Integrated miRNA and mRNA expression profiling of the inflammatory breast cancer subtype

Background:

MicroRNAs (miRNAs) are key regulators of gene expression. In this study, we explored whether altered miRNA expression has a prominent role in defining the inflammatory breast cancer (IBC) phenotype.

Methods:

We used quantitative PCR technology to evaluate the expression of 384 miRNAs in 20 IBC and 50 non-IBC samples. To gain understanding on the biological functions deregulated by aberrant miRNA expression, we looked for direct miRNA targets by performing pair-wise correlation coefficient analysis on expression levels of 10 962 messenger RNAs (mRNAs) and by comparing these results with predicted miRNA targets from TargetScan5.1.

Results:

We identified 13 miRNAs for which expression levels were able to correctly predict the nature of the sample analysed (IBC vs non-IBC). For these miRNAs, we detected a total of 17 295 correlated miRNA–mRNA pairs, of which 7012 and 10 283 pairs showed negative and positive correlations, respectively. For four miRNAs (miR-29a, miR-30b, miR-342-3p and miR-520a-5p), correlated genes were concordant with predicted targets. A gene set enrichment analysis on these genes demonstrated significant enrichment in biological processes related to cell proliferation and signal transduction.

Conclusions:

This study represents, to the best of our knowledge, the first integrated analysis of miRNA and mRNA expression in IBC. We identified a set of 13 miRNAs of which expression differed between IBC and non-IBC, making these miRNAs candidate markers for the IBC subtype.     

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.     

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.     

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.     

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.     

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.     

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.     

Circulating microRNAs in plasma of patients with oesophageal squamous cell carcinoma

Background:

Several recent studies demonstrated that microRNAs (miRNAs) are stably detectable in plasma/serum. We hypothesised that plasma miRNAs concentrations contributed to potential biomarkers in patients with oesophageal squamous cell carcinoma (ESCC).

Methods:

We selected three oncogenic miRNAs (miR-21, miR-184, miR-221) and one tumour suppressive miRNA (miR-375), which are frequently reported in squamous cell carcinoma, as candidate targets for this plasma miRNA assay. This study was divided into three steps: (1) Determination of appropriate plasma miRNAs in preliminary tests. (2) Evaluation of whether the plasma miRNA assays could monitor tumour dynamics. (3) Validation study on the clinical application of plasma miRNA assays in 50 ESCC patients and 20 healthy volunteers.

Results:

(1) In preliminary tests, the plasma level of miR-21 was significantly higher (P=0.0218) and that of miR-375 (P=0.0052) was significantly lower in ESCC patients than controls. (2) The high plasma miR-21 levels reflected tumour levels in all cases (100%). The plasma level of miR-21 was significantly reduced in postoperative samples (P=0.0058). (3) On validation analysis, the plasma level of miR-21 tended to be higher in ESCC patients (P=0.0649), while that of miR-375 was significantly lower (P<0.0001) and the miR-21/miR-375 ratio was significantly higher (P<0.0001) in ESCC patients than in controls. The value of the area under the receiver-operating characteristic curve (AUC) was 0.816 for the miR-21/miR-375 ratio assay. Patients with a high plasma level of miR-21 tended to have greater vascular invasion (P=0.1554) and to show a high correlation with recurrence (P=0.0164).

Conclusion:

Detection of circulating miRNAs might provide new complementary tumour markers for ESCC.     

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 profiling of diagnostic needle aspirates from patients with pancreatic cancer

Background:

A major challenge to the development of biomarkers for pancreatic cancer (PC) is the small amount of tissue obtained at the time of diagnosis. Single-gene analyses may not reliably predict biology of PC because of its complex molecular makeup. MicroRNA (miRNA) profiling may provide a more informative molecular interrogation of tumours. The primary objective of this study was to determine the feasibility of performing miRNA arrays and quantitative real-time PCR (qRT–PCR) from archival formalin-fixed paraffin-embedded (FFPE) cell blocks obtained from fine-needle aspirates (FNAs) that is the commonest diagnostic procedure for suspected PC.

Methods:

MicroRNA expression profiling was performed on FFPE from FNA of suspicious pancreatic masses. Subjects included those who had a pathological diagnosis of pancreatic adenocarcinoma and others with a non-malignant pancreatic histology. Exiqon assay was used to quantify miRNA levels and qRT–PCR was used to validate abnormal expression of selected miRNAs.

Results:

A total of 29 and 15 subjects had pancreatic adenocarcinoma and no evidence of cancer, respectively. The RNA yields per patient varied from 25 to 100 ng. Profiling demonstrated deregulation of over 228 miRNAs in pancreatic adenocarcinoma of which the top 7 were further validated by qRT–PCR. The expression of let-7c, let-7 f, and miR-200c were significantly reduced in most patients whereas the expression of miR-486-5p and miR-451 were significantly elevated in all pancreas cancer patients. MicroRNAs let-7d and miR-423-5p was either downregulated or upregulated with a significant inter-individual variation in their expression.

Conclusion:

This study demonstrated the feasibility of using archival FFPE cell blocks from FNAs to establish RNA-based molecular signatures unique to pancreatic adenocarcinoma with potential applications in clinical trials for risk stratification, patient selection, and target validation.     

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.     

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.     

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.     

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-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.     

Whole blood-derived miRNA profiles as potential new tools for ovarian cancer screening

Background:

Screening is an unsolved problem for ovarian cancer (OvCA). As late detection is equivalent to poor prognosis, we analysed whether OvCA patients show diagnostically meaningful microRNA (miRNA) patterns in blood cells.

Methods:

Blood-borne whole miRNome profiles from 24 patients with OvCA and 15 age- and sex-matched healthy controls were biostatistically evaluated.

Results:

Student''s t-test revealed 147 significantly deregulated miRNAs before and 4 after Benjamini–Hochberg adjustment. Although these included miRNAs already linked to OvCA (e.g., miR-16, miR-155), others had never before been connected to specific diseases. A bioinformatically calculated miRNA profile allowed for discrimination between blood samples of OvCA patients and healthy controls with an accuracy of >76%. When only cancers of the serous subtype were considered and compared with an extended control group (n=39), accuracy, specificity and sensitivity all increased to >85%.

Conclusion:

Our proof-of-principle study strengthens the hypothesis that neoplastic diseases generate characteristic miRNA fingerprints in blood cells. Still, the obtained OvCA-associated miRNA pattern is not yet sensitive and specific enough to permit the monitoring of disease progression or even preventive screening. Microarray-based miRNA profiling from peripheral blood could thus be combined with other markers to improve the notoriously difficult but important screening for OvCA.