The miR-143/-145 cluster regulates plasminogen activator inhibitor-1 in bladder cancer

Background:

Upregulation of the proto-oncogene plasminogen activator inhibitor-1 (PAI-1) is a common hallmark of various solid tumours, but the mechanisms controlling its expression are not fully understood.

Methods:

We investigate microRNAs (miRNAs) regulating PAI-1 in a panel of normal bladder urothelial biopsies, superficial Ta bladder tumours and invasive T1–T4 tumours using expression microarrays and qRT–PCR. The prognostic implications of PAI-1 deregulation are established by tissue microarray staining of non-muscle-invasive bladder tumours. MicroRNA repression of PAI-1 is assayed by ectopic miRNA expression, argonaute immunoprecipitation and luciferase assays.

Results:

We found that the miR-143/-145 cluster is downregulated in all stages of bladder cancer and inversely correlated with PAI-1 expression. Mature miR-143 and miR-145 are coordinately expressed, and both directly target the PAI-1 3′UTR, leading to reduced PAI-1 mRNA and protein levels. Furthermore, we show that PAI-1 and miR-145 levels may serve as useful prognostic markers for non-muscle-invasive bladder tumours for which accurate progressive outcome is currently difficult to predict.

Conclusion:

This report provides the first evidence for direct miRNA regulation of PAI-1 in bladder cancer. We also demonstrate mRNA co-targeting by a cluster of non-family miRNAs, and suggest miR-145 and PAI-1 as clinically relevant biomarkers in bladder cancer.     

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.     

Circulating miR-378 in plasma: a reliable,haemolysis-independent biomarker for colorectal cancer

Background:

Plasma circulating tumour-specific microRNAs (miRNAs) are promising biomarkers of tumour presence and recurrence, especially for diseases whose best chance of successful treatment requires early diagnosis and timely surgery of an already malignant but not yet invasive tumour, such as colorectal cancer (CRC).

Methods:

Expression levels of miRNAs previously found to be differently expressed in tumour vs normal colon tissues were investigated by quantitative real-time PCR in plasma from CRC patients and from healthy donors and confirmed in independent case control series. The validated miRNAs were also measured after surgery. Analyses were repeated on the subsets of haemolysis-free samples.

Results:

We identified four miRNAs differently expressed between the compared groups, two (miR-21 and miR-378) of which were validated. miR-378 expression decreased in non-relapsed patients 4–6 months after surgery and miR-378 ability to discriminate CRC patients from healthy individuals was not influenced by haemolysis levels of plasma samples.

Conclusion:

The miRNA analysis on plasma samples represents a useful non-invasive tool to assess CRC presence as well as tumour-free status at follow-up. Plasma levels of miR-378 could be used to discriminate CRC patients from healthy individuals, irrespective of the level of haemoglobin of plasma samples.     

Identifying microRNAs regulating B7-H3 in breast cancer: the clinical impact of microRNA-29c

Background:

B7-H3, an immunoregulatory protein, is overexpressed in several cancers and is often associated with metastasis and poor prognosis. Here, our aim was to identify microRNAs (miRNAs) regulating B7-H3 and assess their potential prognostic implications in breast cancer.

Methods:

MicroRNAs targeting B7-H3 were identified by transfecting two breast cancer cell lines with a library of 810 miRNA mimics and quantifying changes of B7-H3 protein levels using protein lysate microarrays. For validations we used western immunoblotting and 3′-UTR luciferase assays. Clinical significance of the miRNAs was assayed by analysing whether their expression levels correlated with outcome in two cohorts of breast cancer patients (142 and 81 patients).

Results:

We identified nearly 50 miRNAs that downregulated B7-H3 protein levels. Western immunoblotting validated the impact of the 20 most effective miRNAs. Thirteen miRNAs (miR-214, miR-363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593) targeted B7-H3 directly by binding to its 3′-UTR region. Finally, high expression of miR-29c was associated with a significant reduced risk of dying from breast cancer in both cohorts.

