in disease development and progression.

Methods:

This study was divided into four steps: (1) confirmation of differentially expressed miRNAs using microarray analysis and quantitative PCR validation; (2) comparison of plasma miR-9 levels during NPC progression; (3) evaluation of the predictive performance of plasma miR-9 as a biomarker for NPC metastasis; and (4) comparison of plasma miR-9 levels between pre- and post-treatment samples.

Results:

Plasma microarray profiling identified 33 differentially expressed miRNAs between NPC patients and healthy volunteers. The significantly declined level of miR-9 in NPC patients was confirmed through two-stage validation. The low level of plasma miR-9 was significantly correlated with worse lymphatic invasion and advanced TNM stage. The plasma miR-9 could distinguish locoregional from metastatic NPC cases with a high sensitivity and specificity. Furthermore, the plasma miR-9 level was significantly elevated in post-treatment plasma compared with those pre-treatment samples.

Conclusion:

Our study reports that plasma miR-9 may serve as a useful biomarker to predict NPC metastasis and to monitor tumour dynamics.     

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer

Background:

Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance.

Methods:

We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining.

Results:

The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients.

Conclusions:

miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.     

The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer

Background:

Although the role of human papilloma virus (HPV) in cervical squamous cell carcinoma (CSCC) is well established, the role in head and neck SCC (HNSCC) is less clear. MicroRNAs (miRNAs) have a role in the cancer development, and HPV status may affect the miRNA expression pattern in HNSCC. To explore the influence of HPV in HNSCC, we made a comparative miRNA profile of HPV-positive (HPV+) and HPV-negative (HPV−) HNSCC against CSCC.

Methods:

Fresh frozen and laser microdissected-paraffin-embedded samples obtained from patients with HPV+/HPV− HNSCC, CSCC and controls were used for microarray analysis. Differentially expressed miRNAs in the HPV+ and HPV− HNSCC samples were compared with the differentially expressed miRNAs in the CSCC samples.

Results:

Human papilloma virus positive (+) HNSCC had a distinct miRNA profile compared with HPV− HNSCC. Significantly more similarity was seen between HPV+ HNSCC and CSCC than HPV− and CSCC. A set of HPV core miRNAs were identified. Of these especially the miR-15a/miR-16/miR195/miR-497 family, miR-143/miR-145 and the miR-106-363 cluster appear to be important within the known HPV pathogenesis.

Conclusion:

This study adds new knowledge to the known pathogenic pathways of HPV and substantiates the oncogenic role of HPV in subsets of HNSCCs.     

Differential expression of microRNAs during melanoma progression: miR-200c, miR-205 and miR-211 are downregulated in melanoma and act as tumour suppressors

Background:

The incidence of malignant melanoma is increasing faster than that for any other cancer. Histological examination of skin excision biopsies remains the standard method for melanoma diagnosis and prognosis. Significant morphological overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis.

Methods:

To identify improved molecular markers to support histological examination, we used microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis to identify differentially expressed microRNAs (miRNAs). Differential expression was validated by qRT–PCR, and functional studies were carried out after transfection of miRNA precursors or inhibitors into melanoma cells to modulate miRNA expression.

Results:

In all, 20 miRNAs showed highly significant differential expression between benign naevi and either primary or metastatic melanomas, the majority being downregulated in melanoma, whereas only 2 miRNAs, namely miR-203 and miR-205, were differentially expressed between primary and metastatic melanomas. In functional in vitro assays, overexpression of miR-200c and miR-205 inhibited anchorage-independent colony formation and overexpression of miR-211 inhibited both anchorage-independent colony formation and invasion.

Conclusion:

We have identified a series of differentially expressed miRNAs that could be useful as diagnostic or prognostic markers for melanoma and have shown that three miRNAs (namely miR-200c, miR-205 and miR-211) act as tumour suppressors.     

MicroRNAs miR-371-3 in serum as diagnostic tools in the management of testicular germ cell tumours

Background:

miRNAs are small noncoding RNA molecules that can be released into body fluids. Germ cell tumours (GCTs) overexpress miRNAs of the miR-371-3 cluster. Thus, serum levels of these miRNAs may correlate with tumour load.

Methods:

miRNAs of the miR-371-3 cluster were quantified in cubital vein blood samples of 20 GCT patients with clinical stage 1, and of 4 patients with advanced stages before and after treatment. In six patients testicular vein blood (TVB) was examined additionally. Seventeen healthy males served as controls. Likewise, expression of miRNAs in 15 matching tumour specimens was measured.

Results:

In all patients, serum levels of miRNAs 371-3 were much higher than in controls. In stage 1, levels decreased postoperatively 336.7-fold, 7.4-fold, and 7.7-fold for miRNAs 371a-3p, 372, and 373-3p, respectively (P<0.01). Also, in those cases with advanced disease levels dropped to the normal range after completion of treatment. miR-371-3 levels in TVB exceeded those in peripheral blood in all cases. Expression of miR-371a-3p was also documented in tumour tissue. However, no correlation was found regarding the extent of miRNA expression in tissue and the values measured in matching serum.

Conclusion:

Thus, miR-371a-3p serum level appears to be a useful biomarker in GCTs.     

MicroRNA expression signature of oral squamous cell carcinoma: functional role of microRNA-26a/b in the modulation of novel cancer pathways

Background:

MicroRNAs (miRNAs) have been shown to play major roles in carcinogenesis in a variety of cancers. The aim of this study was to determine the miRNA expression signature of oral squamous cell carcinoma (OSCC) and to investigate the functional roles of miR-26a and miR-26b in OSCC cells.

Methods:

An OSCC miRNA signature was constructed by PCR-based array methods. Functional studies of differentially expressed miRNAs were performed to investigate cell proliferation, migration, and invasion in OSCC cells. In silico database and genome-wide gene expression analyses were performed to identify molecular targets and pathways mediated by miR-26a/b.

Results:

miR-26a and miR-26b were significantly downregulated in OSCC. Restoration of both miR-26a and miR-26b in cancer cell lines revealed that these miRNAs significantly inhibited cancer cell migration and invasion. Our data demonstrated that the novel transmembrane TMEM184B gene was a direct target of miR-26a/b regulation. Silencing of TMEM184B inhibited cancer cell migration and invasion, and regulated the actin cytoskeleton-pathway related genes.

Conclusions:

Loss of tumour-suppressive miR-26a/b enhanced cancer cell migration and invasion in OSCC through direct regulation of TMEM184B. Our data describing pathways regulated by tumour-suppressive miR-26a/b provide new insights into the potential mechanisms of OSCC oncogenesis and metastasis.