Association of the microRNA-499 variants with susceptibility to hepatocellular carcinoma in a Chinese population

microRNAs (miRNAs) are a new class of small non-coding RNAs that function as tumor suppressors or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNA may contribute to cancer development. We hypothesized that genetic variations of the miRNA could be associated with the risk of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). A total of 100 patients with HCC, 100 cases of chronic hepatitis B and 100 health adults were enrolled in this present study. Two common polymorphisms in pre-miRNAs: Homo sapiens miRNA-146a (hsa-mir-146a) (rs291016, guanine to cytosine [G-C]) and hsa-mir-499 (rs3746444; adenine to guanine [C-T]) were genotyped by PCR-Restriction Fragment Length Polymorphism and confirmed by bidirectional DNA sequencing. Significant differences were found in frequency and distribution of the genotypes of miRNA-499 between the HCC and the control group. Compared with miRNA-499 T/T, the odds ratio (OR) of patients with miRNA-499 C/C for developing HCC was 3.630 (95% CI: 1.545–8.532), and OR for developing HBV-related HCC was 3.133 (95% CI: 1.248–7.861). There was no significant association between miRNA-146a polymorphism and the risk of HCC in all subjects. Our results suggested that hsa-mir-499 polymorphism was associated with susceptibility to HBV-related HCC in Chinese population. Further characterization of miRNA SNPs may open new avenue for the study of cancer and therapeutic interventions.     

Cell-Free MicroRNA Expression Profiles in Malignant Effusion Associated with Patient Survival in Non-Small Cell Lung Cancer

Objective

MicroRNAs (miRNAs) expression is altered in cancer cells, and miRNAs could serve as diagnostic and prognostic biomarker for cancer patients. This study was designed to analyze circulating miRNAs expression in the malignant pleural effusion (MPE) and their association with patient survival in non-small cell lung cancer (NSCLC).

Methods

Pleural effusion from 184 patients with NSCLC and MPE were collected. MiRNA microarray and bioinformatics interpretation were used to evaluate miRNA expression profiles in 10 NSCLC patients with different survival prognosis. Associations were validated in 184 patients (randomly classified into training and validation set with equal number in each group) using quantitative RT-PCR. Risk scores were formulated based on the expression signature of miRNAs. Clinical data, such as patient survival, were collected for correlation analysis.

Results

Thirty-three miRNAs were found to be altered more than two-fold by microarray in malignant effusions between longer-survival and shorter-survival groups, and levels of five miRNAs (miRNA-93, miRNA-100, miRNA-134, miRNA-151 and miRNA-345) were significantly associated with overall survival. High expression of miR-100 and low expression of miRNA-93, miRNA-134, miRNA-151 and miRNA-345 were associated with poor survival in both the training and validation cohort. Patients with high risk scores had overall poor survival compared to the patients with low risk scores. Risk score was an independent predictor of patient survival.

Conclusions

Expression patterns of miRNAs are systematically altered in MPE of patient with NSCLC. The five miRNA signature from the effusion may serve as a predictor for the overall survival of patients with lung cancers.     

A Seven-microRNA Expression Signature Predicts Survival in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth common cancer. The differential expression of microRNAs (miRNAs) has been associated with the prognosis of various cancers. However, limited information is available regarding genome-wide miRNA expression profiles in HCC to generate a tumor-specific miRNA signature of prognostic values. In this study, the miRNA profiles in 327 HCC patients, including 327 tumor and 43 adjacent non-tumor tissues, from The Cancer Genome Atlas (TCGA) Liver hepatocellular carcinoma (LIHC) were analyzed. The associations of the differentially expressed miRNAs with patient survival and other clinical characteristics were examined with t-test and Cox proportional regression model. Finally, a tumor-specific miRNA signature was generated and examined with Kaplan–Meier survival, univariate\multivariate Cox regression analyses and KEGG pathway analysis. Results showed that a total of 207 miRNAs were found differentially expressed between tumor and adjacent non-tumor HCC tissues. 78 of them were also discriminatively expressed with gender, race, tumor grade and AJCC tumor stage. Seven miRNAs were significantly associated with survival (P value <0.001). Among the seven significant miRNAs, six (hsa-mir-326, hsa-mir-3677, hsa-mir-511-1, hsa-mir-511-2, hsa-mir-9-1, and hsa-mir-9-2) were negatively associated with overall survival (OS), while the remaining one (hsa-mir-30d) was positively correlated. A tumor-specific 7-miRNAs signature was generated and validated as an independent prognostic predictor. Collectively, we have identified and validated an independent prognostic model based on the expression of seven miRNAs, which can be used to assess patients’ survival. Additional work is needed to translate our model into clinical practice.     