Conclusions:

We identified miRNAs efficiently downregulating B7-H3 expression. The expression of miR-29c correlated with survival in breast cancer patients, suggesting a tumour suppressive role for this miRNA.     

Deregulated microRNAs in gastric cancer tissue-derived mesenchymal stem cells: novel biomarkers and a mechanism for gastric cancer

Background:

MicroRNAs (miRNAs) are involved in gastric cancer development and progression. However, the expression and role of miRNAs in gastric cancer stromal cells are still unclear.

Methods:

The miRNAs differentially expressed in gastric cancer tissue-derived mesenchymal stem cells (GC-MSCs) relative to adjacent non-cancerous tissue-derived MSCs (GCN-MSCs) and in cancer tissues relative to adjacent non-cancerous tissues were screened using miRNA microarray and validated by quantitative RT–PCR. The impact of GC-MSCs on HGC-27 cells was observed in vitro using colony formation and transwell assays, and these cells were subcutaneously co-injected into mice to assess tumour growth in vivo. Exogenous downregulation of miR-221 expression in cells was achieved using an miRNA inhibitor.

Results:

miR-214, miR-221 and miR-222 were found to be commonly upregulated in GC-MSCs and cancer tissues. Their levels were tightly associated with lymph node metastasis, venous invasion and the TNM stage. Gastric cancer tissue-derived mesenchymal stem cells significantly promoted HGC-27 growth and migration and increased the expression of miR-221 via paracrine secretion, and the targeted inhibition of miR-221 in GC-MSCs could block its tumour-supporting role. GC-MSC-derived exosomes were found to deliver miR-221 to HGC-27 cells and promoted their proliferation and migration.

Conclusions:

Gastric cancer tissue-derived mesenchymal stem cells favour gastric cancer progression by transferring exosomal miRNAs to gastric cancer cells, thus providing a novel mechanism for the role of GC-MSCs and new biomarkers for gastric cancer.     

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.     

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.     

Detection of gastric cancer-associated microRNAs on microRNA microarray comparing pre- and post-operative plasma

Background:

Recently, it was reported that plasma microRNAs (miRNAs) are low-invasive useful biomarkers for cancer. We attempted to isolate gastric cancer (GC)-associated miRNAs comparing pre- and post-operative paired plasma, thereby excluding the possible effects of individual variability.

Methods:

This study was divided into four steps: (1) microarray analysis comparing pre- and post-operative plasma; (2) validation of candidate miRNAs by quantitative RT–PCR; (3) validation study of selected miRNAs using paired plasma; and (4) comparison of the levels of selected miRNAs in plasma between healthy controls and patients.

Results:

From the results of microarray analysis, nine candidate miRNAs the levels of which were markedly decreased in post-operative plasma were selected for further studies. After confirmation of their post-operative marked reduction, two candidate miRNAs, miR-451 and miR-486, were selected as plasma biomarkers, considering the abundance in plasma, and marked decrease in post-operative samples. In validation, the two miRNAs were found to decrease in post-operative plasma in 90 and 93% of patients (both P<0.01). In comparison with healthy controls, the levels of both miRNAs were found to be significantly higher in patients, and the area under the curve values were high at 0.96 and 0.92.

Conclusion:

Plasma miR-451 and miR-486 could be useful blood-based biomarkers for screening GC.     

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.     

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.     

An evaluation of urinary microRNA reveals a high sensitivity for bladder cancer

Background:

Urinary biomarkers are needed to improve the care and reduce the cost of managing bladder cancer. Current biomarkers struggle to identify both high and low-grade cancers due to differing molecular pathways. Changes in microRNA (miR) expression are seen in urothelial carcinogenesis in a phenotype-specific manner. We hypothesised that urinary miRs reflecting low- and high-grade pathways could detect bladder cancers and overcome differences in genetic events seen within the disease.

Methods:

We investigated urinary samples (n=121) from patients with bladder cancer (n=68) and age-matched controls (n=53). Fifteen miRs were quantified using real-time PCR.

Results:

We found that miR is stable within urinary cells despite adverse handling and detected differential expression of 10 miRs from patients with cancer and controls (miRs−15a/15b/24-1/27b/100/135b/203/212/328/1224, ANOVA P<0.05). Individually, miR-1224-3p had the best individual performance with specificity, positive and negative predictive values and concordance of 83%, 83%, 75% and 77%, respectively. The combination of miRs-135b/15b/1224-3p detected bladder cancer with a high sensitivity (94.1%), sufficient specificity (51%) and was correct in 86% of patients (concordance).

Conclusion:

The use of this panel in patients with haematuria would have found 94% of urothelial cell carcinoma, while reducing cystoscopy rates by 26%. However, two invasive cancers (3%) would have been missed.     

Circulating microRNAs as prognostic therapy biomarkers in head and neck cancer patients

Background:

The prediction of therapy response in head and neck squamous cell cancer (HNSCC) requires biomarkers, which are also a prerequisite for personalised therapy concepts. The current study aimed to identify therapy-responsive microRNAs (miRNAs) in the circulation that can serve as minimally invasive prognostic markers for HNSCC patients undergoing radiotherapy.

Methods:

We screened plasma miRNAs in a discovery cohort of HNSCC patients before therapy and after treatment. We further compared the plasma miRNAs of the patients to age- and sex-matched healthy controls. All miRNAs identified as biomarker candidates were then confirmed in an independent validation cohort of HNSCC patients and tested for correlation with the clinical outcome.

Results:

We identified a signature of eight plasma miRNAs that differentiated significantly (P=0.003) between HNSCC patients and healthy donors. MiR-186-5p demonstrated the highest sensitivity and specificity to classify HNSCC patients and healthy individuals. All therapy-responsive and patient-specific miRNAs in plasma were also detectable in tumour tissues derived from the same patients. High expression of miR-142-3p, miR-186-5p, miR-195-5p, miR-374b-5p and miR-574-3p in the plasma correlated with worse prognosis.

Conclusions:

Circulating miR-142-3p, miR-186-5p, miR-195-5p, miR-374b-5p and miR-574-3p represent the most promising markers for prognosis and therapy monitoring in the plasma of HNSCC patients. We found strong evidence that the circulating therapy-responsive miRNAs are tumour related and were able to validate them in an independent cohort of HNSCC patients.     

MiR-221/-222 differentiate prognostic groups in advanced breast cancers and influence cell invasion

Background:

MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells.

Methods:

MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT–PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222.

Results:

In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed.

Conclusion:

This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.     

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.     

Genistein downregulates onco-miR-1260b and inhibits Wnt-signalling in renal cancer cells

Background:

Wnt-signalling has an important role in renal cancer and it is modulated by genistein in other cancers. Recently, microRNAs (miRNAs) have emerged as new regulators of gene expression. Thus, we focused on miRNAs to examine the regulatory mechanism of genistein on the Wnt-signalling pathway in renal cell carcinoma (RCC).

Methods:

Initially, we investigated the effect of genistein on Wnt-signalling (TOPflash reporter assay (TCF reporter assays)) in renal cancer cells, and using microarray identified candidate miRNAs whose expression was decreased by genistein. We performed functional analyses and investigated the relationship between miRNA expression and renal cancer patient outcomes. We also did 3′UTR luciferase assays to look at direct miRNA regulation of Wnt-signalling-related genes.

Results:

Genistein promoted apoptosis while inhibiting RCC cell proliferation and invasion. Genistein also decreased TCF reporter activity in RCC cells. We found that miR-1260b was highly expressed and significantly downregulated by genistein in RCC cells. The expression of miR-1260b was significantly higher in renal cancer tissues compared with normal, and significantly related to overall shorter survival. In addition, miR-1260b promoted renal cancer cell proliferation and invasion in RCC cells. The 3′UTR luciferase activity of target genes (sFRP1, Dkk2, Smad4) was significantly decreased and their protein expression significantly upregulated in miR-1260b inhibitor-transfected renal cancer cells.

Conclusion:

Our data suggest that genistein inhibited Wnt-signalling by regulating miR-1260b expression in renal cancer cells.     

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.