Integrated analysis of clinical significance and functional involvement of microRNAs in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common lethal cancers worldwide. To explore the potential prognosis-associated microRNAs (miRNAs) for HCC patients, we performed integrated analyses on the miRNA expression profiles from The Cancer Genome Atlas project. Genome-wide overall survival (OS)- and progression-free survival (PFS)-associated miRNA screening were performed by multivariate Cox proportional hazards regression analyses. A five-miRNA expression signature (miR-148a, miR-3677-3p, miR-744*, miR-210, and miR-3613-5p) was identified as an indicator for HCC OS (p < .0001; hazard ratio [HR] = 2.631). In addition, a seven-miRNA expression signature (miR-127-5p, miR-146a, miR-152, miR-193a-3p, miR-331-5p, miR-500a*, and miR-550a*) was identified as a predictor for HCC PFS (p < .0001; HR = 2.608). This systematic analysis suggested that both the OS- and PFS-associated signatures have better performance in HCC survival prediction than the conventional clinicopathological parameters. Further functional enrichment analysis of the corresponding genes targeted by these signature miRNAs revealed their biological significance in the PI3K-Akt signaling pathway. In conclusion, our present study identified a five-miRNA OS-associated signature and a seven-miRNA PFS-associated signature as HCC prognostic biomarkers with potential clinical significance, which could enable the development of novel targeted therapeutic strategies for HCC treatment.     

miRNA profiling of granulosa cell-derived exosomes reveals their role in promoting follicle development

To explore whether granulosa cell (GC)-derived exosomes (GC-Exos) and follicular fluid-derived exosomes (FF-Exos) have functional similarities in follicle development and to establish relevant experiments to validate whether GC-Exos could serve as a potential substitute for follicular fluid-derived exosomes to improve folliculogenesis. GC-Exos were characterized. MicroRNA (miRNA) profiles of exosomes from human GCs and follicular fluid were analyzed in depth. The signature was associated with folliculogenesis, such as phosphatidylinositol 3 kinases-protein kinase B signal pathway, mammalian target of rapamycin signal pathway, mitogen-activated protein kinase signal pathway, Wnt signal pathway, and cyclic adenosine monophosphate signal pathway. A total of five prominent miRNAs were found to regulate the above five signaling pathways. These miRNAs include miRNA-486-5p, miRNA-10b-5p, miRNA-100-5p, miRNA-99a-5p, and miRNA-21-5p. The exosomes from GCs and follicular fluid were investigated to explore the effect on folliculogenesis by injecting exosomes into older mice. The proportion of follicles at each stage is counted to help us understand folliculogenesis. Exosomes derived from GCs were isolated successfully. miRNA profiles demonstrated a remarkable overlap between the miRNA profiles of FF-Exos and GC-Exos. The shared miRNA signature exhibited a positive influence on follicle development and activation. Furthermore, exosomes derived from GCs and follicular fluid promoted folliculogenesis in older female mice. Exosomes derived from GCs had similar miRNA profiles and follicle-promoting functions as follicular fluid exosomes. Consequently, GC-Exos are promising for replacing FF-Exos and developing new commercial reagents to improve female fertility.     

MicroRNA signature predicts survival in papillary thyroid carcinoma

Papillary thyroid cancer (PTC) accounts for the majority of malignant thyroid tumors. Recently, several microRNA (miRNA) expression profiling studies have used bioinformatics to suggest miRNA signatures as potential prognostic biomarkers in various malignancies. However, a prognostic miRNA biomarker has not yet been established for PTC. The aim of the present study was to identify miRNAs with prognostic value for the overall survival (OS) of patients with PTC by analyzing high-throughput miRNA data and their associated clinical characteristics downloaded from The Cancer Genome Atlas database. From our dataset, 150 differentially expressed miRNAs were identified between tumor and nontumor samples; of these miRNAs, 118 were upregulated and 32 were downregulated. Among the 150 differentially expressed miRNAs, a four miRNA signature was identified that reliably predicts OS in patients with PTC. This miRNA signature was able to classify patients into a high-risk group and a low-risk group with a significant difference in OS (P < .01). The prognostic value of the signature was validated in a testing set ( P < .01). The four miRNA signature was an independent prognostic predictor according to the multivariate analysis and demonstrated good performance in predicting 5-year disease survival with an area under the receiver operating characteristic curve area under the curve (AUC) score of 0.886. Thus, this signature may serve as a novel biomarker for predicting the survival of patients with PTC.     

High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal,Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options.     

microRNA-342, microRNA-191 and microRNA-510 are differentially expressed in T regulatory cells of type 1 diabetic patients

Regulatory T cells (Tregs) are critical regulators of autoimmune diseases, including type 1 diabetes mellitus. It is hypothesised that Tregs’ function can be influenced by changes in the expression of specific microRNAs (miRNAs). Thus, we performed miRNAs profiling in a population of Tregs separated from peripheral blood of five type 1 diabetic patients and six healthy donors. For more detailed molecular characterisation of Tregs, we additionally compared miRNAs expression profiles of Tregs and conventional T cells. Tregs were isolated according to CD3+, CD4+, CD25^hi+ and CD127− by flow cytometry, and miRNA expression profiling was performed using TaqMan Array Human MicroRNA Panel-1 (384-well low density array). In Tregs of diabetic patients we found significantly increased expression of miRNA-510 (p = 0.05) and decreased expression of both miRNA-342 (p < 0.0001) and miRNA-191 (p = 0.0079). When comparing Tregs and T cells, we revealed that Tregs had significant higher expression of miRNA-146a and lower expression of eight specific miRNAs (20b, 31, 99a, 100, 125b, 151, 335, and 365). To our knowledge, this is the first study demonstrating changes in miRNA expression profiles occurring in Tregs of T1D patients and a miRNAs signature of adult Tregs.     

lncRNA RMRP knockdown suppress hepatocellular carcinoma biological activities via regulation miRNA-206/TACR1

Long noncoding RNA, RNA component of mitochondrial RNA processing endoribonuclease (RMRP) plays an important role in cancer development and is closely correlated with prognosis in cancer patients. However, whether RMRP affects prognosis in patients with hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to investigate the expression level of RMRP in HCC and its correlation with prognosis in patients with HCC and explain the effects and associated mechanisms by conducting an in vitro study. The high expression level of RMRP was correlated with poor prognosis in patients with HCC. Using in vitro analysis, RMRP knockdown suppressed HCC cell proliferation, invasion, and migration (P < .05). miRNA-206 overexpression had similar effects in HCC cell lines (Bel-7402 and Huh-7). Using Western blot analysis and cellular immunofluorescence detection, RMRP downregulation significantly suppressed TACR1/Erk1/2 pathway, while miRNA-206 was significantly upregulated (P < .05). RMRP downregulation inhibits HCC-related biological activities by the regulation of miRNA-206/TACR1.     

Characterization of microRNAs from sheep (<Emphasis Type="Italic">Ovis aries</Emphasis>) using computational and experimental analyses

Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA* and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA* had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3′UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA* in sheep.     

A robust six-miRNA prognostic signature for head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) remains a major health problem worldwide. We aimed to identify a robust microRNA (miRNA)-based signature for predicting HNSCC prognosis. The miRNA expression profiles of HNSCC were obtained from The Cancer Genome Atlas (TCGA) database. The TCGA HNSCC cohort was randomly divided into the discovery and validation cohort. A miRNA-based prognostic signature was built up based on TGCA discovery cohort, and then further validated. The downstream targets of prognostic miRNAs were subjected to functional enrichment analyses. The role of miR-1229-3p, a prognosis-related miRNA, in tumorigenesis of HNSCC was further evaluated. A total of 305 significantly differentially expressed miRNAs were found between HNSCC samples and normal tissues. A six-miRNA prognostic signature was constructed, which exhibited a strong association with overall survival (OS) in the TCGA discovery cohort. In addition, these findings were successfully confirmed in TCGA validation cohort and our own independent cohort. The miRNA-based signature was demonstrated as an independent prognostic indicator for HNSCC. A risk signature-based nomogram model was constructed and showed good performance for predicting the OS for HNSCC. The functional analyses revealed that the downstream targets of these prognostic miRNAs were closely linked to cancer progression. Mechanistically, in vitro analysis revealed that miR-1229-3p played a tumor promoting role in HNSCC. In conclusion, our study has developed a robust miRNA-based signature for predicting the prognosis of HNSCC with high accuracy, which will contribute to improve the therapeutic outcome.     

Identification of miRNA targets with stable isotope labeling by amino acids in cell culture

miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection. This repressed set of genes significantly overlaps with miRNA-1 regulated genes that have been identified with DNA array technology and are predicted by computational methods. Moreover, we find that the 3′-untranslated region for the repressed set are enriched in miRNA-1 complementary sites. Our findings demonstrate that SILAC can be used for miRNA target identification and that one highly expressed miRNA can regulate the levels of many different proteins.     

MicroRNA-574-5p Was Pivotal for TLR9 Signaling Enhanced Tumor Progression via Down-Regulating Checkpoint Suppressor 1 in Human Lung Cancer

Accumulating data suggested that functional expression of Toll-like receptors (TLRs) in tumor cells was involved in tumor progression. Our previous study demonstrated that TLR9 signaling could enhance the tumor progression of human lung cancer cells in vitro and in vivo. We further showed that miR-574-5p was the mostly up-regulated miRNA in human lung cancer cells under TLR9 signaling by miRNA array analysis. Here we characterized the potential role of miRNA-574-5p in enhanced tumor progression induced by TLR9 signaling in human lung cancer. We confirmed that TLR9 signaling effectively elevated the expression of miR-574-5p in human lung cancer cells. Notably, we found that down-regulation of miRNA-574-5p using miR-574-5p inhibitor in vitro or miR-574-5p sponge in vivo significantly abrogated the enhanced tumor progression induced by TLR9 signaling. Further studies showed that miR-574-5p was an important player associated with enhanced tumor progression of human lung cancer cells. Notably, we identified checkpoint suppressor 1 (Ches1) as the dominant direct target for miRNA-574-5p to confer the TLR9 signaling enhanced tumor progression. We revealed that over-expression of Ches1 significantly inhibited the cell cycle entry of human lung cancer cells. Finally, we revealed that the expression of miR-574-5p was positively correlated with TLR9 and reversely correlated with Ches1 in lung cancer patients. Our findings not only facilitated the further understanding of the crosstalk between miRNAs and TLRs in tumor biology, but also provided novel potential candidates for treatment of cancer.     

MicroRNAs up-regulated by CagA of Helicobacter pylori induce intestinal metaplasia of gastric epithelial cells

CagA of Helicobacter pylori is a bacterium-derived oncogenic protein closely associated with the development of gastric cancers. MicroRNAs (miRNAs) are a class of widespread non-coding RNAs, many of which are involved in cell growth, cell differentiation and tumorigenesis. The relationship between CagA protein and miRNAs is unclear. Using mammalian miRNA profile microarrays, we found that miRNA-584 and miRNA-1290 expression was up-regulated in CagA-transformed cells, miRNA-1290 was up-regulated in an Erk1/2-dependent manner, and miRNA-584 was activated by NF-κB. miRNA-584 sustained Erk1/2 activities through inhibition of PPP2a activities, and miRNA-1290 activated NF-κB by knockdown of NKRF. Foxa1 was revealed to be an important target of miRNA-584 and miRNA-1290. Knockdown of Foxa1 promoted the epithelial-mesenchymal transition significantly. Overexpression of miRNA-584 and miRNA-1290 induced intestinal metaplasia of gastric epithelial cells in knock-in mice. These results indicate that miRNA-584 and miRNA-1290 interfere with cell differentiation and remodel the tissues. Thus, the miRNA pathway is a new pathogenic mechanism of CagA.     

A ten-microRNA expression signature predicts survival in glioblastoma

Glioblastoma (GBM) is the most common and aggressive primary brain tumor with very poor patient median survival. To identify a microRNA (miRNA) expression signature that can predict GBM patient survival, we analyzed the miRNA expression data of GBM patients (n=222) derived from The Cancer Genome Atlas (TCGA) dataset. We divided the patients randomly into training and testing sets with equal number in each group. We identified 10 significant miRNAs using Cox regression analysis on the training set and formulated a risk score based on the expression signature of these miRNAs that segregated the patients into high and low risk groups with significantly different survival times (hazard ratio [HR]=2.4; 95% CI=1.4-3.8; p<0.0001). Of these 10 miRNAs, 7 were found to be risky miRNAs and 3 were found to be protective. This signature was independently validated in the testing set (HR=1.7; 95% CI=1.1-2.8; p=0.002). GBM patients with high risk scores had overall poor survival compared to the patients with low risk scores. Overall survival among the entire patient set was 35.0% at 2 years, 21.5% at 3 years, 18.5% at 4 years and 11.8% at 5 years in the low risk group, versus 11.0%, 5.5%, 0.0 and 0.0% respectively in the high risk group (HR=2.0; 95% CI=1.4-2.8; p<0.0001). Cox multivariate analysis with patient age as a covariate on the entire patient set identified risk score based on the 10 miRNA expression signature to be an independent predictor of patient survival (HR=1.120; 95% CI=1.04-1.20; p=0.003). Thus we have identified a miRNA expression signature that can predict GBM patient survival. These findings may have implications in the understanding of gliomagenesis, development of targeted therapy and selection of high risk cancer patients for adjuvant therapy.     

MicroRNA-10b pleiotropically regulates invasion, angiogenicity and apoptosis of tumor cells resembling mesenchymal subtype of glioblastoma multiforme

Glioblastoma multiforme (GBM) is a heterogeneous disease despite its seemingly uniform pathology. Deconvolution of The Cancer Genome Atlas''s GBM gene expression data has unveiled the existence of distinct gene expression signature underlying discrete GBM subtypes. Recent conflicting findings proposed that microRNA (miRNA)-10b exclusively regulates glioma growth or invasion but not both. We showed that silencing of miRNA-10b by baculoviral decoy vectors in a glioma cell line resembling the mesenchymal subtype of GBM reduces its growth, invasion and angiogenesis while promoting apoptosis in vitro. In an orthotopic human glioma mouse model, inhibition of miRNA-10b diminishes the invasiveness, angiogenicity and growth of the mesenchymal subtype-like glioma cells in the brain and significantly prolonged survival of glioma-bearing mice. We demonstrated that the pleiotropic nature of miRNA-10b was due to its suppression of multiple tumor suppressors, including TP53, FOXO3, CYLD, PAX6, PTCH1, HOXD10 and NOTCH1. In particular, siRNA-mediated knockdown experiments identified TP53, PAX6, NOTCH1 and HOXD10 as invasion regulatory genes in our mesenchymal subtype-like glioma cells. By interrogating the REMBRANDT, we noted that dysregulation of many direct targets of miRNA-10b was associated with significantly poorer patient survival. Thus, our study uncovers a novel role for miRNA-10b in regulating angiogenesis and suggests that miRNA-10b may be a pleiotropic regulator of gliomagenesis.